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Speech/Language Disorders Database

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Gene / phenotype associations for CYP19A1

  • Click column headers to sort. Click to expand any row to see more details about the particular assertion of an association between variants of CYP19A1 and a particular phenotypic variable.
  • Click the Pubmed IDs in the last column to link out to the primary research article. Click the links in the last column to download the full Genotype-Phenotype record as JSON or XML formatted text.
Entrez Id Symbol Location Disorder Brief Phenotype Reference Year Download
1588 CYP19A1 15q21.1 SSD Nonword repetition Anthoni et al 2012 JSON | XML

Additional Phenotype Details: 
Standardized tests for nonword repetition (NWR) include the Children's Test of Nonword Repetition (CNRep, Gathercole et al 1994), the nonword repetition test (NRT, Dollaghan & Campbell 1998), and the Nonword Repetition subtest of the Comprehensive Test of Phonological Processing (Wagner et al. 1999).

References

Gathercole SE, Willis CS, Baddeley AD, Emslie H. The Children’s Test of Nonword Repetition: a test of phonological working memory. Memory 1994;2:103-27.

Dollaghan, C., & Campbell, T. F. (1998). Nonword repetition and child language impairment. Journal of Speech, Language, and Hearing Research, 41, 1136–1146

Wagner, R. K., Torgesen, J. K., & Rashotte, C. A. (1999). Comprehensive test of phonological processing. Austin, TX: PRO-ED.

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs10046  (P = 0.00005)
rs8034835  (P = 0.00004)
rs700518  (P = 0.02)
rs934634  (P = 0.001)
rs2289105  (P = 0.005)
rs2470152  (P = 0.003)
rs2899472  (P = 0.009)
rs767199  (P = 0.006)
rs11632903  (P = 0.001)
rs936306  (P = 0.03)
rs2470176  (P = 0.04)
rs730154  (P = 0.05)


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1588 CYP19A1 15q21.1 SSD Digit span Anthoni et al 2012 JSON | XML

Additional Phenotype Details: 
May be measured using the digit span subtest of the Wechsler Intelligence Scale for Children

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs10046  (P = 0.04)
rs8034835  (P = 0.004)
rs1065778  (P = 0.02)
rs2289105  (P = 0.02)
rs767199  (P = 0.03)


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1588 CYP19A1 15q21.1 Dyslexia Full scale IQ Anthoni et al 2012 JSON | XML

Additional Phenotype Details: 
Tested using the Wechsler Adult Intelligence Scale (WAIS)

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs10046  (P = 0.02)
rs8034835  (P = 0.02)
rs1065778  (P = 0.02)
rs700518  (P = 0.04)
rs934634  (P = 0.04)
rs2289105  (P = 0.02)


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1588 CYP19A1 15q21.1 Dyslexia Susceptibility to developmental dyslexia Anthoni et al 2012 JSON | XML

Additional Phenotype Details: Typical diagnostic criteria for dyslexia include remarkable deviation from population mean on age-appropriate standardized reading and spelling tests, such as those in the Wechsler intelligence tests. Reading tests may include oral reading and non-word reading.

Usually, a Performance Intelligence Quotient (PIQ) of at least 70 or 80 on the age-appropriate Weschler test is also a criterion.

References (fourth editions of these tests are also now available):

Wechsler D. 1991. Wechsler intelligence scale for children- third edition (WISC-III). San Antonio: The Psychological Corporation.

Wechsler D. 1997. Wechsler Adult Intelligence Scale-III (WAIS-III). San Antonio: The Psychological Corporation.

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs8034835-rs2899472  (P = 0.039)  - G - C
rs11632903-rs1902586  (P = 0.023)  - T - G


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1588 CYP19A1 15q21.1 SSD Repetition of multisyllabic words Anthoni et al 2012 JSON | XML

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs10046  (P = 0.005)
rs8034835  (P = 0.007)
rs1065778  (P = 0.006)
rs700518  (P = 0.009)
rs2289105  (P = 0.002)
rs2470152  (P = 0.002)
rs767199  (P = 0.006)
rs936306  (P = 0.02)
rs2470176  (P = 0.03)
rs730154  (P = 0.001)


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1588 CYP19A1 15q21.1 Dyslexia Phoneme awareness Anthoni et al 2012 JSON | XML

Additional Phenotype Details: 

May be measured by any of several tasks, such as phoneme reversal or phoneme deletion. For example, Newbury et al 2011 used the Spoonerisms Test from the Phonological Assessment battery (PhAB), while Parracchini et al 2008 used a phoneme deletion test from the Auditory Analysis Task.

References

Gallagher A, Frederickson N (1995) The phonological assessment battery (PhAB): an initial assessment of its theoretical and practical utility. Educ Child Psychol 12:53–67

Newbury DF, Paracchini S, Scerri TS, Winchester L, Addis L, Richardson AJ, Walter J, Stein JF, Talcott JB, Monaco AP (2011) Investigation of dyslexia and SLI risk variants in reading- and language-impaired subjects. Behavior Genetics 41, 90-104.

Paracchini S, Steer CD, Buckingham LL, Morris AP, Ring S, Scerri T, Stein J, Pembrey ME, Ragoussis J, Golding J, Monaco AP (2008) Association of the KIAA0319 dyslexia susceptibility gene with reading skills in the general population. The American Journal Of Psychiatry 165, 1576-84.

Rosner J, Simon DP: The Auditory Analysis Test: an initial report. J Learning Disabilities 1971; 4:40–48

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs1065778  (P = 0.02)
rs934634  (P = 0.03)
rs11632903  (P = 0.03)
rs1004984  (P = 0.01)
rs2470144  (P = 0.04)
rs749292  (P = 0.03)


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1588 CYP19A1 15q21.1 SSD Listening comprehension Anthoni et al 2012 JSON | XML

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs2470152  (P = 0.02)
rs2899472  (P = 0.02)


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1588 CYP19A1 15q21.1 Dyslexia Spelling ability Anthoni et al 2012 JSON | XML

Additional Phenotype Details: Possible measures include the spelling subtests of any of a number of intelligence and literacy tests, e.g.:

Peabody Individual Achievement Test (PIAT)
Western Australian Literacy and Numeracy Assessment (WALNA)
Wide Range Achievement Test (WRAT)

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs10046  (P = 0.002)
rs8034835  (P = 0.02)
rs1065778  (P = 0.04)
rs934634  (P = 0.01)
rs2289105  (P = 0.05)
rs11632903  (P = 0.01)
rs1004984  (P = 0.03)
rs749292  (P = 0.02)


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1588 CYP19A1 15q21.1 SSD Reading comprehension Anthoni et al 2012 JSON | XML

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs11632903  (P = 0.005)


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1588 CYP19A1 15q21.1 Dyslexia Phonological processing Anthoni et al 2012 JSON | XML

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs934634  (P = 0.008)
rs2470152  (P = 0.05)
rs1004984  (P = 0.01)
rs2470144  (P = 0.01)


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1588 CYP19A1 15q21.1 SSD Susceptibility to developmental dyslexia Anthoni et al 2012 JSON | XML

Additional Phenotype Details: Typical diagnostic criteria for dyslexia include remarkable deviation from population mean on age-appropriate standardized reading and spelling tests, such as those in the Wechsler intelligence tests. Reading tests may include oral reading and non-word reading.

Usually, a Performance Intelligence Quotient (PIQ) of at least 70 or 80 on the age-appropriate Weschler test is also a criterion.

References (fourth editions of these tests are also now available):

Wechsler D. 1991. Wechsler intelligence scale for children- third edition (WISC-III). San Antonio: The Psychological Corporation.

Wechsler D. 1997. Wechsler Adult Intelligence Scale-III (WAIS-III). San Antonio: The Psychological Corporation.

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs8034835-rs2899472-rs1065778-rs700518  (P = 0.032)  - G - C - A - G
rs1902586-rs936306-rs2470176-rs2470152  (P = 0.023)  - G - C - A - G


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1588 CYP19A1 15q21.1 Dyslexia Reading comprehension Anthoni et al 2012 JSON | XML

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs10046  (P = 0.05)
rs8034835  (P = 0.007)
rs1065778  (P = 0.0006)
rs2470152  (P = 0.01)


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1588 CYP19A1 15q21.1 Dyslexia Oral reading Anthoni et al 2012 JSON | XML

Additional Phenotype Details: May be assessed with the passages reading task of the Gray oral reading test (GORT).

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs10046  (P = 0.002)
rs8034835  (P = 0.002)
rs1065778  (P = 0.003)
rs700518  (P = 0.0004)  - Remained significant at <0.05 level after correction for multiple testing
rs934634  (P = 0.01)
rs2289105  (P = 0.01)
rs2470152  (P = 0.04)
rs3575192  (P = 0.05)


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1588 CYP19A1 15q21.1 SSD Performance IQ Anthoni et al 2012 JSON | XML

Additional Phenotype Details: 
The part of the WAIS-III that tested Perceptual Organization and Processing Speed. The Perceptual Organization Index was determined by Block Design, Matrix Reasoning, and Picture Completion. The Processing Speed Index was determined by Digit Symbol-Coding and Symbol Search.

Reference

Wechsler D. 1997. Wechsler Adult Intelligence Scale-III (WAIS-III). San Antonio: The Psychological Corporation.

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs8034835  (P = 0.001)
rs934634  (P = 0.007)
rs936306  (P = 0.04)


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1588 CYP19A1 15q21.1 SSD Vocabulary Anthoni et al 2012 JSON | XML

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs10046  (P = 0.000007)
rs2289105  (P = 0.00003)
rs936306  (P = 0.003)
rs2470176  (P = 0.02)


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1588 CYP19A1 15q21.1 SSD Rate of repetition of single syllables Anthoni et al 2012 JSON | XML

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs1065778  (P = 0.05)
rs700518  (P = 0.03)
rs2289105  (P = 0.04)
rs1004984  (P = 0.000004)


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1588 CYP19A1 15q21.1 SSD Rate of repetition of double syllables Anthoni et al 2012 JSON | XML

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs10046  (P = 0.05)
rs8034835  (P = 0.007)
rs1065778  (P = 0.00004)
rs700518  (P = 0.0000001)
rs934634  (P = 0.02)
rs2289105  (P = 0.000002)
rs2899472  (P = 0.002)
rs11632903  (P = 0.004)
rs1902586  (P = 0.002)
rs1004984  (P = 0.007)


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1588 CYP19A1 15q21.1 SSD Sentence imitation Anthoni et al 2012 JSON | XML

Basic Study Type:  - FISH (fluorescent in situ hybridization) - Association study - Evolutionary sequence analysis - Candidate gene sequencing in nonhuman primates - Electrophoretic mobility shift assay (EMSA) - Brain expression anaylsis - In vitro study: Rat hippocampal neurons - Aromatase knockout mice

Study Cohort: 
FISH study:

One dyslexic individual with t(2;15)(p12;q21) (Nopola-Hemmi et al. 2000)

Association study:

"Six cohorts of family-based material of Caucasian origin (Table 1) were genotyped for 16–20 SNPs located in the CYP19A1 gene." Four were dyslexia cohorts: Finland, Germany, Colorado (US), Georgia (US). One was an SLI cohort (Iowa, US) and one was an SSD cohort (Ohio, US).

The quantitative trait analysis used data from all six cohorts. The categorical association study used data from the Finnish, German, and Georgia dyslexia cohorts, and the Ohio SSD cohort.

Finnish dyslexia cohort (FI, DYS)
"Nineteen Finnish three-generation families (130 subjects; Table 1), of Caucasian origin."

German dyslexia cohort (GER, DYS)
"A total of 411 trios of German Caucasian origin (1,233 individuals totally; Table 1) were genotyped. . .The families were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Wu¨rzburg.

Colorado dyslexia cohort (CO, US, DYS)
"This population was recruited through the Colorado Learning Disabilities Research Center and included 216 nuclear families with a total of 880 genotyped individuals (Table 1). . .This sample set is made up of ~89% Caucasian, ~3% African-American, Asian, or Native American, and ~8% self-identified as mixed."

Georgia dyslexia cohort (GA, US, DYS)
"Seventeen Caucasian families of US Caucasian origin and consisting of 57 subjects (Table 1) were studied. Families were recruited and referred through schools, physicians, and community announcements at the Center for Clinical and Developmental Neuropsychology (CCDN) at the University of Georgia."

Specific language impairment cohort (IA, US, SLI)
"The Iowa cohort consisted of 573 participants of Caucasian origin, all members of an ongoing longitudinal study of children with SLI (Table 1) and a control group of typically developing age mates. The longitudinal cohort was obtained from a large population sample (N = 7,206) of monolingual English speaking kindergarten-age children from Iowa, who participated in a cross-sectional epidemiologic study of SLI. . .The members of this longitudinal cohort initially consisted of 604 children and slightly more than one-third presented with language impairment as 6-year-olds, and the remaining represented a random sample of typically developing age-mates."

Speech sound disorder cohort (OH, US, SSD)
"One-hundred-and-eighteen Caucasian families of US origin consisting of 550 subjects (80 affected with both SSD and dyslexia, 147 affected with only SSD, 41 affected with only dyslexia, 274 unaffected with either SSD or dyslexia and eight of unknown phenotype) were genotyped (Table 1)."

Genotyping Methods: 
FISH and Southern blotting

"For the mapping of the translocation breakpoints, 10 BAC clones from chromosome 2 (RP11-502A5,
-419E14,-332A19, -89C12, -236I9, -521O14, -351F21, -1290B4, -548D17 and -513019; BACPAC Resource Center (BPRC) at Children’s Hospital Oakland Research Institute, Oakland, CA, USA) and 12 clones from chromosome 15 (RP11-10D13, -13H19, -56B16, -96N2, -108K3, -145A4, -209K10, -394B5, -430B1, -519C12, -522G20 and -540E17; Genome Systems, St Louis, MO, USA) were used as probes in FISH. Bacterial cultures and DNA isolation were performed according to standard protocols and probes were labeled by nick translation with FITC-dUTP (NEN Life Science Products, Boston, MA, USA), SpectrumOrange-dUTP (Vysis Inc, Downers Grove, IL), or biotin-14-dATP (detection with avidin conjugated FITC). FISH-analyses were performed according to standard protocols and the slides were analyzed on a Zeiss Axioplan 2 epifluorescence microscope (Carl Zeiss, Go¨ttingen, Germany). Images were captured using a cooled CCD camera (Sensys Photometrics, Mu¨nchen, Germany) and Smart-Capture 2 (DigitalScientific Ltd., Cambridge, UK) or ISIS software (Metasystems GmbH, Altlussheim, Germany).
"Genomic DNA (15 lg) from the individual carrying the translocation and from an unrelated control were digested with BamHI, EcoRI, HindIII, KpnI, SacI, ScaI and SphI and subjected to electrophoresis and Southern hybridization as previously described (Taipale et al. 2003). PCRamplified genomic fragments from non-repetitive regions of the BAC clone RP11-108K3 were used as hybridization probes. PCR and labelling reactions were performed as previously described (Hannula-Jouppi et al. 2005). Putative genes/exons from the 200 kb BAC clone spanning the breakpoint on chromosome 2 were in silico predicted using Genscan (genes.mit.edu/GENSCAN.html) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of each of the 19 predicted genes/exons were tested by PCR on human cDNA libraries from fetal brain (cat. No. HL5504u, Clontech and cat. No. 052001b, Stratagene) and from leukocytes (cat. No. HL5509u and HL5019t, Clontech)."

Association study:

"In the Finnish (FI, DYS) and Georgia (GA, US, DYS) dyslexia cohorts, 20 SNPs were genotyped using matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry as previously described (Peyrard-Janvid et al. 2004). PCR assays and extension primers were designed using the SpectroDESIGNER software (Sequenom). The same procedure was applied to the German (GER, DYS) cohort for 16 of those 20 SNPs (all except rs934634, rs700519, rs749292 and rs3575192). For the Colorado dyslexia (CO, US, DYS), the SSD (OH, US, SSD) and the SLI (IA, US, SLI) cohorts, genotype data for 16 of those 20 SNPs (all except rs700519, rs6493494, rs749292 and rs3575192) were successfully generated using the 50 exonuclease TaqMan Assay by Design or Assays in Demand from Applied Biosystems (Foster City, CA, USA). Real-time PCR was conducted using the ABI 7700HT system. Genotypes were assigned with the SDS 2.0 software (Applied Biosystems).
"CEPH genomic DNA, negative controls and replicates of some samples were included on each plate to assure consistency of the genotype calls. Discrepancies in genotype calls and Mendelian errors were identified using the PEDCHECK (O’Connell and Weeks 1998) and the MARKERINFO from the S.A.G.E. program package. All genotypes were independently confirmed by two investigators. Genotyping results were also cross-validated by duplicate genotyping of 10–96 samples across the different laboratories. Allele frequencies were also checked to match across the different data sets."

Analysis Methods: 
- Categorical association study: Pedigree disequilibrium test (PDTPHASE)

"Testing for Hardy–Weinberg equilibrium was done via a Chi-squared goodness-of-fit test using only the founders to eliminate the non-independence owing to family data. Intermarker LD was visualized and pairwise R2 values were determined using the Haploview v3.2 software (Barrett et al. 2005).
"PDTPHASE v2.4 from the software package UNPHASED (Dudbridge 2003) was used to test for both single SNP and haplotype association with binary traits in all three populations, i.e. Finnish, German and North-American. This program is an implementation of the original PDT (Martin et al. 2000) but allowing missing data. Haplotypes were looked at in two- to four-marker sliding window."

- Quantitative trait analysis: Variance component test of association (likelihood ratio test)

"A variance-component model developed for family-based association was used to assess single SNP significance of QTs in the GA, US, DYS and OH, US, SSD cohorts, as well as in both cohorts combined. This method assesses association between a marker and phenotype, while simultaneously estimating residual and multifactorial (polygenic, familial, and marital) variance components. Age was found to be significant in both populations and therefore was included in the baseline model as a covariate. At each SNP and for each trait, we tested for an additive, a dominant or a recessive allele effect. These three tests are correlated with each other and, because any two of these null hypotheses imply the third, they effectively count as two independent tests (Elston et al. 1999). Therefore, in each population and for each trait, the total number of independent tests performed is equal to twice the number of SNPs genotyped. To account for these multiple tests when determining allelic association to a trait, Sidak’s correction was used (Sidak 1967).
"Because the same reading test (WRMT-R, see Supplementary Table 9) was administered to participants in the GA, US, DYS and the OH, US, SSD cohorts, and the definition of dyslexia used to classify participants as affected was identical across the two cohorts, we combined p-values from tests of allelic association using Fisher’s method (Fisher 1948)."

- Evolutionary sequence analysis: GenomeVISTA alignment

- Candidate gene sequencing: Coding exons of CYP10A1 and brain specific exon/promoter I.f sequenced in chimpanzee, bonobo, gorilla, and orangutan

-EMSA experiments: Used nuclear and whole-cell extracts from human neuroblastoma cell line (SH-SY5Y)

- Brain expression study: Correlation between CYP19A1 and other dyslexia-associated genes

- In vitro effect of testosterone on rat hippocampal neuron process outgrowth (dependence on aromatase): ANOVA, t-test

- Aromatase knockout mice: Student's t-test.

Other Details: 
Diagnostic and inclusion criteria:

Finnish dyslexia cohort
See Nopola-Hemmi et al. 2001.

German dyslexia cohort
"The diagnostic inclusion criteria and phenotypic measures have been described in detail previously (Schulte-Korne et al. 1996, 2001, 2007; Ziegler et al. 2005; Schumacher et al. 2006). Briefly, the diagnosis of dyslexia was based on the spelling score using the T distribution of the general population. Based on the correlation between IQ and spelling of 0.4 (Schulte-Korne et al. 2001), an anticipated spelling score was calculated. The child was classified as dyslexic if the discrepancy between the anticipated and the observed spelling score was at least one standard deviation. Probands and all siblings fulfilling the inclusion criteria were assessed with several psychometric tests. These tests targeted different aspects of the dyslexia, i.e. word reading, phonological awareness and short term memory (see Supplementary Table 9)."

Colorado dyslexia cohort
"Ascertainment and evaluation of this population has been described previously (DeFries et al. 1997). Briefly, families were selected through twins living in Colorado, at least one of whom had a history of reading problems by school report and confirmed by school records. Exclusion criteria included a full scale IQ score less than 80 and any sensory or medical problems that would interfere with reading. The twins and available siblings were given an extensive battery of assessments of reading, spelling, phonology, orthography, rapid naming, and intelligence."

Georgia dyslexia cohort
"All qualifying families had at least one proband between the ages of 8 and 12 years with significant reading problems and no history of neurological impairment, traumatic brain injury, psychiatric disorders, or severe pre- and/or perinatal complications. . .The test battery consisted of measures designed to assess intelligence, academic achievement, receptive and expressive language, phonological processing, memory, reading, spelling, visual-spatial ability, executive functioning, handedness, and social-emotional functioning (see Supplementary Table 9 for a full description of the tests used)."

Iowa SLI cohort
"All children had normal hearing and no diagnosis of neurodevelopmental disorders. A description of the sampling methods for the original cross-sectional sample and selection of the longitudinal sample have been described previously (Tomblin et al. 1997, 2000). . .The phenotypic data for the current study were collected when the participants were in kindergarten and later in second grade. The speech sound production data were obtained when the children were in kindergarten. The remaining behavioral phenotypic measures including receptive and expressive language were obtained in second grade at which time the children had been receiving reading instruction for approximately 2 years."

Ohio SSD cohort
"Probands were enrolled in speech-language therapy for a moderate to severe speech sound production disorder. Children were also required to have normal hearing, intelligence, and speech mechanism (adequate oral structures for producing speech sounds). An extensive battery of standardized speech sound production, receptive and expressive language, reading decoding and comprehension, spelling, oral-motor skills, memory, and phonological processing measures were administered to all probands and their siblings of 4 years of age and older (see Supplementary Table 9 for a listing of the specific measures)."

Associated Markers:
rs934634  (P = 0.03)
rs2899472  (P = 0.02)


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