SLDB

Speech/Language Disorders Database

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Gene / phenotype associations for GCFC2

  • Click column headers to sort. Click to expand any row to see more details about the particular assertion of an association between variants of GCFC2 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
6936 GCFC2 2p12 Dyslexia Susceptibility to developmental dyslexia Anthoni et al 2007 JSON | XML

Conflicting Studies:

  • Newbury et al (2011) - Failed to replicate association with 4 SNPs in MRPL19/C2ORF3 (aka GCFC2) region and dyslexia in a group of 188 probands and 331 unrelated UK dyslexic cases
  • Newbury et al (2011) - Failed to replicate association with 4 SNPs in MRPL19/C2ORF3 (aka GCFC2) region and dyslexia in a group of 188 probands and 331 unrelated UK dyslexic cases
  • Paracchini et al (2011) - Tested association of SNPs within MRPL19/C2ORF3 (aka GCFC2) and quantitative measures of reading and spelling in a sample of 520 Australians (the Raine cohort). No significant association was detected for any individual SNPs nor for haplotype analysis.
  • Scerri et al (2011) - No evidence of association with dyslexia categorical variable for rs917235 in pure dyslexia or dyslexia / SLI cases vs. controls in UK ALSPAC cohort
  • 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:  - Association study - Brain expression analysis - Comparison of expression levels across individuals - Sequencing in non-human primates

    Study Cohort: 
    Summary:

    (1) Finnish sample set: 19 families, 130 individuals (50 affected, 63 healthy, 17 unknown)

    (2) German sample set: 251 families, 1050 individuals (429 affected, 119 healthy sibs and 502 parents of unknown phenotype)

    Details:

    "Eleven Finnish three-generation pedigrees consisting of 83 subjects (34 affected, 41 healthy, 8 phenotype unknown) were genotyped in the first round of fine mapping. Kindreds have been partly described previously (16), but because of sample and/or phenotype availability, 13 more subjects were included. In the second round of fine mapping, eight additional families (47 individuals, 16 affected, 22 healthy, nine unknown) were added to the analysis. This expanded the Finnish sample set to 130 individuals (50 affected, 63 healthy, 17 unknown). All phenotypes were ascertained as previously described (29).

    "The replication set contained 251 German families. The sample set consisted of altogether 1050 individuals, with 429 affected (251 probands and 178 sibs), 119 healthy sibs and 502 of unknown phenotype (all parents). The samples were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg (59%) and Wu¨rzburg (41%). The diagnostic criteria and phenotypic measures have been described in detail previously (30)."

    Genotyping Methods: 
    Genotyping

    "In the first stage of fine mapping, eight microsatellite markers (D2S2109, D2S438, D2S1262, D2S253, D2S289, D2S2162, D2S435, D2S394) and 24 SNPs with an average spacing of 225 kb (range 42–556 kb) were genotyped in the Finnish kindreds. Twenty of these SNPs were genotyped in the German samples. In the second stage of fine mapping, 15 additional SNPs were selected over the 157 kb candidate region and genotyped in the full Finnish and German sample sets. We also tagged the LRRTM4 gene using three additional SNPs (rs654148, rs2901848, rs2178759) and moreover, 11 cSNPs with minor allele frequency >5% were genotyped in the full sample set of Finnish and German families. Altogether, nine SNPs with low success rates were removed from analysis.

    "All SNPs were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF, Sequenom) as described previously (19), by sequencing, or by PCR amplification and visualization in agarose gels (19 bp DIP ss49855073). The Sequenom assays were designed using the SpectroDESIGNER software and are available upon request. A genotyping success rate of 80% was required for inclusion in analyses. All genotypes were independently confirmed by two investigators. Data were checked for Mendelian consistency using Pedcheck (33), and unresolved inconsistencies were assigned as missing genotypes."

    PCR and sequencing reactions

    "PCRs (all primer sequences available upon request) were carried out in 10–25 µl reactions containing 0.5–1 ng/µl of genomic DNA, 1.5–3 mM MgCl2, 0.4 mM of each dNTP, 0.8 µM of each primer and 0.03 U/µl of HotStarTaq DNA polymerase (Qiagen). We used a touch-down protocol with 42 cycles of amplification with 18°C of decrease in annealing temperature at each round; two cycles at 63°C and at 62°C, respectively, three cycles at 61°C and 56°C, respectively and 10 cycles at 55°C and 54°C, respectively. PCR cycles had an initial denaturation at 95°C for 15 min, 30 s at each annealing temperature and 30 s to 1 min 30 s elongation at 72°C with a final extension of 10 min at 72°C. Primate DNA PCR was carried out following the touch-down protocol but ending at 55°C for 25 cycles.

    "PCR products were dephosphorylated by 0.4 U/µl shrimp alkaline phosphatase (Amersham Biosciences/GE) and 2 U/µl exonuclease I (New England BioLabs) and were further sequenced using DYEnamic ET Dye terminator kit (Amersham Biosciences/GE) following the manufacturer’s instructions. Each fragment was sequenced in both directions using the amplification primers. Purified sequencing products were resolved using a MegaBACE 1000 instrument and Mega-BACE long-read matrix (Amersham Biosciences/GE), visualized using the Sequence Analyzer v3.0 software (Amersham Biosciences/GE), and assembled and analyzed using the Pregap and Gap4 software (www.cbi.pku.edu.cn/tools/staden), comparing to the sequence NT_022184.14, build 35 (www.ncbi.nih.gov). Sequences were verified visually by two independent readers.

    "Genomic sequencing was performed for two affected individuals homozygous for the susceptibility haplotype, one of German and one of Finnish descent, and one affected individual (German) homozygous for the opposite non-risk haplotype. The ~86 kb genomic sequence was first masked for repeats (woody.embl-heidelberg.de/repeatmask) and the unique segments (~46 kb) were sequenced. In total, 109 fragments (200–1000 bp, with 100–200 bp overlaps) were amplified by PCR and sequenced as described above.

    "Four candidate genes, LRRTM4, CTNNA2, MRPL19 and C2ORF3 were screened for polymorphisms by direct sequencing of all their coding exons and exon–intron junctions. Nineteen affected individuals were sequenced (11 for CTNNA2). The human primers were used to sequence MRPL19 and C2ORF3 in Gorilla (Gorilla gorilla), chimpanzee (Pan troglodytes), pigmy chimpanzee (Pan paniscus) and orangutan (Pongo pygmaeus) DNA samples (Primate panel PRP00001 IPBIR, Camden, New Jersey, USA)."

    Analysis Methods: 
    "TDT (34) was used to test for single marker as well as for haplotype (two to four markers) associations. Phased haplotypes and global P-values were obtained using TDTHAP (35). To assess global P-values, 50 000 permutations were run.

    "Intermarker LD was visualized and haplotype blocks were constructed using the Haploview3.2 software (36).

    Other Details: 
    For details on diagnostic criteria on the Finnish sample, see Nopola-Hemmi et al (2001), "A dominant gene for developmental dyslexia on chromosome 3," J. Med. Genet., 38, 658–664.

    For details on diagnosis of the German sample, see Ziegler et al 2004, "Developmental dyslexia-Recurrence risk estimates from a German bi-center study using the single proband sib pair design," Hum. Hered., 59, 136-143.

    For the Finnish sample, the following tests were used for adults and children, respectively:

    Leinonen S, Muller K, Leppänen P, Aro M, Ahonen T, Lyytinen H. Heterogeneity in adult dyslexic readers: relating processing skills to the speed and accuracy of oral test reading. Reading and Writing:an Interdisciplinary Journal (in press).


    Häyrinen T, Serenius-Sirve S, Korkman M. Lukilasse. Lukemisen, kirjoittamisen ja laskemisen seulontatestistö peruskoulun ala-asteen luokille 1-6. (Reading and writing test designed
    for and normated in Finnish elementary school.) Helsinki: Psykologien kustannus Oy, 1999.


    For the German sample:

    Schulte-Körne G: Legasthenie und Sprachwahrnehmung. Münster, Waxmann, 2001.


    Gene characterization and expression analysis of
    FLJ13391, MRPL19 and C2ORF3

    "The gene structures were verified and improved by fully sequencing I.M.A.G.E. clones BC030144 (primary B-cells from tonsils) for MRPL19; BF665321 (primitive neuroectoderm), AI816424 (fetal frontal lobe), BI457763 (hypothalamus) and BF966531 (hippocampus) for C2ORF3; and BC063016 (pooled pancreas and spleen) for FLJ13391. To confirm the 5' end of MRPL19 and C2ORF3 genes, we performed 5' RACE experiments using Marathon-Ready cDNA from fetal brain tissue (cat. no. 639302; Clontech) following the manufacturer’s instructions.

    "The expression of the three genes was studied by PCR on human cDNA libraries from fetal brain (human fetal brain large-insert cDNA library; cat. no. HL5504u, Clontech; human fetal brain Uni-ZAP XR library, cat. no. 052001b; Stratagene) and from leukocytes (human leukocyte large-insert
    cDNA library, cat. no. HL5509u, Clontech; human leukocyte 5' STRETCH PLUS cDNA library, cat. no. HL5019t, Clontech). PCR products were visualized by agarose gel electrophoresis and further sequenced.

    "Putative new genes/exons from the 86 kb sequence between FLJ13391 and MRPL19 were predicted in silico with Genscan (genes.mit.edu/GENSCAN.html and vega.sanger.ac.uk/Homo_sapiens) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of all 27 predicted genes/exons was then tested by PCR on the four human brain and leukocyte cDNA libraries. One gene prediction (GENSCAN59094, vega.sanger.ac.uk/Homo_sapiens) was thoroughly tested by screening >1 000 000 clones from each of two human fetal brain cDNA libraries because of its overlap with the risk haplotype at rs1000585-rs917235-rs714939.

    "Ready-made TaqMan gene expression assays for FLJ13391 (Hs00259924_m1), MRPL19 (Hs00608519_m1), C2ORF3 (Hs00162632_m1), DYX1C1 (Hs00370049_m1), DCDC2 (Hs00393203_m1), KIAA0319 (Hs00207788_m1), ROBO1 (Hs00268049_m1), GAPDH (4310884E) and 18S rRNA (4319413E) were purchased from Applied Biosystems. We assayed expression levels for these genes in total RNA from nine different areas of adult human brain: thalamus, hypothalamus, frontal-, occipital-, parietal-, temporal cortex (cat. nos 6762, 6864, 6810, 6812, 6814, 6816; Ambion), hippocampus, paracentral- and post-central gyrus (cat. nos 636565, 636574, 636573; Clontech), and from whole adult and fetal brain (cat. nos 636530, 636526; Clontech). For each tissue, three independent cDNA syntheses (500 ng total RNA per reaction) were performed using the SuperScript III first-strand synthesis kit (cat. no. 18080-051; Invitrogen). From each cDNA synthesis, quantitative real-time PCR was performed in quadruplets, using 5 ng of RNA per gene assay and run on ABI PRISM 7700 Sequence Detection PCR System (Applied Biosystems). All assays were performed in 10 µl reactions according to the manufacturer’s instructions. Relative standard expression curves were drawn for 18S rRNA and all tested genes. Relative quantification of the data was performed using the comparative Ct (threshold cycle) method (Sequence Detection System bulletin 2, Applied Biosystems). Ct values were adjusted to 18S rRNA and thereafter normalized to the whole brain sample.

    "To quantify mRNA expression levels from each allele of MRPL19 and C2ORF3, we analyzed individuals heterozygous for rs17689863 (Ser277Ser) in MRPL19 and rs1803196 (Val536Val) in C2ORF3. Five Finnish dyslexic and six normal readers, and three German dyslexic and one normal reader were studied. Total RNA was extracted from EBV-transformed lymphocyte cell lines using the RNeasy purification kit (cat. no. 74004; Qiagen) and cDNA synthesis (500 ng of total RNA per reaction) was performed using the SuperScript III first-strand synthesis kit (cat. nos 18080-051 and 12371-019; Invitrogen; for the Finnish and the German samples, respectively). Both cDNA and genomic DNA from each individual were sequenced in six independent reactions, originating from at least two separate PCR amplifications. Peak heights were compared and an allelic ratio was calculated for each sequence. The cDNA ratio values (unknown proportions) were normalized by dividing with the genomic values (1:1 proportion by definition). Data were pooled by genotype (risk haplotype heterozygotes versus non-risk haplotype heterozygotes) to evaluate (by two-tailed t-test) whether the normalized value differed from equal expression."

    "Evolutionary analysis of the MRPL19 and C2ORF3 genes was performed with an LRT using the CODEML program of the paml3.15 package (22). Mouse sequence (ENSMUSP-00000032124) was used as out-group for MRPL19, and dog (XP_540209) for C2ORF3."

    Associated Markers:
    rs1000585-rs917235-rs714939  (P = 0.005)  - Pooled across Finnish and German populations
    rs917235-rs714939-rs6732511  (P = 0.001)  - Pooled across Finnish and German populations


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    6936 GCFC2 2p12 Dyslexia Attenuated expression in heterozygous carriers of the dyslexia risk haplotype Anthoni et al 2007 JSON | XML

    Basic Study Type:  - Association study - Brain expression analysis - Comparison of expression levels across individuals - Sequencing in non-human primates

    Study Cohort: 
    Summary:

    (1) Finnish sample set: 19 families, 130 individuals (50 affected, 63 healthy, 17 unknown)

    (2) German sample set: 251 families, 1050 individuals (429 affected, 119 healthy sibs and 502 parents of unknown phenotype)

    Details:

    "Eleven Finnish three-generation pedigrees consisting of 83 subjects (34 affected, 41 healthy, 8 phenotype unknown) were genotyped in the first round of fine mapping. Kindreds have been partly described previously (16), but because of sample and/or phenotype availability, 13 more subjects were included. In the second round of fine mapping, eight additional families (47 individuals, 16 affected, 22 healthy, nine unknown) were added to the analysis. This expanded the Finnish sample set to 130 individuals (50 affected, 63 healthy, 17 unknown). All phenotypes were ascertained as previously described (29).

    "The replication set contained 251 German families. The sample set consisted of altogether 1050 individuals, with 429 affected (251 probands and 178 sibs), 119 healthy sibs and 502 of unknown phenotype (all parents). The samples were recruited from the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg (59%) and Wu¨rzburg (41%). The diagnostic criteria and phenotypic measures have been described in detail previously (30)."

    Genotyping Methods: 
    Genotyping

    "In the first stage of fine mapping, eight microsatellite markers (D2S2109, D2S438, D2S1262, D2S253, D2S289, D2S2162, D2S435, D2S394) and 24 SNPs with an average spacing of 225 kb (range 42–556 kb) were genotyped in the Finnish kindreds. Twenty of these SNPs were genotyped in the German samples. In the second stage of fine mapping, 15 additional SNPs were selected over the 157 kb candidate region and genotyped in the full Finnish and German sample sets. We also tagged the LRRTM4 gene using three additional SNPs (rs654148, rs2901848, rs2178759) and moreover, 11 cSNPs with minor allele frequency >5% were genotyped in the full sample set of Finnish and German families. Altogether, nine SNPs with low success rates were removed from analysis.

    "All SNPs were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF, Sequenom) as described previously (19), by sequencing, or by PCR amplification and visualization in agarose gels (19 bp DIP ss49855073). The Sequenom assays were designed using the SpectroDESIGNER software and are available upon request. A genotyping success rate of 80% was required for inclusion in analyses. All genotypes were independently confirmed by two investigators. Data were checked for Mendelian consistency using Pedcheck (33), and unresolved inconsistencies were assigned as missing genotypes."

    PCR and sequencing reactions

    "PCRs (all primer sequences available upon request) were carried out in 10–25 µl reactions containing 0.5–1 ng/µl of genomic DNA, 1.5–3 mM MgCl2, 0.4 mM of each dNTP, 0.8 µM of each primer and 0.03 U/µl of HotStarTaq DNA polymerase (Qiagen). We used a touch-down protocol with 42 cycles of amplification with 18°C of decrease in annealing temperature at each round; two cycles at 63°C and at 62°C, respectively, three cycles at 61°C and 56°C, respectively and 10 cycles at 55°C and 54°C, respectively. PCR cycles had an initial denaturation at 95°C for 15 min, 30 s at each annealing temperature and 30 s to 1 min 30 s elongation at 72°C with a final extension of 10 min at 72°C. Primate DNA PCR was carried out following the touch-down protocol but ending at 55°C for 25 cycles.

    "PCR products were dephosphorylated by 0.4 U/µl shrimp alkaline phosphatase (Amersham Biosciences/GE) and 2 U/µl exonuclease I (New England BioLabs) and were further sequenced using DYEnamic ET Dye terminator kit (Amersham Biosciences/GE) following the manufacturer’s instructions. Each fragment was sequenced in both directions using the amplification primers. Purified sequencing products were resolved using a MegaBACE 1000 instrument and Mega-BACE long-read matrix (Amersham Biosciences/GE), visualized using the Sequence Analyzer v3.0 software (Amersham Biosciences/GE), and assembled and analyzed using the Pregap and Gap4 software (www.cbi.pku.edu.cn/tools/staden), comparing to the sequence NT_022184.14, build 35 (www.ncbi.nih.gov). Sequences were verified visually by two independent readers.

    "Genomic sequencing was performed for two affected individuals homozygous for the susceptibility haplotype, one of German and one of Finnish descent, and one affected individual (German) homozygous for the opposite non-risk haplotype. The ~86 kb genomic sequence was first masked for repeats (woody.embl-heidelberg.de/repeatmask) and the unique segments (~46 kb) were sequenced. In total, 109 fragments (200–1000 bp, with 100–200 bp overlaps) were amplified by PCR and sequenced as described above.

    "Four candidate genes, LRRTM4, CTNNA2, MRPL19 and C2ORF3 were screened for polymorphisms by direct sequencing of all their coding exons and exon–intron junctions. Nineteen affected individuals were sequenced (11 for CTNNA2). The human primers were used to sequence MRPL19 and C2ORF3 in Gorilla (Gorilla gorilla), chimpanzee (Pan troglodytes), pigmy chimpanzee (Pan paniscus) and orangutan (Pongo pygmaeus) DNA samples (Primate panel PRP00001 IPBIR, Camden, New Jersey, USA)."

    Analysis Methods: 
    "TDT (34) was used to test for single marker as well as for haplotype (two to four markers) associations. Phased haplotypes and global P-values were obtained using TDTHAP (35). To assess global P-values, 50 000 permutations were run.

    "Intermarker LD was visualized and haplotype blocks were constructed using the Haploview3.2 software (36).

    Other Details: 
    For details on diagnostic criteria on the Finnish sample, see Nopola-Hemmi et al (2001), "A dominant gene for developmental dyslexia on chromosome 3," J. Med. Genet., 38, 658–664.

    For details on diagnosis of the German sample, see Ziegler et al 2004, "Developmental dyslexia-Recurrence risk estimates from a German bi-center study using the single proband sib pair design," Hum. Hered., 59, 136-143.

    For the Finnish sample, the following tests were used for adults and children, respectively:

    Leinonen S, Muller K, Leppänen P, Aro M, Ahonen T, Lyytinen H. Heterogeneity in adult dyslexic readers: relating processing skills to the speed and accuracy of oral test reading. Reading and Writing:an Interdisciplinary Journal (in press).


    Häyrinen T, Serenius-Sirve S, Korkman M. Lukilasse. Lukemisen, kirjoittamisen ja laskemisen seulontatestistö peruskoulun ala-asteen luokille 1-6. (Reading and writing test designed
    for and normated in Finnish elementary school.) Helsinki: Psykologien kustannus Oy, 1999.


    For the German sample:

    Schulte-Körne G: Legasthenie und Sprachwahrnehmung. Münster, Waxmann, 2001.


    Gene characterization and expression analysis of
    FLJ13391, MRPL19 and C2ORF3

    "The gene structures were verified and improved by fully sequencing I.M.A.G.E. clones BC030144 (primary B-cells from tonsils) for MRPL19; BF665321 (primitive neuroectoderm), AI816424 (fetal frontal lobe), BI457763 (hypothalamus) and BF966531 (hippocampus) for C2ORF3; and BC063016 (pooled pancreas and spleen) for FLJ13391. To confirm the 5' end of MRPL19 and C2ORF3 genes, we performed 5' RACE experiments using Marathon-Ready cDNA from fetal brain tissue (cat. no. 639302; Clontech) following the manufacturer’s instructions.

    "The expression of the three genes was studied by PCR on human cDNA libraries from fetal brain (human fetal brain large-insert cDNA library; cat. no. HL5504u, Clontech; human fetal brain Uni-ZAP XR library, cat. no. 052001b; Stratagene) and from leukocytes (human leukocyte large-insert
    cDNA library, cat. no. HL5509u, Clontech; human leukocyte 5' STRETCH PLUS cDNA library, cat. no. HL5019t, Clontech). PCR products were visualized by agarose gel electrophoresis and further sequenced.

    "Putative new genes/exons from the 86 kb sequence between FLJ13391 and MRPL19 were predicted in silico with Genscan (genes.mit.edu/GENSCAN.html and vega.sanger.ac.uk/Homo_sapiens) and GrailEXP (grail.lsd.ornl.gov/grailexp). The expression of all 27 predicted genes/exons was then tested by PCR on the four human brain and leukocyte cDNA libraries. One gene prediction (GENSCAN59094, vega.sanger.ac.uk/Homo_sapiens) was thoroughly tested by screening >1 000 000 clones from each of two human fetal brain cDNA libraries because of its overlap with the risk haplotype at rs1000585-rs917235-rs714939.

    "Ready-made TaqMan gene expression assays for FLJ13391 (Hs00259924_m1), MRPL19 (Hs00608519_m1), C2ORF3 (Hs00162632_m1), DYX1C1 (Hs00370049_m1), DCDC2 (Hs00393203_m1), KIAA0319 (Hs00207788_m1), ROBO1 (Hs00268049_m1), GAPDH (4310884E) and 18S rRNA (4319413E) were purchased from Applied Biosystems. We assayed expression levels for these genes in total RNA from nine different areas of adult human brain: thalamus, hypothalamus, frontal-, occipital-, parietal-, temporal cortex (cat. nos 6762, 6864, 6810, 6812, 6814, 6816; Ambion), hippocampus, paracentral- and post-central gyrus (cat. nos 636565, 636574, 636573; Clontech), and from whole adult and fetal brain (cat. nos 636530, 636526; Clontech). For each tissue, three independent cDNA syntheses (500 ng total RNA per reaction) were performed using the SuperScript III first-strand synthesis kit (cat. no. 18080-051; Invitrogen). From each cDNA synthesis, quantitative real-time PCR was performed in quadruplets, using 5 ng of RNA per gene assay and run on ABI PRISM 7700 Sequence Detection PCR System (Applied Biosystems). All assays were performed in 10 µl reactions according to the manufacturer’s instructions. Relative standard expression curves were drawn for 18S rRNA and all tested genes. Relative quantification of the data was performed using the comparative Ct (threshold cycle) method (Sequence Detection System bulletin 2, Applied Biosystems). Ct values were adjusted to 18S rRNA and thereafter normalized to the whole brain sample.

    "To quantify mRNA expression levels from each allele of MRPL19 and C2ORF3, we analyzed individuals heterozygous for rs17689863 (Ser277Ser) in MRPL19 and rs1803196 (Val536Val) in C2ORF3. Five Finnish dyslexic and six normal readers, and three German dyslexic and one normal reader were studied. Total RNA was extracted from EBV-transformed lymphocyte cell lines using the RNeasy purification kit (cat. no. 74004; Qiagen) and cDNA synthesis (500 ng of total RNA per reaction) was performed using the SuperScript III first-strand synthesis kit (cat. nos 18080-051 and 12371-019; Invitrogen; for the Finnish and the German samples, respectively). Both cDNA and genomic DNA from each individual were sequenced in six independent reactions, originating from at least two separate PCR amplifications. Peak heights were compared and an allelic ratio was calculated for each sequence. The cDNA ratio values (unknown proportions) were normalized by dividing with the genomic values (1:1 proportion by definition). Data were pooled by genotype (risk haplotype heterozygotes versus non-risk haplotype heterozygotes) to evaluate (by two-tailed t-test) whether the normalized value differed from equal expression."

    "Evolutionary analysis of the MRPL19 and C2ORF3 genes was performed with an LRT using the CODEML program of the paml3.15 package (22). Mouse sequence (ENSMUSP-00000032124) was used as out-group for MRPL19, and dog (XP_540209) for C2ORF3."

    Associated Markers:
    rs1803196   (P = 0.002)  - Significance assessed with a two-tailed t test


    Comments

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    6936 GCFC2 2p12 SLI Susceptibility to Specific Language Impairment Scerri et al 2011 JSON | XML

    Additional Phenotype Details: 
    Specific Language Impairment may be diagnosed based on an expressive and/or receptive language score less than a certain number of SDs (e.g. 1.5) below the mean for the individual's age on a test such as the Clinical Evaluation of Language Fundamentals (CELF). Usually, a Performance Intelligence Quotient (PIQ) of at least 70 or 80 on the age-appropriate Weschler test is also a criterion. See Tomblin et al 1996 for a specific system for diagnosing SLI.


    References

    Semel EM, Wiig EH, Secord W (1992) Clinical evaluation of language fundamentals—revised. Phychological Corporation, San Antonio.

    Tomblin JB, Records NL, Zhang X (1996) A system for the diagnosis of specific language impairment in kindergarten children. J Speech Hear Res 39:1284–1294.

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

    Associated Markers:
    rs917235  (P = 0.033)  - Suggestive evidence in pure SLI cases vs. controls


    Comments


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    Negative evidence for association with GCFC2

    Conflicting Ref Year Original Report Failed Association Comment
    Newbury et al 2011 Anthoni et al (2007) Susceptibility to developmental dyslexia (Dyslexia) Failed to replicate association with 4 SNPs in MRPL19/C2ORF3 (aka GCFC2) region and dyslexia in a group of 188 probands and 331 unrelated UK dyslexic cases

    Basic Study Type: 

    Study Cohort: 
    Summary: SLI cohort consists of 780 individuals from 181 families. Dyslexia cohort consists of 634 probands and siblings from 264 families. For case-control analysis, also had 331 unrelated dyslexic individuals.


    SLI cohort

    "The SLI families used in this study were provided by the SLI Consortium (SLIC) cohort which has previously been described in detail (SLIC 2002, 2004; Falcaro et al. 2008). This family-based sample included 780 individuals from 181 2-generation families and had a child male:female ratio of 1.6:1. . . The samples were assessed by one of five separate centres across the UK and were derived from both clinical and epidemiological studies. The Newcomen Centre at Guy’s Hospital, London, the Cambridge Language and Speech Project (CLASP—(Burden et al. 1996)), the Child Life and Health Department at the University of Edinburgh (Clark et al. 2007), the Department of Child Health at the University of Aberdeen and the Manchester Language Study (Conti-Ramsden and Botting 1999; Conti-Ramsden et al. 1997)."

    Dyslexia cohort

    "The collection of families used for quantitative trait association has been extensively described previously (Francks et al. 2004). Briefly, all probands and siblings from our complete Oxford set of 264 unrelated nuclear families (a total of 634 siblings with a male:female ratio of 1.5:1), were identified from the dyslexia clinic at the Royal Berkshire Hospital (Reading, U.K)."

    For case-control analyses only: "In addition to the dyslexia family-based sample, we analysed a collection of 331 UK unrelated cases which have not been investigated in previous studies for any of the loci under study here. These samples were recruited through the Dyslexia Research Centre clinics in Oxford and Reading, and the Aston Dyslexia and Development clinic in Birmingham. The cases are between 8 and 18.5 years of age, have a BAS2 single-word reading score B100 (at chronological age) and >1.5 SDs below that predicted by their IQ scores. Since these individuals were collected as a case cohort, we did not investigate quantitative measures for them."

    Genotyping Methods: 
    "In total 31 SNPs were genotyped. These included four SNPs from MRPL19/C2ORF3, four SNPs from DCDC2, six SNPs from KIAA0319, two SNPs from DYX1C1, five SNPs from CNTNAP2, five SNPs from CMIP and five SNPs from ATP2C2 (Table 1). SLI and dyslexia individuals were genotyped in separate experiments, therefore the SNP data quality varied between these cohorts. In addition, control data were not available for all SNPs, therefore case–control analyses were not performed for all 31 SNPs. SNPs were genotyped using the Sequenom iPLEX assay. Genotypes in the form of marker clusters were checked manually in the MassArray TyperAnalyser software. Any SNP with a success rate of <80% or any SNP that showed consistent bad inheritances (>10 errors after data clean-up) within each independent cohort were removed from the association analyses. Probabilities of Hardy–Weinberg Equilibrium (HWE) were calculated for all SNPs within PEDSTATS (Wigginton and Abecasis 2005) and any SNP with a HWE-P of <0.0001 was excluded."

    Analysis Methods: 
    - QTDT (Quantitative Transmission Disequilibrium Test) performed independently within SLI and dyslexia families; IBD (Identity By Descent) calculated in MERLIN

    - Also conducted case-control analyses (allelic association test, within PLINK)

    Other Details: 
    SLI cohort, diagnostic criteria:

    "All families were ascertained on the basis of a single proband with receptive and/or expressive language skills, either currently or in the past, more than 1.5 SD below the normative mean for their chronological age."

    SLI cohort, other inclusion criteria

    "Any child reported to have a nonverbal IQ of below 80 was excluded from the study. Other exclusion criteria included monozygotic twinning, chronic illness requiring multiple hospital visits or admissions, deafness, a clinical diagnosis of autism, English being a second language, children with known neurological disorders and children under local authority care."

    SLI cohort, phenotypic measures:

    "Language abilities of all available SLIC children (regardless of language ability) were assessed using the expressive (ELS, n = 392) and receptive (RLS, n = 392) scales of the Clinical Evaluation of Language Fundamentals (CELF-R) battery (Semel et al. 1992) and a 28-item nonword repetition (NWR, n = 451) test (Gathercole et al. 1994). Reading aptitude was measured using the single word reading (Read, n = 312), single-word spelling (Spell, n = 310) and reading comprehension (Comp, n = 276) tests from the Wechsler Objective Reading Dimensions (WORD) (Rust et al. 1993). Verbal and non-verbal IQ were examined, primarily for exclusion purposes, using the Wechsler Intelligence Scales for Children (WISC-III) (Wechsler 1992). Due to the age constraints of standardised tests, all phenotypic data were collected for children only. All scores were normalised for age effects. Due to logistical constraints, the reading tests were only performed for a subset of SLI individuals."

    Dyslexia cohort, diagnostic criteria:

    "Families were ascertained if the proband had a British Abilities Scales (BAS) single-word reading score >2 SDs below that predicted by their intelligence quotient (IQ) and if at least one other sibling had a history of reading problems. These criteria identified some probands with high IQ scores and BAS reading scores within the ‘normal’ range. Therefore, after collecting 173 UK families the ascertainment conditions were changed such that the only required criterion was that the probands’ difference in their BAS single-word reading score had to be >=1 SD below the population mean for their age (and not IQ), along with an IQ >= 90."


    Dyslexia cohort, other inclusion criteria
    "Probands were excluded if they had been diagnosed with co-occuring developmental disorders such as SLI, autism or attention deficit-hyperactivity disorder (ADHD)."

    Dyslexia cohort, phenotypic measures

    "We administered a battery of psychometric tests to all probands and siblings in each family, and we age-adjusted and standardized their scores against a normative control data set, as described elsewhere (Marlow et al. 2001; Fisher et al. 2002). These included measures of single-word reading ability (READ; n = 634) and spelling ability (SPELL; n = 603) from the British Ability Scales (BAS) (Elliot et al. 1983) or Wide Range Achievement Test (WRAT-R) for children older than 14.5 (Jastak and Wilkson 1984), phonological decoding ability (PD; n = 629) (use of letter to sound relationship rules to read pseudowords) (Castles and Coltheart 1993), phonemic awareness (PA; n = 591) (awareness of the phonemic structure of language; test which required the individuals to orally move phonemes either within the same word or between words, also known as ‘‘spoonerism’’) (Gallagher and Frederickson 1995), orthographic coding (OC-irreg; n = 625) (the ability to read real words that do not follow conventional spelling to sound rules e.g. yacht) (Castles and Coltheart 1993) and orthographic coding assessed by a forced word choice test (OC-choice; n = 548) (identification of a correctly spelt word from two phonologically equivalent options, e.g. rane vs rain) between (Olson et al. 1994). Tests of verbal and non-verbal reasoning were assessed using the BAS similarities (SIM; n = 620) or BAS matrices (MAT; n = 588) tests respectively (Elliot et al. 1983). The Similarities sub-scale of the Wechsler Adult Intelligence Scales (WAIS), which is analogous to the BAS similarities test, was used when age was >17.5 years (Wechsler 1981). Note that the measures of reading and spelling were derived from different tests of the same constructs to those utilized in the SLI cohort."

    Newbury et al 2011 Anthoni et al (2007) Susceptibility to developmental dyslexia (Dyslexia) Failed to replicate association with 4 SNPs in MRPL19/C2ORF3 (aka GCFC2) region and dyslexia in a group of 188 probands and 331 unrelated UK dyslexic cases

    Basic Study Type: 

    Study Cohort: 
    Summary: SLI cohort consists of 780 individuals from 181 families. Dyslexia cohort consists of 634 probands and siblings from 264 families. For case-control analysis, also had 331 unrelated dyslexic individuals.


    SLI cohort

    "The SLI families used in this study were provided by the SLI Consortium (SLIC) cohort which has previously been described in detail (SLIC 2002, 2004; Falcaro et al. 2008). This family-based sample included 780 individuals from 181 2-generation families and had a child male:female ratio of 1.6:1. . . The samples were assessed by one of five separate centres across the UK and were derived from both clinical and epidemiological studies. The Newcomen Centre at Guy’s Hospital, London, the Cambridge Language and Speech Project (CLASP—(Burden et al. 1996)), the Child Life and Health Department at the University of Edinburgh (Clark et al. 2007), the Department of Child Health at the University of Aberdeen and the Manchester Language Study (Conti-Ramsden and Botting 1999; Conti-Ramsden et al. 1997)."

    Dyslexia cohort

    "The collection of families used for quantitative trait association has been extensively described previously (Francks et al. 2004). Briefly, all probands and siblings from our complete Oxford set of 264 unrelated nuclear families (a total of 634 siblings with a male:female ratio of 1.5:1), were identified from the dyslexia clinic at the Royal Berkshire Hospital (Reading, U.K)."

    For case-control analyses only: "In addition to the dyslexia family-based sample, we analysed a collection of 331 UK unrelated cases which have not been investigated in previous studies for any of the loci under study here. These samples were recruited through the Dyslexia Research Centre clinics in Oxford and Reading, and the Aston Dyslexia and Development clinic in Birmingham. The cases are between 8 and 18.5 years of age, have a BAS2 single-word reading score B100 (at chronological age) and >1.5 SDs below that predicted by their IQ scores. Since these individuals were collected as a case cohort, we did not investigate quantitative measures for them."

    Genotyping Methods: 
    "In total 31 SNPs were genotyped. These included four SNPs from MRPL19/C2ORF3, four SNPs from DCDC2, six SNPs from KIAA0319, two SNPs from DYX1C1, five SNPs from CNTNAP2, five SNPs from CMIP and five SNPs from ATP2C2 (Table 1). SLI and dyslexia individuals were genotyped in separate experiments, therefore the SNP data quality varied between these cohorts. In addition, control data were not available for all SNPs, therefore case–control analyses were not performed for all 31 SNPs. SNPs were genotyped using the Sequenom iPLEX assay. Genotypes in the form of marker clusters were checked manually in the MassArray TyperAnalyser software. Any SNP with a success rate of <80% or any SNP that showed consistent bad inheritances (>10 errors after data clean-up) within each independent cohort were removed from the association analyses. Probabilities of Hardy–Weinberg Equilibrium (HWE) were calculated for all SNPs within PEDSTATS (Wigginton and Abecasis 2005) and any SNP with a HWE-P of <0.0001 was excluded."

    Analysis Methods: 
    - QTDT (Quantitative Transmission Disequilibrium Test) performed independently within SLI and dyslexia families; IBD (Identity By Descent) calculated in MERLIN

    - Also conducted case-control analyses (allelic association test, within PLINK)

    Other Details: 
    SLI cohort, diagnostic criteria:

    "All families were ascertained on the basis of a single proband with receptive and/or expressive language skills, either currently or in the past, more than 1.5 SD below the normative mean for their chronological age."

    SLI cohort, other inclusion criteria

    "Any child reported to have a nonverbal IQ of below 80 was excluded from the study. Other exclusion criteria included monozygotic twinning, chronic illness requiring multiple hospital visits or admissions, deafness, a clinical diagnosis of autism, English being a second language, children with known neurological disorders and children under local authority care."

    SLI cohort, phenotypic measures:

    "Language abilities of all available SLIC children (regardless of language ability) were assessed using the expressive (ELS, n = 392) and receptive (RLS, n = 392) scales of the Clinical Evaluation of Language Fundamentals (CELF-R) battery (Semel et al. 1992) and a 28-item nonword repetition (NWR, n = 451) test (Gathercole et al. 1994). Reading aptitude was measured using the single word reading (Read, n = 312), single-word spelling (Spell, n = 310) and reading comprehension (Comp, n = 276) tests from the Wechsler Objective Reading Dimensions (WORD) (Rust et al. 1993). Verbal and non-verbal IQ were examined, primarily for exclusion purposes, using the Wechsler Intelligence Scales for Children (WISC-III) (Wechsler 1992). Due to the age constraints of standardised tests, all phenotypic data were collected for children only. All scores were normalised for age effects. Due to logistical constraints, the reading tests were only performed for a subset of SLI individuals."

    Dyslexia cohort, diagnostic criteria:

    "Families were ascertained if the proband had a British Abilities Scales (BAS) single-word reading score >2 SDs below that predicted by their intelligence quotient (IQ) and if at least one other sibling had a history of reading problems. These criteria identified some probands with high IQ scores and BAS reading scores within the ‘normal’ range. Therefore, after collecting 173 UK families the ascertainment conditions were changed such that the only required criterion was that the probands’ difference in their BAS single-word reading score had to be >=1 SD below the population mean for their age (and not IQ), along with an IQ >= 90."


    Dyslexia cohort, other inclusion criteria
    "Probands were excluded if they had been diagnosed with co-occuring developmental disorders such as SLI, autism or attention deficit-hyperactivity disorder (ADHD)."

    Dyslexia cohort, phenotypic measures

    "We administered a battery of psychometric tests to all probands and siblings in each family, and we age-adjusted and standardized their scores against a normative control data set, as described elsewhere (Marlow et al. 2001; Fisher et al. 2002). These included measures of single-word reading ability (READ; n = 634) and spelling ability (SPELL; n = 603) from the British Ability Scales (BAS) (Elliot et al. 1983) or Wide Range Achievement Test (WRAT-R) for children older than 14.5 (Jastak and Wilkson 1984), phonological decoding ability (PD; n = 629) (use of letter to sound relationship rules to read pseudowords) (Castles and Coltheart 1993), phonemic awareness (PA; n = 591) (awareness of the phonemic structure of language; test which required the individuals to orally move phonemes either within the same word or between words, also known as ‘‘spoonerism’’) (Gallagher and Frederickson 1995), orthographic coding (OC-irreg; n = 625) (the ability to read real words that do not follow conventional spelling to sound rules e.g. yacht) (Castles and Coltheart 1993) and orthographic coding assessed by a forced word choice test (OC-choice; n = 548) (identification of a correctly spelt word from two phonologically equivalent options, e.g. rane vs rain) between (Olson et al. 1994). Tests of verbal and non-verbal reasoning were assessed using the BAS similarities (SIM; n = 620) or BAS matrices (MAT; n = 588) tests respectively (Elliot et al. 1983). The Similarities sub-scale of the Wechsler Adult Intelligence Scales (WAIS), which is analogous to the BAS similarities test, was used when age was >17.5 years (Wechsler 1981). Note that the measures of reading and spelling were derived from different tests of the same constructs to those utilized in the SLI cohort."

    Paracchini et al 2011 Anthoni et al (2007) Susceptibility to developmental dyslexia (Dyslexia) Tested association of SNPs within MRPL19/C2ORF3 (aka GCFC2) and quantitative measures of reading and spelling in a sample of 520 Australians (the Raine cohort). No significant association was detected for any individual SNPs nor for haplotype analysis.

    Basic Study Type: 

    Study Cohort: 
    "The Raine study is a pregnancy cohort that was recruited prior to 18 weeks’ gestation from the public antenatal clinic at King Edward Memorial Hospital (KEMH) or surrounding private clinics in Perth, Western Australia (WA) (Newnham et al . 1993). Approximately 100 unselected antenatal patients per month were enrolled during this period from August 1989 to April 1992, with a final sample of 2979 women. . .From this original cohort of women, 2868 of their children have been followed over the last two decades with detailed assessments performed every 2–3 years."

    Genotyping Methods: 
    "In the Raine study, DNA was collected using standardized procedures from 74% of adolescence who attended the 14-year follow-up on and a further 5% at the 16-year follow-up. Genome-wide data were generated using an Illumina 660 Quad array for each individual. Only SNPs that passed quality control (QC) criteria (call rate >= 95%, minor allele frequency >0.05 and Hardy–Weinberg disequilibrium P-value >0.01) were retained for genetic analysis."

    Analysis Methods: 
    "Our first analysis examined selected SNPs within the DYX1C1, KIAA0319, DCDC2 and MRPL19/C2ORF3 genes that have been previously found to be associated with dyslexia. Fourteen of these SNPs were available for the Raine sample (Table S2). These SNPs were tested for association with quantitative measures of reading and spelling scores (Table 1) using an allelic test of also tested for association haplotypes derived from the markers at theMRPL19/C2ORF3 locus, as previously reported (Anthoni et al. 2007). Haplotypes were inferred using SIMHAP version 1.0.2. In all analyses, gender was specified as a covariate. Principal components analysis with Eigenstrat (Price et al . 2006) showed evidence of population stratification and the first two principal components were also included in all analyses.
    ". . .The LD among DYX1C1 SNPs in this sample was determined with HAPLOVIEW version 4.2 (http://www.broadinstitute.org/haploview/haploview) (Barrett et al. 2005). Power calculations were computed using QUANTO version 4.02 (Gauderman et al. 2002) (Fig. S1)."

    Other Details: 
    Raine study inclusion criteria

    "The inclusion criteria were (1) English language skills sufficient to understand the study demands, (2) an expectation to deliver at KEMH and (3) an intention to remain in WA to enable future follow-up of their child."

    Sample inclusion criteria

    "Inclusion criteria for the current study were no known intellectual disability; a nonverbal IQ within normal limits as assessed by the Raven’s Colored Progressive Matrices (CPM; i.e. a score >=16th percentile, corresponding to approximately >-1 SD the population average of the 50th centile) and biological parents who were both of white European origin. Furthermore, because the current study had an interest in literacy development of the birth cohort, only those children who spoke English at home were included for analysis."

    Reading and spelling assessment of children at age 10 years

    "The Western Australian Literacy and Numeracy Assessment (WALNA) is administered annually to all students across WA in school grades three (age 8), five (age 10) and seven (age 12). The WALNA on whether children have reached theminimum standards of reading, writing, spelling and numeracy. The tests have been written to cater for the diverse range of students in Australian schools, and to ensure that there is no systematic bias associated with factors such as gender, culture or geographic location. Every year the WALNA is evaluated by expert judges for content and construct validity and scrutinized by psychometricians for misfitting items, precision and bias. The current study concerned performance on the reading and spelling subtests of the WALNA, completed by the Raine cohort during school grade 5 (between 2000 and 2002).
    "For the reading test, children were given a magazine and required to answer 33 multiple-choice questions on its contents. A further two questions required a short answer of one to two sentences each. For each item, children were directed to the relevant page and article title (e.g. ‘read Helicopter on page 2 of the magazine and answer questions 1–5’). The spelling subtest consisted of two tasks. In the first task, participants were provided with a written paragraph that included 10 spelling mistakes, each of which were circled. The passage was first read aloud by the teacher from beginning to end. The teacher then read through the passage again, this time pausing at each circled word (spelling mistake), upon which children were required to write down the correct spelling of the word. The second spelling task was similar to the first; however, rather than spelling mistakes, the written passage given to children included 14 blank spaces for missing words. The passage, including the missing words, was then read aloud by the teacher twice: the first time, children were instructed to follow the text with their finger, and the second time, children were required to write down each missing word. For items in both the spelling and reading tests, a score (of 1) was awarded for correct answers only. Raw scores for the reading and spelling tests were summed and then converted via a Rasch measurement model (Doig & Groves 2006) into an interval scale to enable easier interpretation of the results. Scores on both the reading and spelling subscales could range from -100 to 800, with higher scores indicating better performance.
    "These data, collected by the WA Department of Education and Training, were then linked to the Raine study dataset by the WA Data Linkage System using a probabilistic method of matching based on a full name, date of birth, gender and address (Kelman et al . 2002). Western Australian Literacy and Numeracy Assessment records were linked for 1038 Raine study children who were in grade five and attending government schools at the time of assessment."

    Scerri et al 2011 Anthoni et al (2007) Susceptibility to developmental dyslexia (Dyslexia) No evidence of association with dyslexia categorical variable for rs917235 in pure dyslexia or dyslexia / SLI cases vs. controls in UK ALSPAC cohort