Strong Genetic Evidence of DCDC2 as a Susceptibility Gene for Dyslexia
2005; Elsevier BV; Volume: 78; Issue: 1 Linguagem: Inglês
10.1086/498992
ISSN1537-6605
AutoresJohannes Schumacher, Heidi Anthoni, Faten Dahdouh, Inke R. König, Axel M. Hillmer, Nadine Kluck, Malou Manthey, Ellen Plume, Andreas Warnke, Helmut Remschmidt, Jutta Hülsmann, Sven Cichon, Cecilia M. Lindgren, Peter Propping, Marco Zucchelli, Andreas Ziegler, Myriam Peyrard‐Janvid, Gerd Schulte‐Körne, Markus M. Nöthen, Juha Kere,
Tópico(s)Autism Spectrum Disorder Research
ResumoWe searched for linkage disequilibrium (LD) in 137 triads with dyslexia, using markers that span the most-replicated dyslexia susceptibility region on 6p21-p22, and found association between the disease and markers within the VMP/DCDC2/KAAG1 locus. Detailed refinement of the LD region, involving sequencing and genotyping of additional markers, showed significant association within DCDC2 in single-marker and haplotype analyses. The association appeared to be strongest in severely affected patients. In a second step, the study was extended to include an independent sample of 239 triads with dyslexia, in which the association—in particular, with the severe phenotype of dyslexia—was confirmed. Our expression data showed that DCDC2, which contains a doublecortin homology domain that is possibly involved in cortical neuron migration, is expressed in the fetal and adult CNS, which—together with the hypothesized protein function—is in accordance with findings in dyslexic patients with abnormal neuronal migration and maturation. We searched for linkage disequilibrium (LD) in 137 triads with dyslexia, using markers that span the most-replicated dyslexia susceptibility region on 6p21-p22, and found association between the disease and markers within the VMP/DCDC2/KAAG1 locus. Detailed refinement of the LD region, involving sequencing and genotyping of additional markers, showed significant association within DCDC2 in single-marker and haplotype analyses. The association appeared to be strongest in severely affected patients. In a second step, the study was extended to include an independent sample of 239 triads with dyslexia, in which the association—in particular, with the severe phenotype of dyslexia—was confirmed. Our expression data showed that DCDC2, which contains a doublecortin homology domain that is possibly involved in cortical neuron migration, is expressed in the fetal and adult CNS, which—together with the hypothesized protein function—is in accordance with findings in dyslexic patients with abnormal neuronal migration and maturation. Dyslexia (MIM 600202) is a specific developmental disorder characterized by severe difficulties in learning to read and spell, despite adequate schooling, normal visual acuity, and a mental age that is within a normal range (ICD-10) (Dilling et al. Dilling et al., 1991Dilling H Mombour W Schmidt MH International classification of mental diseases, ICD-10. German edition. Hans Huber, Bern1991Google Scholar). This disorder is more frequent in boys than in girls (Rutter et al. Rutter et al., 2004Rutter M Caspi A Fergusson D Horwood LJ Goodman R Maughan B Moffitt TE Meltzer H Carroll J Sex differences in developmental reading disability: new findings from 4 epidemiological studies.JAMA. 2004; 291: 2007-2012Crossref PubMed Scopus (309) Google Scholar) and affects 5%–12% of school-aged children (Katusic et al. Katusic et al., 2001Katusic SK Colligan RC Barbaresi WJ Schaid DJ Jacobsen SJ Incidence of reading disability in a population-based birth cohort, 1976-1982, Rochester, Minn.Mayo Clin Proc. 2001; 76: 1081-1092Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Although reading and spelling disorders characterize the core dyslexia phenotype, related abilities—for example, phoneme awareness (PA) (analyzing and discriminating phonemes), verbal memory, and orthographic processing—are correlates of a broader definition of the dyslexia phenotype. In recent years, linkage studies have identified chromosomal regions likely to contain genes contributing to dyslexia. Nine regions (DYX1–DYX9) have been suggested to date and are listed by the Human Gene Nomenclature Committee. Of these loci, DYX2 on chromosome 6p21-p22 should be considered one of the most promising candidate regions, since several groups have independently reported linkage between DYX2 and dyslexia (Cardon et al. Cardon et al., 1994Cardon LR Smith SD Fulker DW Kimberling WJ Pennington BF DeFries JC Quantitative trait locus for reading disability on chromosome 6.Science. 1994; 266: 276-279Crossref PubMed Scopus (462) Google Scholar; Grigorenko et al. Grigorenko et al., 1997Grigorenko EL Wood FB Meyer MS Hart LA Speed WC Shuster A Pauls DL Susceptibility loci for distinct components of developmental dyslexia on chromosomes 6 and 15.Am J Hum Genet. 1997; 60: 27-39PubMed Google Scholar, Grigorenko et al., 2000Grigorenko EL Wood FB Meyer MS Pauls DL Chromosome 6p influences on different dyslexia-related cognitive processes: further confirmation.Am J Hum Genet. 2000; 66: 715-723Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, Grigorenko et al., 2003Grigorenko EL Wood FB Golovyan L Meyer M Romano C Pauls D Continuing the search for dyslexia genes on 6p.Am J Med Genet B Neuropsychiatr Genet. 2003; 118: 89-98Crossref Scopus (40) Google Scholar; Fisher et al. Fisher et al., 1999Fisher SE Marlow AJ Lamb J Maestrini E Williams DF Richardson AJ Weeks DE Stein JF Monaco AP A quantitative-trait locus on chromosome 6p influences different aspects of developmental dyslexia.Am J Hum Genet. 1999; 64: 146-156Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar; Gayán et al. Gayán et al., 1999Gayán J Smith SD Cherny SS Cardon LR Fulker DW Brower AM Olson RK Pennington BF DeFries JC Quantitative-trait locus for specific language and reading deficits on chromosome 6p.Am J Hum Genet. 1999; 64: 157-164Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar; Kaplan et al. Kaplan et al., 2002Kaplan DE Gayán J Ahn J Won T-W Pauls D Olson RK DeFries JC Wood F Pennington BF Page GP Smith SD Gruen JR Evidence for linkage and association with reading disability, on 6p21.3-22.Am J Hum Genet. 2002; 70: 1287-1298Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). According to National Center for Biotechnology Information (NCBI) Build 35, the region spans ∼16.4 Mb between STR markers D6S109 and D6S291. At the association level, however, the situation is less clear, and positive results have been reported for two independent gene clusters (Deffenbacher et al. Deffenbacher et al., 2004Deffenbacher KE Kenyon JB Hoover DM Olson RK Pennington BF DeFries JC Smith SD Refinement of the 6p21.3 quantitative trait locus influencing dyslexia: linkage and association analyses.Hum Genet. 2004; 115: 128-138Crossref PubMed Scopus (135) Google Scholar; Francks et al. Francks et al., 2004Francks C Paracchini S Smith SD Richardson AJ Scerri TS Cardon LR Marlow AJ MacPhie IL Walter J Pennington BF Fisher SE Olson RK DeFries JC Stein JF Monaco AP A 77-kilobase region of chromosome 6p22.2 is associated with dyslexia in families from the United Kingdom and from the United States.Am J Hum Genet. 2004; 75: 1046-1058Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar; Cope et al. Cope et al., 2005Cope N Harold D Hill G Moskvina V Stevenson J Holmans P Owen MJ O'Donovan MC Williams J Strong evidence that KIAA0319 on chromosome 6p is a susceptibility gene for developmental dyslexia.Am J Hum Genet. 2005; 76: 581-591Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). In the present study, we aimed to explore the contribution of the chromosome 6 locus to dyslexia and related phenotypes in the German population. In a first step, we searched for association in a sample of 137 triads (initial sample), employing a combination of STR and SNP-marker genotyping as well as sequencing. In a second step, the study was extended to include an independent sample of 239 triads with dyslexia (replication sample), to confirm the association obtained in the initial sample. Finally, extensive mutation and expression analysis was employed for further characterization of the susceptibility locus. All families of the two samples were of German descent and were recruited through the Departments of Child and Adolescent Psychiatry and Psychotherapy at the Universities of Marburg and Würzburg. All individuals (or the parents of children aged <14 years) gave written informed consent for participation in the study. The study was approved by the ethics committees of the Universities of Marburg and Würzburg. The diagnostic inclusion criteria and phenotypic measures have been described in detail elsewhere (Schulte-Körne et al. Schulte-Körne et al., 1996Schulte-Körne G Deimel W Muller K Gutenbrunner C Remschmidt H Familial aggregation of spelling disability.J Child Psychol Psychiatry. 1996; 37: 817-822Crossref PubMed Scopus (65) Google Scholar, Schulte-Körne et al., 2001Schulte-Körne G Deimel W Remschmidt H Diagnosis of reading and spelling disorder.Z Kinder Jugendpsychiatr Psychother. 2001; 29: 113-116Crossref PubMed Scopus (39) Google Scholar; Schumacher et al. Schumacher et al., 2005Schumacher J König IR Plume E Propping P Warnke A Manthey M Duell M Kleensang A Repsilber D Preis M Remschmidt H Ziegler A Nöthen MM Schulte-Körne G Linkage analyses of chromosomal region 18p11-q12 in dyslexia.J Neural Transm. 2005; (electronically published August 3, 2005; accessed November 16, 2005)(http://www.springerlink.com/(clxfmy45ptvarl553jtgalbs)/app/home/contribution.asp?referrer=parent&backto=searcharticlesresults,1,2;)PubMed Google Scholar; Ziegler et al. Ziegler et al., 2005Ziegler A König IR Deimel W Plume E Nöthen MM Propping P Kleensang A Müller-Myhsok B Warnke A Remschmidt H Schulte-Körne G Developmental dyslexia—recurrence risk estimates from a German bi-center study using the single proband sib pair design.Hum Hered. 2005; 59: 136-143Crossref PubMed Scopus (46) Google Scholar) and are shown in table 1. In brief, potential probands who had difficulty learning to spell or who had received the diagnosis of dyslexia were referred to the investigators by parents, teachers, special educators, or practitioners. Because clinical studies of dyslexia in Germany usually base sample selection on spelling disorder and because our previous findings all rest on this selection criterion (see Schulte-Körne et al. Schulte-Körne et al., 1996Schulte-Körne G Deimel W Muller K Gutenbrunner C Remschmidt H Familial aggregation of spelling disability.J Child Psychol Psychiatry. 1996; 37: 817-822Crossref PubMed Scopus (65) Google Scholar, Schulte-Körne et al., 1998Schulte-Körne G Grimm T Nöthen MM Müller-Myhsok B Cichon S Vogt IR Propping P Remschmidt H Evidence for linkage of spelling disability to chromosome 15.Am J Hum Genet. 1998; 63: 279-282Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar; Schumacher et al. Schumacher et al., 2005Schumacher J König IR Plume E Propping P Warnke A Manthey M Duell M Kleensang A Repsilber D Preis M Remschmidt H Ziegler A Nöthen MM Schulte-Körne G Linkage analyses of chromosomal region 18p11-q12 in dyslexia.J Neural Transm. 2005; (electronically published August 3, 2005; accessed November 16, 2005)(http://www.springerlink.com/(clxfmy45ptvarl553jtgalbs)/app/home/contribution.asp?referrer=parent&backto=searcharticlesresults,1,2;)PubMed Google Scholar; Ziegler et al. Ziegler et al., 2005Ziegler A König IR Deimel W Plume E Nöthen MM Propping P Kleensang A Müller-Myhsok B Warnke A Remschmidt H Schulte-Körne G Developmental dyslexia—recurrence risk estimates from a German bi-center study using the single proband sib pair design.Hum Hered. 2005; 59: 136-143Crossref PubMed Scopus (46) Google Scholar), the probands' spelling ability was selected for inclusion (for diagnostic criterion, see the following subsection). Since a spelling disorder cannot be reliably diagnosed at an earlier age, only those children attending a regular primary school (no special school, e.g., for learning-disabled children) who had reached at least the middle of second grade were included in the study. All children were investigated at one of the Departments of Child and Adolescent Psychiatry, by use of standardized and unstandardized tests as described below, and family and medical history were taken.Table 1Sample CharacteristicsPhenotypeInitial Sample (n=137)Replication Sample (n=239)No. of subjects (M:F)99:38176:63IQ (±SD)109±12.6109±12.7SpellingaMean ± SD.30.5±5.729.2±5.7ReadingaMean ± SD.35.9±1134.1±10.6PAaMean ± SD.40.7±8.939.2±9.3PDaMean ± SD.38.5±10.837.7±10.2Orthographic processingaMean ± SD.33.9±11.232.1±11.6RN: letteraMean ± SD.41.9±13.640.1±13.5RN: numberaMean ± SD.44.1±14.342.9±13.9RN: objects and colorsaMean ± SD.44.2±10.343.5±10.1a Mean ± SD. Open table in a new tab To exclude families in which the proband or a sibling showed symptoms of attention deficit–hyperactivity disorder (ADHD [MIM 143465]), a standardized clinical interview (Unnewehr et al. Unnewehr et al., 1995Unnewehr S Schneider S Margraf J Kinder-DIPS—Diagnostisches Interview bei psychischen Störungen im Kindes-und Jugendalter. Hogrefe, Göttingen1995Google Scholar) was performed with the mother. The reasons for exclusion of comorbid children with dyslexia and ADHD or of children who had siblings with ADHD were, first, that the traits might overlap (Willcutt et al. Willcutt et al., 2002Willcutt EG Pennington BF Smith SD Cardon LR Gayán J Knopik VS Olson RK DeFries JC Quantitative trait locus for reading disability on chromosome 6p is pleiotropic for attention-deficit/hyperactivity disorder.Am J Med Genet. 2002; 114: 260-268Crossref PubMed Scopus (125) Google Scholar) and, second, that symptoms of inattention and hyperactivity might influence child behavior during the neuropsychological examinations. Additional exclusionary criteria were a bilingual education, intelligence quotient (IQ) <85, and an uncorrected peripheral hearing or vision disorder or a psychiatric or neurological disorder influencing the development of reading and spelling ability. The diagnosis of dyslexia was based on the spelling score, with use of the T distribution of the general population. For inclusion in the trial, the following discrepancy criterion had to be fulfilled by the proband: on the basis of an assumed correlation between the IQ and spelling of 0.4 (Schulte-Körne et al. Schulte-Körne et al., 1996Schulte-Körne G Deimel W Muller K Gutenbrunner C Remschmidt H Familial aggregation of spelling disability.J Child Psychol Psychiatry. 1996; 37: 817-822Crossref PubMed Scopus (65) Google Scholar, Schulte-Körne et al., 2001Schulte-Körne G Deimel W Remschmidt H Diagnosis of reading and spelling disorder.Z Kinder Jugendpsychiatr Psychother. 2001; 29: 113-116Crossref PubMed Scopus (39) Google Scholar), an expected spelling score was estimated. The child was classified as "affected" if the discrepancy between the expected and the observed spelling score was ≥1σ. Spelling was measured using age-appropriate spelling tests (writing to dictation) that render T scores that are distributed as N(50,100) in unaffected children (Schulte-Körne et al. Schulte-Körne et al., 2001Schulte-Körne G Deimel W Remschmidt H Diagnosis of reading and spelling disorder.Z Kinder Jugendpsychiatr Psychother. 2001; 29: 113-116Crossref PubMed Scopus (39) Google Scholar). The IQ was assessed using the Culture Fair Test (CFT-1) (Weiß and Osterland Weiß and Osterland, 1997Weiß RH Osterland J Grundintelligenztest Skala 1 CFT 1. Hogrefe, Göttingen1997Google Scholar) or CFT-20 (WeißWeiß, 1998Weiß RH Grundintelligenztest Skala 2 CFT 20. Hogrefe, Göttingen1998Google Scholar), depending on the proband's age. Probands fulfilling the inclusion criteria were assessed by use of several psychometric tests, none of which was administered to the parents. These tests targeted different aspects of the dyslexia phenotype, with single-word reading, PA, phonological decoding (PD), rapid naming (RN), and orthographic coding (OC). All probands and their siblings performed a single-word and nonword reading test (Salzburger Lese- und Rechtschreibtest [Landerl et al. Landerl et al., 1997Landerl K Wimmer H Moser E SLRT—Salzburger Lese-und Rechtschreibtest. Hans Huber, Bern1997Google Scholar]). This test also renders T scores that are distributed as N(50,100) in unaffected children (Landerl et al. Landerl et al., 1997Landerl K Wimmer H Moser E SLRT—Salzburger Lese-und Rechtschreibtest. Hans Huber, Bern1997Google Scholar). Because there are no standardized German reading or PD tests for children at or above the fifth grade, an unstandardized reading test was administered to the children in our study who had reached that grade (Schumacher et al. Schumacher et al., 2005Schumacher J König IR Plume E Propping P Warnke A Manthey M Duell M Kleensang A Repsilber D Preis M Remschmidt H Ziegler A Nöthen MM Schulte-Körne G Linkage analyses of chromosomal region 18p11-q12 in dyslexia.J Neural Transm. 2005; (electronically published August 3, 2005; accessed November 16, 2005)(http://www.springerlink.com/(clxfmy45ptvarl553jtgalbs)/app/home/contribution.asp?referrer=parent&backto=searcharticlesresults,1,2;)PubMed Google Scholar). The test requires children to read a list of 48 words and 48 pronounceable nonwords as accurately and as quickly as possible. The dependent variables were the number of words and nonwords read correctly in 1 min. Population data and age corrections were not available for this test. Three tests were administered to measure the spectrum of phonological awareness of study children in grades 2–4. The tests were presented verbally, and subjects were to respond orally. The tests included a phoneme-segmentation task, a phoneme-deletion task, and a phoneme-reversal task. For children at or above the fifth grade, a phoneme-segmentation test, a phoneme-reversal test, and a phoneme-binding and word-reversal test were administered. A pseudohomophone test was administered, which assesses the ability to discriminate real words from pseudohomophones (OC). These pseudohomophones were generated by substituting or adding graphemes in a real word, which results in a pseudohomophone that sounds very similar to the real word but is orthographically wrong. This test is considered a measure of orthographic processing, because pseudowords and real words sound the same, and the phonological analysis of the word cannot discriminate between them. The children heard single words, with headphones, at a sound pressure of 70 dB. After this, a word or a pseudoword corresponding to the word or nonword presented via headphones appeared on the computer screen. The subjects were asked to press the right button for a pseudoword and the left button for a real word. Thirty-five words or pseudowords were presented one after another in a random order. The number of correct responses (i.e., number of real words that were recognized as correctly spelled, maximally 20) was recorded by the computer. The test was started after four practice trials. The rapid-naming test used for this study was developed on the basis of the work of Denckla and Rudel (Denckla and Rudel, 1974Denckla MB Rudel RG Rapid automatized naming of pictured objects, colors, letters and numbers by normal children.Cortex. 1974; 10: 186-202Crossref PubMed Scopus (491) Google Scholar). Four trials—naming objects, number, letters, and colors—were conducted. Each trial consisted of items that were arrayed in consecutive rows. Each row consisted of five items that were repeated in a different order, for a total of 10 rows. The trials were printed on a sheet of paper, and children were asked to read them as quickly as they could without making mistakes. A stopwatch was used to measure the time taken by the child to name all stimuli on the entire list. A practice item was given before starting the tests. Color naming was measured by rectangles of five different colors (red, green, brown, blue, and black). Each color was presented in a random order, with the provision that no item appear twice in succession. Number naming was measured with 1-digit numbers (e.g., 7, 2, 9, 6, 4) in the same way as color naming. Object naming was measured with colored line drawings of common objects (e.g., scissors, candle, comb, clock, key); for letter naming, single consonants or vowels (e.g., p, s, o, a, d) were presented. For STR-marker genotyping, one oligonucleotide of each primer pair was fluorescein labeled, and PCRs were performed on MJ Research thermocyclers. The resulting amplified products were separated on denaturing polyacrylamide gels on an automated DNA sequencer (Model 377 [Applied Biosystems]). Allele sizes were determined relative to an internal size standard in each lane by use of Genescan Analysis Version 2.1.1 and Genotyper Version 2.0 software (Applied Biosystems). All gels were scored independently by two individuals who were blind to the disease status. SNP-marker genotyping was performed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom). PCR assays and associated extension reactions were performed on MJ Research thermocyclers. Cleaned extension products were analyzed by a Mass ARRAY mass spectrometer (Bruker Daltonik), and peaks were identified using the SpectroTYPER RT 2.0 software (Sequenom). All genotypes (1) were scored independently by two individuals who were blind to the disease status and (2) were tested for Mendelian inheritance, in every triad, by use of PedCheck (O'Connell and Weeks O'Connell and Weeks, 1998O'Connell JR Weeks DE PedCheck: a program for identification of genotype incompatibilities in linkage analysis.Am J Hum Genet. 1998; 63: 259-266Abstract Full Text Full Text PDF PubMed Scopus (1806) Google Scholar). PCR was performed on MJ Research thermocyclers. All products were cleaned of unincorporated primers and dNTPs by use of shrimp alkaline phosphatase and exonuclease I and were further sequenced using DYEnamic ET Dye terminator kit (Amersham Biosciences). Sequencing products were electrophoresed using a MegaBACE 1000 instrument and MegaBACE long-read matrix, were visualized using the Sequence Analyzer v3.0 software (Amersham Biosciences), and were further aligned using the Pregap and Gap4 software (Staden Package). In addition, a separate viewer compared each FASTA output from sequencing results with corresponding genomic sequences (GenBank accession number NT_007592) with use of Blast 2 sequences. A DCDC2 probe was generated, by touch-down PCR, with oligonucleotides DCDC2-probeF (5′-GCAAGTCGAGGAGATTCTGG-3′) and DCDC2-probeR (5′-CAAGTGGCAATTGTCTTCCA-3′) at annealing temperatures of 63°C–55°C. The PCR product was [32P]-labeled by the use of Ready-to-Go DNA labeling beads (Amersham) and was purified by ProbeQuant G-50 Micro Columns (Amersham), in accordance with the manufacturer's recommendations. Hybridization of human brain tissue northern-blot panels II and V and human fetal tissues II (CLONTECH) was performed overnight in ExpressHyb Solution (CLONTECH) at 68°C. Blots were washed in accordance with standard protocol, and autoradiographs were exposed 3–8 d at −70°C. The CLONTECH human multiple tissue cDNA panels I and II and human-blood fractions were used as templates for PCRs with primers DCDC2-2F (5′-CCTCATAAACCCAGCTTCTCG-3′) and DCDC2-2R (5′-CAGGTTGAGGTTCCAGTCGAT-3′) for the analysis of DCDC2 "long," DCDC2-smallF (5′-ATTCAGCCACACGATGTCAC-3′) and DCDC2-probeR (5′-CAAGTGGCAATTGTCTTCCA-3′) for analysis of DCDC2 "short," and KAAG1-F2 (5′-CCCAACTGCAACGAGAGTTT-3′) and KAAG1-R2 (5′-GTGTGTGTGCGTCCTCCTC-3′) for the analysis of KAAG1. PCR reactions were performed with annealing temperatures of 60°C (DCDC2-2F/-2R), 56°C (DCDC2-smallF/-probeR), and 63°C (KAAG1-F2/-R2) in 35 cycles. PCR products were separated by electrophoresis on 1.5% agarose gels and were visualized by UV and ethidium-bromide staining. In both samples, transmitted and untransmitted alleles were determined, and the transmission/disequilibrium test (TDT) was performed to test for genetic association in the triads. To allow for multiple alleles, we used the marginal homogeneity test (Spielman and Ewens Spielman and Ewens, 1996Spielman RS Ewens WJ The TDT and other family-based tests for linkage disequilibrium and association.Am J Hum Genet. 1996; 59: 983-989PubMed Google Scholar). If an asymptotic P value was <.1, an exact P value was calculated as implemented in S.A.G.E., v4.6. In the initial, replication, and pooled samples, genotypic relative risks (GRR) with 95% CIs were estimated for single loci on the basis of the methods of Scherag et al. (Scherag et al., 2002Scherag A Dempfle A Hinney A Hebebrand J Schafer H Confidence intervals for genotype relative risks and allele frequencies from the case parent trio design for candidate-gene studies.Hum Hered. 2002; 54: 210-217Crossref PubMed Scopus (11) Google Scholar) and Franke et al. (Franke et al., 2005Franke D Philippi A Tores F Hager J Ziegler A König IR On confidence intervals for genotype relative risks and attributable risks from case parent trio design for candidate-gene studies.Hum Hered. 2005; 60: 81-88Crossref PubMed Scopus (5) Google Scholar). Since this assumes diallelic markers, STR alleles were summarized on the basis of their transmission frequencies. Two-locus haplotype frequencies were estimated using the expectation-maximization algorithm, as implemented in UNPHASED v2.403 (Dudbridge Dudbridge, 2003Dudbridge F Pedigree disequilibrium tests for multilocus haplotypes.Genet Epidemiol. 2003; 25: 115-121Crossref PubMed Scopus (1044) Google Scholar). The respective GRRs with 95% CIs were obtained as indicated above. To test for association with additionally assessed quantitative component processes in the initial sample, the approach of Rabinowitz (Rabinowitz, 1997Rabinowitz D A transmission disequilibrium test for quantitative trait loci.Hum Hered. 1997; 47: 342-350Crossref PubMed Scopus (195) Google Scholar), implemented in QTDT v2.4.6 (Abecasis et al. Abecasis et al., 2000Abecasis GR Cardon LR Cookson WOC A general test of association for quantitative traits in nuclear families.Am J Hum Genet. 2000; 66: 279-292Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar), was utilized with estimation of P values from 10,000 permutations. To identify LD within the linkage region DYX2, we first analyzed 16 STR markers encompassing an ∼24-Mb region between D6S289 and D6S1610 (see table 2). STR-marker positions and distances were extracted from the Marshfield map (Center for Medical Genetics) and from the UCSC Genome Browser (UCSC Genome Bioinformatics). In our initial sample, comprising 137 triads with dyslexia, we found most-significant association between the disease status and STR marker D6S276 (P=.004) (see table 3). Alleles 5 and 6 were more frequently transmitted to the affected children (53 transmissions vs. 33 nontransmissions and 13 transmissions vs. 1 nontransmission, respectively). D6S276 is located within the doublecortin-domain-containing-2 gene (DCDC2 [MIM 605755]) and in close proximity to the kidney-associated-antigen-1 gene (KAAG1 [MIM 608211]) and the vesicular-membrane-protein-p24 gene (VMP) (fig. 1A).Table 2Location of STR Markers Used for Systematic TDT Analysis of Chromosomal Region 6p21-p22PositionSTR MarkerAccession NumberNCBI Build 35 (bp)Marshfield Map (cM)D6S289AFM200WC91528991829.93D6S1567AFMA219ZD91745273633.43D6S109D6S1092001783334.23D6S422AFM234XA32037801635.66D6S1665AFMC017XG12099619136.37D6S506…21868089…D6S1660AFMB355WG52332175640.14D6S299AFM217XG72393332442.27D6S276AFM158YE92419378144.41D6S2439GATA163B102431467142.27D6S1571AFMA223XD92497222842.98D6S1281GATA89B072530492744.41D6S105MFD612777925344.41D6S258AFM031YH122902877844.41D6S1560AFMA205YD93356266447.71D6S1610AFMB024YG13926752553.81 Open table in a new tab Table 3Systematic TDT Analysis: Results of the Initial Dyslexia Sample (n=137 Triads)TDT ResultsSTR Marker and AlleleNo. TransmittedNo. Not TransmittedPaGlobal P values are shown in bold italics.D6S289:.2985 11311.6831 24741.5224 34346.7505 44451.4726 54442.8292 68465.1196 74349.5316 81023.0236 10111.0000D6S1567:.2103 126261.0000 22317.3428 3917.1167 4119.6547 58672.2654 61729.0768 7116.2252 82223.8815 98688.8795 103141.2386 11126.1573D6S109:.5942 11819.8694 228281.0000 31213.8414 421211.0000 54027.1122 66664.8608 78089.4887 84030.2320 93350.0620 1069.4386 1184.2482 1220.1573 13111.0000D6S422:.5780 15762.6467 22011.1060 35157.5637 4813.2752 52116.4111 689.8083 7881.0000 812.5637 969.4386 1057.5637 11105.1967 1201.3173 1310.3173 1420.1573 1510.3173 1610.3173D6S1665:.0166 1186.0143 22737.2113 35278.0226 475.5637 53519.0295 62115.3173 74047.4530 852.2568 943.7055 1010.3173 1120.1573D6S506:.2022 17469.6759 23342.2987 32624.7773 44135.4913 51120.1060 62112.1172 703.0833 801.3173D6S1660:.8653 12120.8759 22127.3865 324241.0000 47278.6242 59682.2940 67178.5663 73128.6961 832.6547 9111.0000D6S299:.8469 11719.7389 297.6171 32019.8728 47582.5764 51412.6949 62515.1138 76967.8638 812.5637 93843.5785 106858.3730 113751.1356 1220.1573D6S276:.0046 166661.0000 2514.0389 32534.2413 41711.2568 55333.0310 6131.0013 71114.5485 8715.0881 986.5930 102322.8815 116580.2129 122520.4561 1357.5637D6S2439:.3267 13946.4477 23642.4969 32329.4054 42616.1228 53238.4733 63625.1590 7771.0000 865.7630 921.5637 1030.0833 1121.5637 1203.0833 1310.3173D6S1571:.8699 16066.5930 238.1317 36761.5959 4129.5127 53839.9093 6221.0000 7118.4913 843.7055 912.5637D6S1281:.7641 12725.7815 298.8084 31825.2858 44133.3524 55760.7815 66971.8658 78475.4754 83138.3994 9331.0000 1001.3173D6S105:.2693 186.5930 22114.2367 31838.0075 414141.0000 54842.5271 62734.3701 78990.9404 85647.3752 93933.4795 101523.1944 111312.8415 1241.1797 1320.1573D6S258:.7786 1139.3938 23029.8964 316161.0000 424241.0000 59594.9420 69388.7102 72022.7576 8413.0290 91720.6219 101110.8273 1154.7389 1232.6547D6S1560:.6115 16064.7194 25843.1356 36656.3653 447.3657 52939.2253 63128.6961 71417.5900 81817.8658 92842.0943 101713.4652 111917.7389 1212.5637 13111.0000D6S1610:.2612 12216.3304 23537.8137 377771.0000 43558.0171 56461.7884 66648.0918 76062.8563 81720.6219 950.0253 10221.0000 110
Referência(s)