Genome-wide analyses of individual differences in quantitatively assessed reading- and language-related skills in up to 34,000 people.

The use of spoken and written language is a fundamental human capacity. Individual differences in reading- and language-related skills are influenced by genetic variation, with twin-based heritability estimates of 30 to 80% depending on the trait. The genetic architecture is complex, heterogeneous, and multifactorial, but investigations of contributions of single-nucleotide polymorphisms (SNPs) were thus far underpowered. We present a multicohort genome-wide association study (GWAS) of five traits assessed individually using psychometric measures (word reading, nonword reading, spelling, phoneme awareness, and nonword repetition) in samples of 13,633 to 33,959 participants aged 5 to 26 y. We identified genome-wide significant association with word reading (rs11208009, P = 1.098 × 10-8) at a locus that has not been associated with intelligence or educational attainment. All five reading-/language-related traits showed robust SNP heritability, accounting for 13 to 26% of trait variability. Genomic structural equation modeling revealed a shared genetic factor explaining most of the variation in word/nonword reading, spelling, and phoneme awareness, which only partially overlapped with genetic variation contributing to nonword repetition, intelligence, and educational attainment. A multivariate GWAS of word/nonword reading, spelling, and phoneme awareness maximized power for follow-up investigation. Genetic correlation analysis with neuroimaging traits identified an association with the surface area of the banks of the left superior temporal sulcus, a brain region linked to the processing of spoken and written language. Heritability was enriched for genomic elements regulating gene expression in the fetal brain and in chromosomal regions that are depleted of Neanderthal variants. Together, these results provide avenues for deciphering the biological underpinnings of uniquely human traits.

Identification of brain cell types underlying genetic association with word reading and correlated traits.

Neuroimaging studies implicate multiple cortical regions in reading ability/disability. However, the neural cell types integral to the reading process are unknown. To contribute to this gap in knowledge, we integrated genetic results from genome-wide association studies for word reading (n = 5054) with gene expression datasets from adult/fetal human brain. Linkage disequilibrium score regression (LDSC) suggested that variants associated with word reading were enriched in genes expressed in adult excitatory neurons, specifically layer 5 and 6 FEZF2 expressing neurons and intratelencephalic (IT) neurons, which express the marker genes LINC00507, THEMIS, or RORB. Inhibitory neurons (VIP, SST, and PVALB) were also found. This finding was interesting as neurometabolite studies previously implicated excitatory-inhibitory imbalances in the etiology of reading disabilities (RD). We also tested traits that shared genetic etiology with word reading (previously determined by polygenic risk scores): attention-deficit/hyperactivity disorder (ADHD), educational attainment, and cognitive ability. For ADHD, we identified enrichment in L4 IT adult excitatory neurons. For educational attainment and cognitive ability, we confirmed previous studies identifying multiple subclasses of adult cortical excitatory and inhibitory neurons, as well as astrocytes and oligodendrocytes. For educational attainment and cognitive ability, we also identified enrichment in multiple fetal cortical excitatory and inhibitory neurons, intermediate progenitor cells, and radial glial cells. In summary, this study supports a role of excitatory and inhibitory neurons in reading and excitatory neurons in ADHD and contributes new information on fetal cell types enriched in educational attainment and cognitive ability, thereby improving our understanding of the neurobiological basis of reading/correlated traits.

Language and reading impairments are associated with increased prevalence of non-right-handedness.

Handedness has been studied for association with language-related disorders because of its link with language hemispheric dominance. No clear pattern has emerged, possibly because of small samples, publication bias, and heterogeneous criteria across studies. Non-right-handedness (NRH) frequency was assessed in N = 2503 cases with reading and/or language impairment and N = 4316 sex-matched controls identified from 10 distinct cohorts (age range 6-19 years old; European ethnicity) using a priori set criteria. A meta-analysis (Ncases  = 1994) showed elevated NRH % in individuals with language/reading impairment compared with controls (OR = 1.21, CI = 1.06-1.39, p = .01). The association between reading/language impairments and NRH could result from shared pathways underlying brain lateralization, handedness, and cognitive functions.

Hypothesis-driven genome-wide association studies provide novel insights into genetics of reading disabilities.

Reading Disability (RD) is often characterized by difficulties in the phonology of the language. While the molecular mechanisms underlying it are largely undetermined, loci are being revealed by genome-wide association studies (GWAS). In a previous GWAS for word reading (Price, 2020), we observed that top single-nucleotide polymorphisms (SNPs) were located near to or in genes involved in neuronal migration/axon guidance (NM/AG) or loci implicated in autism spectrum disorder (ASD). A prominent theory of RD etiology posits that it involves disturbed neuronal migration, while potential links between RD-ASD have not been extensively investigated. To improve power to identify associated loci, we up-weighted variants involved in NM/AG or ASD, separately, and performed a new Hypothesis-Driven (HD)-GWAS. The approach was applied to a Toronto RD sample and a meta-analysis of the GenLang Consortium. For the Toronto sample (n = 624), no SNPs reached significance; however, by gene-set analysis, the joint contribution of ASD-related genes passed the threshold (p~1.45 × 10-2, threshold = 2.5 × 10-2). For the GenLang Cohort (n = 26,558), SNPs in DOCK7 and CDH4 showed significant association for the NM/AG hypothesis (sFDR q = 1.02 × 10-2). To make the GenLang dataset more similar to Toronto, we repeated the analysis restricting to samples selected for reading/language deficits (n = 4152). In this GenLang selected subset, we found significant association for a locus intergenic between BTG3-C21orf91 for both hypotheses (sFDR q < 9.00 × 10-4). This study contributes candidate loci to the genetics of word reading. Data also suggest that, although different variants may be involved, alleles implicated in ASD risk may be found in the same genes as those implicated in word reading. This finding is limited to the Toronto sample suggesting that ascertainment influences genetic associations.

Language Difficulties in School-Age Children With Developmental Dyslexia.

Developmental dyslexia (DD) is a common reading disability, affecting 5% to 11% of children in North America. Children classified as having DD often have a history of early language delay (ELD) or language impairments. Nevertheless, studies have reported conflicting results as to the association between DD-ELD and the extent of current language difficulties in children with DD. To examine these relationships, we queried the parents of school-age children with reading difficulties on their child's early and current language ability. Siblings were also examined. Children were directly assessed using quantitative tests of language and reading skills. To compare this study with the literature, we divided the sample (N = 674) into three groups: DD, intermediate readers (IR), and skilled readers (SR). We found a significant association between DD and ELD, with parents of children in the DD/IR groups reporting their children put words together later than the SR group. We also found a significant association between DD and language difficulties, with children with low reading skills having low expressive/receptive language abilities. Finally, we identified early language predicted current language, which predicted reading skills. These data contribute to research indicating that children with DD experience language difficulties, suggesting early recognition may help identify reading problems.

DNA Variant in the RPGRIP1L Gene Influences Alternative Splicing.

The retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) gene encodes a ciliary protein that is critical for processes related to brain development, including development of left-right asymmetry, sonic hedgehog signaling, and neural tube formation. RPGRIP1L is a risk factor for retinal degeneration, and rare, deleterious variants in the RPGRIP1L gene cause Joubert syndrome and Meckel syndrome, both autosomal recessive disorders. These syndromes are characterized by dysfunctional primary cilia that result in abnormal development - and even lethality in the case of Meckel syndrome. Genetic studies have also implicated RPGRIP1L in psychiatric disorders by suggestive findings from genome-wide association studies and findings from rare-variant exome analyses for bipolar disorder and de novo mutations in autism. In this study we identify a common variant in RPGRIP1L, rs7203525, that influences alternative splicing, increasing the inclusion of exon 20 of RPGRIP1L. We detected this alternative splicing association in human postmortem brain tissue samples and, using a minigene assay combined with in vitro mutagenesis, confirmed that the alternative splicing is attributable to the alleles of this variant. The predominate RPGRIP1L isoform expressed in adult brains does not contain exon 20; thus, a shift to include this exon may impact brain function.

Genome-wide association study of word reading: Overlap with risk genes for neurodevelopmental disorders.

Reading disabilities (RD) are the most common neurocognitive disorder, affecting 5% to 17% of children in North America. These children often have comorbid neurodevelopmental/psychiatric disorders, such as attention deficit/hyperactivity disorder (ADHD). The genetics of RD and their overlap with other disorders is incompletely understood. To contribute to this, we performed a genome-wide association study (GWAS) for word reading. Then, using summary statistics from neurodevelopmental/psychiatric disorders, we computed polygenic risk scores (PRS) and used them to predict reading ability in our samples. This enabled us to test the shared aetiology between RD and other disorders. The GWAS consisted of 5.3 million single nucleotide polymorphisms (SNPs) and two samples; a family-based sample recruited for reading difficulties in Toronto (n = 624) and a population-based sample recruited in Philadelphia [Philadelphia Neurodevelopmental Cohort (PNC)] (n = 4430). The Toronto sample SNP-based analysis identified suggestive SNPs (P ~ 5 × 10-7 ) in the ARHGAP23 gene, which is implicated in neuronal migration/axon pathfinding. The PNC gene-based analysis identified significant associations (P < 2.72 × 10-6 ) for LINC00935 and CCNT1, located in the region of the KANSL2/CCNT1/LINC00935/SNORA2B/SNORA34/MIR4701/ADCY6 genes on chromosome 12q, with near significant SNP-based analysis. PRS identified significant overlap between word reading and intelligence (R2 = 0.18, P = 7.25 × 10-181 ), word reading and educational attainment (R2 = 0.07, P = 4.91 × 10-48 ) and word reading and ADHD (R2 = 0.02, P = 8.70 × 10-6 ; threshold for significance = 7.14 × 10-3 ). Overlap was also found between RD and autism spectrum disorder (ASD) as top-ranked genes were previously implicated in autism by rare and copy number variant analyses. These findings support shared risk between word reading, cognitive measures, educational outcomes and neurodevelopmental disorders, including ASD.

Chromosome 5 and Gilles de la Tourette syndrome: Linkage in a large pedigree and association study of six candidates in the region.

Gilles de la Tourette Syndrome (TS) is a neuropsychiatric disorder characterized by both motor and vocal tics. In our previous genome scan for TS we identified evidence for linkage to the centromeric region of chromosome 5 in a single large family of 32 individuals with 10 family members with TS or chronic multiple tics (CMT). In this paper we report further analyses of the 5p-centromeric region in this pedigree. An additional 11 family members were identified and screened for TS. Using a set of 14 microsatellite markers we refined the linked region to a approximately 28 Mb interval between the markers D5S1506 and D5S76. A set of six candidate genes located in this region were selected to be tested for genetic association with TS. These genes were GDNF, ITGA1, ISL1, FGF10, HCN1 and SLC1A3. The TDT statistic was used for the association tests in a sample of 171 independent nuclear families with 241 affected children with TS. We found no evidence for an association between TS and markers in these genes in this sample of families. This study represents the first efforts to narrow the linkage region in the extended pedigree and the first tests of candidate genes in the chromosome 5 region linked to TS.

Tagging SNP association study of the IL-1beta gene (IL1B) and childhood-onset mood disorders.

Given substantial evidence for IL-1beta involvement in the etiology of depression, the IL1B gene is a strong candidate for involvement in susceptibility to depressive disorders. However, association studies investigating this, to date, have been limited to just two polymorphisms (rs1143627[-31T/C] and rs16944[-511C/T]) that constitute only a fraction of the genetic variation that is actually present across this gene in the population. Here, in a family-based association study of childhood-onset mood disorders (COMD), characterized by onset of depression before the age of 15, we have used a gene-wide approach, employing a panel of five tagging SNPs spanning the entire gene. Based on TDT analyses of both individual alleles and haplotypes, in a study sample of 646 families (with 782 affected children), none of the SNPs, including those implicated in transcriptional regulation of the gene, showed evidence for association with COMD. This is the largest and most comprehensive study of IL1B in relation to mood disorders that has been carried out, to date. The results do not support the involvement of IL1B as a major factor in genetic risk for early-onset mood disorders.

Relations between multi-informant assessments of ADHD symptoms, DAT1, and DRD4.

Researchers conducting candidate gene studies of attention-deficit/hyperactivity disorder (ADHD) typically obtain symptom ratings from multiple informants (i.e., mothers, fathers, and teachers) and use a psychologist's best estimate or a simple algorithm, such as taking the highest symptom ratings across informants, to construct diagnostic phenotypes for estimating association. Nonetheless, these methods have never been empirically validated in the context of a molecular genetic study. In the current study, the authors systematically evaluated several methods of operationalizing phenotypes and the resulting evidence for association between ADHD and the candidate genes: dopamine transporter gene (DAT1) and dopamine D4 receptor gene (DRD4). Use of symptom scores as continuous scales in regression analysis suggested that the combination of mother and teacher ratings yielded the strongest evidence for association between hyperactive-impulsive ADHD symptoms and DAT1 and between inattentive ADHD symptoms and DRD4. Teacher ratings alone were sufficient for evaluating the association between inattentive symptoms and DAT1. Further, this regression-based method consistently yielded stronger evidence for association among ADHD symptoms, DAT1, and DRD4 than did three simple algorithms (i.e., the and, or, and averaging rules). The implications of these results for future molecular genetic studies of ADHD are discussed.

Association of the dopamine transporter gene and ADHD symptoms in a Canadian population-based sample of same-age twins.

Attention deficit hyperactivity disorder (ADHD) is the most prevalent psychiatric disorder emerging during childhood. Psychostimulant medications (e.g., methylphenidate) noticeably reduce ADHD symptoms in most children. Since methylphenidate inhibits dopamine transporter activity, the dopamine transporter gene (DAT1) was considered to be the prime candidate risk gene in ADHD. Several studies found evidence for an association between the 10-repeat allele of the variable number of tandem repeat (VNTR) located in the 3' untranslated region and ADHD and/or ADHD symptoms in clinical and population-based samples. However, this finding was not replicated in all samples. In this study, we investigated the association between the DAT1 gene and ADHD symptoms in a population-based twin sample from Québec (Canada). We used two polymorphisms, the VNTR and rs27072, the last providing the most significant results in a clinical sample from Toronto (Ontario, Canada). No association was noted between the VNTR and ADHD symptoms in children at 6 and 7 years of age, as reported by teachers. However, a significant association was found for the rs27072 polymorphism and symptoms of inattention and hyperactivity/impulsivity. These findings indicate that the DAT1 gene contributes to ADHD symptoms in this sample and further suggest that the VNTR may not be the optimal polymorphism for study in all populations.

The gene for synapsin III and attention-deficit hyperactivity disorder.

OBJECTIVE: Recent studies have implicated the involvement of proteins regulating neurotransmitter release in the etiology of attention deficit hyperactivity disorder. On the basis of the role of synapsin III in the modulation of neurotransmitter release, we tested this gene as a candidate contributing to the genetic susceptibility of attention deficit hyperactivity disorder. METHOD: In this study, we genotyped five markers across the gene on 177 small, nuclear families consisting of an attention deficit hyperactivity disorder proband, their parents, and 43 affected siblings. We examined the transmission of the alleles at each one of these sites and the haplotypes of the polymorphisms using the transmission disequilibrium test. RESULT: Our observations did not yield any evidence of biased transmission of the alleles at any polymorphism or haplotype. On the basis of the evidence for synapsins in learning and memory from animal models, we also investigated the relationship of this gene to verbal short-term and working memory as measured by digit span forward and backwards. No evidence was found for an association of this gene to these traits. CONCLUSION: Our findings with this particular sample do not support the synapsin III locus as a major susceptibility locus contributing to attention deficit hyperactivity disorder.

Association study of the SLITRK5 gene and Tourette syndrome.

Tourette syndrome (TS) and obsessive-compulsive disorder commonly occur together. Family studies indicate shared genetic risk factors. SLITRK5, one of a family of six SLITRK genes, has been suggested as a possible candidate gene contributing towards obsessive-compulsive disorder on the basis of the mouse knockout model that shows excessive grooming behaviours that are alleviated with fluoxetine. In this study, we tested the SLITRK5 gene as a candidate for TS in a family-based sample with 377 affected children. Using single nucleotide polymorphisms tagging the gene, we did not find any evidence supporting the association of TS and SLITRK5.

Analysis of the dopamine beta hydroxylase gene in Gilles de la Tourette syndrome.

Numerous lines of evidence support the role of the catecholamines in the development of tics and Gilles de la Tourette syndrome (GTS). Dopamine-beta-hydroxylase (DBH) is the key enzyme in the conversion of dopamine to norepinephrine and the alleles of several polymorphisms of the DBH gene are correlated with individual variation in serum levels of the enzyme. We investigated the genetic relationship of the gene for DBH to GTS in two samples, one collected in Canada and one collected in Turkey. In total 106 affected probands and siblings in 71 nuclear pedigrees and 40 affected individuals and 71 family members in five multi-generational pedigrees were genotyped for three polymorphisms in the DBH locus. In the Canadian pedigrees we found no convincing evidence for linkage either in the multi-generational pedigrees or association in the nuclear families. We found significant evidence for association in the Turkish pedigrees (n = 29) for the 19 bp insertion/deletion markers; however, there was no supporting evidence for association with the other two markers. Based on the small sample size and low number of informative transmissions, we conclude that the results from the 19 bp insertion/deletion markers may be a chance false positive finding. These findings, in total, suggest that the DBH locus is unlikely to be a major gene influencing the susceptibility to DBH.

Gene for the serotonin transporter and ADHD: no association with two functional polymorphisms.

Evidence from both human and animal studies implicates the serotonergic system in the development of attention-deficit hyperactivity disorder (ADHD) including positive association studies for several key serotonergic genes. The serotonin transporter (HTT) regulates the availability of serotonin by reuptake of the neurotransmitter from the synaptic cleft. Several studies have reported an association of this gene to ADHD, specifically the long variant of a common insertion/deletion polymorphism located in the promoter of this gene that results in increased transcription and higher HTT expression. An additional study found no evidence for an association with this polymorphism. Recently, an A/G single nucleotide polymorphism (SNP) was found within the promoter polymorphism with functional studies indicating that the long variant containing the G allele at this site behaves like the short variant. This previously unidentified functional change may have confounded earlier association studies. We investigated the relationship of several variants to ADHD: the promoter polymorphisms, SNP in the 3' untranslated region (3'UTR) with a reported association to ADHD and a rare, non-synonymous coding SNP. These polymorphisms were genotyped in 209 ADHD families identified through an affected proband. We did not find evidence for an association of these polymorphisms, or haplotypes of these polymorphisms, to ADHD in this sample.

Sequence variation in the 3'-untranslated region of the dopamine transporter gene and attention-deficit hyperactivity disorder (ADHD).

The dopamine transporter gene (DAT1) has been reported to be associated with attention-deficit hyperactivity disorder (ADHD) in a number of studies [Cook et al. (1995): Am J Human Genet 56(4):9993-998; Gill et al. (1997): Mol Psychiatry 2(4):311-313; Waldman et al. (1998): Am J Human Genet 63(6):1767-1776; Barr et al. (2001): Biol Psychiatry 49(4):333-339; Curran et al. (2001): Mol Psychiatry 6(4):425-428; Chen et al. (2003): Mol Psychiatry 8(4):393-396]. Specifically, the 10-repeat allele of the 40-bp variable number of tandem repeats (VNTR) polymorphism located in the 3' untranslated region (UTR) of the gene has been found to be associated with ADHD. There is evidence from in vitro studies indicating that variability in the repeat number, and sequence variation in the 3'-UTR of the DAT1 gene may influence the level of the dopamine transporter protein [Fuke et al. (2001): Pharmacogenomics J 1(2):152-156; Miller and Madras (2002): Mol Psychiatry 7(1):44-55]. In this study, we investigated whether DNA variation in the DAT1 3'UTR contributed to ADHD by genotyping DNA variants around the VNTR region in a sample of 178 ADHD families. These included a MspI polymorphism (rs27072), a DraI DNA change (T/C) reported to influence DAT1 expression levels, and a BstUI polymorphism (rs3863145) in addition to the VNTR. We also screened the VNTR region by direct resequencing to determine if there was sequence variation within the repeat units that could account for the association. Our results indicate that DAT1 is associated with ADHD in our sample but not with alleles of the VNTR polymorphism. We did not find any variation in the sequence for either the 10- or 9-repeat alleles in the probands screened nor did we observe the reported DraI (T/C) variation. Our results therefore refute the possibility of the reported DraI variation or alleles of the VNTR as the functional variants contributing to the disorder.

Association study of neurotrophic tyrosine kinase receptor type 2 (NTRK2) and childhood-onset mood disorders.

Childhood-onset mood disorders (COMD) are often familial, and twin studies of COMD provide compelling evidence that genetic factors are involved. Deficits in neural plasticity have been suggested to underlie the development of depression. The receptor tropomyosin related kinase B (TrkB) and its ligand, brain derived neurotrophic factor (BDNF), play essential roles in neural plasticity, and mRNA expression of both of these genes has been shown to be influenced by stress and chronic antidepressant treatment. In addition, TrkB knock-out mice display inappropriate stress coping mechanisms. Having previously shown that BDNF is associated with COMD, in this study we investigated the gene encoding TrkB, neurotrophic tyrosine kinase, receptor, type 2 (NTRK2) as a susceptibility factor in COMD. We tested for association of NTRK2 with COMD in two independent samples: (a) a case-control sample matched on ethnicity and gender, consisting of 120 cases who met DSM III/IV criteria for major depressive or dysthymic disorder before age 14 or bipolar I/II before the age of 18, and controls, and (b) a family based control sample of 113 families collected in Hungary, identified by a proband between the age of 7 and 14 who met DSM IV criteria for major depressive disorder or bipolar I/II disorder. There was no evidence for an allelic or genotypic association of three polymorphisms of NTRK2 with COMD in the case-control sample. Also, in the family based sample, using the transmission disequilibrium test (TDT), we did not identify any evidence of allelic association for each marker individually or when haplotypes were analyzed. Based on these results, using these three polymorphisms, we do not find support for NTRK2 as a susceptibility gene for COMD.

Joint analysis of the DRD5 marker concludes association with attention-deficit/hyperactivity disorder confined to the predominantly inattentive and combined subtypes.

Attention-deficit/hyperactivity disorder (ADHD) is a highly heritable, heterogeneous disorder of early onset, consisting of a triad of symptoms: inattention, hyperactivity, and impulsivity. The disorder has a significant genetic component, and theories of etiology include abnormalities in the dopaminergic system, with DRD4, DAT1, SNAP25, and DRD5 being implicated as major susceptibility genes. An initial report of association between ADHD and the common 148-bp allele of a microsatellite marker located 18.5 kb from the DRD5 gene has been followed by several studies showing nonsignificant trends toward association with the same allele. To establish the postulated association of the (CA)(n) repeat with ADHD, we collected genotypic information from 14 independent samples of probands and their parents, analyzed them individually and, in the absence of heterogeneity, analyzed them as a joint sample. The joint analysis showed association with the DRD5 locus (P=.00005; odds ratio 1.24; 95% confidence interval 1.12-1.38). This association appears to be confined to the predominantly inattentive and combined clinical subtypes.