Next-gen sequencing identifies non-coding variation disrupting miRNA-binding sites in neurological disorders.

Understanding the genetic factors underlying neurodevelopmental and neuropsychiatric disorders is a major challenge given their prevalence and potential severity for quality of life. While large-scale genomic screens have made major advances in this area, for many disorders the genetic underpinnings are complex and poorly understood. To date the field has focused predominantly on protein coding variation, but given the importance of tightly controlled gene expression for normal brain development and disorder, variation that affects non-coding regulatory regions of the genome is likely to play an important role in these phenotypes. Herein we show the importance of 3 prime untranslated region (3'UTR) non-coding regulatory variants across neurodevelopmental and neuropsychiatric disorders. We devised a pipeline for identifying and functionally validating putatively pathogenic variants from next generation sequencing (NGS) data. We applied this pipeline to a cohort of children with severe specific language impairment (SLI) and identified a functional, SLI-associated variant affecting gene regulation in cells and post-mortem human brain. This variant and the affected gene (ARHGEF39) represent new putative risk factors for SLI. Furthermore, we identified 3'UTR regulatory variants across autism, schizophrenia and bipolar disorder NGS cohorts demonstrating their impact on neurodevelopmental and neuropsychiatric disorders. Our findings show the importance of investigating non-coding regulatory variants when determining risk factors contributing to neurodevelopmental and neuropsychiatric disorders. In the future, integration of such regulatory variation with protein coding changes will be essential for uncovering the genetic causes of complex neurological disorders and the fundamental mechanisms underlying health and disease.

Lack of replication for the myosin-18B association with mathematical ability in independent cohorts.

Twin studies indicate that dyscalculia (or mathematical disability) is caused partly by a genetic component, which is yet to be understood at the molecular level. Recently, a coding variant (rs133885) in the myosin-18B gene was shown to be associated with mathematical abilities with a specific effect among children with dyslexia. This association represents one of the most significant genetic associations reported to date for mathematical abilities and the only one reaching genome-wide statistical significance. We conducted a replication study in different cohorts to assess the effect of rs133885 maths-related measures. The study was conducted primarily using the Avon Longitudinal Study of Parents and Children (ALSPAC), (N = 3819). We tested additional cohorts including the York Cohort, the Specific Language Impairment Consortium (SLIC) cohort and the Raine Cohort, and stratified them for a definition of dyslexia whenever possible. We did not observe any associations between rs133885 in myosin-18B and mathematical abilities among individuals with dyslexia or in the general population. Our results suggest that the myosin-18B variant is unlikely to be a main factor contributing to mathematical abilities.

Genome-wide screening for DNA variants associated with reading and language traits.

Reading and language abilities are heritable traits that are likely to share some genetic influences with each other. To identify pleiotropic genetic variants affecting these traits, we first performed a genome-wide association scan (GWAS) meta-analysis using three richly characterized datasets comprising individuals with histories of reading or language problems, and their siblings. GWAS was performed in a total of 1862 participants using the first principal component computed from several quantitative measures of reading- and language-related abilities, both before and after adjustment for performance IQ. We identified novel suggestive associations at the SNPs rs59197085 and rs5995177 (uncorrected P ≈ 10(-7) for each SNP), located respectively at the CCDC136/FLNC and RBFOX2 genes. Each of these SNPs then showed evidence for effects across multiple reading and language traits in univariate association testing against the individual traits. FLNC encodes a structural protein involved in cytoskeleton remodelling, while RBFOX2 is an important regulator of alternative splicing in neurons. The CCDC136/FLNC locus showed association with a comparable reading/language measure in an independent sample of 6434 participants from the general population, although involving distinct alleles of the associated SNP. Our datasets will form an important part of on-going international efforts to identify genes contributing to reading and language skills.

Genome-wide association analyses of child genotype effects and parent-of-origin effects in specific language impairment.

Specific language impairment (SLI) is a neurodevelopmental disorder that affects linguistic abilities when development is otherwise normal. We report the results of a genome-wide association study of SLI which included parent-of-origin effects and child genotype effects and used 278 families of language-impaired children. The child genotype effects analysis did not identify significant associations. We found genome-wide significant paternal parent-of-origin effects on chromosome 14q12 (P = 3.74 × 10(-8)) and suggestive maternal parent-of-origin effects on chromosome 5p13 (P = 1.16 × 10(-7)). A subsequent targeted association of six single-nucleotide-polymorphisms (SNPs) on chromosome 5 in 313 language-impaired individuals and their mothers from the ALSPAC cohort replicated the maternal effects, albeit in the opposite direction (P = 0.001); as fathers' genotypes were not available in the ALSPAC study, the replication analysis did not include paternal parent-of-origin effects. The paternally-associated SNP on chromosome 14 yields a non-synonymous coding change within the NOP9 gene. This gene encodes an RNA-binding protein that has been reported to be significantly dysregulated in individuals with schizophrenia. The region of maternal association on chromosome 5 falls between the PTGER4 and DAB2 genes, in a region previously implicated in autism and ADHD. The top SNP in this association locus is a potential expression QTL of ARHGEF19 (also called WGEF) on chromosome 1. Members of this protein family have been implicated in intellectual disability. In summary, this study implicates parent-of-origin effects in language impairment, and adds an interesting new dimension to the emerging picture of shared genetic etiology across various neurodevelopmental disorders.

Investigation of dyslexia and SLI risk variants in reading- and language-impaired subjects.

Dyslexia (or reading disability) and specific language impairment (or SLI) are common childhood disorders that show considerable co-morbidity and diagnostic overlaps and have been suggested to share some genetic aetiology. Recently, genetic risk variants have been identified for SLI and dyslexia enabling the direct evaluation of possible shared genetic influences between these disorders. In this study we investigate the role of variants in these genes (namely MRPL19/C20RF3, ROBO1, DCDC2, KIAA0319, DYX1C1, CNTNAP2, ATP2C2 and CMIP) in the aetiology of SLI and dyslexia. We perform case-control and quantitative association analyses using measures of oral and written language skills in samples of SLI and dyslexic families and cases. We replicate association between KIAA0319 and DCDC2 and dyslexia and provide evidence to support a role for KIAA0319 in oral language ability. In addition, we find association between reading-related measures and variants in CNTNAP2 and CMIP in the SLI families.

Genetic advances in the study of speech and language disorders.

Developmental speech and language disorders cover a wide range of childhood conditions with overlapping but heterogeneous phenotypes and underlying etiologies. This characteristic heterogeneity hinders accurate diagnosis, can complicate treatment strategies, and causes difficulties in the identification of causal factors. Nonetheless, over the last decade, genetic variants have been identified that may predispose certain individuals to different aspects of speech and language difficulties. In this review, we summarize advances in the genetic investigation of stuttering, speech-sound disorder (SSD), specific language impairment (SLI), and developmental verbal dyspraxia (DVD). We discuss how the identification and study of specific genes and pathways, including FOXP2, CNTNAP2, ATP2C2, CMIP, and lysosomal enzymes, may advance our understanding of the etiology of speech and language disorders and enable us to better understand the relationships between the different forms of impairment across the spectrum.

A locus for an auditory processing deficit and language impairment in an extended pedigree maps to 12p13.31-q14.3.

Despite the apparent robustness of language learning in humans, a large number of children still fail to develop appropriate language skills despite adequate means and opportunity. Most cases of language impairment have a complex etiology, with genetic and environmental influences. In contrast, we describe a three-generation German family who present with an apparently simple segregation of language impairment. Investigations of the family indicate auditory processing difficulties as a core deficit. Affected members performed poorly on a nonword repetition task and present with communication impairments. The brain activation pattern for syllable duration as measured by event-related brain potentials showed clear differences between affected family members and controls, with only affected members displaying a late discrimination negativity. In conjunction with psychoacoustic data showing deficiencies in auditory duration discrimination, the present results indicate increased processing demands in discriminating syllables of different duration. This, we argue, forms the cognitive basis of the observed language impairment in this family. Genome-wide linkage analysis showed a haplotype in the central region of chromosome 12 which reaches the maximum possible logarithm of odds ratio (LOD) score and fully co-segregates with the language impairment, consistent with an autosomal dominant, fully penetrant mode of inheritance. Whole genome analysis yielded no novel inherited copy number variants strengthening the case for a simple inheritance pattern. Several genes in this region of chromosome 12 which are potentially implicated in language impairment did not contain polymorphisms likely to be the causative mutation, which is as yet unknown.

Sixteen New Cases Contributing to the Characterization of Patients with Distal 22q11.2 Microduplications.

The chromosome region 22q11.2 has long been recognized to be susceptible to genomic rearrangement. More recently, this genomic instability has been shown to extend distally (involving LCR22E-H) to the commonly deleted/duplicated region. To date, 21 index cases with 'distal' 22q11.2 duplications have been reported. We report on the clinical and molecular characterization of 16 individuals with distal 22q11.2 duplications identified by DNA microarray analysis. Two of the individuals have been partly described previously. The clinical phenotype varied among the patients in this study, although the majority displayed various degrees of developmental delay and speech disturbances. Other clinical features included behavioral problems, hypotonia, and dysmorphic facial features. Notably, none of the patients was diagnosed with a congenital heart defect. We found a high degree of inherited duplications. Additional copy number changes of unclear clinical significance were identified in 5 of our patients, and it is possible that these may contribute to the phenotypic expression in these patients as has been suggested recently in a 2-hit 'digenic' model for 16p12.1 deletions. The varied phenotypic expression and incomplete penetrance observed for distal 22q11.2 duplications makes it exceedingly difficult to ascribe pathogenicity for these duplications. Given the observed enrichment of the duplication in patient samples versus healthy controls, it is likely that distal 22q11.2 duplications represent a susceptibility/risk locus for speech and mild developmental delay.

Detection, breakpoint identification and detailed characterisation of a CNV at the FRA16D site using SNP assays.

Copy Number Variants (CNV) and other submicroscopic structural changes are now recognised to be widespread across the human genome. We show that SNP data generated for association study can be utilised for the identification of deletion CNVs. During analysis of data for an SNP association study for Specific Language Impairment (SLI) a deletion was identified. SLI adversely affects the language development of children in the absence of any obvious cause. Previous studies have found linkage to a region on chromosome 16. The deletion was located in a known fragile site FRA16D in intron 5-6 of the WWOX gene (also known as FOR). Changes in the FRA16D site have been previously linked to cancer and are often characterised in cell lines. A long-range PCR assay was used to confirm the existence of the deletion. We also show the breakpoint identification and large-scale characterisation of this CNV in a normal human sample set.

Genetic and phenotypic effects of phonological short-term memory and grammatical morphology in specific language impairment.

Deficits in phonological short-term memory and aspects of verb grammar morphology have been proposed as phenotypic markers of specific language impairment (SLI) with the suggestion that these traits are likely to be under different genetic influences. This investigation in 300 first-degree relatives of 93 probands with SLI examined familial aggregation and genetic linkage of two measures thought to index these two traits, non-word repetition and tense marking. In particular, the involvement of chromosomes 16q and 19q was examined as previous studies found these two regions to be related to SLI. Results showed a strong association between relatives' and probands' scores on non-word repetition. In contrast, no association was found for tense marking when examined as a continuous measure. However, significant familial aggregation was found when tense marking was treated as a binary measure with a cut-off point of -1.5 SD, suggestive of the possibility that qualitative distinctions in the trait may be familial while quantitative variability may be more a consequence of non-familial factors. Linkage analyses supported previous findings of the SLI Consortium of linkage to chromosome 16q for phonological short-term memory and to chromosome 19q for expressive language. In addition, we report new findings that relate to the past tense phenotype. For the continuous measure, linkage was found on both chromosomes, but evidence was stronger on chromosome 19. For the binary measure, linkage was observed on chromosome 19 but not on chromosome 16.

FOXP2 is not a major susceptibility gene for autism or specific language impairment.

The FOXP2 gene, located on human 7q31 (at the SPCH1 locus), encodes a transcription factor containing a polyglutamine tract and a forkhead domain. FOXP2 is mutated in a severe monogenic form of speech and language impairment, segregating within a single large pedigree, and is also disrupted by a translocation in an isolated case. Several studies of autistic disorder have demonstrated linkage to a similar region of 7q (the AUTS1 locus), leading to the proposal that a single genetic factor on 7q31 contributes to both autism and language disorders. In the present study, we directly evaluate the impact of the FOXP2 gene with regard to both complex language impairments and autism, through use of association and mutation screening analyses. We conclude that coding-region variants in FOXP2 do not underlie the AUTS1 linkage and that the gene is unlikely to play a role in autism or more common forms of language impairment.