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.

Copy number variations of chromosome 16p13.1 region associated with schizophrenia.

Deletions and reciprocal duplications of the chromosome 16p13.1 region have recently been reported in several cases of autism and mental retardation (MR). As genomic copy number variants found in these two disorders may also associate with schizophrenia, we examined 4345 schizophrenia patients and 35,079 controls from 8 European populations for duplications and deletions at the 16p13.1 locus, using microarray data. We found a threefold excess of duplications and deletions in schizophrenia cases compared with controls, with duplications present in 0.30% of cases versus 0.09% of controls (P=0.007) and deletions in 0.12 % of cases and 0.04% of controls (P>0.05). The region can be divided into three intervals defined by flanking low copy repeats. Duplications spanning intervals I and II showed the most significant (P = 0.00010) association with schizophrenia. The age of onset in duplication and deletion carriers among cases ranged from 12 to 35 years, and the majority were males with a family history of psychiatric disorders. In a single Icelandic family, a duplication spanning intervals I and II was present in two cases of schizophrenia, and individual cases of alcoholism, attention deficit hyperactivity disorder and dyslexia. Candidate genes in the region include NTAN1 and NDE1. We conclude that duplications and perhaps also deletions of chromosome 16p13.1, previously reported to be associated with autism and MR, also confer risk of schizophrenia.

Population-based linkage analysis of schizophrenia and bipolar case-control cohorts identifies a potential susceptibility locus on 19q13.

Population-based linkage analysis is a new method for analysing genomewide single nucleotide polymorphism (SNP) genotype data in case-control samples, which does not assume a common disease, common variant model. The genome is scanned for extended segments that show increased identity-by-descent sharing within case-case pairs, relative to case-control or control-control pairs. The method is robust to allelic heterogeneity and is suited to mapping genes which contain multiple, rare susceptibility variants of relatively high penetrance. We analysed genomewide SNP datasets for two schizophrenia case-control cohorts, collected in Aberdeen (461 cases, 459 controls) and Munich (429 cases, 428 controls). Population-based linkage testing must be performed within homogeneous samples and it was therefore necessary to analyse the cohorts separately. Each cohort was first subjected to several procedures to improve genetic homogeneity, including identity-by-state outlier detection and multidimensional scaling analysis. When testing only cases who reported a positive family history of major psychiatric disease, consistent with a model of strongly penetrant susceptibility alleles, we saw a distinct peak on chromosome 19q in both cohorts that appeared in meta-analysis (P=0.000016) to surpass the traditional level for genomewide significance for complex trait linkage. The linkage signal was also present in a third case-control sample for familial bipolar disorder, such that meta-analysing all three datasets together yielded a linkage P=0.0000026. A model of rare but highly penetrant disease alleles may be more applicable to some instances of major psychiatric diseases than the common disease common variant model, and we therefore suggest that other genome scan datasets are analysed with this new, complementary method.

Genome-wide association study of recurrent major depressive disorder in two European case-control cohorts.

Major depressive disorder (MDD) is a highly prevalent disorder with substantial heritability. Heritability has been shown to be substantial and higher in the variant of MDD characterized by recurrent episodes of depression. Genetic studies have thus far failed to identify clear and consistent evidence of genetic risk factors for MDD. We conducted a genome-wide association study (GWAS) in two independent datasets. The first GWAS was performed on 1022 recurrent MDD patients and 1000 controls genotyped on the Illumina 550 platform. The second was conducted on 492 recurrent MDD patients and 1052 controls selected from a population-based collection, genotyped on the Affymetrix 5.0 platform. Neither GWAS identified any SNP that achieved GWAS significance. We obtained imputed genotypes at the Illumina loci for the individuals genotyped on the Affymetrix platform, and performed a meta-analysis of the two GWASs for this common set of approximately half a million SNPs. The meta-analysis did not yield genome-wide significant results either. The results from our study suggest that SNPs with substantial odds ratio are unlikely to exist for MDD, at least in our datasets and among the relatively common SNPs genotyped or tagged by the half-million-loci arrays. Meta-analysis of larger datasets is warranted to identify SNPs with smaller effects or with rarer allele frequencies that contribute to the risk of MDD.

Alpha-5/alpha-3 nicotinic receptor subunit alleles increase risk for heavy smoking.

Twin studies indicate that additive genetic effects explain most of the variance in nicotine dependence (ND), a construct emphasizing habitual heavy smoking despite adverse consequences, tolerance and withdrawal. To detect ND alleles, we assessed cigarettes per day (CPD) regularly smoked, in two European populations via whole genome association techniques. In these approximately 7500 persons, a common haplotype in the CHRNA3-CHRNA5 nicotinic receptor subunit gene cluster was associated with CPD (nominal P=6.9 x 10(-5)). In a third set of European populations (n= approximately 7500) which had been genotyped for approximately 6000 SNPs in approximately 2000 genes, an allele in the same haplotype was associated with CPD (nominal P=2.6 x 10(-6)). These results (in three independent populations of European origin, totaling approximately 15 000 individuals) suggest that a common haplotype in the CHRNA5/CHRNA3 gene cluster on chromosome 15 contains alleles, which predispose to ND.

Evaluation of results from genome-wide studies of language and reading in a novel independent dataset.

Recent genome-wide association scans (GWAS) for reading and language abilities have pin-pointed promising new candidate loci. However, the potential contributions of these loci remain to be validated. In this study, we tested 17 of the most significantly associated single nucleotide polymorphisms (SNPs) from these GWAS studies (P < 10(-6) in the original studies) in a new independent population dataset from the Netherlands: known as Familial Influences on Literacy Abilities. This dataset comprised 483 children from 307 nuclear families and 505 adults (including parents of participating children), and provided adequate statistical power to detect the effects that were previously reported. The following measures of reading and language performance were collected: word reading fluency, nonword reading fluency, phonological awareness and rapid automatized naming. Two SNPs (rs12636438 and rs7187223) were associated with performance in multivariate and univariate testing, but these did not remain significant after correction for multiple testing. Another SNP (rs482700) was only nominally associated in the multivariate test. For the rest of the SNPs, we did not find supportive evidence of association. The findings may reflect differences between our study and the previous investigations with respect to the language of testing, the exact tests used and the recruitment criteria. Alternatively, most of the prior reported associations may have been false positives. A larger scale GWAS meta-analysis than those previously performed will likely be required to obtain robust insights into the genomic architecture underlying reading and language.

A sibling-pair based approach for mapping genetic loci that influence quantitative measures of reading disability.

Family and twin studies consistently demonstrate a significant role for genetic factors in the aetiology of the reading disorder dyslexia. However, dyslexia is complex at both the genetic and phenotypic levels, and currently the nature of the core deficit or deficits remains uncertain. Traditional approaches for mapping disease genes, originally developed for single-gene disorders, have limited success when there is not a simple relationship between genotype and phenotype. Recent advances in high-throughput genotyping technology and quantitative statistical methods have made a new approach to identifying genes involved in complex disorders possible. The method involves assessing the genetic similarity of many sibling pairs along the lengths of all their chromosomes and attempting to correlate this similarity with that of their phenotypic scores. We are adopting this approach in an ongoing genome-wide search for genes involved in dyslexia susceptibility, and have already successfully applied the method by replicating results from previous studies suggesting that a quantitative trait locus at 6p21.3 influences reading disability.

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.

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.

A genome-wide search for quantitative trait loci affecting the cortical surface area and thickness of Heschl's gyrus.

Heschl's gyrus (HG) is a core region of the auditory cortex whose morphology is highly variable across individuals. This variability has been linked to sound perception ability in both speech and music domains. Previous studies show that variations in morphological features of HG, such as cortical surface area and thickness, are heritable. To identify genetic variants that affect HG morphology, we conducted a genome-wide association scan (GWAS) meta-analysis in 3054 healthy individuals using HG surface area and thickness as quantitative traits. None of the single nucleotide polymorphisms (SNPs) showed association P values that would survive correction for multiple testing over the genome. The most significant association was found between right HG area and SNP rs72932726 close to gene DCBLD2 (3q12.1; P=2.77 × 10(-7) ). This SNP was also associated with other regions involved in speech processing. The SNP rs333332 within gene KALRN (3q21.2; P=2.27 × 10(-6) ) and rs143000161 near gene COBLL1 (2q24.3; P=2.40 × 10(-6) ) were associated with the area and thickness of left HG, respectively. Both genes are involved in the development of the nervous system. The SNP rs7062395 close to the X-linked deafness gene POU3F4 was associated with right HG thickness (Xq21.1; P=2.38 × 10(-6) ). This is the first molecular genetic analysis of variability in HG morphology.

LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia.

Left-right asymmetrical brain function underlies much of human cognition, behavior and emotion. Abnormalities of cerebral asymmetry are associated with schizophrenia and other neuropsychiatric disorders. The molecular, developmental and evolutionary origins of human brain asymmetry are unknown. We found significant association of a haplotype upstream of the gene LRRTM1 (Leucine-rich repeat transmembrane neuronal 1) with a quantitative measure of human handedness in a set of dyslexic siblings, when the haplotype was inherited paternally (P=0.00002). While we were unable to find this effect in an epidemiological set of twin-based sibships, we did find that the same haplotype is overtransmitted paternally to individuals with schizophrenia/schizoaffective disorder in a study of 1002 affected families (P=0.0014). We then found direct confirmatory evidence that LRRTM1 is an imprinted gene in humans that shows a variable pattern of maternal downregulation. We also showed that LRRTM1 is expressed during the development of specific forebrain structures, and thus could influence neuronal differentiation and connectivity. This is the first potential genetic influence on human handedness to be identified, and the first putative genetic effect on variability in human brain asymmetry. LRRTM1 is a candidate gene for involvement in several common neurodevelopmental disorders, and may have played a role in human cognitive and behavioral evolution.

Bivariate linkage scan for reading disability and attention-deficit/hyperactivity disorder localizes pleiotropic loci.

BACKGROUND: There is a growing interest in the study of the genetic origins of comorbidity, a direct consequence of the recent findings of genetic loci that are seemingly linked to more than one disorder. There are several potential causes for these shared regions of linkage, but one possibility is that these loci may harbor genes with manifold effects. The established genetic correlation between reading disability (RD) and attention-deficit/hyperactivity disorder (ADHD) suggests that their comorbidity is due at least in part to genes that have an impact on several phenotypes, a phenomenon known as pleiotropy. METHODS: We employ a bivariate linkage test for selected samples that could help identify these pleiotropic loci. This linkage method was employed to carry out the first bivariate genome-wide analysis for RD and ADHD, in a selected sample of 182 sibling pairs. RESULTS: We found evidence for a novel locus at chromosome 14q32 (multipoint LOD=2.5; singlepoint LOD=3.9) with a pleiotropic effect on RD and ADHD. Another locus at 13q32, which had been implicated in previous univariate scans of RD and ADHD, seems to have a pleiotropic effect on both disorders. 20q11 is also suggested as a pleiotropic locus. Other loci previously implicated in RD or ADHD did not exhibit bivariate linkage. CONCLUSIONS: Some loci are suggested as having pleiotropic effects on RD and ADHD, while others might have unique effects. These results highlight the utility of this bivariate linkage method to study pleiotropy.

Renal effects of continuous infusion of recombinant interleukin-2 in children.

Recombinant interleukin-2 (rIL-2) is a new promising treatment for cancer, but is associated with severe renal toxicity. This study is the first to analyse the renal effects of rIL-2 in children. Twenty-one cycles of continuous rIL-2 infusion were studied in 15 patients; mean age was 6.9 years and average weight 18.9 kg. Interstitial fluid retention and oliguria (baseline, 1.7 ml/kg per hour; nadir, 0.5 mg/kg per hour) were associated with hypotension (baseline, 101/56 mm Hg; nadir, 85/43 mm Hg) and decreased intravascular volume (plasma renin activity increased x 10). Weight gain (+7.9%) was observed in 13 cycles whereas weight loss (-6.3%) was shown in 8 cycles because of digestive and cutaneous losses, mainly in the youngest patients. This prerenal azotaemia was characterized by a decrease in creatinine clearance (from 101 to 36 ml/min per 1.73 m2) and a low fractional excretion of sodium (FENa) (from 0.70% to 0.09%). Hypotension and hypovolaemia needed vascular filling (n = 12), dopamine (n = 7) and interruption of rIL-2 (n = 2). Most abnormalities occurred as early as day 2 of therapy and were always reversible after a short period with sodium leakage (diuresis = 2.2 ml/kg per hour, FENa = 2.01%). Hypophosphataemia was associated with low urinary excretion of phosphorus, suggesting an increased uptake of inorganic phosphorus by rapidly proliferating lymphoid cells.

Investigation of quantitative measures related to reading disability in a large sample of sib-pairs from the UK.

We describe a family-based sample of individuals with reading disability collected as part of a quantitative trait loci (QTL) mapping study. Eighty-nine nuclear families (135 independent sib-pairs) were identified through a single proband using a traditional discrepancy score of predicted/actual reading ability and a known family history. Eight correlated psychometric measures were administered to each sibling, including single word reading, spelling, similarities, matrices, spoonerisms, nonword and irregular word reading, and a pseudohomophone test. Summary statistics for each measure showed a reduced mean for the probands compared to the co-sibs, which in turn was lower than that of the population. This partial co-sib regression back to the mean indicates that the measures are influenced by familial factors and therefore, may be suitable for a mapping study. The variance of each of the measures remained largely unaffected, which is reassuring for the application of a QTL approach. Multivariate genetic analysis carried out to explore the relationship between the measures identified a common factor between the reading measures that accounted for 54% of the variance. Finally the familiality estimates (range 0.32-0.73) obtained for the reading measures including the common factor (0.68) supported their heritability. These findings demonstrate the viability of this sample for QTL mapping, and will assist in the interpretation of any subsequent linkage findings in an ongoing genome scan.

Genome-wide scan of reading ability in affected sibling pairs with attention-deficit/hyperactivity disorder: unique and shared genetic effects.

Attention-deficit/hyperactivity disorder (ADHD) and reading disability (RD) are common highly heritable disorders of childhood, which frequently co-occur. Data from twin and family studies suggest that this overlap is, in part, due to shared genetic underpinnings. Here, we report the first genome-wide linkage analysis of measures of reading ability in children with ADHD, using a sample of 233 affected sibling pairs who previously participated in a genome-wide scan for susceptibility loci in ADHD. Quantitative trait locus (QTL) analysis of a composite reading factor defined from three highly correlated reading measures identified suggestive linkage (multipoint maximum lod score, MLS>2.2) in four chromosomal regions. Two regions (16p, 17q) overlap those implicated by our previous genome-wide scan for ADHD in the same sample: one region (2p) provides replication for an RD susceptibility locus, and one region (10q) falls approximately 35 cM from a modestly highlighted region in an independent genome-wide scan of siblings with ADHD. Investigation of an individual reading measure of Reading Recognition supported linkage to putative RD susceptibility regions on chromosome 8p (MLS=2.4) and 15q (MLS=1.38). Thus, the data support the existence of genetic factors that have pleiotropic effects on ADHD and reading ability--as suggested by shared linkages on 16p, 17q and possibly 10q--but also those that appear to be unique to reading--as indicated by linkages on 2p, 8p and 15q that coincide with those previously found in studies of RD. Our study also suggests that reading measures may represent useful phenotypes in ADHD research. The eventual identification of genes underlying these unique and shared linkages may increase our understanding of ADHD, RD and the relationship between the two.