Leveraging base-pair mammalian constraint to understand genetic variation and human disease.

Thousands of genomic regions have been associated with heritable human diseases, but attempts to elucidate biological mechanisms are impeded by an inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function, agnostic to cell type or disease mechanism. Single-base phyloP scores from 240 mammals identified 3.3% of the human genome as significantly constrained and likely functional. We compared phyloP scores to genome annotation, association studies, copy-number variation, clinical genetics findings, and cancer data. Constrained positions are enriched for variants that explain common disease heritability more than other functional annotations. Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

Leveraging Base Pair Mammalian Constraint to Understand Genetic Variation and Human Disease.

Although thousands of genomic regions have been associated with heritable human diseases, attempts to elucidate biological mechanisms are impeded by a general inability to discern which genomic positions are functionally important. Evolutionary constraint is a powerful predictor of function that is agnostic to cell type or disease mechanism. Here, single base phyloP scores from the whole genome alignment of 240 placental mammals identified 3.5% of the human genome as significantly constrained, and likely functional. We compared these scores to large-scale genome annotation, genome-wide association studies (GWAS), copy number variation, clinical genetics findings, and cancer data sets. Evolutionarily constrained positions are enriched for variants explaining common disease heritability (more than any other functional annotation). Our results improve variant annotation but also highlight that the regulatory landscape of the human genome still needs to be further explored and linked to disease.

DeepGWAS: Enhance GWAS Signals for Neuropsychiatric Disorders via Deep Neural Network.

Genetic dissection of neuropsychiatric disorders can potentially reveal novel therapeutic targets. While genome-wide association studies (GWAS) have tremendously advanced our understanding, we approach a sample size bottleneck (i.e., the number of cases needed to identify >90% of all loci is impractical). Therefore, computationally enhancing GWAS on existing samples may be particularly valuable. Here, we describe DeepGWAS, a deep neural network-based method to enhance GWAS by integrating GWAS results with linkage disequilibrium and brain-related functional annotations. DeepGWAS enhanced schizophrenia (SCZ) loci by ~3X when applied to the largest European GWAS, and 21.3% enhanced loci were validated by the latest multi-ancestry GWAS. Importantly, DeepGWAS models can be transferred to other neuropsychiatric disorders. Transferring SCZ-trained models to Alzheimer's disease and major depressive disorder, we observed 1.3-17.6X detected loci compared to standard GWAS, among which 27-40% were validated by other GWAS studies. We anticipate DeepGWAS to be a powerful tool in GWAS studies.

Rare tandem repeat expansions associate with genes involved in synaptic and neuronal signaling functions in schizophrenia.

Tandem repeat expansions (TREs) are associated with over 60 monogenic disorders and have recently been implicated in complex disorders such as cancer and autism spectrum disorder. The role of TREs in schizophrenia is now emerging. In this study, we have performed a genome-wide investigation of TREs in schizophrenia. Using genome sequence data from 1154 Swedish schizophrenia cases and 934 ancestry-matched population controls, we have detected genome-wide rare (<0.1% population frequency) TREs that have motifs with a length of 2-20 base pairs. We find that the proportion of individuals carrying rare TREs is significantly higher in the schizophrenia group. There is a significantly higher burden of rare TREs in schizophrenia cases than in controls in genic regions, particularly in postsynaptic genes, in genes overlapping brain expression quantitative trait loci, and in brain-expressed genes that are differentially expressed between schizophrenia cases and controls. We demonstrate that TRE-associated genes are more constrained and primarily impact synaptic and neuronal signaling functions. These results have been replicated in an independent Canadian sample that consisted of 252 schizophrenia cases of European ancestry and 222 ancestry-matched controls. Our results support the involvement of rare TREs in schizophrenia etiology.

Increased Prevalence of Rare Copy Number Variants in Treatment-Resistant Psychosis.

BACKGROUND: It remains unknown why ~30% of patients with psychotic disorders fail to respond to treatment. Previous genomic investigations of treatment-resistant psychosis have been inconclusive, but some evidence suggests a possible link between rare disease-associated copy number variants (CNVs) and worse clinical outcomes in schizophrenia. Here, we identified schizophrenia-associated CNVs in patients with treatment-resistant psychotic symptoms and then compared the prevalence of these CNVs to previously published schizophrenia cases not selected for treatment resistance. METHODS: CNVs were identified using chromosomal microarray (CMA) and whole exome sequencing (WES) in 509 patients with treatment-resistant psychosis (a lack of clinical response to ≥3 adequate antipsychotic medication trials over at least 5 years of psychiatric hospitalization). Prevalence of schizophrenia-associated CNVs in this sample was compared to that in a previously published large schizophrenia cohort study. RESULTS: Integrating CMA and WES data, we identified 47 cases (9.2%) with at least one CNV of known or possible neuropsychiatric risk. 4.7% (n = 24) carried a known neurodevelopmental risk CNV. The prevalence of well-replicated schizophrenia-associated CNVs was 4.1%, with duplications of the 16p11.2 and 15q11.2-q13.1 regions, and deletions of the 22q11.2 chromosomal region as the most frequent CNVs. Pairwise loci-based analysis identified duplications of 15q11.2-q13.1 to be independently associated with treatment resistance. CONCLUSIONS: These findings suggest that CNVs may uniquely impact clinical phenotypes beyond increasing risk for schizophrenia and may potentially serve as biological entry points for studying treatment resistance. Further investigation will be necessary to elucidate the spectrum of phenotypic characteristics observed in adult psychiatric patients with disease-associated CNVs.

The impact of educational attainment, intelligence and intellectual disability on schizophrenia: a Swedish population-based register and genetic study.

Schizophrenia (SCZ) is highly heterogenous and no subtypes characterizing treatment response or longitudinal course well. Cognitive impairment is a core clinical feature of SCZ and a determinant of poorer outcome. Genetic overlap between SCZ and cognitive traits is complex, with limited studies of comprehensive epidemiological and genomic evidence. To examine the relation between SCZ and three cognitive traits, educational attainment (EDU), premorbid cognitive ability, and intellectual disability (ID), we used two Swedish samples: a national cohort (14,230 SCZ cases and 3,816,264 controls) and a subsample with comprehensive genetic data (4992 cases and 6009 controls). Population-based analyses confirmed worse cognition as a risk factor for SCZ, and the pedigree and SNP-based genetic correlations were comparable. In the genotyped cases, those with high EDU and premorbid cognitive ability tended to have higher polygenetic risk scores (PRS) of EDU and intelligence and fewer rare exonic variants. Finally, by applying an empirical clustering method, we dissected SCZ cases into four replicable subgroups characterized by EDU and ID. In particular, the subgroup with higher EDU in the national cohort had fewer adverse outcomes including long hospitalization and death. In the genotyped subsample, this subgroup had higher PRS of EDU and no excess of rare genetic burdens than controls. In conclusion, we found extensive evidence of a robust relation between cognitive traits and SCZ, underscoring the importance of cognition in dissecting the heterogeneity of SCZ.

Elevated common variant genetic risk for tourette syndrome in a densely-affected pedigree.

Tourette syndrome (TS) is a highly heritable neuropsychiatric disorder with complex patterns of genetic inheritance. Recent genetic findings in TS have highlighted both numerous common variants with small effects and a few rare variants with moderate or large effects. Here we searched for genetic causes of TS in a large, densely-affected British pedigree using a systematic genomic approach. This pedigree spans six generations and includes 122 members, 85 of whom were individually interviewed, and 53 of whom were diagnosed as "cases" (consisting of 28 with definite or probable TS, 20 with chronic multiple tics [CMT], and five with obsessive-compulsive behaviors [OCB]). A total of 66 DNA samples were available (25 TS, 15 CMT, 4 OCB cases, and 22 unaffecteds) and all were genotyped using a dense single nucleotide polymorphism (SNP) array to identify shared segments, copy number variants (CNVs), and to calculate genetic risk scores. Eight cases were also whole genome sequenced to test whether any rare variants were shared identical by descent. While we did not identify any notable CNVs, single nucleotide variants, indels or repeat expansions of near-Mendelian effect, the most distinctive feature of this family proved to be an unusually high load of common risk alleles for TS. We found that cases within this family carried a higher load of TS common variant risk similar to that previously found in unrelated TS cases. Thus far, the strongest evidence from genetic data for contribution to TS risk in this family comes from multiple common risk variants rather than one or a few variants of strong effect.

Treatment-resistant psychotic symptoms and early-onset dementia: A case report of the 3q29 deletion syndrome.

The 3q29 deletion is a rare copy number variant associated with neurodevelopmental and psychiatric disorders, including a >40-fold increased risk for schizophrenia. Current understanding of the clinical phenotype is derived primarily from published cases of patients in childhood or early adolescence. Symptoms include mild to moderate learning disability, developmental delay, facial dysmorphism, microcephaly, ocular disorders, and gastrointestinal abnormalities. There is, however, a lack of detailed longitudinal case studies describing 3q29 deletion syndrome in adults with psychosis. In this case report, we describe the lifetime clinical portrait of a 57-year-old woman with 3q29 deletion syndrome, treatment-resistant psychotic symptoms, multiple medical comorbidities, and a previously unreported co-occurrence of early-onset dementia.

Association test using Copy Number Profile Curves (CONCUR) enhances power in rare copy number variant analysis.

Copy number variants (CNVs) are the gain or loss of DNA segments in the genome that can vary in dosage and length. CNVs comprise a large proportion of variation in human genomes and impact health conditions. To detect rare CNV associations, kernel-based methods have been shown to be a powerful tool due to their flexibility in modeling the aggregate CNV effects, their ability to capture effects from different CNV features, and their accommodation of effect heterogeneity. To perform a kernel association test, a CNV locus needs to be defined so that locus-specific effects can be retained during aggregation. However, CNV loci are arbitrarily defined and different locus definitions can lead to different performance depending on the underlying effect patterns. In this work, we develop a new kernel-based test called CONCUR (i.e., copy number profile curve-based association test) that is free from a definition of locus and evaluates CNV-phenotype associations by comparing individuals' copy number profiles across the genomic regions. CONCUR is built on the proposed concepts of "copy number profile curves" to describe the CNV profile of an individual, and the "common area under the curve (cAUC) kernel" to model the multi-feature CNV effects. The proposed method captures the effects of CNV dosage and length, accounts for the numerical nature of copy numbers, and accommodates between- and within-locus etiological heterogeneity without the need to define artificial CNV loci as required in current kernel methods. In a variety of simulation settings, CONCUR shows comparable or improved power over existing approaches. Real data analyses suggest that CONCUR is well powered to detect CNV effects in the Swedish Schizophrenia Study and the Taiwan Biobank.

Increased burden of ultra-rare structural variants localizing to boundaries of topologically associated domains in schizophrenia.

Despite considerable progress in schizophrenia genetics, most findings have been for large rare structural variants and common variants in well-imputed regions with few genes implicated from exome sequencing. Whole genome sequencing (WGS) can potentially provide a more complete enumeration of etiological genetic variation apart from the exome and regions of high linkage disequilibrium. We analyze high-coverage WGS data from 1162 Swedish schizophrenia cases and 936 ancestry-matched population controls. Our main objective is to evaluate the contribution to schizophrenia etiology from a variety of genetic variants accessible to WGS but not by previous technologies. Our results suggest that ultra-rare structural variants that affect the boundaries of topologically associated domains (TADs) increase risk for schizophrenia. Alterations in TAD boundaries may lead to dysregulation of gene expression. Future mechanistic studies will be needed to determine the precise functional effects of these variants on biology.

Treatment-resistant psychotic symptoms and the 15q11.2 BP1-BP2 (Burnside-Butler) deletion syndrome: case report and review of the literature.

The 15q11.2 BP1-BP2 (Burnside-Butler) deletion is a rare copy number variant impacting four genes (NIPA1, NIPA2, CYFIP1, and TUBGCP5), and carries increased risks for developmental delay, intellectual disability, and neuropsychiatric disorders (attention-deficit/hyperactivity disorder, autism, and psychosis). In this case report (supported by extensive developmental information and medication history), we present the complex clinical portrait of a 44-year-old woman with 15q11.2 BP1-BP2 deletion syndrome and chronic, treatment-resistant psychotic symptoms who has resided nearly her entire adult life in a long-term state psychiatric institution. Diagnostic and treatment implications are discussed.

Correction: Common-variant associations with fragile X syndrome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of Single Gene Copy Number Variants in Schizophrenia.

BACKGROUND: Genetic studies of schizophrenia have implicated numerous risk loci including several copy number variants (CNVs) of large effect and hundreds of loci of small effect. In only a few cases has a specific gene been clearly identified. Rare CNVs affecting a single gene offer a potential avenue to discovering schizophrenia risk genes. METHODS: CNVs were generated from exome sequencing of 4913 schizophrenia cases and 6188 control subjects from Sweden. We integrated two CNV calling methods (XHMM and ExomeDepth) to expand our set of single-gene CNVs and leveraged two different approaches for validating these variants (quantitative polymerase chain reaction and NanoString). RESULTS: We found a significant excess of all rare CNVs (deletions: p = .0004, duplications: p = .0006) and single-gene CNVs (deletions: p = .04, duplications: p = .03) in schizophrenia cases compared with control subjects. An expanded set of CNVs generated from integrating multiple approaches showed a significant burden of deletions in 11 of 21 gene sets previously implicated in schizophrenia and across all genes in those sets (p = .008), although no tests survived correction. We performed an extensive validation of all deletions in the significant set of voltage-gated calcium channels among CNVs called from both exome sequencing and genotyping arrays. In total, 4 exonic, single-gene deletions were validated in schizophrenia cases and none in control subjects (p = .039), of which all were identified by exome sequencing. CONCLUSIONS: These results point to the potential contribution of single-gene CNVs to schizophrenia, indicate that the utility of exome sequencing for CNV calling has yet to be maximized, and note that single-gene CNVs should be included in gene-focused studies using other classes of variation.

The genomics of major psychiatric disorders in a large pedigree from Northern Sweden.

We searched for genetic causes of major psychiatric disorders (bipolar disorder, schizoaffective disorder, and schizophrenia) in a large, densely affected pedigree from Northern Sweden that originated with three pairs of founders born around 1650. We applied a systematic genomic approach to the pedigree via karyotyping (N = 9), genome-wide SNP arrays (N = 418), whole-exome sequencing (N = 26), and whole-genome sequencing (N = 10). Comprehensive analysis did not identify plausible variants of strong effect. Rather, pedigree cases had significantly higher genetic risk scores compared to pedigree and community controls.

Common-variant associations with fragile X syndrome.

Fragile X syndrome is rare but a prominent cause of intellectual disability. It is usually caused by a de novo mutation that occurs on multiple haplotypes and thus would not be expected to be detectible using genome-wide association (GWA). We conducted GWA in 89 male FXS cases and 266 male controls, and detected multiple genome-wide significant signals near FMR1 (odds ratio = 8.10, P = 2.5 × 10-10). These findings withstood robust attempts at falsification. Fine-mapping yielded a minimum P = 1.13 × 10-14, but did not narrow the interval. Comprehensive functional genomic integration did not provide a mechanistic hypothesis. Controls carrying a risk haplotype had significantly longer FMR1 CGG repeats than controls with the protective haplotype (P = 4.75 × 10-5), which may predispose toward increases in CGG number to the premutation range over many generations. This is a salutary reminder of the complexity of even "simple" monogenetic disorders.

A randomized approach to speed up the analysis of large-scale read-count data in the application of CNV detection.

BACKGROUND: The application of high-throughput sequencing in a broad range of quantitative genomic assays (e.g., DNA-seq, ChIP-seq) has created a high demand for the analysis of large-scale read-count data. Typically, the genome is divided into tiling windows and windowed read-count data is generated for the entire genome from which genomic signals are detected (e.g. copy number changes in DNA-seq, enrichment peaks in ChIP-seq). For accurate analysis of read-count data, many state-of-the-art statistical methods use generalized linear models (GLM) coupled with the negative-binomial (NB) distribution by leveraging its ability for simultaneous bias correction and signal detection. However, although statistically powerful, the GLM+NB method has a quadratic computational complexity and therefore suffers from slow running time when applied to large-scale windowed read-count data. In this study, we aimed to speed up substantially the GLM+NB method by using a randomized algorithm and we demonstrate here the utility of our approach in the application of detecting copy number variants (CNVs) using a real example. RESULTS: We propose an efficient estimator, the randomized GLM+NB coefficients estimator (RGE), for speeding up the GLM+NB method. RGE samples the read-count data and solves the estimation problem on a smaller scale. We first theoretically validated the consistency and the variance properties of RGE. We then applied RGE to GENSENG, a GLM+NB based method for detecting CNVs. We named the resulting method as "R-GENSENG". Based on extensive evaluation using both simulated and empirical data, we concluded that R-GENSENG is ten times faster than the original GENSENG while maintaining GENSENG's accuracy in CNV detection. CONCLUSIONS: Our results suggest that RGE strategy developed here could be applied to other GLM+NB based read-count analyses, i.e. ChIP-seq data analysis, to substantially improve their computational efficiency while preserving the analytic power.

Exploration of large, rare copy number variants associated with psychiatric and neurodevelopmental disorders in individuals with anorexia nervosa.

Anorexia nervosa (AN) is a serious and heritable psychiatric disorder. To date, studies of copy number variants (CNVs) have been limited and inconclusive because of small sample sizes. We conducted a case-only genome-wide CNV survey in 1983 female AN cases included in the Genetic Consortium for Anorexia Nervosa. Following stringent quality control procedures, we investigated whether pathogenic CNVs in regions previously implicated in psychiatric and neurodevelopmental disorders were present in AN cases. We observed two instances of the well-established pathogenic CNVs in AN cases. In addition, one case had a deletion in the 13q12 region, overlapping with a deletion reported previously in two AN cases. As a secondary aim, we also examined our sample for CNVs over 1 Mbp in size. Out of the 40 instances of such large CNVs that were not implicated previously for AN or neuropsychiatric phenotypes, two of them contained genes with previous neuropsychiatric associations, and only five of them had no associated reports in public CNV databases. Although ours is the largest study of its kind in AN, larger datasets are needed to comprehensively assess the role of CNVs in the etiology of AN.

Implication of LRRC4C and DPP6 in neurodevelopmental disorders.

We performed whole-genome sequencing on an individual from a family with variable psychiatric phenotypes that had a sensory processing disorder, apraxia, and autism. The proband harbored a maternally inherited balanced translocation (46,XY,t(11;14)(p12;p12)mat) that disrupted LRRC4C, a member of the highly specialized netrin G family of axon guidance molecules. The proband also inherited a paternally derived chromosomal inversion that disrupted DPP6, a potassium channel interacting protein. Copy Number (CN) analysis in 14,077 cases with neurodevelopmental disorders and 8,960 control subjects revealed that 60% of cases with exonic deletions in LRRC4C had a second clinically recognizable syndrome associated with variable clinical phenotypes, including 16p11.2, 1q44, and 2q33.1 CN syndromes, suggesting LRRC4C deletion variants may be modifiers of neurodevelopmental disorders. In vitro, functional assessments modeling patient deletions in LRRC4C suggest a negative regulatory role of these exons found in the untranslated region of LRRC4C, which has a single, terminal coding exon. These data suggest that the proband's autism may be due to the inheritance of disruptions in both DPP6 and LRRC4C, and may highlight the importance of the netrin G family and potassium channel interacting molecules in neurodevelopmental disorders. © 2016 Wiley Periodicals, Inc.