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Background: Bipolar disorder is a highly heritable psychiatric condition for which specific genetic factors remain largely unknown. In the present study, we used combined whole-exome sequencing and linkage analysis to identify risk loci and dissect the contribution of common and rare variants in families with a high density of illness., Methods: Overall, 117 participants from 15 Australian extended families with bipolar disorder (72 with affective disorder, including 50 with bipolar disorder type I or II, 13 with schizoaffective disorder-manic type and 9 with recurrent unipolar disorder) underwent whole-exome sequencing. We performed genome-wide linkage analysis using MERLIN and conditional linkage analysis using LAMP. We assessed the contribution of potentially functional rare variants using a genebased segregation test., Results: We identified a significant linkage peak on chromosome 10q11-q21 (maximal single nucleotide polymorphism = rs10761725; exponential logarithm of the odds [LODexp] = 3.03; empirical p = 0.046). The linkage interval spanned 36 protein-coding genes, including a gene associated with bipolar disorder, ankyrin 3 (ANK3). Conditional linkage analysis showed that common ANK3 risk variants previously identified in genome-wide association studies - or variants in linkage disequilibrium with those variants - did not explain the linkage signal (rs10994397 LOD = 0.63; rs9804190 LOD = 0.04). A family-based segregation test with 34 rare variants from 14 genes under the linkage interval suggested rare variant contributions of 3 brain-expressed genes: NRBF2 (p = 0.005), PCDH15 (p = 0.002) and ANK3 (p = 0.014)., Limitations: We did not examine non-coding variants, but they may explain the remaining linkage signal., Conclusion: Combining family-based linkage analysis with next-generation sequencing data is effective for identifying putative disease genes and specific risk variants in complex disorders. We identified rare missense variants in ANK3, PCDH15 and NRBF2 that could confer disease risk, providing valuable targets for functional characterization., Competing Interests: P. Mitchell reports personal fees from Sanofi (Hangzhou) and Janssen-Cilag, outside the submitted work., (© 2021 Joule Inc. or its licensors.)

Bipolar disorder (BD) is a complex psychiatric condition with high heritability, the genetic architecture of which likely comprises both common variants of small effect and rare variants of higher penetrance, the latter of which are largely unknown. Extended families with high density of illness provide an opportunity to map novel risk genes or consolidate evidence for existing candidates, by identifying genes carrying pathogenic rare variants. We performed whole-exome sequencing (WES) in 15 BD families (117 subjects, of whom 72 were affected), augmented with copy number variant (CNV) microarray data, to examine contributions of multiple classes of rare genetic variants within a familial context. Linkage analysis and haplotype reconstruction using WES-derived genotypes enabled exclusion of false-positive single-nucleotide variants (SNVs), CNV inheritance estimation, de novo variant identification and candidate gene prioritization. We found that rare predicted pathogenic variants shared among ≥3 affected relatives were overrepresented in postsynaptic density (PSD) genes (P = 0.002), with no enrichment in unaffected relatives. Genome-wide burden of likely gene-disruptive variants was no different in affected vs. unaffected relatives (P = 0.24), but correlated significantly with age of onset (P = 0.017), suggesting that a high disruptive variant burden may expedite symptom onset. The number of de novo variants was no different in affected vs. unaffected offspring (P = 0.89). We observed heterogeneity within and between families, with the most likely genetic model involving alleles of modest effect and reduced penetrance: a possible exception being a truncating X-linked mutation in IRS4 within a family-specific linkage peak. Genetic approaches combining WES, CNV and linkage analyses in extended families are promising strategies for gene discovery.

Objective: Bipolar disorder (BD) is one of the most heritable psychiatric conditions and is associated with high suicide risk. To explore the reasons for this link, this study examined the interaction between traumatic stress and BD polygenic risk score in relation to suicidal ideation, suicide attempt, and nonsuicidal self-injury (NSSI) in adolescent and young adult offspring and relatives of persons with BD (BD-relatives) compared with adolescent and young adult offspring of individuals without psychiatric disorders (controls)., Method: Data were collected from 4 sites in the United States and 1 site in Australia from 2006 through 2012. Generalized estimating equation models were used to compare rates of ideation, attempts, and NSSI between BD-relatives (n = 307) and controls (n = 166) and to determine the contribution of demographic factors, traumatic stress exposure, lifetime mood or substance (alcohol/drug) use disorders, and BD polygenic risk score., Results: After adjusting for demographic characteristics and mood and substance use disorders, BD-relatives were at increased risk for suicidal ideation and attempts but not for NSSI. Independent of BD-relative versus control status, demographic factors, or mood and substance use disorders, exposure to trauma within the past year (including bullying, sexual abuse, and domestic violence) was associated with suicide attempts (p = .014), and BD polygenic risk score was marginally associated with attempts (p = .061). Importantly, the interaction between BD polygenic risk score and traumatic event exposures was significantly associated with attempts, independent of demographics, relative versus control status, and mood and substance use disorders (p = .041)., Conclusion: BD-relatives are at increased risk for suicide attempts and ideation, especially if they are exposed to trauma and have evidence of increased genetic vulnerability., (Copyright © 2017 American Academy of Child and Adolescent Psychiatry. Published by Elsevier Inc. All rights reserved.)

Alpha-2,8-sialyltransferase 2 (ST8SIA2) is an enzyme responsible for the transfer of polysialic acid (PSA) to glycoproteins, principally the neuronal cell adhesion molecule (NCAM1), and is involved in neuronal plasticity. Variants within ST8SIA2 have previously shown association with bipolar disorder, schizophrenia and autism. In addition, altered PSA-NCAM expression in brains of patients with schizophrenia or bipolar disorder indicates a functional dysregulation of glycosylation in mental illness. To explore the role of sequence variation affecting PSA-NCAM formation, we conducted a targeted re-sequencing study of a ∼ 100 kb region--including the entire ST8SIA2 gene and its region of interaction with NCAM1--in 48 Caucasian cases with bipolar disorder using the Roche 454 platform. We identified over 400 DNA variants, including 47 putative novel variants not described in dbSNP. Validation of a subset of variants via Sequenom showed high reliability of Roche 454 genotype calls (97% genotype concordance, with 80% of novel variants independently verified). We did not observe major loss-of-function mutations that would affect PSA-NCAM formation, either by ablating ST8SIA2 function or by affecting the ability of NCAM1 to be glycosylated. However, we identified 13 SNPs in the UTRs of ST8SIA2, a synonymous coding SNP in exon 5 (rs2305561, P207P) and many additional non-coding variants that may influence splicing or regulation of ST8SIA2 expression. We calculated nucleotide diversity within ST8SIA2 on specific haplotypes, finding that the diversity on the specific "risk" and "protective" haplotypes was lower than other non-disease-associated haplotypes, suggesting that putative functional variation may have arisen on a spectrum of haplotypes. We have identified common and novel variants (rs11074064, rs722645, 15:92961050) that exist on a spectrum of haplotypes, yet are plausible candidates for conferring the effect of risk and protective haplotypes via multiple enhancer elements. A Galaxy workflow/pipeline for sequence analysis used herein is available at: https://main.g2.bx.psu.edu/u/a-shaw-neura/p/next-generation-resources.

The contactin-associated protein-like 2 (CNTNAP2) gene is a member of the neurexin superfamily. CNTNAP2 was first implicated in the cortical dysplasia-focal epilepsy (CDFE) syndrome, a recessive disease characterized by intellectual disability, epilepsy, language impairments and autistic features. Associated SNPs and heterozygous deletions in CNTNAP2 were subsequently reported in autism, schizophrenia and other psychiatric or neurological disorders. We aimed to comprehensively examine evidence for the role of CNTNAP2 in susceptibility to psychiatric disorders, by the analysis of multiple classes of genetic variation in large genomic datasets. In this study we used: i) summary statistics from the Psychiatric Genomics Consortium (PGC) GWAS for seven psychiatric disorders; ii) examined all reported CNTNAP2 structural variants in patients and controls; iii) performed cross-disorder analysis of functional or previously associated SNPs; and iv) conducted burden tests for pathogenic rare variants using sequencing data (4,483 ASD and 6,135 schizophrenia cases, and 13,042 controls). The distribution of CNVs across CNTNAP2 in psychiatric cases from previous reports was no different from controls of the database of genomic variants. Gene-based association testing did not implicate common variants in autism, schizophrenia or other psychiatric phenotypes. The association of proposed functional SNPs rs7794745 and rs2710102, reported to influence brain connectivity, was not replicated; nor did predicted functional SNPs yield significant results in meta-analysis across psychiatric disorders at either SNP-level or gene-level. Disrupting CNTNAP2 rare variant burden was not higher in autism or schizophrenia compared to controls. Finally, in a CNV mircroarray study of an extended bipolar disorder family with 5 affected relatives we previously identified a 131kb deletion in CNTNAP2 intron 1, removing a FOXP2 transcription factor binding site. Quantitative-PCR validation and segregation analysis of this CNV revealed imperfect segregation with BD. This large comprehensive study indicates that CNTNAP2 may not be a robust risk gene for psychiatric phenotypes. [ABSTRACT FROM AUTHOR]

Alpha-2,8-sialyltransferase 2 (ST8SIA2) is an enzyme responsible for the transfer of polysialic acid (PSA) to glycoproteins, principally the neuronal cell adhesion molecule (NCAM1), and is involved in neuronal plasticity. Variants within ST8SIA2 have previously shown association with bipolar disorder, schizophrenia and autism. In addition, altered PSA-NCAM expression in brains of patients with schizophrenia or bipolar disorder indicates a functional dysregulation of glycosylation in mental illness. To explore the role of sequence variation affecting PSA-NCAM formation, we conducted a targeted re-sequencing study of a ∼100 kb region – including the entire ST8SIA2 gene and its region of interaction with NCAM1 – in 48 Caucasian cases with bipolar disorder using the Roche 454 platform. We identified over 400 DNA variants, including 47 putative novel variants not described in dbSNP. Validation of a subset of variants via Sequenom showed high reliability of Roche 454 genotype calls (97% genotype concordance, with 80% of novel variants independently verified). We did not observe major loss-of-function mutations that would affect PSA-NCAM formation, either by ablating ST8SIA2 function or by affecting the ability of NCAM1 to be glycosylated. However, we identified 13 SNPs in the UTRs of ST8SIA2, a synonymous coding SNP in exon 5 (rs2305561, P207P) and many additional non-coding variants that may influence splicing or regulation of ST8SIA2 expression. We calculated nucleotide diversity within ST8SIA2 on specific haplotypes, finding that the diversity on the specific “risk” and “protective” haplotypes was lower than other non-disease-associated haplotypes, suggesting that putative functional variation may have arisen on a spectrum of haplotypes. We have identified common and novel variants (rs11074064, rs722645, 15∶92961050) that exist on a spectrum of haplotypes, yet are plausible candidates for conferring the effect of risk and protective haplotypes via multiple enhancer elements. A Galaxy workflow/pipeline for sequence analysis used herein is available at: https://main.g2.bx.psu.edu/u/a-shaw-neura/p/next-generation-resources. [ABSTRACT FROM AUTHOR]

Bipolar Disorder (BD) is a common, complex psychiatric condition characterized by recurrent fluctuations of mood. Historically, linkage approaches implicated a number of genetic loci, but signals typically failed to be replicated due to heterogeneity of family samples in individual studies and lack of power. Recent Genome-Wide Association Studies (GWAS) have implicated common variants in CACNA1C, ODZ4 and ANK3 as increasing risk of BD, although those individual variants have small effect sizes. Sequencing studies are underway to characterise the impact of common and rare variants in these and other genes. Here we perform a combined whole exome sequencing and linkage study, with the aim of identifying linkage signals and dissecting the contribution of common and rare variants. Whole Exome Sequencing (WES) was performed in 117 subjects from 15 Australian multiplex BD families of European origin (72 affected, of whom 62 had diagnoses of BD type-1 or schizoaffective disorder-manic type). Genotypes were called using samtools pileup and filtered to include haplotype-informative markers (HapMap CEU) using LINKDATAGEN. Familial relationships were confirmed by pair-wise identity-by-descent in PLINK. Genome-wide linkage analysis was performed (using ~6,000 WES-derived SNPs) under non-parametric Kong & Cox linear and exponential models in Merlin. To reduce clinical heterogeneity we considered patients with severe forms of illness as affected (n=62), and subjects with BD-II or depression considered unknown. Regions with suggestive linkage (LOD>2) were fine mapped to reduce inter-marker intervals to <1 cM. Empirical significance of linkage was calculated using 10,000 simulated data sets and permuting phenotypes. Linkage analysis conditioning on GWAS-associated SNPs was performed using LAMP. To assess contribution of potentially damaging functional variation, an allele frequency weighted gene-based rareFBAT test was used. Linkage analysis across 15 extended BD families found a peak on chromosome 10q11-21 (maxSNP=rs10761725; LODlinear=2.52, empirical p=0.045; LODexp=3.03, empirical p=0.046). The 95% confidence interval spanned 15.9 cM (chr10:51,588-67,727 Mb; hg19) and included top GWAS-associated gene ANK3 and 38 other protein-coding genes captured by WES. Conditional linkage analysis with ANK3 GWAS-associated SNP (rs10994397, MAF=0.076 in CEU) demonstrated that this risk variant did not explain the linkage signal, with only a small contribution (LOD=0.63, p=0.089). Examination of haplotype breakpoints did not establish a clear localisation of the gene/s driving the signal. We identified 56 missense or truncating variants (regardless of frequency) informative for association analysis across 23 protein-coding genes, including 5 in ANK3. Preliminary analysis using rare FBAT gene-based tests find no significant evidence for association with any of the 23 genes after multiple testing correction. We found no evidence that variants increasing risk to BD are constrained within any single gene in the 10q11-21 region, including ANK3. Our findings are consistent with the 10q11-21 region containing multiple functional variants that contribute to BD susceptibility with modest effects. Our study suggests heterogeneity within 10q11-21, and highlights the need for examination of genetic variants beyond the ANK3 common risk variants previously identified. [ABSTRACT FROM AUTHOR]