De Novo Gene Variants and Familial Bipolar Disorder

The detection of de novo variants (DNVs) by next-generation sequencing has facilitated the identification of candidate genes in psychiatric disorders.1,2 Spontaneous DNV mutations are estimated to explain approximately 5% of genetic liability in autism and schizophrenia.1,2 In bipolar disorder (BD), common genetic variants explain approximately 30% of the heritability,3 and rare inherited variants also contribute to disease risk.4 However, the involvement of DNVs in BD is largely unexplored, with only 1 independent study5 reporting 71 DNVs from 79 singleton BD families. We present a DNV study in 18 multiplex bipolar families, combining 32 individuals previously reported4 with 29 additional participants.


Introduction
The detection of de novo variants (DNVs) by next-generation sequencing has facilitated the identification of candidate genes in psychiatric disorders. 1,2 Spontaneous DNV mutations are estimated to explain approximately 5% of genetic liability in autism and schizophrenia. 1,2 In bipolar disorder (BD), common genetic variants explain approximately 30% of the heritability, 3 and rare inherited variants also contribute to disease risk. 4 However, the involvement of DNVs in BD is largely unexplored, with only 1 independent study 5 reporting 71 DNVs from 79 singleton BD families. We present a DNV study in 18 multiplex bipolar families, combining 32 individuals previously reported 4 with 29 additional participants.

Methods
This study was approved by the University of New South Wales Ethics Committee. Participants provided written consent. This study follows the Strengthening the Reporting of Genetic Association Studies (STREGA) reporting guideline.
We performed whole-genome sequencing of 9 multiplex families (47 participants, including 29 offspring; there were 8 unaffected offspring and 21 case offspring with BD type I, BD type II, or schizoaffective-disorder bipolar type) in this case-control study. Identification of DNVs used 2 variant calling software packages, GATK version 3.4.0 (Broad Institute) and RTG-Core sequencing software version 3.7.1 (Real Time Genomics Ltd). All variants were validated by Sanger sequencing and were combined with variants previously identified by whole-exome sequencing in 32 offspring, 4

Results
Thirty-two DNVs from 12 female and 17 male offspring were validated and combined with 31 DNVs from our previous report, 4 5 and rates observed in autism (36%) and schizophrenia (34%). 2 Gene-based association tests for the 42 DNV-bearing genes implicated haptoglobin (HP) and pyruvate carboxylase (PC), 2 brain-expressed genes associated across 3 psychiatric disorders that carry potentially pathogenic DNVs. The PC gene encodes a mitochondrial enzyme involved in glucose metabolism and neurotransmitter synthesis, which lies within one of 30 BD-associated loci recently reported, 3 and it had the strongest gene-based association (P = 7.61 × 10 −7 ; z statistic = 4.808; Cohen d = 0.042) (Table).  To identify relevant networks, 218 genes from BD-significant loci 3 were combined with 112 genes carrying DNVs from the current and previous BD studies. 4,5 Protein-protein interaction network analysis implicated 4 genes: microtubule associated protein 4 (MAP4), which promotes microtubule assembly and is reported to carry DNV in autism; WD repeat and HMG-box DNA binding protein 1 (WDHD1), a DNA replication initiation factor; eukaryotic translation initiation factor 4E (EIF4E), which interacts with cytoplasmic Fragile X Mental Retardation interaction protein and is implicated in autism; and striatin (STRN), a largely unknown calmodulin-binding protein with a potential role in dendritic Ca2 + signaling and striatal neuron maturation (Figure). Of these 4 genes, MAP4 and WDHD1 harbored predicted-pathogenic variants with nominal gene-based association in schizophrenia (Table).

Discussion
Examination of DNVs in psychiatric disorders has traditionally focused on singleton families. These findings suggest that DNVs may also contribute to mutational load in multiplex BD families, as previously observed for multiplex autism families. 6 Although this study is limited by the small sample size, the overall de novo mutation rate was comparable in cases and unaffected offspring, whereas deleterious DNVs were observed more frequently in participants with BD, which is consistent with previous reports in autism and schizophrenia. 2 This study highlighted HP, PC, MAP4, and WDHD1 as potential susceptibility genes for BD. Additional sequencing studies in larger cohorts are needed to further delineate the impact of DNVs in BD.