exome sequencing in mood and psychotic disorders.

doi:10.1111/pcn.12247

Whole genome/exome sequencing in mood and psychotic disorders Tadafumi Kato, MD, PhD* Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science Institute, Wako, Japan

Recent developments in DNA sequencing technologies have allowed for genetic studies using whole genome or exome analysis, and these have been applied in the study of mood and psychotic disorders, including bipolar disorder, depression, schizophrenia, and schizoaffective disorder. In this review, the current situation, recent findings, methodological problems, and future directions of whole genome/ exome analysis studies of these disorders are summarized. Whole genome/exome studies of bipolar disorder have included pedigree analysis and case– control studies, demonstrating the role of previously implicated pathways, such as calcium signaling, cyclic adenosine monophosphate response element binding protein (CREB) signaling, and potassium channels. Extensive analysis of trio families and case– control studies showed that de novo mutations play a role in the genetic architecture of schizophrenia and

indicated that mutations in several molecular pathways, including chromatin regulation, activityregulated cytoskeleton, post-synaptic density, N-methyl-D-aspartate receptor, and targets of fragile X mental retardation protein, are associated with this disorder. Depression is a heterogeneous group of diseases and studies using exome analysis have been conducted to identify rare mutations causing Mendelian diseases that accompany depression. In the near future, clarification of the genetic architecture of bipolar disorder and schizophrenia is expected. Identification of causative mutations using these new technologies will facilitate neurobiological studies of these disorders.

MONG THE MOOD and psychotic disorders, bipolar disorder and schizophrenia each affect approximately 1% of the population, causing severe psychosocial disturbance and requiring life-long treatment.1,2 On the other hand, major depression is a common disease with lifetime prevalence of around 10%, but its diagnosis and treatment is challenging,3

thus necessitating biology-based classification. The social burden due to these disorders is large but current treatments are still insufficient and more studies to clarify their causes are needed to improve diagnostic methods and treatment. As genetic factors play an important role in these disorders, identification of causative genes will be the entry-point of neurobiological studies on these mental disorders. Identification of the neurobiological basis of these disorders will also be helpful to eliminate the social stigma surrounding these disorders.4 In the last decade, numerous single nucleotide polymorphisms (SNP) and de novo or inherited copy number variations (CNV) have been found to be

A

*Correspondence: Tadafumi Kato, MD, PhD, Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Email: [email protected] Accepted 9 October 2014.

Key words: bipolar disorder, de novo mutations, depression, next generation sequencing, schizophrenia.

© 2014 The Author Psychiatry and Clinical Neurosciences © 2014 Japanese Society of Psychiatry and Neurology

1

2 T. Kato

associated with these disorders by genome-wide association studies (GWAS) and CNV analysis using DNA microarray. However, the results of these analyses could be a mixture of true and false positive results and genome-wide significantly associated SNP and CNV that are validated by independent studies so far can explain only a part of the heritability. Further studies are required to explain the genetic architecture of these disorders. Recent progress in next-generation sequencing, or massively parallel sequencing technology, enables the sequencing of whole genome or exome. These technologies were initially applied to identify the causative mutations of inherited diseases with unknown genetic mutations.5 The sharp decrease in the cost of sequencing has allowed whole-genome or exome sequencing to be more frequently used as methods of analysis. In autism spectrum disorder, the role of de novo and inherited point mutations has been clarified by exome analysis of more than 1000 patient families, including trios (proband and parents) and quartets (proband, sibling, and parents).6 These discoveries prompted the application of whole-genome or exome analysis to mood and psychotic disorders. In this review, the current status and future directions of whole-genome/exome analysis in bipolar disorder, schizophrenia, and depression are discussed.

METHODS A PubMed search was performed using the following terms: ‘((bipolar disorder) OR (schizophrenia) OR (depression) OR (depressive disorder)) AND ((whole genome) OR (exome) OR (next generation sequencing) OR (next generation sequencer))’ (August/ 2014). The search results were manually reviewed and original reports on whole-genome or exome sequencing in bipolar disorder, major depressive disorder, or schizophrenia were selected. Other relevant papers found by reference searches were also included.

BIPOLAR DISORDER Goes et al. are currently investigating the genetic basis of bipolar disorder using whole-exome sequencing by a case–control analysis of 937 cases and 912 controls as well as linkage analysis of eight

Psychiatry and Clinical Neurosciences 2014

multiplex pedigrees.7 A preliminary analysis of this ongoing project was published by Chen et al., who applied their new method of analysis termed ‘Burden or Mutation Position test (BOMP)’, a modified version of the burden test, to the data from 191 bipolar cases and 107 controls.8 Seven gene-sets involved in MAPK signaling, axon guidance, neurological system process, metabolism of proteins, neuroactive ligand receptor interaction, Huntington’s disease, and calcium signaling, were enriched among the mutations (Table 1).8–13 Cruceanu et al. are currently investigating variants that confer susceptibility to bipolar disorder.9 To reduce phenotypic heterogeneity, the group has focused on large pedigrees of lithium-responsive bipolar disorder, analyzing 250 individuals by exome analysis, which include three to seven affected individuals across one to three generations. A preliminary analysis of this ongoing study revealed that a missense mutation (F387L) of ZNF259 was associated with bipolar disorder in one family. In the other pedigree, several candidate mutations, including a mutation on the splicing site of MTOR, were partially linked with bipolar disorder. In a study of a small bipolar pedigree, Kerner et al. performed exome sequencing in three siblings suffering from bipolar disorder and one healthy sibling from one family to identify a mutation linked with bipolar disorder.10 Both parents were healthy and a recessive model did not show any mutation. The dominant model, on the other hand, showed an association with eight genes, IQUB, JMJD1C, GADD45A, GOLGB1, PLSCR5, VRK2, MESDC2, and FGGY, which were expressed in the brain and related to cyclic adenosine monophosphate response element binding protein (CREB)-regulated intracellular signaling. Georgi et al. analyzed one large, Amish bipolar pedigree. They performed linkage analysis by genotyping SNP in 388 of 497 members of this large pedigree. In addition, they sequenced the whole genome of 50 members of this family, including 23 members with bipolar disorder.11 They first identified several nominally significant linkage loci and deleterious variants in these regions. They found that 42 genes of five linkage peaks were nominally associated, but none survived the correction of multiple testing. Strauss et al. performed exome sequencing in seven of 14 patients in four Amish pedigrees and 10 variants were selected for association analysis.12 Among



Whole genome/exome in mental disorders 3

Table 1. Summary of whole genome/exome studies in bipolar disorder Study design

Sample

Platform

Findings

Chen et al.,8 2013

Case–control study

191 BD, 107 controls

Exome

Nimblegen v1.0/ Illumina GAII, Numblegen v2.0/Illumina HiSeq2000 SureSelect Human All Exon v4/HiSeq2000 TruSeq/Illumina

Seven gene-sets were nominally associated with BD

Cruceanu et al.,9 2013 Kerner et al.,10 2013

Large pedigrees

250 individuals from 25 families

Exome

Single family

3 affected siblings and an unaffected sibling

Exome

Georgi et al.,11 2014

Large pedigree

50 subjects of a pedigree consisting of 497 individuals

Whole genome sequencing with linkage analysis

Complete Genomics

Strauss et al.,12 2014

Large pedigrees

7 subjects of 4 families

Exome

Fiorentino et al.,13 2014

Case only

99 patients with BD

Whole genome

SureSelect Human All Exon 37Mb(v1)/ Illumina GAII Complete Genomics

ZNF259 F387L variant showed completely linkage with BD in one pedigree Linkage with eight genes (IQUB, JMJD1C, GADD45A, GOLGB1, PLSCR5, VRK2, MESDC2, FGGY) related to CREB signaling Each nominally significant linkage peak had several deleterious variants, but no robust risk loci were identified KCNH7 R394H mutation was associated with BD

CACNA1C rs79398153 and ANK3 N2643S were associated with BD

BD, bipolar disorder.

these, KCNH7 R394H (rs78247304) was shared by all 14 patients and associated in larger pedigrees (n = 340). This mutation altered the potassium channel function. GWAS also suggested an association of bipolar disorder with KCNH714 and other potassium channels, such as KCNMB215 and KCNQ2.16 Fiorentino et al. performed whole genome sequencing in 99 subjects with bipolar I disorder.13 From the resultant huge dataset, they specifically focused on ANK3 and CACNA1C, which were found to be associated with bipolar disorder by GWAS,17 and the obtained results were followed up in 1510 patients with bipolar disorder and 1095 controls. An intron variant of CACNA1C (rs79398153) and a missense mutation of ANK3 (N2643S) was associated with bipolar disorder. However, the association of bipolar disorder with N2643S mutation of ANK3 was not supported in a previous study.18

In summary, several large projects of wholegenome/exome sequencing are ongoing in North America and Europe, with some preliminary data already reported. Many of these projects focus on linkage analysis in multiply affected, large pedigrees, possibly due to the high heritability of this disease and the availability of DNA resources of large Amish bipolar pedigrees from the National Institute of Mental Health Human Genetics Initiative (https:// www.nimhgenetics.org/). Several other studies have focused on case–control analysis. Although it is too premature to interpret these studies, preliminary analyses have supported some of the previously implicated pathways in bipolar disorder, such as calcium signaling, CREB signaling, and potassium channels. Unlike studies on autism and schizophrenia, there has been no report of analysis of trio families with bipolar disorder. We are currently investigating de novo and transmitted mutations in trio families with bipolar disorder.19


4 T. Kato

DEPRESSION Compared with bipolar disorder, heritability is lower in depression, and GWAS in more than 9000 cases did not show any genome-wide association signals.20 This might be due to the heterogeneity of this disorder. Considering the complexity of depression, it is plausible that genes known to cause Mendelian diseases can also confer the risk of a complex disease, such as depression, in a small subgroup of patients by a pleiotropic effect.21 Indeed, an extensive investigation of medical records of 110 million patients demonstrated associations between Mendelian diseases and complex diseases, including depression, bipolar disorder, and schizophrenia.22 Thus, it would be meaningful to focus on Mendelian diseases that accompany depression by whole-genome/exome analysis. Exome sequencing is particularly effective to identify recessive mutations.5 Thus, we focused on a patient with progressive external ophthalmoplegia (PEO) and comorbid depression resulting from a consanguineous marriage. We looked for homozygous mutations causative for these phenotypes by exome sequencing and identified a novel missense mutation of RRM2B,23 which was previously identified as a causative gene for autosomal dominantly inherited PEO.24,25 In a patient of neuromuscular disorder with depression and multiple mitochondrial DNA (mtDNA) deletions, exome sequencing showed a compound heterozygous mutation of MPV encoding a mitochondrial inner-membrane protein with unknown function.26 These findings added new genes, in addition to ANT1, Twinkle, and POLG1, among the causative genes for mitochondrial diseases with mtDNA deletions that accompany depression as one of the phenotypes.25 In a case with cerebellar ataxia and severe depression responsive to CoQ10 treatment, exome analysis showed compound heterozygous mutation of ADCK3/CABC1, a causative gene for ubiquinone deficiency.27 In multiple families of hereditary diffuse leukoencephalopathy with spheroids, characterized by white matter disease with variable psychiatric phenotypes, including depression, the causative mutation was found to be colony-stimulating factor 1 receptor (CSF1R) by exome analysis.28 Tammiste et al. applied exome sequencing to a pharmacogenomic study of antidepressants. They sequenced the exomes of five responders and five non-responders after 12-week treatment with


escitalopram. The selected 38 variants were genotyped into two independent sample sets treated with escitalopram (n = 116 and n = 394). They found that a common polymorphism (rs41271330) of bone morphogenetic protein 5 (BMP5) was associated with treatment response in both samples.29 Absence of genome-wide association signals in depression suggests that it would not be meaningful to perform large-scale genetic studies considering depression as a single disease entity. Therefore, no large whole-genome/exome project related to depression has been reported. Depression should be treated as a mixture of diseases caused by various genetic factors. However, there might be a common neural circuit responsible for depression even if causative genetic factors are different. Neurobiological investigations in animal models carrying mutations causative for Mendelian diseases with comorbid depression might be a useful strategy to elucidate the neural circuit responsible for depression and bipolar disorder.30

SCHIZOPHRENIA Trio analysis In initial applications of exome analysis of patients with schizophrenia, Girard et al. sequenced the exomes of 14 trios of schizophrenia31 and found significantly more nonsense de novo mutations (four of 15 families) than expected (Table 2).31–44 On the other hand, Xu et al. sequenced 53 trios of sporadic schizophrenia and 23 control trios and identified 40 de novo mutations in the patient group.32 The rate of non-synonymous to synonymous variants was higher in schizophrenia trios than in control trios. The nonsynonymous variants included a damaging mutation in DGCR2, within 22q11.2 microdeletion region. In a subsequent study, Xu et al. increased the number of trios to 231 schizophrenia trios and 34 control trios.33 They replicated the excess of de novo disruptive, non-synonymous variants in probands with schizophrenia. Recurrent de novo events were found in four genes (LAMA2, DPYD, TRRAP, and VPS39), which is unlikely to be a chance finding. Genes with functional mutations in schizophrenia were enriched with the genes expressed during the early prenatal period in the hippocampus and dorsolateral prefrontal cortex. By closely analyzing the insertion/ deletions in these datasets, Takata et al. found two de novo, loss-of-function mutations of SETD1A




Table 2. Summary of whole genome/exome studies in schizophrenia Study design

Sample

Platform

Findings

Trio analysis

14 trios

Exome

SureSelect v1/Illumina GAII

53 SCZ trios, 23 control trios

Exome

SureSelect/Illumina HiSeq2000

Trio analysis

231 schizophrenia trios, 34 control trios

Exome

Takata et al.,34 2014 Ionita-Laza et al.,35 2014

Trio analysis

Same as above

Exome

SureSelect v2, NimbleGen SeqCap EZ v2/Illumina HiSeq2000 Same as above

Significantly more nonsense mutations (4 of 15 families) than expected Enrichment of non-synonymous mutations among de novo mutations Recurrent de novo mutations in LAMA2, DPYD, TRRAP and VPS

Xu et al.,32 2011

Trio analysis

Xu et al.,33 2012

Trio analysis

Same as above

Exome

Same as above

McCarthy et al.,36 2014 Glusuner et al.,37 2013

Trio analysis

57 trios

Exome

SeqCap EZ/HiSeq2000

Quartet analysis

Exome

SeqCap EZ v2/HiSeq2000

Fromer et al.,38 2014

Trio analysis

84 quartets (affected proband, unaffected sibling, and parents) and 21 trios 1844 samples from 623 families (mostly trios)

Exome

SureSelect v2, SeqCapEZ v2, SureSelect 50Mb/ Illumina HiSeq

Case only

166 cases with SCZ or schizoaffective disorder

Exome

SureSelect Human All Exon 37Mb or 50Mb/ Ilumina GAII or HiSeq

Case–control

2536 cases and 2543 controls

Exome

SureSelect 29Mb or v2 (33Mb)/Illumina GAII or HiSeq2000

Pedigree analysis Timms et al.,41 2013

Large pedigrees

12 patients from 5 pedigrees

Exome

SureSelect/SOLiD

Missense and frame-shift mutations of GRM5 were linked with schizophrenia in one family each

Pharmacogenomics Drogemoller et al.,42 2014

Cases only

11 cases responsive or non-responsive to antipsychotics

Exome

SureSelect 50Mb/ Illumina HiSeq2000

rs11368509 in UPP2 was associated with response to antipsychotics

Somatic mutations Bundo et al.,43 2014

Postmortem brains

Brain and liver from 3 patients and 3 controls

Whole genome

Complete Genomics

Brain-specific LINE1 retrotransposition in SCZ was found in synapse-related genes

One with SCZ and the other without SCZ

Exome

SureSelect/Illumina HiSeq

Mutations of EFCAB11, CLVS2, KAT8, APOH and SNX31 were implicated

Trio analysis Girard et al.,31 2011

Case–control analysis Need et al.,39 2012

Purcell et al.,40 2012

Combination with CNV analysis Subjects with 22q11.2 Balan et al.,44 2014 deletion

Recurrent loss of function mutations in SETD1A. Mutations were clustered within FAN1 at 15q13.3. Most of the mutation carriers had comorbid depression Mutations related to chromatin remodeling are enriched De novo mutations formed protein network and gene co-expression network in prefrontal cortex De novo mutations are overrepresented among glutamatergic postsynaptic proteins (ARC and NMDAR) Though there is no study-wide significant association, several candidate variants were found only in cases Genes related to voltage gated calcium channels, activity-regulated cytoskeleton, PSD-95, and NMDA receptor network were enriched

ARC, activity-regulated cytoskeleton; CNV, copy number variations; NMDAR, N-methyl-D-aspartate receptor; SCZ, schizophrenia.


6 T. Kato

encoding a subunit of histone methyltransferase.34 They also found that loss-of-function variants were more significantly transmitted in schizophrenia trios. This is in accordance with a recent finding that nonsense alleles tended to be more frequent in patients with schizophrenia than controls.45 Applying a new approach to identify mutations within schizophrenia-related CNV regions to the same dataset, Ionita-Laza et al. found that rare nonsynonymous mutations were significantly enriched in Fanconi-associated nuclease 1 (FAN1) within 15q13.3, one of the schizophrenia-related CNV regions.35 This gene is close to the imprinting center and the potential role of the parent-of-origin effect in psychoses has been studied.46 It was found that this association was limited to maternally transmitted alleles, thus supporting the possible role of imprinting. McCarthy et al.36 sequenced 171 individuals of 42 sporadic and 15 familial trios; 50 with schizophrenia, four with schizoaffective disorder, two with bipolar disorder, and one with psychotic disorder not otherwise specified and found that nonsense de novo mutations were enriched in sporadic trios. Recently, Petrovski and colleagues used exome data of 6503 individuals to assess whether each gene has more or less functional variations than expected. Causative genes of Mendelian diseases had a lower number of mutations than expected, which is termed as ‘intolerant’.47 By analyzing their dataset based on this finding, McCarthy et al.36 found that nonsense de novo mutations were significantly intolerant. The genes with de novo mutations overlapped with autism and enriched in chromatin modifiers, such as CHD8, MECP2, and HUWE1.36 Gulsuner et al.37 sequenced the exomes of 399 individuals from 105 trio families, including 84 unaffected siblings. Probands carried significantly more damaging de novo mutations than siblings. Probands of older fathers had a larger number of de novo mutations. Probands had more damaging mutations than healthy siblings and it was assumed that 21% of sporadic schizophrenia might be attributable to de novo damaging mutations. It was found that damaging mutations found in the probands were functionally related, evidenced by more nodes and edges in protein networks from genes carrying the mutations compared with siblings. Genes carrying the damaging de novo mutations in schizophrenia formed a network enriched for transcriptional coexpression in dorsolateral and ventrolateral prefrontal cortex.


In the largest trio study of schizophrenia to date, Fromer et al. comprehensively investigated de novo mutations in 623 families with schizophrenia (534 with schizophrenia and 89 with schizoaffective disorder) from Bulgaria. This study group majorly consisted of trios; however, it also included 12 quartets and one multigenerational family.38 In total, 1844 samples were sequenced. Because this study is substantially larger than all prior studies, the evidence obtained from this study should be given the highest weight. In contrast to previous studies, de novo nonsynonymous or loss-of-function mutations were not statistically significantly enriched in schizophrenia. This finding contradicts the findings of the previous studies in a much smaller number of subjects showing increased rates of de novo mutations in schizophrenia. De novo mutation rate correlated with higher paternal age, which was consistent with the results of a previous report.48 Among the de novo mutations, genes related to postsynaptic density, activity-regulated cytoskeleton (ARC) complex and N-methyl-D-aspartate receptor (NMDAR) complex were significantly enriched in non-synonymous mutations. The latter two were also enriched in lossof-function mutations. Enrichment of these categories was also found in intellectual disability, while NMDAR was enriched in autism in published datasets. Genes whose mRNAs are targeted by fragile X mental retardation protein (FMRP) were also enriched in non-synonymous mutations. Importantly, they identified recurrent de novo loss-offunction mutations in the TAF13 gene, which is statistically significant at the genome-wide level. TAF13 encodes a subunit of transcription factor IID (TFIID) transcription initiation complex.

Case–control analysis Need et al. sequenced the exomes of 166 cases with schizophrenia or schizoaffective disorder and selected 5788 variants for case–control analysis.39 Among these, 5155 were successfully genotyped by custom bead arrays. Although none of them were significantly associated after the correction of multiple testing, several mutations, including KL, encoding Klotho, were found only in patients, suggesting a pathophysiological role. The most comprehensive case–control study of schizophrenia using exome analysis to date was published by Purcell et al.40 They sequenced the exomes of 2536 Swedish patients with schizophrenia as well




as those of 2543 controls. Gene-sets related to voltage-gated calcium channels, activity-regulated cytoskeleton-associated scaffold protein (ARC), PSD95, and NMDA receptor network were enriched in the mutations found in patients. No statistically significant enrichment of singleton mutations of a single gene was revealed even by this large sample set. Among the disruptive variants, 10 variants of KYNU encoding kynureninase were found among patients and none among controls. This is particularly interesting, considering the kynurenine hypothesis of psychoses.49 They also observed the enrichment of mutations in FMRP targets. Many of the findings are similar to those reported in a study conducted by the same group that investigated de novo mutations.38

to chromatin regulation, ARC-associated scaffold protein, PSD-95, NMDA receptor, and FMRP targets. These findings were replicated in two studies. As a whole, exome studies clearly showed that rare de novo and transmitted mutations within these specific molecular pathways contribute to the genetic architecture of schizophrenia. However, trio and case–control studies so far have rarely shown significant association with a gene, but have mostly shown association with gene-sets or previously defined pathways. Moreover, the penetrance of de novo mutation is not known and it is not understood yet whether de novo and inherited mutations have different penetrance.

Pedigree analysis

SOMATIC MUTATIONS

Timms et al. performed CNV analysis and SNP genotyping in five large pedigrees with schizophrenia, and sequenced the exome of 12 subjects among these pedigrees.41 There was no linkage between any CNV and schizophrenia, while 22 variants were seen in all affected members. The group especially focused on a novel missense mutation and an additional frame-shift mutation of GRM5, which were found in one family each. They also reported a mutation of PPEF2, encoding a protein phosphatase affecting the level of GRM5, in one pedigree.

Past studies have focused on de novo mutations that possibly occurred during gametogenesis and inherited mutations. However, somatic mutations that occur during development can also cause disease. A representative example of such somatic mutations is cancer. However, various somatic (mosaic) mutations have also been reported in neuropsychiatric diseases,50 such as heteroplasmic mitochondrial DNA mutations, triplet repeat expansion, aneuploidy, brain-specific CNV, and retrotransposition of long interspersed nuclear element-1 (LINE1), which is a retrotransposon. Brain-specific, de novo, somatic point-mutations of AKT3,51 PIK3CA, and MTOR52 are reportedly causative for hemimegalencephaly, a neurodevelopmental disease. We recently found that LINE1 copy number increased in the postmortem brains of patients with schizophrenia.43 To comprehensively identify LINE1 insertion sites, we sequenced the whole genome in the brain and liver in three patients with schizophrenia and three controls. Mobile element insertion was detected by the identification of mate pairs in which one end uniquely maps to the reference genome and the other end maps to mobile element sequences. We identified that brain-specific LINE1 insertion in patients with schizophrenia was significantly enriched in synaptic and schizophreniarelated genes.43 Together with the increased copy number of LINE1 in neurodevelopmental animal models of schizophrenia and induced pluripotent cells-derived neurons of patients with schizophrenia, the results suggest a role of LINE1 retrotransposition in schizophrenia.

Pharmacogenomics To identify genetic traits related to responsiveness to antipsychotics, Drogemoller et al.42 sequenced exomes of 11 patients with first-episode schizophrenia that was responsive or nonresponsive to antipsychotics. One variant, rs11368509 on the splice site of uridine phosphorylase 2 (UPP2), was associated with better response in the original samples (n = 103) and an independent cohort (n = 222).

Summary In summary, though initial small trio studies showed enrichment of nonsense mutations, the largest study by Fromer et al.38 did not show the enrichment of de novo non-synonymous or loss-offunction mutations. De novo and transmitted mutations were enriched in the gene-sets related


8 T. Kato

TECHNICAL CONSIDERATIONS Exome analysis and CNV Before the era of sequencing, studies of CNV using DNA microarrays have demonstrated the role of de novo and inherited CNV in autism spectrum disorder53 and schizophrenia.54 The effect of CNV on bipolar disorder was relatively modest.55 To examine possible roles of point mutations in CNV regions associated with schizophrenia, Ionita-Laza et al. applied a new statistical approach to find clusters of rare variants associated with a disease by setting a 20-kb sliding window using the exome sequence data of schizophrenia trios. As described in the previous section on schizophrenia, they found that rare non-synonymous mutations were enriched in FAN1 within 15q13.3. It is well established that the 22q11.2 deletion is a strong risk factor for schizophrenia. However, the penetrance of 22q11.2 deletion for schizophrenia is not 100% and there might be some additional genetic factors causing schizophrenia. To identify these additional genetic factors, Balan et al. applied exome analysis to two cases of 22q11.2 deletion, with or without schizophrenia. They found nonsense mutations of EFCAB11 and CLVS2 as well as frame-shift mutations of KAT8, APOH, and SNX31, and suggested that they might be such additional genetic risk factors for schizophrenia.44 Past CNV studies used DNA microarray techniques. However, CNV data would also be available from whole genome or exome data.56 Myles-Worsley et al. found a CNV containing SLC1A12, encoding glutamate transporter in a pedigree containing both schizophrenia and bipolar disorder cases by DNA microarray. Using whole-genome sequencing, they successfully identified the precise location of the deletion overlapping at exon1.57

Candidate gene studies Exome/whole genome analyses show numerous candidate variants and such data are difficult to test using conventional SNP genotyping techniques in an independent sample set. For this purpose, a targeted resequencing method was recently introduced.58 Based on the data of the 1000 Genomes project, Takata et al.59 selected 47 candidate single nucleotide variants from among candidate genes of schizophrenia and bipolar disorder that are rare (less than 5%),


Asian-specific, and damaging, and genotyped using MassARRAY. They found that a missense variant of GRIN3A (R480G) encoding a subunit of NMDA receptor was more frequent in schizophrenia with study-wide significance. Hu et al.45 selected 101 genes as candidate genes of schizophrenia based on previous studies and performed deep sequencing in a discovery cohort (525 patients with schizophrenia and 619 controls) and a replication cohort (455 patients and 336 controls). They found that stop codon mutations of NRXN1 were found in two patients and mutations of TCF4 (Transcription factor 4) were frequently seen in patients. Crowley et al.60 sequenced 10 traditional candidate genes in the discovery set (727 patients with schizophrenia and 733 controls) by Sanger method and next-generation sequencing and verified the finding in an independent sample set. None of the genes, including DISC1 (disrupted schizophrenia 1), had single nucleotide variants showing association with schizophrenia at the genome-wide significant level.

Reverse phenotyping Previous genetic studies in complex diseases, such as bipolar disorder and schizophrenia, were primarily based on the ethical framework that the results of genetic analysis are not delivered to the participants. In this situation, it was difficult to follow up the phenotype of the disease in detail based on genetic findings. However, recent progress in genome analysis technologies made it easier to start with genotyping rather than extensive phenotyping. In this situation, it would be more efficient to start with genotyping, which is followed by detailed phenotyping based on genotype information.61 By refining the phenotype definition by reverse phenotyping, more robust genetic findings are expected.62 This approach also identified a new candidate gene for bipolar disorder. In a study of neurodevelopmental disorders, CNV were identified that disrupted methyl-CpG-binding domain protein 5 (MBD5) at 2q23.1.63 The authors focused on 22 individuals whose MBD5 gene was disrupted and 11 of them were verified as de novo mutations. Though major neuropsychiatric phenotypes of these subjects were intellectual disability, epilepsy, and autism, one with MBD5 disruption and one with 2q23.1 deletion were found to have bipolar disorder,63 suggesting that MBD5 is a candidate gene for bipolar disorder.



SHANK family genes encode scaffold proteins of the post-synaptic density. Duplication of 22q13 containing SHANK3 reportedly causes autism spectrum disorder, attention deficit/hyperactivity disorder (AD/HD), and schizophrenia. To identify the role of SHANK3 in 22q13 duplication syndrome, Han et al. generated Shank3 transgenic mice.64 As the mice displayed hyperactivity, the group hypothesized that amphetamine, which is effective in AD/HD, would have a sedative effect; however, the hyperactivity worsened. Based on this finding, the authors performed a telephonic interview of the two patients carrying the SHANK3 duplication. Whereas one patient, an 11-year old girl, was diagnosed with AD/HD and epilepsy, the other male patient was diagnosed with bipolar disorder and epilepsy. The Shank3 transgenic mice also showed spontaneous epileptic seizures. Hyperactivity in the mice did not improve with lithium but rather with valproate treatment. Thus, duplication of SHANK3 might be a genetic risk factor for bipolar disorder and Shank3overexpressing mice could be used as a model for valproate-responsive mania. De novo mutation of CHD8, encoding chromodomain helicase DNA-binding protein 8 was identified in a trio exome study of psychoses.36 Reverse phenotyping of carriers of de novo or inherited loss-of-function mutations of CHD8 showed that autism with macrocephaly, distinct faces, and gastrointestinal complaints was a major phenotype, which was supported by a phenotype of a zebrafish model.65 In addition, three out of 15 cases were found to have mood or psychotic symptoms.

Analysis methods Data from whole-genome/exome analysis should be analyzed by optimized methods, different from those used for GWAS using common Tag SNP. While GWAS deals with common (≥5%) variants that are in linkage disequilibrium with responsible variants, rare (≤1%) variants that directly have functional roles are the major target for whole-genome/exome analysis. Due to their rarity, application of conventional association tests for each single variant is underpowered in a realistic sample size. The simplest statistical approach to overcome this problem is the burden test, in which all putative causative variants in a gene are collapsed into one and subjected to standard association tests, such as


Fisher’s exact probability test.66 In this test, each single variant is assumed to be causal67 based on single major gene hypothesis. Such methods are powerful when most of the variants are causal in the same direction. When there are protective mutations or additive effects of multiple variants, the assumption is not true and such violations reduce the power of the analysis. To overcome such challenges, a number of methods have been proposed. In adaptive burden tests, the weight and threshold of each mutation is considered. In the sequence kernel association test (SKAT)68 and C-alpha,69 both risk and protective alleles can be considered. The other approach is to identify the enrichment of specific molecular pathways, protein interaction, and gene co-expression patterns among the identified mutations, as described in many of the abovementioned studies. In general, statistical approaches should be selected based on the hypothesis and statistical power and sample size. It is difficult to judge pathogenicity of each de novo mutation observed by trio analysis. Samocha et al.70 proposed a framework to interpret de novo mutation based on the precise estimate of mutability of each gene and the identification of genes under selective constraint. Using this method, they found that loss of function mutations were more significantly seen in cases with autism spectrum disorder (ASD) than unaffected siblings, whereas such a finding was not seen in cases with an IQ above 100. They also showed that nonsense, splice site, and frame-shift mutations of DYRK1A and SCN2A were significantly more frequently seen in ASD. On the other hand, identification of four de novo mutations of TTN in ASD was not statistically significant. One matter of debate is: which is more useful, exome analysis71 or whole genome sequencing?72 Technically, exome sequencing needs the process of exome capture, while the experimental method of whole genome analysis is straightforward. The data size is approximately 50 times larger for whole genome at the same read depth, which causes higher sequencing cost of whole genome analysis. However, the recent cost reduction of sequencing makes the cost of whole genome sequencing dramatically lower. In this situation, major problems might be the cost and burden of computing and bioinformatics, including the difficulty of annotating non-coding regions. Thus, it is still controversial which is better suited for clinical studies.


10

T. Kato

CONCLUSION In just a few years since the first paper on exome analysis of schizophrenia was published, huge amounts of data have been generated and the results of these analyses have been relatively consistent. On the other hand, whole-genome/exome studies of bipolar disorder are still ongoing. In the coming years, the genetic architecture of bipolar disorder and schizophrenia will soon be clarified. Genetic analysis of mood and psychotic disorders lays the foundation for further neurobiological studies. Considering the rapid progress of genetic studies in bipolar disorder and schizophrenia, studies in the next few years will provide valuable implications for neurobiological research.

ACKNOWLEDGMENT The author has received honoraria for lectures, manuscripts, and/or consultancy, from Kyowa Hakko Kirin Co., Ltd, Eli Lilly Japan K.K., Otsuka Pharmaceutical Co., Ltd, GlaxoSmithKline K.K., Taisho Toyama Pharmaceutical Co., Ltd, Dainippon Sumitomo Pharma Co., Ltd, Meiji Seika Pharma Co., Ltd, Pfizer Japan Inc., Mochida Pharmaceutical Co., Ltd, Shionogi & Co., Ltd, Janssen Pharmaceutical K.K., Pfizer, Yoshitomiyakuhin, Agilent Technologies, and Astellas Pharma Inc. within the last 3 years. T.K. also received a research grant from Takeda Pharmaceutical Co., Ltd.

REFERENCES 1. Kanba S, Kato T, Terao T, Yamada K, Committee for Treatment Guidelines of Mood Disorders JSoMD. Guideline for treatment of bipolar disorder by the Japanese Society of Mood Disorders, 2012. Psychiatry Clin. Neurosci. 2013; 67: 285–300. 2. Stahl SM, Morrissette DA, Citrome L et al. ‘Metaguidelines’ for the management of patients with schizophrenia. CNS Spectr. 2013; 18: 150–162. 3. Motomura K, Kanba S. Lost in translation: Confusion about depression and antidepressant therapy in Japan. Psychiatry Clin. Neurosci. 2013; 67: 1–2. 4. Ando S, Yamaguchi S, Aoki Y, Thornicroft G. Review of mental-health-related stigma in Japan. Psychiatry Clin. Neurosci. 2013; 67: 471–482. 5. Roach JC, Glusman G, Smit AF et al. Analysis of genetic inheritance in a family quartet by whole-genome sequencing. Science 2010; 328: 636–639.


6. Krumm N, O’Roak BJ, Shendure J, Eichler EE. A de novo convergence of autism genetics and molecular neuroscience. Trends Neurosci. 2014; 37: 95–105. 7. Goes F, Parla J, Pirooznia M et al. Exome sequencing in bipolar disorder: Family and case-control results implicate a set of ten interacting genes. 16th Annual Conference of the International Society for Bipolar Disorders. Seoul, 2014. 8. Chen YC, Carter H, Parla J et al. A hybrid likelihood model for sequence-based disease association studies. PLoS Genet. 2013; 9: e1003224. 9. Cruceanu C, Ambalavanan A, Spiegelman D et al. Familybased exome-sequencing approach identifies rare susceptibility variants for lithium-responsive bipolar disorder. Genome 2013; 56: 634–640. 10. Kerner B, Rao AR, Christensen B, Dandekar S, Yourshaw M, Nelson SF. Rare genomic variants link bipolar disorder with anxiety disorders to CREB-regulated intracellular signaling pathways. Front. Psychiatry 2013; 4: 154. 11. Georgi B, Craig D, Kember RL et al. Genomic view of bipolar disorder revealed by whole genome sequencing in a genetic isolate. PLoS Genet. 2014; 10: e1004229. 12. Strauss KA, Markx S, Georgi B et al. A population-based study of KCNH7 p.Arg394His and bipolar spectrum disorder. Hum. Mol. Genet. 2014; (forthcoming). 13. Fiorentino A, O’Brien NL, Locke DP et al. Analysis of ANK3 and CACNA1C variants identified in bipolar disorder whole genome sequence data. Bipolar Disord. 2014; 16: 583–591. 14. Kuo PH, Chuang LC, Liu JR et al. Identification of novel loci for bipolar I disorder in a multi-stage genome-wide association study. Prog. Neuropsychopharmacol Biol. Psychiatry 2014; 51: 58–64. 15. Hattori E, Toyota T, Ishitsuka Y et al. Preliminary genomewide association study of bipolar disorder in the Japanese population. Am. J. Med. Genet. B Neuropsychiatr. Genet. 2009; 150B: 1110–1117. 16. Judy JT, Seifuddin F, Pirooznia M et al. Converging evidence for epistasis between ANK3 and potassium channel gene KCNQ2 in bipolar disorder. Front. Genet. 2013; 4: 87. 17. Ferreira MA, O’Donovan MC, Meng YA et al. Collaborative genome-wide association analysis supports a role for ANK3 and CACNA1C in bipolar disorder. Nat. Genet. 2008; 40: 1056–1058. 18. Doyle GA, Lai AT, Chou AD et al. Re-sequencing of ankyrin 3 exon 48 and case-control association analysis of rare variants in bipolar disorder type I. Bipolar Disord. 2012; 14: 809–821. 19. Matoba N, Kataoka M, Fujii K, Kato T. Whole exome sequencing of trio families of bipolar disorder. XXIst World Congress of Psychiatric Genetics. Boston, 2013. 20. Flint J, Kendler KS. The genetics of major depression. Neuron 2014; 81: 484–503. 21. Zhu X, Need AC, Petrovski S, Goldstein DB. One gene, many neuropsychiatric disorders: Lessons from Mendelian diseases. Nat. Neurosci. 2014; 17: 773–781.



22. Blair DR, Lyttle CS, Mortensen JM et al. A nondegenerate code of deleterious variants in Mendelian loci contributes to complex disease risk. Cell 2013; 155: 70–80. 23. Takata A, Kato M, Nakamura M et al. Exome sequencing identifies a novel missense variant in RRM2B associated with autosomal recessive progressive external ophthalmoplegia. Genome Biol. 2011; 12: R92. 24. Tyynismaa H, Ylikallio E, Patel M, Molnar MJ, Haller RG, Suomalainen A. A heterozygous truncating mutation in RRM2B causes autosomal-dominant progressive external ophthalmoplegia with multiple mtDNA deletions. Am. J. Hum. Genet. 2009; 85: 290–295. 25. Kato T. Mitochondrial dysfunction as the molecular basis of bipolar disorder: therapeutic implications. CNS Drugs 2007; 21: 1–11. 26. Garone C, Rubio JC, Calvo SE et al. MPV17 mutations causing adult-onset multisystemic disorder with multiple mitochondrial DNA deletions. Arch. Neurol. 2012; 69: 1648–1651. 27. Blumkin L, Leshinsky-Silver E, Zerem A, Yosovich K, Lerman-Sagie T, Lev D. Heterozygous mutations in the ADCK3 gene in siblings with cerebellar atrophy and extreme phenotypic variability. JIMD Rep. 2014; 12: 103– 107. 28. Rademakers R, Baker M, Nicholson AM et al. Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids. Nat. Genet. 2012; 44: 200–205. 29. Tammiste A, Jiang T, Fischer K et al. Whole-exome sequencing identifies a polymorphism in the BMP5 gene associated with SSRI treatment response in major depression. J. Psychopharmacol. 2013; 27: 915–920. 30. Kato T. Molecular neurobiology of bipolar disorder: A disease of ‘mood-stabilizing neurons’? Trends Neurosci. 2008; 31: 495–503. 31. Girard SL, Gauthier J, Noreau A et al. Increased exonic de novo mutation rate in individuals with schizophrenia. Nat. Genet. 2011; 43: 860–863. 32. Xu B, Roos JL, Dexheimer P et al. Exome sequencing supports a de novo mutational paradigm for schizophrenia. Nat. Genet. 2011; 43: 864–868. 33. Xu B, Ionita-Laza I, Roos JL et al. De novo gene mutations highlight patterns of genetic and neural complexity in schizophrenia. Nat. Genet. 2012; 44: 1365–1369. 34. Takata A, Xu B, Ionita-Laza I, Roos JL, Gogos JA, Karayiorgou M. Loss-of-function variants in schizophrenia risk and SETD1A as a candidate susceptibility gene. Neuron 2014; 82: 773–780. 35. Ionita-Laza I, Xu B, Makarov V et al. Scan statistic-based analysis of exome sequencing data identifies FAN1 at 15q13.3 as a susceptibility gene for schizophrenia and autism. Proc. Natl Acad. Sci. U.S.A. 2014; 111: 343– 348. 36. McCarthy SE, Gillis J, Kramer M et al. De novo mutations in schizophrenia implicate chromatin remodeling and


37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

support a genetic overlap with autism and intellectual disability. Mol. Psychiatry 2014; 19: 652–658. Gulsuner S, Walsh T, Watts AC et al. Spatial and temporal mapping of de novo mutations in schizophrenia to a fetal prefrontal cortical network. Cell 2013; 154: 518– 529. Fromer M, Pocklington AJ, Kavanagh DH et al. De novo mutations in schizophrenia implicate synaptic networks. Nature 2014; 506: 179–184. Need AC, McEvoy JP, Gennarelli M et al. Exome sequencing followed by large-scale genotyping suggests a limited role for moderately rare risk factors of strong effect in schizophrenia. Am. J. Hum. Genet. 2012; 91: 303–312. Purcell SM, Moran JL, Fromer M et al. A polygenic burden of rare disruptive mutations in schizophrenia. Nature 2014; 506: 185–190. Timms AE, Dorschner MO, Wechsler J et al. Support for the N-methyl-D-aspartate receptor hypofunction hypothesis of schizophrenia from exome sequencing in multiplex families. JAMA Psychiatry 2013; 70: 582–590. Drogemoller BI, Niehaus DJ, Chiliza B et al. Patterns of variation influencing antipsychotic treatment outcomes in South African first-episode schizophrenia patients. Pharmacogenomics 2014; 15: 189–199. Bundo M, Toyoshima M, Okada Y et al. Increased l1 retrotransposition in the neuronal genome in schizophrenia. Neuron 2014; 81: 306–313. Balan S, Iwayama Y, Toyota T, Toyoshima M, Maekawa M, Yoshikawa T. 22q11.2 deletion carriers and schizophreniaassociated novel variants. Br. J. Psychiatry 2014; 204: 398– 399. Hu X, Zhang B, Liu W et al. A survey of rare coding variants in candidate genes in schizophrenia by deep sequencing. Mol. Psychiatry 2014; 19: 857–858. Kato T, Winokur G, Coryell W, Keller MB, Endicott J, Rice J. Parent-of-origin effect in transmission of bipolar disorder. Am. J. Med. Genet. 1996; 67: 546–550. Petrovski S, Wang Q, Heinzen EL, Allen AS, Goldstein DB. Genic intolerance to functional variation and the interpretation of personal genomes. PLoS Genet. 2013; 9: e1003709. Kong A, Frigge ML, Masson G et al. Rate of de novo mutations and the importance of father’s age to disease risk. Nature 2012; 488: 471–475. Altamura AC, Buoli M, Pozzoli S. Role of immunological factors in the pathophysiology and diagnosis of bipolar disorder: Comparison with schizophrenia. Psychiatry Clin. Neurosci. 2014; 68: 21–36. Kato T, Iwamoto K, Kakiuchi C, Kuratomi G, Okazaki Y. Genetic or epigenetic difference causing discordance between monozygotic twins as a clue to molecular basis of mental disorders. Mol. Psychiatry 2005; 10: 622–630. Poduri A, Evrony GD, Cai X et al. Somatic activation of AKT3 causes hemispheric developmental brain malformations. Neuron 2012; 74: 41–48.


12

T. Kato

52. Lee JH, Huynh M, Silhavy JL et al. De novo somatic mutations in components of the PI3K-AKT3-mTOR pathway cause hemimegalencephaly. Nat. Genet. 2012; 44: 941– 945. 53. Shishido E, Aleksic B, Ozaki N. Copy-number variation in the pathogenesis of autism spectrum disorder. Psychiatry Clin. Neurosci. 2014; 68: 85–95. 54. Tam GW, Redon R, Carter NP, Grant SG. The role of DNA copy number variation in schizophrenia. Biol. Psychiatry 2009; 66: 1005–1012. 55. Georgieva L, Rees E, Moran JL et al. De novo CNVs in bipolar affective disorder and schizophrenia. Hum. Mol. Genet. 2014; (forthcoming). 56. Fromer M, Moran JL, Chambert K et al. Discovery and statistical genotyping of copy-number variation from whole-exome sequencing depth. Am. J. Hum. Genet. 2012; 91: 597–607. 57. Myles-Worsley M, Tiobech J, Browning SR et al. Deletion at the SLC1A1 glutamate transporter gene co-segregates with schizophrenia and bipolar schizoaffective disorder in a 5-generation family. Am. J. Med. Genet. B Neuropsychiatr. Genet. 2013; 162B: 87–95. 58. O’Roak BJ, Vives L, Fu W et al. Multiplex targeted sequencing identifies recurrently mutated genes in autism spectrum disorders. Science 2012; 338: 1619–1622. 59. Takata A, Iwayama Y, Fukuo Y et al. A population-specific uncommon variant in GRIN3A associated with schizophrenia. Biol. Psychiatry 2013; 73: 532–539. 60. Crowley JJ, Hilliard CE, Kim Y et al. Deep resequencing and association analysis of schizophrenia candidate genes. Mol. Psychiatry 2013; 18: 138–140. 61. Stessman HA, Bernier R, Eichler EE. A genotype-first approach to defining the subtypes of a complex disease. Cell 2014; 156: 872–877.


62. Schulze TG, McMahon FJ. Defining the phenotype in human genetic studies: Forward genetics and reverse phenotyping. Hum. Hered. 2004; 58: 131–138. 63. Hodge JC, Mitchell E, Pillalamarri V et al. Disruption of MBD5 contributes to a spectrum of psychopathology and neurodevelopmental abnormalities. Mol. Psychiatry 2014; 19: 368–379. 64. Han K, Holder JL, Jr, Schaaf CP et al. SHANK3 overexpression causes manic-like behaviour with unique pharmacogenetic properties. Nature 2013; 503: 72–77. 65. Bernier R, Golzio C, Xiong B et al. Disruptive CHD8 mutations define a subtype of autism early in development. Cell 2014; 158: 263–276. 66. Li B, Leal SM. Methods for detecting associations with rare variants for common diseases: Application to analysis of sequence data. Am. J. Hum. Genet. 2008; 83: 311–321. 67. Lee S, Abecasis GR, Boehnke M, Lin X. Rare-variant association analysis: Study designs and statistical tests. Am. J. Hum. Genet. 2014; 95: 5–23. 68. Wu MC, Lee S, Cai T, Li Y, Boehnke M, Lin X. Rare-variant association testing for sequencing data with the sequence kernel association test. Am. J. Hum. Genet. 2011; 89: 82–93. 69. Neale BM, Rivas MA, Voight BF et al. Testing for an unusual distribution of rare variants. PLoS Genet. 2011; 7: e1001322. 70. Samocha KE, Robinson EB, Sanders SJ et al. A framework for the interpretation of de novo mutation in human disease. Nat. Genet. 2014; 46: 944–950. 71. Chilamakuri CS, Lorenz S, Madoui MA et al. Performance comparison of four exome capture systems for deep sequencing. BMC Genomics 2014; 15: 449. 72. Lam HY, Clark MJ, Chen R et al. Performance comparison of whole-genome sequencing platforms. Nat. Biotechnol. 2012; 30: 78–82.


Exome sequencing: new insights into lipoprotein disorders.

Clinical exome sequencing in neurogenetic and neuropsychiatric disorders.

Exome sequencing in unspecific intellectual disability and rare disorders.

Exome Sequencing and the Management of Neurometabolic Disorders.

Anhedonia in psychotic and non psychotic disorders.

A Review of Biomarkers in Mood and Psychotic Disorders: A Dissection of Clinical vs. Preclinical Correlates.

Role of social media and the Internet in pathways to care for adolescents and young adults with psychotic disorders and non-psychotic mood disorders.

An exome sequencing strategy to diagnose lethal autosomal recessive disorders.

Targeted exome sequencing for mitochondrial disorders reveals high genetic heterogeneity.

Clinical exome sequencing for genetic identification of rare Mendelian disorders.

Whole-exome sequencing: discovering genetic causes of orthopaedic disorders.

"Secondary" and drug-induced mood, anxiety, psychotic, catatonic, and personality disorders: a review of the literature.

Integrated analysis of whole-exome sequencing and transcriptome profiling in males with autism spectrum disorders.

Mood and affect disorders.

Mood disorders in Asians.

Whole exome sequencing diagnosis of inborn errors of metabolism and other disorders in United Arab Emirates.

[Prevention of psychotic disorders].

Whole exome sequencing of extreme morbid obesity patients: translational implications for obesity and related disorders.

Leveraging ancestry to improve causal variant identification in exome sequencing for monogenic disorders.

Effectiveness of exome and genome sequencing guided by acuity of illness for diagnosis of neurodevelopmental disorders.

Exome sequencing of extended families with autism reveals genes shared across neurodevelopmental and neuropsychiatric disorders.

Addendum: Mood and affect disorders.

Exome sequencing for gene discovery in lethal fetal disorders--harnessing the value of extreme phenotypes.

Recent Advances in Autism Spectrum Disorders: Applications of Whole Exome Sequencing Technology.