公募シンポジウム

公募シンポジウム次世代シークエンサーを用いた最新の精神疾患の病態解明

1B-公募1-1 The molecular analysis of psychiatric diseases based on common disease―rare variant hypothesis 黒滝直弘¹，森本芳郎¹，今村明¹，小野慎治¹，三嶋博之²，木下晃²，小澤寛樹¹，吉浦孝一朗² ¹長崎大院・医・精神神経科学，²長崎大院・医・原研遺伝 Psychiatric diseases including schizophrenia（SCZ）, mood disorders, and gender identity dysfunction are considered to the genetic background. A lot of from genome-wide association study（GWAS）have been performed based on common disease-common variant hypothesis so far. Susceptibility genes directly linked to the appropriate treatment, however, have not been discovered. In this situation, we analyzed familial cases struggled with schizophrenia based on common disease-rare variant hypothesis. One family we found in relatively isolated local area in Nagasaki, Japan is over three generations and has 13 patients with SCZ among of 35 individuals. They are almost severe and some patients are requested long-term hospitalization. Among them, elderly patients show noticeable negative symptoms, without physical minor anomalies. G-band staining of a patient does show normal karyotype, 46,XY, and microarray analysis dose not detect structural abnormality such as chromosomal deletion and duplication. But, interestingly, by exome sequencing using next generation sequencer and going through filtering step, just two genes have been detected to have gene alterations. One is relatively common variant of the gene（Gene A）, while another（Gene B）have not been reported as a responsible of any diseases. Both mutations are completely segregated with disease in the family. We hypothesize that gene B would be possible responsible for the familial cases and we are now analyzing the function of Gene B.		1B-公募1-2 De novo single-nucleotide variants in sporadic schizophrenia 沼田周助徳島大学病院精神科神経科 Object：It is well known that schizophrenia have a strong genetic component. Despite its high heritability, there are many patients with no family history of schizophrenia, which are called sporadic cases. The purpose of the present study was to detect de novo non-synonymous mutations in schizophrenia by a trio-based exome sequencing in the Japanese population. Methods：We collected 18 family trios consisting of a patient with schizophrenia and two unaffected parents. Exome enrichment was conducted by TruSeq DNA Sample Prep Kits and TruSeq Exome Enrichment Kit（Illumina）. We sequenced their exomes by using HiSeq1000/1500（Illumina）. Raw sequencing data for each individual was mapped to the human reference genome（build hg19）by using the Burrows-Wheeler Aligner（BWA v0.5.9）. We analyzed mapped sequence data with the Genome Analysis Toolkit（GATK, v2.6-4 and v2.6-5）software. Candidate de novo mutations were validated by Sanger sequencing. Result：On average, we obtained 15.4 GB of raw sequence data per sample, and 96.9% of these data were mapped to the reference genome（hg19）. On detection of de novo mutations, we found 82 de novo SNVs in 18 trios. Of these 82 de novo SNVs, 17 were predicted to be non-synonymous mutations. Of these 17 de novo non-synonymous SNVs, we validated nine mutations in eight trios by Sanger sequencing. Conclusion：Our results suggest that de novo non-synonymous mutations may be involved in the pathology of schizophrenia.

1B-公募1-3 Rare missense variations and risk of autism：whole-exome sequencing in affected sib-pair families. 江川純，渡部雄一郎，染矢俊幸新潟大学大学院医歯学総合研究科精神医学 Genetic risk of autism spectrum disorder（ASD）has been suggested to involve the combined effects of many common variations of small effect, as well as rare variations of large effect. To investigate the role of rare variations, we performed whole-exome sequencing（WES）in two families, each with three affected siblings. We also performed a two-stage follow-up case-control study in a Japanese population. WES of the six affected siblings identified six novel rare missense variations. Among these variations, CLN8 R24H was inherited in one family by three affected siblings from an affected father and thus co-segregated with ASD（Egawa et al., 2015 PLoS One）. Subsequently, to further investigate the role of CLN8 in the genetic etiology of ASD, we performed resequencing and association analysis of CLN8 with ASD in a Japanese population. The first sample consisted of 256 patients with ASD and 667 control individuals. The second sample consisted of 312 patients with ASD and 352 control individuals. The CLN8 coding region was resequenced in 256 ASD patients of the first sample by direct sequencing. Rare non-synonymous variations with mutant allele identified by resequencing, were genotyped in the first and second samples using the TaqMan 5′-exonuclease assay. Resequencing the CLN8 coding region in 256 ASD patients identified five rare missense variations：g.1719291G＞A（R24H）, rs201670636（F39L）, rs116605307（R97H）, rs143701028（T108M）and rs138581191（N152S）. These variations were genotyped in 568 patients and 1017 controls. However, no significant association between these variations and ASD was identified. This study does not support a contribution of rare missense CLN8 variations to ASD susceptibility in the Japanese population（Inoue et al., 2015 PLoS One）. We possess another 6 affected sib-pair samples and continue WES of the samples to identify rare risk variations for ASD.		1B-公募1-4 Next-generation sequencing of candiate genes for schizophrenia and autism Aleksic Branko，久島周，木村大樹，尾崎紀夫名古屋大学大学院医学系研究科名古屋大学医学部精神医学教室 Schizophrenia and autism are severe, lifelong brain disorders with complex etiology and high prevalence. A strong link has been established between both disorders and de novo copy number variants, but the culprit genes remain unknown. Numerous previous studies have shown an association between the genes that encode these proteins and schizophrenia（SZ）and autism spectrum disorders（ASD）, which share a substantial portion of genetic risks. We sequenced the protein-encoding regions of neurodevelopmentally relevant genes in 562 cases（370 SZ and 192 ASD patients）on the Ion PGM platform. Candidate variants were selected for association analysis using large Japanese population based sample. Our results suggest that rare missense mutations may increase susceptibility to SZ and/or ASD. These findings may strengthen the theory that rare, non-synonymous variants confer substantial genetic risks for these disorders.