HLA and schizophrenia: a high-throughput sequencing study

描述(申请人提供)：精神分裂症是一种遗传复杂的疾病，遗传率为70%-80%，患病率为0.5%-1.0%。罕见的拷贝数变异和常见的SNP之间有很强的关联性。最强的共同SNP信号跨越主要组织相容性复合体(MHC，染色体6p21)，该复合体包括人类白细胞抗原(HLA)基因座。随着国际样本量的增加，这种关联性变得越来越强，目前的最低p=2.2x10-12(精神病学GWAS联合会)。SNP优势比不大，由于广泛的连锁不平衡(LD)，本地化很困难。MHC中的大多数复杂疾病关联至少部分地映射到经典的HLAI和II类基因座的影响。我们假设人类白细胞抗原的影响在病因学上具有重要意义。 精神分裂症病例的子集，并与自身免疫过程、感染和/或MHC I类基因座在神经元可塑性中的作用有关。因此，分析人类白细胞抗原变异对精神分裂症风险的影响是至关重要的。人类白细胞抗原效应的直接演示将为精神分裂症的发病机制、治疗和预防开辟一条重要的新途径。新的治疗和预防策略将代表着这种慢性和致残性疾病的重大突破，即使它们被证明只与与人类白细胞抗原相关的病例子集有关。在任何大的精神分裂症样本中都没有直接对人类白细胞抗原基因进行分型。对人类白细胞抗原效应的剖析通常需要测试大量的发现样本和来自其他种族人群的样本，以检测针对不同模式的LD的真实信号(等位基因和单倍型)。该区域高度重复的序列使得大多数方法难以进行准确的测序和单倍型分析。我们建议使用一种新的经济有效的方法对基于NIMH库的欧洲人、非裔美国人和中国人血统的大型精神分裂症病例对照NIMH库样本中8个最多态的HLA11个基因进行测序。8个基因座(A、B、C、DRB1/3/4/5、DQA1、DQB1、DPA1、DPB1)的11个基因的几乎所有外显子和内含子的全部靶向片段将通过长距离PCR扩增，然后进行非常深的第二代测序(Illumina HiSeq2000平台，100bp成对末端阅读)，以检测已知和新的等位基因。这种方法允许对每个基因内的多态进行完全分阶段，避免等位基因调用中的歧义，并准确确定可能改变基因表达的蛋白质和内含子变体之间的功能差异。提出了一个持续的质量控制策略。建议研究21,900人：7000名欧洲血统(EA)病例和7000名对照来自精神分裂症的分子遗传学(MGS)和基因组精神病学队列(GPC)样本；2000名非洲裔美国人病例和2000名对照(祖先匹配)来自MGS和PAARTNERS样本；1300名中国病例-父母三人来自NIMH台湾样本。将分析与精神分裂症风险有关的单个HLA基因座上的单倍型等位基因，然后分析包括单一氨基酸变化的“序列特征”，以及更复杂的特征(可以更强地预测疾病风险)，如预测表达蛋白质中相同结构或功能变化的变异组。将进行条件分析，以确定主要的人类白细胞抗原关联以及次要影响。将进一步剖析主要基因座上最强的易感和保护作用，以确定关键的序列、结构和功能变异。数据将与NIMH储存库和免疫基因组数据分析工作组(IDAWG，www.igdawg.org)共享，该工作组正在制定纳入新序列数据的标准。因此，这将是第一次在非常大的样本中对多态位点外显子和内含子进行测序和准确单倍型的疾病关联研究。所提出的分析将使我们能够剖析人类白细胞抗原与SCZ的关联，并将对理解人类白细胞抗原序列变异作出重大贡献。 与公共卫生相关：最近，有强有力的证据表明，个体在6号染色体特定部分的DNA序列上的差异，称为人类白细胞抗原(HL A)区域，对精神分裂症的发生有显著的预测作用。许多其他疾病与该地区的差异有关，在大多数情况下，这表明自身免疫和/或感染在疾病中发挥了作用。这项研究将使用一种新的DNA测序方法对2.19万名欧洲人、非裔美国人或中国血统的人进行研究，以准确地确定人类白细胞抗原基因的哪些变异使人们容易患上精神分裂症，从而更深入地研究这种关联的原因。了解这个问题可能会导致治疗甚至预防精神分裂症的新方法，至少对一些人来说是这样。

DESCRIPTION (provided by applicant): Schizophrenia is a genetically complex disease, with a heritability of 70-80% and prevalence of 0.5-1.0%. There are robust associations for rare copy number variants and for common SNPs. The strongest common- SNP signal spans the Major Histocompatibility Complex (MHC, chromosome 6p21) which includes the human leukocyte antigen (HLA) loci. The association has grown stronger each time the international sample size has increased, with the current lowest p=2.2x10-12 (Psychiatric GWAS Consortium). SNP odds ratios are modest and localization is difficult due to extensive linkage disequilibrium (LD). Most of the complex disease associations in the MHC map at least in part to the effects of the classical HLA class I and II loci. We hypothesize that HLA effects are etiologically important in a subset of schizophrenia cases, and are related to auto-immune processes, infection, and/or the role of MHC Class I loci in neuronal plasticity. It is therefore critical to dissect the effects ofHLA variants on schizophrenia risk. Direct demonstration of HLA effects would open a major new line of research into mechanisms, treatment and prevention of schizophrenia. New treatment and prevention strategies would represent a major breakthrough for this chronic and disabling disease, even if they proved to be relevant only to an HLA-linked subset of cases. HLA alleles have not been directly typed in any large schizophrenia sample. Dissection of HLA effects typically requires testing of a large discovery sample and of samples from additional ethnic populations, to detect true signals (alleles and haplotypes) against diverse patterns of LD. The highly repetitive sequences in this region make accurate sequencing and haplotyping difficult by most methods. We propose to sequence the 11 genes in the 8 most polymorphic HLA loci in large schizophrenia case-control NIMH repository-based samples of European, African-American and Chinese ancestry using a new cost-effective method. The entire targeted block of almost all exons and introns in 11 HLA genes in 8 loci (A, B, C, DRB1/3/4/5, DQA1, DQB1, DPA1, DPB1) will be amplified by long-range PCR, followed by very deep second-generation sequencing (Illumina HiSeq2000 platform, 100bp paired-end reads) to detect known and novel alleles. This method allows full phasing of polymorphisms within each gene, avoiding ambiguity in allele calls, and yields accurate determination of functional differences among proteins and intronic variants that can alter gene expression. An ongoing QC strategy is proposed. It is proposed to study 21,900 individuals: 7000 European-ancestry (EA) cases and 7000 controls from the Molecular Genetics of Schizophrenia (MGS) and Genomic Psychiatric Cohort (GPC) samples; 2000 African- American cases and 2000 controls (ancestry-matched) from the MGS and PAARTNERS samples; and 1300 Chinese case-parent trios from the NIMH Taiwanese sample. Association with schizophrenia risk will be analyzed for haplotyped alleles at the individual HLA loci, and then for the "sequence features" which include single amino acid changes, and more complex features (which can more strongly predict disease risk) such as sets of variants which predict the same structural or functional changes in the expressed protein. Conditional analyses will be performed to determine the primary HLA association as well as secondary effects. The most strongly predisposing and protective effects at the primary locus will be further dissected to identify the crucial sequence, structural and functional variations. Data will be shared with the NIMH repository and with the Immunogenomics Data Analysis Working Group (IDAWG, www.igdawg.org) which is developing standards to incorporate new sequence data. Thus, this will be the first disease association study to sequence and accurately haplotype the exons and introns of polymorphic HLA loci in very large samples. The proposed analyses will allow us to dissect HLA associations with SCZ and will make major contributions to the understanding of HLA sequence variation. PUBLIC HEALTH RELEVANCE: Recently, strong evidence has been reported that individuals' differences in the DNA sequence in a specific part of chromosome 6, called the human leukocyte antigen (HLA) region, are significantly predictive of developing schizophrenia. Many other diseases are associated with differences in this region, and in most cases, this indicates that auto-immunity and/or infections are playing a role in the disease. This study will use a new DNA sequencing method to study 21,900 people of European, African-American or Chinese ancestry to determine exactly which variations in the HLA genes predispose people to develop schizophrenia, so that the reasons for this association can be studied in more depth. Understanding this problem could lead to new approaches to treatment or even prevention of schizophrenia, at least for some individuals.