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%。对于罕见的拷贝数变异和常见的SNP，存在稳健的关联。最强的共同SNP信号跨越主要组织相容性复合体（MHC，染色体6p 21），其包括人白细胞抗原（HLA）基因座。随着国际样本量的增加，这种关联越来越强，目前最低的p=2.2x10-12（精神病学GWAS联盟）。SNP优势比是适度的，并且由于广泛的连锁不平衡（LD）而难以定位。MHC图谱中的大多数复杂疾病关联至少部分地与经典HLA I类和II类基因座的作用有关。我们假设HLA的影响是病因学上重要的， 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HLA变异对精神分裂症风险的影响至关重要。HLA效应的直接证明将为精神分裂症的机制、治疗和预防开辟一条重要的新研究路线。新的治疗和预防策略将代表这种慢性和致残性疾病的重大突破，即使它们被证明仅与HLA相关的病例子集有关。HLA等位基因尚未在任何大型精神分裂症样本中直接分型。HLA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LD模式的真实信号（等位基因和单倍型）。该区域的高度重复序列使得大多数方法难以进行精确测序和单体型分析。我们建议使用一种新的具有成本效益的方法，对欧洲、非洲裔美国人和中国血统的大型精神分裂症病例对照NIMH库样本中8个最具多态性的HLA位点的11个基因进行测序。将通过长距离PCR扩增8个基因座（A、B、C、DRB 1/3/4/5、DQA 1、DQB 1、DPA 1、DPB 1）中11个HLA基因中几乎所有外显子和内含子的整个靶向区块，然后进行非常深的第二代测序（Illumina HiSeq 2000平台，100 bp配对末端读数）以检测已知和新的等位基因。该方法允许每个基因内的多态性的完全定相，避免等位基因调用中的模糊性，并产生可以改变基因表达的蛋白质和内含子变体之间的功能差异的准确测定。提出了持续的QC策略。拟研究21，900名个体：来自精神分裂症分子遗传学（MGS）和基因组精神病队列（GPC）样本的7000名欧洲血统（EA）病例和7000名对照;来自MGS和PAARTNERS样本的2000名非洲裔美国人病例和2000名对照（血统匹配）;以及来自NIMH台湾样本的1300名中国病例-父母三人组。与精神分裂症风险的关联将针对单个HLA基因座的单倍型等位基因进行分析，然后针对“序列特征”进行分析，所述“序列特征”包括单个氨基酸变化和更复杂的特征（其可以更强地预测疾病风险），例如预测表达蛋白中相同结构或功能变化的变体集。将进行条件分析，以确定主要HLA相关性以及次要效应。在主要位点的最强烈的易感性和保护作用将进一步解剖，以确定关键的序列，结构和功能的变化。数据将与NIMH数据库和免疫基因组学数据分析工作组（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，这将是第一个疾病关联研究测序和准确的单倍型多态性HLA基因座的外显子和内含子在非常大的样本。建议的分析将使我们能够解剖HLA与SCZ的关联，并将作出重大贡献的HLA序列变异的理解。 公共卫生相关性：最近，有强有力的证据表明，个体在第6号染色体特定部分（称为人类白细胞抗原（HLA）区域）的DNA序列差异显著预测精神分裂症的发展。许多其他疾病与该区域的差异有关，在大多数情况下，这表明自身免疫和/或感染在疾病中起作用。这项研究将使用一种新的DNA测序方法，对21,900名欧洲人、非洲裔美国人或中国人血统的人进行研究，以准确确定HLA基因中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新的方法来治疗甚至预防精神分裂症，至少对一些人来说。

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.