Identification of Genetic and Epigenetic Alterations in Spondyloarthritis

脊柱关节炎遗传和表观遗传改变的鉴定

• 批准号: 8435256
• 负责人: TIBOR T. GLANT
• 金额: $ 32.51万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8435256
• 关键词: Affect; Alleles; Animal Model; Animals; Ankylosing spondylitis; Ankylosis; Anterior uveitis; Area; Arthritis; Autoimmune Diseases; Autoimmune Responses; Biological Models; Candidate Disease Gene; Cartilage; Cells; Chromosomes; Chromosomes, Human, Pair 17; Chromosomes, Human, Pair 2; Chronic; Clinical assessments; Code; Congenic Mice; Congenic Strain; DBA/2 Mouse; DNA Methylation; Databases; Development; Disease; Epigenetic Process; Epistatic Gene; Etiology; Figs - dietary; Gene Cluster; Gene Expression; Gene Expression Regulation; Gene Mutation; Generations; Genes; Genetic; Genetic Heterogeneity; Genetic Polymorphism; Genetic Predisposition to Disease; Genetic Recombination; Genetic Risk; Genome; Genomics; HLA Antigens; HLA-B27 Antigen; Histopathology; Human; Human Genome; Hybrids; Immune; Immunization; Inbred BALB C Mice; Inbreeding; Individual; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Intercistronic Region; Intergenic Sequence; Intervertebral disc structure; Joints; Knockout Mice; Knowledge; LacZ Genes; Ligaments; Localized Disease; Lymphocyte; Major Histocompatibility Complex; Methods; Methylation; Modeling; Modification; Monitor; Mononuclear; Mouse Strains; Mus; Mutate; Mutation; Nucleic Acid Regulatory Sequences; Nucleotides; Other Genetics; Parents; Patients; Peripheral; Play; Polyarthritides; Population; Predisposing Factor; Predisposition; Proteoglycan; Quantitative Trait Loci; Reaction; Regulation; Research; Research Personnel; Resistance; Role; Sacroiliac joint structure; Sampling; Severities; Single Nucleotide Polymorphism; Site; Skeleton; Source; Spleen; Spondylarthritis; Spondylitis; Tendon structure; Testing; Transgenic Mice; Triplet Multiple Birth; Uveitis; Vertebral column; aggrecan; base; chemical group; congenic; density; epigenome; epigenomics; experience; gene function; genetic association; genetic manipulation; genome wide association study; histocompatibility gene; immune function; in vivo; insertion/deletion mutation; mouse genome; next generation; peripheral blood; prevent; promoter; protective effect; public health relevance; screening; skeletal; soft tissue; tool; transcription factor

DESCRIPTION (provided by applicant): Ankylosing spondylitis (AS) is a polygenic autoimmune disease of the axial skeleton affecting 0.3-0.5 % of the human population and causing a debilitating condition. AS starts with inflammation around ligament and tendon attachments (enthesitis) and is frequently associated with inflammation of the peripheral joints. Also, AS is often associated with extra-skeletal manifestations such as anterior uveitis and inflammatory bowel disease. Although the etiology of AS is unknown, genetic and environmental components have been prominently implicated as predisposing factors. The major genetic contributor is the major histocompatibility complex (MHC)-encoded (human leukocyte antigen) HLA-B27 first described in 1973. However, the presence of HLA-B27 alone is not sufficient for disease development. A few non-MHC candidate genes have also been implicated in AS. However, progress in AS-related genomic research has been hampered by the extreme genetic heterogeneity of the human population, and the fact that human individuals cannot be genetically manipulated. Animal models are invaluable tools for understanding the mechanisms of human (autoimmune) disorders. Proteoglycan [PG]-induced spondyloarthritis (PGISpA), which develops in genetically homogeneous inbred BALB/c mice upon immunization with cartilage PG, is permissive to genetic manipulation. PGISpA is the only inducible model of AS in which spine involvement is associated with sacroiliitis, uveitis and frequently but not always peripheral arthritis. Using genome-wide screening, we identified Pgis2 (on chromosome [Chr] 2, syntenic with human Chr9:SPA locus) as one of the most prominent quantitative trait loci (QTLs) that affects both the onset and severity of spondylitis. We generated congenic lines in which relevant Chr2 intervals from PGISpA-resistant DBA/2 were inserted into the PGISpA-susceptible BALB/c genome. A special value of this combination is that both mouse strains carry the same MHC (H2d), thus, the major genetic predisposition genetic factor (~50%) is "silenced". These congenic strains were tested for association of PGISpA susceptibility. The protective/suppressive regions of DBA/2 origin were stepwise reduced to ~3.0 mega basepair (Mbp) size, and 4 parents (DBA/2 and BALB/c) and 6 PGISpA-resistant and PGISpA-susceptible congenic mice were sequenced using a next-generation high-throughput sequencing method. The 10 genomic sequences were aligned to each other and to the reference (database) C57Bl/6 (B6) genomic sequence. Over 93% of indels (mutations: polymorphisms, deletions, insertions found in DBA/2 alleles) were localized in 3 relatively small genomic regions affecting a cluster of 3 (Gpr107-Nsc1-Hmcn2) genes and 2 other genes (St6galnac6 and Lmx1b) and their intergenic regions. Although non-sense mutations were not found in the coding sequences of these 5 genes, an unusually high number of mutations occurred in their intronic and intergenic sequences. We hypothesize that these genes contribute to PGISpA in BALB/c, and protect disease development in DBA/2 mice or in congenic strains carrying DBA/2 alleles of these regions. In vivo results with IVSC strains also suggest that genes in the three mutated Chr2 subregions have gene-gene (epistatic) interactions, i.e., they may act synergistically to completely prevent the development of PGISpA. Moreover, St6galnac6 was highly expressed in inflamed soft tissues of the spine (in areas of peridiscitis and enthesitis) in BALB/c mice, and the St6galnac6 promoter was heavily methylated (silenced) in PGISpA- resistant DBA/2 mice, indicating that epigenetic alterations have profound effects on the regulation of expression of this gene. We further hypothesize that both genetic mutations and epigenetic alterations are involved in the pathological mechanisms of PGISpA. In Aim 1, we will narrow, and (if possible) separate genes of the Gpr107-Nsc1-Hmcn2 cluster via new recombinations by generating new IVSC mice (with even smaller Chr intervals or single genes), which will be intercrossed with St6galnac6- and/or Lmx1b-specific IVSC mice to test epistatic effect among the mutated genes. In Aim 1B, we will generate transgenic mice carrying (non-mutated) Hmcn2 alone or in combination with Gpr107 and Nsc1 of non-DBA/2 origin (B6) to reverse the protective effect (i.e., restore susceptibility) of any of this region on PGISpA. In Ai 2, we will use St6galnac6-deficient and viable Lmx1b+/- mice to test the independent or combined effects of these genes on PGISpA, and Lmx1b-LacZ transgenic mice to detect expression of the Lmx1b transcription factor during the progression of SpA. Because epigenetic alterations may be important etiologic components of autoimmune diseases, and St6galnac6 expression appears to play a key role in early inflammatory reactions, in Aim 3 we will focus on how the altered methylation of the St6galnac6 gene affects the development of inflammation of in PGISpA.

描述（由申请人提供）： 强直性脊柱炎 (AS) 是一种中轴骨骼的多基因自身免疫性疾病，影响 0.3-0.5% 的人口并导致衰弱状况。 AS 始于韧带和肌腱附件周围的炎症（附着点炎），并且通常与周围关节的炎症有关。此外，AS 常常与骨骼外表现相关，例如前葡萄膜炎和炎症性肠病。尽管 AS 的病因尚不清楚，但遗传和环境因素已被认为是诱发因素。主要的遗传贡献者是 1973 年首次描述的主要组织相容性复合体 (MHC) 编码的（人类白细胞抗原）HLA-B27。然而，HLA-B27 的单独存在不足以导致疾病的发展。一些非 MHC 候选基因也与 AS 相关。然而，由于人类的极端遗传异质性以及人类个体无法进行基因操纵，AS相关基因组研究的进展受到了阻碍。动物模型是了解人类（自身免疫）疾病机制的宝贵工具。蛋白多糖 [PG] 诱导的脊柱关节炎 (PGISpA) 是在用软骨 PG 免疫后在遗传同质的近交 BALB/c 小鼠中发生的，允许进行基因操作。 PGISpA 是唯一一种可诱导的 AS 模型，其中脊柱受累与骶髂关节炎、葡萄膜炎以及经常但不总是外周关节炎相关。通过全基因组筛选，我们确定 Pgis2（位于染色体 [Chr]2 上，与人类 Chr9:SPA 位点同线）是影响脊柱炎发病和严重程度的最显着的数量性状位点 (QTL) 之一。我们生成了同类系，其中来自 PGISpA 抗性 DBA/2 的相关 Chr2 区间被插入到 PGISpA 敏感的 BALB/c 基因组中。这种组合的一个特殊价值是，两种小鼠品系都携带相同的 MHC (H2d)，因此，主要的遗传易感性遗传因素 (~50%) 被“沉默”。测试了这些同源菌株与 PGISpA 易感性的关联。 DBA/2 起源的保护/抑制区域逐步减小至约 3.0 兆碱基对 (Mbp) 大小，并使用测序仪对 4 个亲本（DBA/2 和 BALB/c）和 6 个 PGISpA 抗性和 PGISpA 敏感同源小鼠进行了测序。下一代高通量测序方法。将 10 个基因组序列相互比对并与参考（数据库）C57Bl/6 (B6) 基因组序列比对。超过 93% 的 indel（突变：DBA/2 等位基因中发现的多态性、缺失、插入）位于 3 个相对较小的基因组区域，影响 3 个 (Gpr107-Nsc1-Hmcn2) 基因和 2 个其他基因（St6galnac6 和 Lmx1b）的簇及其基因间区域。尽管在这5个基因的编码序列中没有发现无义突变，但它们的内含子和基因间序列中发生了异常多的突变。我们假设这些基因有助于 BALB/c 中的 PGISpA，并保护 DBA/2 小鼠或携带这些区域的 DBA/2 等位基因的同源品系的疾病发展。 IVSC 菌株的体内结果还表明，三个突变 Chr2 亚区中的基因具有基因-基因（上位）相互作用，即它们可能协同作用以完全阻止 PGISpA 的发展。此外，St6galnac6 在 BALB/c 小鼠的脊柱发炎软组织（椎周炎和附着点炎区域）中高度表达，并且 St6galnac6 启动子在 PGISpA 抗性 DBA/2 小鼠中严重甲基化（沉默），表明表观遗传改变对该基因的表达调控具有深远的影响。我们进一步假设基因突变和表观遗传改变都参与 PGISpA 的病理机制。在目标 1 中，我们将通过新的重组缩小并（如果可能）分离 Gpr107-Nsc1-Hmcn2 簇的基因，生成新的 IVSC 小鼠（具有更小的 Chr 间隔或单个基因），这些小鼠将与 St6galnac6- 和/或Lmx1b特异性IVSC小鼠来测试突变基因之间的上位效应。在目标 1B 中，我们将生成单独携带（非突变）Hmcn2 或与非 DBA/2 来源的 Gpr107 和 Nsc1 (B6) 组合的转基因小鼠，以逆转任何该区域的保护作用（即恢复易感性）在 PGISpA 上。在Ai 2中，我们将使用St6galnac6缺陷且存活的Lmx1b+/-小鼠来测试这些基因对PGISpA的独立或联合作用，并使用Lmx1b-LacZ转基因小鼠来检测SpA进展过程中Lmx1b转录因子的表达。由于表观遗传改变可能是自身免疫性疾病的重要病因，并且 St6galnac6 表达似乎在早期炎症反应中发挥关键作用，因此在目标 3 中，我们将重点关注 St6galnac6 基因甲基化的改变如何影响 PGISpA 炎症的发展。