Identification of Genetic and Epigenetic Alterations in Spondyloarthritis

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%的人群，并导致衰弱。AS开始于韧带和肌腱附件周围的炎症（附着点炎），并且经常与外周关节的炎症相关。此外，AS通常与骨骼外表现相关，如前葡萄膜炎和炎症性肠病。虽然AS的病因尚不清楚，但遗传和环境成分已被显著地暗示为易感因素。主要的遗传贡献者是主要组织相容性复合体（MHC）编码的（人类白细胞抗原）HLA-B27，1973年首次描述。然而，仅HLA-B27的存在不足以导致疾病的发展。一些非MHC候选基因也与AS有关。然而，AS相关基因组研究的进展受到了人类群体极端遗传异质性的阻碍，以及人类个体不能被遗传操纵的事实。动物模型是理解人类（自身免疫）疾病机制的宝贵工具。蛋白聚糖[PG]诱导的脊椎关节炎（PGISpA），这是在遗传上同质的近交系BALB/c小鼠与软骨PG免疫后，是允许的遗传操作。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、葡萄膜炎和经常但不总是外周关节炎相关。使用全基因组筛选，我们确定Pgis 2（在染色体[Chr] 2上，与人类Chr 9：SPA基因座同线）作为影响脊柱炎发病和严重程度的最突出的数量性状基因座（QTL）之一。我们产生了同源系，其中来自PGISpA抗性DBA/2的相关Chr 2间隔被插入到PGISpA敏感的BALB/c基因组中。这种组合的特殊价值在于两种小鼠品系携带相同的MHC（H2 d），因此，主要遗传易感性遗传因子（~50%）被“沉默”。检测这些同源菌株的PGISpA敏感性。将DBA/2起源的保护/抑制区逐步减少至约3.0兆碱基对（Mbp）大小，并使用下一代高通量测序方法对4只亲本（DBA/2和BALB/c）和6只PGISpA抗性和PGISpA敏感的同类小鼠进行测序。将10个基因组序列彼此比对并与参考（数据库）C57 B1/6（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（Gpr 107-Nsc 1-Hmcn 2）基因和2个其他基因（St 6 galnac 6和Lmx 1b）及其基因间区域的簇。虽然在这5个基因的编码序列中没有发现无义突变，但在其内含子和基因间序列中发生了异常高的突变。我们假设这些基因对BALB/c中的PGISpA有贡献，并保护DBA/2小鼠或携带这些区域的DBA/2等位基因的同类品系中的疾病发展。IVSC菌株的体内结果还表明，三个突变的Chr 2亚区中的基因具有基因-基因（上位性）相互作用，即，它们可以协同作用以完全阻止PGISpA的发展。此外，St 6 galnac 6在BALB/c小鼠的脊柱的发炎软组织（在骨膜炎和附着点炎的区域中）中高度表达，并且St 6 galnac 6启动子在PGISpA抗性DBA/2小鼠中高度甲基化（沉默），表明表观遗传改变对该基因的表达调控具有深远影响。我们进一步假设，基因突变和表观遗传改变都参与了PGISpA的病理机制。在目标1中，我们将通过产生新的IVSC小鼠（具有更小的Chr间隔或单个基因），通过新的重组缩小并（如果可能）分离Gpr 107-Nsc 1-Hmcn 2簇的基因，将其与St 6 galnac 6和/或Lmx 1b特异性IVSC小鼠杂交，以测试突变基因之间的上位效应。在目标1B中，我们将产生单独携带（非突变的）Hmcn 2或与非DBA/2来源的Gpr 107和Nsc 1组合的转基因小鼠（B6），以逆转保护作用（即，恢复敏感性）。在Ai 2中，我们将使用St 6 galnac 6缺陷和存活的Lmx 1b +/-小鼠来测试这些基因对PGISpA的独立或联合作用，并使用Lmx 1b-LacZ转基因小鼠来检测SpA进展期间Lmx 1b转录因子的表达。由于表观遗传学改变可能是自身免疫性疾病的重要病因组成部分，并且St 6 galnac 6表达似乎在早期炎症反应中起关键作用，因此在目标3中，我们将关注St 6 galnac 6基因的甲基化改变如何影响PGISpA中炎症的发展。

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.