Identification of Genetic and Epigenetic Alterations in Spondyloarthritis

描述（由申请人提供）：强直性脊柱炎（AS）是一种轴骨多基因自身免疫性疾病，影响0.3- 0.5%的人口，并导致衰弱状态。AS开始于韧带和肌腱附着物周围的炎症（肌腱炎），并且经常与周围关节的炎症相关。此外，AS常伴有骨外表现，如前葡萄膜炎和炎症性肠病。虽然AS的病因尚不清楚，遗传和环境因素已显著牵连为易感因素。主要的遗传因子是主要组织相容性复合体（MHC）编码的（人类白细胞抗原）HLA-B27，于1973年首次被描述。然而，HLA-B27的单独存在并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，由于人类群体的极端遗传异质性，以及人类个体不能被基因操纵，与自闭症相关的基因组研究进展受到阻碍。动物模型是理解人类（自身免疫性）疾病机制的宝贵工具。蛋白多糖[PG]诱导的脊椎炎（PGISpA），在遗传同质的近交系BALB/c小鼠中免疫软骨PG后发生，允许基因操作。PGISpA是唯一的AS诱导模型，其中脊柱受累与骶髂炎，葡萄膜炎和经常但不总是周围性关节炎有关。通过全基因组筛选，我们发现ppgis2（位于染色体[Chr] 2上，与人类Chr9:SPA位点一致）是影响脊柱炎发病和严重程度的最重要的数量性状位点（qtl）之一。我们将pgispa抗性DBA/2的相关Chr2片段插入到pgispa易感的BALB/c基因组中，生成了同源系。这种组合的一个特殊价值是，两种小鼠品系携带相同的MHC (H2d)，因此，主要的遗传易感性遗传因子（~50%）被“沉默”。检测了这些同源菌株对PGISpA的敏感性。采用新一代高通量测序方法对4只亲本（DBA/2和BALB/c）和6只pgispa耐药和易感基因小鼠进行测序，将DBA/2基因的保护/抑制区域逐步缩小至~3.0兆碱基对（Mbp）大小。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indels（突变：在DBA/2等位基因中发现的多态性、缺失、插入）定位于3个相对较小的基因组区域，影响3个基因簇（Gpr107-Nsc1-Hmcn2）和另外2个基因（St6galnac6和Lmx1b）及其基因间区域。虽然在这5个基因的编码序列中未发现非义突变，但在它们的内含子和基因间序列中发生了异常多的突变。我们假设这些基因参与了BALB/c中的PGISpA，并保护DBA/2小鼠或携带这些区域DBA/2等位基因的同源菌株的疾病发展。IVSC菌株的体内实验结果也表明，三个突变的Chr2亚区基因具有基因-基因（上位性）相互作用，即它们可能协同作用以完全阻止PGISpA的发展。此外，在BALB/c小鼠中，St6galnac6在脊柱炎症软组织（骨膜炎和腹膜炎区域）中高表达，而在PGISpA-抗性DBA/2小鼠中，St6galnac6启动子重度甲基化（沉默），表明表观遗传改变对该基因的表达调控有深远的影响。我们进一步假设基因突变和表观遗传改变都参与了PGISpA的病理机制。在Aim 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），通过新的重组来缩小和（如果可能的话）分离Gpr107-Nsc1-Hmcn2集群的基因，这些小鼠将与St6galnac6和/或lmx1b特异性IVSC小鼠杂交，以测试突变基因之间的遗传效应。在Aim 1B中，我们将产生单独携带（未突变的）Hmcn2或与非dba /2来源的Gpr107和Nsc1 （B6）结合的转基因小鼠，以逆转该区域任何一个对PGISpA的保护作用（即恢复易感性）。在Ai 2中，我们将使用st6galnac6缺失和活的Lmx1b+/-小鼠来检测这些基因对PGISpA的独立或联合作用，并使用Lmx1b- lacz转基因小鼠来检测Lmx1b转录因子在SpA进展过程中的表达。由于表观遗传改变可能是自身免疫性疾病的重要病因学成分，而St6galnac6的表达似乎在早期炎症反应中起关键作用，在Aim 3中，我们将重点关注St6galnac6基因甲基化改变如何影响PGISpA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.