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: 8716676
• 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/8716676
• 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; High-Throughput Nucleotide Sequencing; 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; risk variant; 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(申请人提供)：强直性脊柱炎(AS)是一种多基因的轴骨自身免疫性疾病，影响0.3-0.5%的人口，并导致虚弱的情况。AS始于韧带和肌腱附着物周围的炎症(终端炎)，并经常与周围关节的炎症有关。此外，AS通常与骨骼外的表现有关，如前葡萄膜炎和炎症性肠病。虽然AS的病因尚不清楚，但遗传和环境因素已被显著地认为是诱因。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)人类白细胞抗原-B27。然而，仅有人类白细胞抗原B27的存在对于疾病的发展是不够的。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的事实，与AS相关的基因组研究的进展一直受到阻碍。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)是一种允许基因操作的疾病，它发生在基因同源的近交系BALB/c小鼠中，通过软骨PG免疫。PGISpA是唯一可诱导的AS模型，在该模型中，脊柱受累与骶髂炎、葡萄膜炎和频繁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基因座同义)是影响脊柱炎发病和严重程度的最显著的数量性状基因座(QTL)之一。我们构建了同源基因系，将抗PGISpA的DBA/2的相应ChR2区间插入到对PGISpA敏感的BALB/c基因组。这一组合的一个特殊价值是，两个小鼠品系携带相同的MHC(H_2d)，因此，主要的遗传易感性遗传因素(~50%)被“沉默”。检测这些同源菌株与PGISpA易感性的关系。DBA/2基因的保护区和抑制区被逐步缩小到3.0兆碱基对(MBP)大小，4个亲本(DBA/2和BALB/c)和6个PGISpA抗性和PGISpA敏感的同基因小鼠被用下一代高通量测序方法进行了测序。将10个基因组序列相互比对，并与参考(数据库)C57BL/6(B6)基因组序列进行比对。超过93%的INDELs(DBA/2等位基因的突变：多态、缺失、插入)位于3个相对较小的基因组区域，影响一个由3个(Gpr107-Nsc1-Hmcn2)基因和2个其他基因(St6galnac6和Lmx1b)及其基因间隔区组成的簇。虽然在这5个基因的编码序列中没有发现无义突变，但它们的内含子和基因间序列发生了异常多的突变。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等位基因的同源菌株的疾病发展。IVSC菌株的体内实验结果也表明，这三个突变的ChR2亚区中的基因存在基因-基因(上位性)相互作用，即它们可能协同作用，完全阻止PGISpA的发展。此外，St6galnac6在BALB/c小鼠脊椎炎症软组织(盘周和末端炎区)中高表达，在PGISpA抗性的DBA/2小鼠中St6galnac6启动子高度甲基化(沉默)，表明表观遗传改变对该基因的表达调控有深远影响。我们进一步假设，基因突变和表观遗传学改变都参与了PGISpA的病理机制。在目标1中，我们将通过新的重组来缩小并(如果可能)分离Gpr107-Nsc1-Hmcn2簇的基因，方法是产生新的IVSC小鼠(具有更小的Chr间隔或单基因)，这些小鼠将与St6galnac6和/或Lmx1b特异的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)Hmcn2的转基因小鼠，或与非DBA/2起源的Gpr107和Nsc1(B6)联合使用，以逆转该区域中任何一个区域对PGISpA的保护作用(即恢复易感性)。在AI 2中，我们将使用St6galnac6缺陷和存活的Lmx1b+/-小鼠来测试这些基因对PGISpA的单独或联合作用，以及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。由于表观遗传学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发挥关键作用，在目标3中，我们将重点关注St6galnac6基因甲基化改变如何影响IN PGISpA的炎症发展。