Genetics Of Familial Mediterranean Fever and Related Conditions

家族性地中海热及相关病症的遗传学

• 批准号: 7964891
• 负责人: Daniel Kastner
• 金额: $ 120.05万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7964891
• 关键词: 16p13.3; Acne; Acute suppurative arthritis due to bacteria; Adenitis; Affect; Alleles; American; Amino Acids; Amyloidosis; Animal Model; Animals; Aphthous Stomatitis; Apoptosis; Arthritis; Autoimmune Process; Base Pairing; Binding; Biochemical; Biochemistry; Biology; Blood; Bone Marrow; Bone Marrow Cells; Canada; Capillary Electrophoresis; Caspase-1; Caucasians; Caucasoid Race; Cell Line; Cells; Cellular biology; Cervical; Child; Childhood; Chimera organism; Chromosomes; Chronic; Classification; Clinical; Complement; Cutaneous; Cytoskeletal Proteins; DNA; Data; Dermatitis; Development; Disease; Embryo; Exhibits; Exons; Familial Mediterranean Fever; Family; Fever; Flare; Frameshift Mutation; Gene Expression; Genes; Genetic; Genetic Polymorphism; Genomics; Goals; Granulocyte Colony-Stimulating Factor; Growth; Hand; Haplotypes; Hematopoietic; Hereditary Periodic Fever Syndromes; Human; ITGAM gene; Immunophenotyping; In Vitro; Inborn Genetic Diseases; Inflammation; Inflammatory; Inherited; Initiator Codon; Interferons; Interleukin-1; Interleukin-1 Receptors; Interleukin-1 beta; Journals; Knock-out; Laboratories; Lead; Leukocytes; Lymphocyte; Manuscripts; Maps; Measures; Mediating; Medicine; Modeling; Molecular; Molecular Profiling; Mouse Strains; Mus; Mutation; N-terminal; Neonatal; Netherlands; New England; Newfoundland; Nonsense Mutation; Nucleic Acid Regulatory Sequences; PTPRC gene; Pathogenesis; Pathway interactions; Patients; Penetrance; Periostitis; Peritoneal Macrophages; Pharyngitis; Phenotype; Population; Population Genetics; Positioning Attribute; Principal Component Analysis; Process; Proteins; Publishing; Puerto Rican; Puerto Rico; Pyoderma; Pyoderma Gangrenosum; Recurrence; Regulation; Rheumatism; Role; Sampling; Sampling Studies; Screening procedure; Serum; Sterility; Syndrome; Systems Biology; T-Lymphocyte; Technology; Terminator Codon; Transcript; Variant; Western Blotting; Whole Blood; Wild Type Mouse; Yeasts; anakinra; animal model development; base; caspase-8; chemokine; cohort; congenic; cytokine; granulocyte; human disease; knockin animal; mRNA Expression; marenostrin; mouse model; mutant; neutrophil; novel; overexpression; patient population; protein expression; response; transmission process; yeast two hybrid system

Background Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by self-limited attacks of fever with serosal, synovial, or cutaneous inflammation, sometimes complicated by systemic amyloidosis. In 1992 our laboratory mapped the FMF locus to chromosome 16p13.3, and in 1997 we isolated the underlying gene, MEFV, and demonstrated that it is highly expressed in granulocytes. We have subsequently studied FMF population genetics, the regulation of FMF gene expression in leukocyte subpopulations, the biochemistry and cell biology of pyrin (the FMF protein), and the development of animal models of FMF. The N-terminal 90 amino acids of pyrin comprise a motif, commonly called the PYRIN domain, found in approximately 20 human proteins. The cognate interaction of the PYRIN domain of pyrin with the homologous domain of the apoptosis-associated specklike protein with a CARD (ASC) places pyrin upstream in the regulation caspase-1-mediated interleukin-1 (IL-1) beta activation. In studies of peritoneal macrophages from a mouse strain expressing a truncated form of pyrin, we found increased caspase-1 activation and IL-1 beta processing, and impaired apoptosis through a caspase-8-dependent, IL-1 beta-independent pathway. By yeast two-hybrid studies, we demonstrated that pyrin interacts with the cytoskeletal protein PSTPIP1, and that PSTPIP1 mutations associated with the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) lead to markedly increased pyrin-binding and IL-1 beta activation. Results of the Last Year Mutational studies in FMF patients: We completed studies of a cohort of 46 patients with clinical FMF but only one demonstrable high-penetrance MEFV mutation, and published the findings in Arthritis and Rheumatism. Using standard capillary electrophoresis sequencing, 46 FMF patients with 1 MEFV mutation were screened for a second disease-associated mutation in all 10 exons of MEFV, including the exon-flanking regions. No new mutation was identified in any of the patients analyzed. A subset of 10 DNA samples was selected for additional sequencing of the entire 15-kb MEFV genomic region using hybridization-based chip technology. A novel heterozygous mutation was identified in the putative regulatory region 1 kb upstream of the start codon at position c.-888G->A in a North American patient, but population screening demonstrated that this variant is a polymorphism in the Caucasian population. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between patients with a single mutation and those with a double mutation. However, FMF patients of both types showed higher protein expression as compared with controls and with non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare mutations in a small number of patients, suggesting the possibility of digenic inheritance. Overall our data indicate that a single mutation in MEFV may, in some circumstances, be sufficient for clinical FMF. Studies of knockin (KI) models of FMF: To study the role of FMF-associated B30.2 domain mutations in the molecular pathogenesis of disease, we generated KI mouse models by inserting the B30.2 domain of wild-type (WT) or FMF-associated M680I, M694V, and V726A mutant pyrin into mouse pyrin, which ordinarily does not include a B30.2 domain. While the WT human B30.2 was embryonic lethal, the mice homozygous for human FMF mutations exhibited a phenotype similar to the human disease, but that was chronic rather than episodic, and generally more severe. KI mice exhibited growth retardation, spontaneous dermatitis and arthritis, and increased CD11b+ cells (especially Ly-6G+ neutrophils) in the blood. Bone marrow (BM) cells of KI mice transferred the KI phenotype into WT mice, and WT BM cells rescued the diseased KI mice, suggesting that BM-derived cells are necessary and sufficient for the disease. Lymphocytes are not required for inflammation since Rag-1 deficient KI mice showed phenotypes similar to Rag-1 sufficient KI mice. In CD45 congenic mixed bone marrow chimeras we found evidence that KI hematopoietic cells could induce WT CD11b expansion. Proinflammatory cytokines, such as IL-1beta, are significantly increased in KI mouse sera, and CD11b+ cells secrete active IL-1beta when stimulated with LPS alone without ATP in vitro. Moreover, the inflammatory phenotype did not appear when KI mice were crossed with IL-1 receptor KO or ASC KO mice. On the other hand, NLRP3-deficient KI mice exhibited the autoinflammatory phenotype. These data suggest that in FMF KI mice there is activation of an ASC-dependent NLRP3-independent inflammasome. We are currently preparing a manuscript describing these findings. Discovery of a novel recessively-inherited autoinflammatory syndrome: During the last year we completed collaborative studies with Dr. Raphaela Goldbach-Mansky establishing the existence of a novel disorder we term the deficiency in IL-1 receptor antagonist (DIRA), and published the initial description of this illness in the New England Journal of Medicine. The clinical findings of this disease include neonatal-onset sterile multifocal osteomyelits, periostitis, and pustulosis. We identified homozygous mutations in IL1RN in a total of nine affected children. One child from a family from Newfoundland, Canada, harbored a two base-pair deletion that caused a frame-shift mutation, N52KfsX25, followed by the incorporation of 24 amino aberrant amino acids and a termination codon. Five children from three unrelated families from the Netherlands were homozygous for a nonsense mutation (E77X). Two children from a consanguineous Lebanese family were homozygous for a second nonsense mutation (Q54X). A ninth patient, from Puerto Rico, was homozygous for a deletion of approximately 175 kb on chromosome 2q that includes six genes IL1RN and five interleukin-1 related genes. The Canadian, Dutch, and Lebanese mutations result in a truncated protein that is not secreted, while the Puerto Rican mutation results in no IL-1 receptor antagonist protein at all. In all cases, this renders patient leukocytes hyperresponsive to IL-1beta stimulation. Patients treated with anakinra responded rapidly. Systems biology analysis of the syndrome of periodic fever with aphthous stomatitis, pharyngitis, and/or cervical adenitis (PFAPA): PFAPA is perhaps the most common recurrent fever syndrome of childhood. The precise pathogenesis is unknown. We therefore undertook a systematic analysis in which we compared whole blood gene expression, serum inflammatory measures, and multicolor lymphocyte immunophenotyping in PFAPA patients during and between flares, healthy controls, and patients with hereditary periodic fever (HPF) syndromes. Principal components analysis revealed the overall mRNA expression profile during PFAPA flares to be remarkably distinct from that in asymptomatic intervals and HPF flares. PFAPA attacks were characterized by a significant overexpression of complement, interleukin-1-related, and interferon-induced genes, whereas T cell-associated transcripts were downregulated. PFAPA flares were most strongly associated with increased serum levels of chemokines for activated T lymphocytes, as well as granulocyte colony-stimulating factor. Activated T lymphocytes correlated inversely with serum concentrations of IP-10/CXCL10. All of five PFAPA patients subsequently treated with anakinra during febrile attacks showed a prompt clinical response. We are currently preparing a manuscript describing our findings.

背景 家族性地中海热（FMF）是一种隐性遗传疾病，其特征是伴有浆膜，滑膜或皮肤炎症的自限性发作，有时会因全身性淀粉样变性而复杂。 1992年，我们的实验室将FMF基因座映射到16p13.3染色体，并在1997年分离了基础基因MEFV，并证明它在粒细胞中高度表达。随后，我们研究了FMF种群遗传学，白细胞亚群中FMF基因表达的调节，吡啶（FMF蛋白）的生物化学和细胞生物学以及FMF动物模型的发展。吡啶的N末端90氨基酸包含一个基序，通常称为吡啶结构域，在大约20种人体蛋白中发现。吡啶的吡啶结构域与凋亡相关的斑点蛋白的同源结构域与卡（ASC）的同源结构域的同源相互作用（在调节caspase-1介导的介导的白介素-1（IL-1）β激活中。在表达曲线截断形式的小鼠菌株的腹膜巨噬细胞的研究中，我们发现CASPASE-1激活和IL-1β处理增加，并通过caspase-8依赖性IL-1β独立途径损害了凋亡。通过酵母的两杂交研究，我们证明吡啶与细胞骨架蛋白PSTPIP1相互作用，并且与脓毒性关节炎综合征与脓毒性性腺肿症和痤疮（PAPA）相关的PSTPIP1突变（PAPA）显着增加了吡啶结合和IL-1 beta beta beta beta beta beta beta beta激活。 去年的结果 FMF患者的突变研究：我们完成了46例临床FMF患者的研究，但只有一项可证明的高渗透率MEFV突变，并发表了关节炎和风湿病的发现。 使用标准毛细管电泳测序，将46例MEFV突变的FMF患者在包括外显子频型区域在内的所有10个外显子中进行了第二个与疾病相关的突变。 在分析的任何患者中均未发现新的突变。 使用基于杂交的芯片技术选择了10个DNA样品的子集，以对整个15 kB MEFV基因组区域进行其他测序。 在北美患者的位置C.-888G-> A上游的推定调节区域中发现了一种新型的杂合突变，但人群筛查表明，这种变体是高加索人群中的多态性。 单倍型分析未识别可能与第二个FMF等位基因的传输相关的常见单倍型。 蛋白质印迹并未表现出单个突变患者和患有双突变患者的吡啶水平有显着差异。 然而，与对照组和非FMF患者相比，两种类型的FMF患者均显示出更高的蛋白质表达。 筛选编码吡啶相互作用蛋白的基因鉴定出少数患者的罕见突变，这表明二氮遗传的可能性。总体而言，我们的数据表明，在某些情况下，MEFV中的单个突变可能足以容纳临床FMF。 FMF的敲除模型的研究：研究与FMF相关的B30.2结构域突变在疾病的分子发病机理中的作用，我们通过插入野生型（WT）的B30.2结构域来产生Ki小鼠模型B30.2域。 尽管WT人B30.2是胚胎致死的，但人类FMF突变纯合的小鼠表现出类似于人类疾病的表型，但这是慢性的，而不是偶发性，通常更为严重。 Ki小鼠在血液中表现出生长迟缓，自发性皮炎和关节炎，并增加了CD11b+细胞（尤其是LY-6G+中性粒细胞）。 Ki小鼠的骨髓（BM）细胞将Ki表型转移到WT小鼠中，WT BM细胞营救了患病的Ki小鼠，这表明BM衍生的细胞对于该疾病是必需的并且足够。 炎症不需要淋巴细胞，因为RAG-1缺乏的Ki小鼠显示出与RAG-1足够Ki小鼠相似的表型。 在CD45中，我们发现的证据表明，Ki造血细胞可以诱导WT CD11b膨胀。 促炎细胞因子（例如IL-1BETA）在Ki小鼠血清中显着增加，CD11b+细胞单独使用没有ATP的LPS刺激ATP，分泌活性IL-1Beta。 此外，当Ki小鼠与IL-1受体KO或ASC KO小鼠交叉时，没有出现炎症表型。 另一方面，NLRP3缺陷的Ki小鼠表现出自发性表型。 这些数据表明，在FMF Ki小鼠中，有依赖ASC的NLRP3独立炎症体的激活。 我们目前正在准备一个描述这些发现的手稿。 发现了一种新颖的隐性自身炎症综合症：在去年，我们完成了与Raphaela Goldbach-Mansky博士的合作研究，确立了一种新型疾病的存在，我们称IL-1受体拮抗剂（DIRA）的缺乏症，并在新英格兰医学杂志上发表了这种疾病的最初描述。 该疾病的临床发现包括新生儿发作的无菌多灶性骨髓性，骨膜炎和脓疱病。 我们确定了总共九名受影响儿童的IL1RN中的纯合突变。 来自加拿大纽芬兰的一个家庭的一个孩子藏有两个基本对删除，导致框架变速突变，即N52KFSX25，然后掺入24个氨基异常氨基酸和终止密码子。 来自荷兰的三个无关家庭的五个孩子是纯合的，因为它是胡说八道的突变（E77X）。 来自黎巴嫩一家人的两个孩子是纯合的，因为第二个废话突变（Q54X）。 一名来自波多黎各的第九名患者是纯合的，因为在2q染色体上删除了约175 kb，其中包括六个基因IL1RN和五个Interleukin-1相关基因。加拿大，荷兰和黎巴嫩突变导致截短的蛋白质，而不是分泌的，而波多黎各人突变根本导致没有IL-1受体拮抗剂蛋白。 在所有情况下，这都使患者白细胞对IL-1BETA刺激的反应过度。 接受Anakinra治疗的患者反应迅速。 系统生物学分析伴有腹膜炎，咽炎和/或宫颈腺炎（PFAPA）的周期性热综合征：PFAPA也许是最常见的童年复发综合症。精确的发病机理未知。 因此，我们进行了系统分析，在该分析中，我们比较了PFAPA患者在耀斑，健康对照组和遗传性周期性发烧（HPF）综合征中的PFAPA患者中的全血基因表达，血清炎症措施和多色淋巴细胞免疫表型。 主成分分析揭示了PFAPA耀斑期间的总体mRNA表达谱，在无症状间隔和HPF耀斑中与之明显不同。 PFAPA攻击的特征是补体，白细胞介素1相关和干扰素诱导的基因的明显过表达，而T细胞相关的转录本下调。 PFAPA耀斑与激活T淋巴细胞的血清趋化因子水平升高以及粒细胞刺激因子最密切相关。 活化的T淋巴细胞与IP-10/CXCL10的血清浓度成反比。随后在高热攻击期间接受Anakinra治疗的五名PFAPA患者中的所有患者均表现出迅速的临床反应。 我们目前正在准备一个描述我们发现的手稿。