GENETICS OF FAMILIAL MEDITERRANEAN FEVER

家族性地中海热的遗传学

• 批准号: 6100519
• 负责人: Daniel L Kastner
• 金额: --
• 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/6100519
• 关键词: amyloidosis; antisense nucleic acid; autosomal recessive trait; child (0-11); clinical research; complementary DNA; family genetics; gene mutation; genetic disorder; genetic library; genetic markers; genetic polymorphism; human genetic material tag; human subject; linkage mapping; molecular cloning; molecular pathology; nucleic acid probes; phenotype; restriction fragment length polymorphism; rheumatism; solution hybridization; transcription factor

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by episodes of fever with serositis and/or synovitis, and a massive influx of granulocytes into the affected anatomic compartments. Some patients also develop systemic AA amyloidosis, which can lead to renal failure and death. FMF is frequent among non-Ashkenazi Jews, Armenians, Arabs, and Turks. Our laboratory set out several years ago to identify the gene causing FMF (designated MEFV) by positional cloning. In 1992 we mapped MEFV to chromosome 16p13.3; in 1997 we identified the FMF gene itself. MEFV is comprised of 10 exons spanning about 14 kb of genomic DNA; it encodes a 3.7 kb transcript observed predominantly in granulocytes. Pyrin, the 781 aa predicted protein product, is homologous to a number of transcription factors and nucleic acid- binding proteins. Pyrin shares with some of these proteins an approximately 400 aa tripartite motif consisting of a B-box zinc finger, an alpha-helical (coiled-coil) domain, and a domain of unknown function designated B30.2. Pyrin also has a bZIP basic DNA-binding motif and a Robbins-Dingwall nuclear targeting signal. We originally described 3 conservative missense mutations (M680I, M694V, and V726A) in the B30.2 domain that were present in FMF patients, but not in a large panel of normal chromosomes. Microsatellite and single nucleotide polymorphism (SNP) haplotype analysis demonstrated ancestral relationships among populations that have been separated for many centuries, suggesting that these mutations are very old. During the 1998 reporting period, we have focused on 5 areas: a) assessing the spectrum of MEFV mutations and associated clinical phenotypes; b) studies of MEFV expression in leukocyte development and activation; c) development of antibodies to facilitate direct studies of the pyrin protein; d) cloning the murine homolog of MEFV and establishing a knockout mouse model of FMF; and e) initiating therapeutic trials for patients not responding fully to colchicine. Progress in each of these areas is summarized below. Mutational analysis: A large percentage of the carrier chromosomes in our initial panel of non-Ashkenazi Jewish and Arab families were attributable to the 3 aforementioned mutations. We subsequently focused our sequencing efforts on the small number of patients in whom we had not identified mutations, and an ethnically much more diverse cohort of 274 new referrals. Earlier this year we reported 4 novel mutations (E148Q, K695R, A744S, and R761H), and we have subsequently submitted a manuscript with 2 additional novel mutations (P369S and a second nucleotide substitution causing M680I). Altogether, there are now 16 known MEFV mutations in FMF; 14 are missense mutations and the other 2 are short in-frame deletions. Eleven fall within the B30.2 domain, while one each is within or near the bZIP basic domain, B-box zinc finger, and coiled-coil domain. Among the mutations identified after the cloning of MEFV, E148Q is the most common, occuring in several ethnic groups. SNP haplotype analysis strongly suggests a common ancestral origin among E148Q chromosomes. Our studies revealed a higher than expected number of mutation-positive patients of Ashkenazi Jewish and Italian ancestry, underscoring the need for clinical awareness in these populations. A survey of DNAs from approximately 200 anonymous but asymptomatic Ashkenazi Jewish subjects from New York demonstrated a carrier frequency of 21% in this population. MEFV mutations, particularly E148Q, P369S, and K695R, are probably not fully penetrant among the Ashkenazim. Our work also provides important data on the sensitivity and specificity of genetic testing for FMF in the U.S. MEFV Expression Studies: Northern blot and RT-PCR have confirmed that MEFV is expressed in peripheral blood neutrophils but not lymphocytes. We have also found message by RT-PCR in peripheral blood eosinophils. We have observed considerable variation in MEFV expression in monocytes, with some normal subjects exhibiting high levels. Normal human bone marrow, either by in situ hybridization or RT-PCR, exhibits MEFV message. The myelomonocytic HL60 cell line showed no expression at baseline, but MEFV message was induced when cells were stimulated by retinoic acid or alkaline pH to differentiate towards neutrophils or eosinophils, respectively. Treatment with PMA, which drives HL60 cells towards monocytic differentiation, was associated with low-level MEFV expression. Cultures of CD34+ human pleuripotent progenitors with cytokines that induce myeloid differentiation was associated with message induction at relatively early stages. RNase protection assays demonstrated marked increases in MEFV expression in monocytes within 1 hour of stimulation with LPS and the Th1 cytokine interferon gamma. These effects were blocked by the Th2 cytokine IL4. Expression studies with stimulated granulocytes are in progress. Antibodies against pyrin: Polyclonal rabbit antibodies have been raised against several pyrin peptides. One of these, directed against residues 435-452, identifies a band of the predicted size (about 85 kDa) on Western blots from in vitro transcribed and translated MEFV, and from a cell line transfected with an MEFV expression construct. This antibody also detects an 85 kDa band in lysates from neutrophils and activated monocytes. The activity of this serum for monocyte (but not granulocyte) lysates can be adsorbed with recombinant pyrin protein. Microsequencing is in progress to confirm the identity of the granulocyte band. Mouse model: Using PCR primers derived from human MEFV, we have cloned the mouse homolog. The exon-intron structure of mouse Mefv is very similar to the human gene. There are several splice variants in the mouse, but the predominant form encodes a predicted protein of 797 aa. Like human pyrin, the predicted mouse protein has a Robbins-Dingwall nuclear targeting sequence, a bZIP basic domain, a B-box zinc finger, and an alpha-helical domain. Due to a frame shift early in exon 10, the B30.2 domain is truncated. At the protein level, there is 47.6% identity and 65.5% similarity between human and mouse proteins.

家族性地中海热（FMF）是常染色体 隐性疾病的特征是脊髓膜炎发烧发作 和/或滑膜炎，以及大量的粒细胞涌入 影响解剖区。一些患者也会发展 全身性AA淀粉样变性，可能导致肾衰竭和死亡。 在非阿什肯纳兹犹太人，亚美尼亚人，阿拉伯人， 和土耳其人。我们的实验室几年前出发，以确定 通过位置克隆引起FMF（指定的MEFV）的基因。在 1992年，我们将MEFV映射到16p13.3染色体； 1997年我们 确定了FMF基因本身。 MEFV由10个外显子组成 跨越约14 kb的基因组DNA；它编码3.7 kb 转录本主要在粒细胞中观察到。 Pyrin，781 AA预测的蛋白质产品与许多 转录因子和核酸结合蛋白。 Pyrin股票 其中一些蛋白质大约为400 aa三方基序 由b-box锌指组成，是α-螺旋（盘绕螺旋） 域，以及指定B30.2的未知函数域。 吡啶还具有BZIP碱性DNA结合基序和A Robbins-Dingwall核靶向信号。我们最初描述 3保守的错义突变（M680i，M694V和V726A） 在FMF患者中存在的B30.2域中，但不在 正常染色体的大面板。微卫星和单一 核苷酸多态性（SNP）单倍型分析证明了 分离的人群之间的祖先关系 许多世纪以来，表明这些突变非常古老。 在1998年的报告期间，我们专注于5个领域：a） 评估MEFV突变和相关临床的光谱 表型； b）研究白细胞中MEFV表达的研究 发展和激活； c）开发抗体 促进对吡啶蛋白的直接研究； d）克隆鼠 MEFV的同源物并建立淘汰鼠标模型 FMF; e）针对未反应的患者进行治疗试验 完全到秋水仙碱。总结了这些领域的每个领域的进展 以下。 突变分析：很大一部分载体 我们的非阿什克纳兹犹太人和阿拉伯的初始面板中的染色体 家族归因于上述3个突变。我们 随后，我们的测序工作集中在少量 我们尚未确定突变的患者，一个种族 274个新推荐人群多样化。今年早些时候 报道了4种新型突变（E148Q，K695R，A744S和R761H）， 随后，我们提交了一个手稿，还有2个 新型突变（P369和第二个核苷酸取代 导致M680i）。总共有16个已知MEFV FMF中的突变； 14个是错义突变，另外两个是 短框内删除。 11落在B30.2域内，而 一个在BZIP基本域内或附近，B盒锌指， 和盘绕螺旋域。在确定的突变之后 MEFV的克隆，E148Q是最常见的，发生在几个 族裔。 SNP单倍型分析强烈建议常见 E148Q染色体之间的祖先来源。我们的研究揭示了 高于预期的突变阳性患者的数量 Ashkenazi犹太人和意大利血统，强调了需要 这些人群的临床意识。来自 大约200个匿名但无症状的Ashkenazi犹太人 来自纽约的受试者的载体频率为21％ 在这个人群中。 MEFV突变，特别是E148Q，P369S， 和K695R，可能并不完全渗透 Ashkenazim。我们的工作还提供了有关 在美国，基因检测的敏感性和特异性 MEFV表达研究：北印迹和RT-PCR具有 确认MEFV在外周血中性粒细胞中表达 但不是淋巴细胞。我们还发现了RT-PCR的消息 外周血嗜酸性粒细胞。我们已经观察到了很大的 单核细胞中MEFV表达的变化，一些正常 受试者表现出高水平。 正常的人骨髓，是 通过原位杂交或RT-PCR，可以显示MEFV消息。这 脊髓细胞细胞HL60细胞系在基线时没有表达 但是，当细胞被刺激时，MEFV消息被诱导 视黄酸或碱性pH分化为中性粒细胞或 嗜酸性粒细胞分别。用PMA处理，驱动HL60 细胞朝着单核细胞分化，与 低级MEFV表达。 CD34+人类的文化 伴有细胞因子诱导髓样的祖先祖细胞 差异化与相对的消息诱导有关 早期阶段。 标有RNase保护测定法 在1小时内1小时内，单核细胞中的MEFV表达增加 LPS和Th1细胞因子干扰素伽马刺激。 这些作用被Th2细胞因子IL4阻塞。表达 刺激粒细胞的研究正在进行中。 抗体 针对吡啶：多克隆兔抗体已升高 几种吡啶肽。其中之一，针对残留物 435-452，识别一个预测尺寸（约85 kDa）的频带 体外转录和翻译MEFV的蛋白质印迹，以及 从用MEFV表达构建体转染的细胞系。 该抗体还检测到来自裂解物中的85 kDa频带 中性粒细胞和活化的单核细胞。该血清的活性 单核细胞（但不是粒细胞）裂解液可以吸附 重组吡啶蛋白。 Microsequencing正在进行 确认粒细胞带的身份。 鼠标模型：使用 源自人MEFV的PCR引物，我们克隆了 鼠标同源物。鼠标MEFV的外显子内结构非常 类似于人类基因。有几个剪接变体 小鼠，但主要形式编码了一种预测的蛋白质 797 AA。像人吡啶一样，预测的小鼠蛋白具有 Robbins-Dingwall核靶向序列，BZIP基本域， B盒锌指和一个α-螺旋域。由于框架 在外显子10的早期，B30.2域被截断。在蛋白质上 水平，人之间的身份有47.6％和65.5％ 和小鼠蛋白。