GENETICS OF THE DOMINANTLY INHERITED PERIODIC FEVER SYNDROMES

显性遗传性周期性发热综合征的遗传学

• 批准号: 6431754
• 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/6431754
• 关键词: autosomal dominant trait; clinical research; gene expression; genetic disorder; genetic mapping; human genetic material tag; human subject; hyperthermia; phenotype; syndrome; tumor necrosis factor alpha

BackgroundThe dominantly inherited periodic fever syndromes are a group of disorders characterized by episodes of fever with serosal, synovial, conjunctival, or cutaneous inflammation; over time some patients also develop systemic AA amyloidosis. These conditions can be distinguished clinically from the recessively inherited familial Mediterranean fever (FMF) by virtue of their prolonged (greater than 1 week) attacks, periorbital edema/conjunctivitis, migratory erythema, and poor therapeutic response to colchicine. Prior to our discovery of a common genetic etiology, the dominantly inherited periodic fever syndromes subsumed several diagnostic entities, including familial Hibernian fever (FHF), benign autosomal dominant familial periodic fever, and autosomal dominant periodic fever with amyloidosis. In early 1998 a collaborator in Adelaide established genetic linkage to chromosome 12p13 for benign autosomal dominant familial periodic fever, and shortly thereafter a collaborator in London established linkage to the same region for FHF. Objective of Present StudiesDuring the 1999 reporting period our objective was to establish a positional cloning project that would ultimately identify the periodic fever gene on chromosome 12p13. Since we were able to achieve this objective in a shorter time than anticipated, we established additional objectives: a) to investigate how mutations in this gene cause disease; b) to perform mutational screening on selected referrals to the Clinical Center, and to study genotype-phenotype relationships; and c) to undertake pilot studies on the treatment of the dominantly inherited periodic fever syndromes.Results of the Last YearIdentification of the gene causing dominantly inherited periodic fevers: Pooling pedigrees from the NIH and our collaborators, we were able to define an approximately 8 cM candidate interval that included the gene (TNFRSF1A, formerly TNFR1) encoding the 55 kDa tumor necrosis factor (TNF) receptor, as well as several other plausible candidates. This TNF receptor protein is variously denoted as TNFR1, p55, and CD120a. By genomic sequencing, we identified 6 different mutations in TNFRSF1A in 7 different families with dominantly inherited periodic fevers. Five of the 6 mutations involve missense substitutions in cysteines that would directly disrupt extracellular disulfide bonds, while the sixth mutation is adjacent to one of these cysteines and may indirectly inhibit disulfide bonding. Families with mutations were not restricted to Irish or Scottish ancestry. We proposed the acronym TRAPS (TNF receptor-associated periodic syndrome) to recognize the common pathogenesis in all of these families of varied ethnic backgrounds and clinical phenotypes. The fact that TNFR1 associates into homotrimers may account for the dominant mode of inheritance, since a mutation in even one of the three chains could lead to significant alterations in the complex.Studies of the molecular pathogenesis of disease: In collaboration with the Lymphocyte Cell Biology Section of the Arthritis and Rheumatism Branch, we investigated the possible mechanisms by which these mutations might cause a hyperinflammatory phenotype. There was no increase in the binding affinity of radiolabeled TNF for leukocytes from patients as compared with controls. We also considered the possibility that disease-associated mutations might cause constitutive activation, perhaps by permitting intermolecular disulfide homodimerization and ligand-independent activation. This was tested by studying the in vitro production of IL-6, a TNF-responsive cytokine, by leukocytes from patients. Baseline IL-6 production was not increased, and the dose- response for purified mononuclear cells stimulated with TNF was similar to controls, thus arguing against constitutive activation.We did, however, find evidence that these mutations lead to a specific defect in the activation-induced shedding of the 55 kDa TNF receptor. The first suggestion that this might be the case was the observation that 27 mutation-positive individuals with 4 different mutations had about 50% of the normal level of circulating soluble p55, and a markedly blunted increase in soluble p55 during attacks (relative to patients with other inflammatory conditions). An intensive study of leukocytes from 3 patients with the C52F mutation showed increased resting levels of membrane TNFR1 relative to normal controls. Stimulation with phorbol ester (PMA) normally induces metalloprotease mediated cleavage of TNFR1 and several other receptors from the cell surface. By flow cytometry, PMA-induced clearance of TNFR1 from the surfaces of both granulocytes and monocytes was markedly reduced relative to normal controls. In contrast, clearance of the 75 kDa TNF receptor (p75) in C52F patients was comparable to controls, thus arguing against a generalized defect in receptor clearance. Moreover, when patient or control leukocytes were cultured with PMA, levels of soluble p55 in the supernatants were much lower for the C52F patients than for controls, while the levels of soluble p75 were similar in patient and control supernatants. Taken together, these data indicate that one important physiologic consequence of the C52F mutation is impaired cleavage of the TNFR1 ectodomain, thus subverting normal homeostasis by permitting repeated stimulation through the cell-bound receptor, and leading to diminished levels of potentially antagonistic circulating p55. TRAPS is the first human disease shown to be caused by a defect in the cleavage of cytokine receptors. Mutational screening and genotype-phenotype studies: Subsequent to our initial report describing the first six TNFRSF1A mutations, we have screened additional families referred for evaluation of periodic fever. Through these efforts we have identified an additional 7 mutations, all of which affect the extracellular domain of the p55 TNF receptor. Six of the 7 are missense mutations, and 2 affect cysteines involved in disulfide bond formation. The seventh mutation is a single base substitution that creates a splice acceptor site in intron 2, resulting in the addition of 4 amino acids to the extracellular domain. This mutation was initially detected by genomic sequencing, and the effect on splicing has been confirmed by cDNA sequencing of RT-PCR products. It is noteworthy that among the 16 families in which we have found TNFRSF1A mutations, there are 13 different mutations. The aforementioned splicing mutation was seen in 2 families, and the T50M mutation was observed in 3 families. However, by microsatellite haplotype analysis, even these represent independent mutational events. This is in stark contrast to FMF, where several founder mutations/haplotypes account for the majority of carrier chromosomes in several populations. It is also remarkable that, despite the fact that TRAPS is seen in a variety of ethnic groups, fully 60% of our families are of Scottish or Irish ancestry, raising the possibility of a permissive genotype at a second locus in the Irish and Scottish populations. Careful observation of families with TNFRSF1A mutations reveals a much broader clinical spectrum than had been described for the prototypic FHF. Systemic AA amyloidosis has been documented in families with the C30R, C33Y, C33G, C52F, and C88Y mutations. A rash similar to that seen in dermatomyositis has been observed in one member of a family with the C30S mutation. Consistent with the known biologic effects of TNF in promoting anorexia and cachexia, TRAPS patients often show significant weight loss during their febrile attacks, and one patient with the R92Q mutation has marked wasting and lipoatrophy. Given the expression of TNF receptors in the central nervous system, and the presence of neurologic abnormalities in TNF transgenic mice, we are actively investigating the potential for neurologic symptoms in TRAPS.Therapeutic studies: During the last 6 months we have begun a pilot study of the use of recombinant p75 TNFR:Fc fusion protein (etanercept) in the treatment of TRAPS. This agent would be expected to reduce TNF-dependent activation of inflammation, and might make up for the observed deficiency of circulating soluble p55. A total of 7 TRAPS patients have been treated with etanercept; 3 have the C52F mutation, 2 have the C33G mutation, and 1 each have the C30S and T50M mutations. The drug has been well-tolerated in all 7 patients. The clinical response in the best studied case, a patient with the C52F mutation, has been dramatic, and the preliminary experience with the remaining 6 has been favorable. Conclusions and SignificanceDuring the last year we substantially increased our understanding of the dominantly inherited periodic fever syndromes. First, we discovered that these disorders are caused by mutations in TNFRSF1A, the gene that encodes the 55 kDa receptor for TNF. We have suggested the new acronym TRAPS (TNF receptor-associated periodic syndrome) to include all mutation-positive patients, regardless of their ethnic backgrounds or the specific features of their periodic inflammation. Five of the 6 initial mutations directly disrupt intrachain disulfide bonds in the extracellular domain, and functional studies of leukocytes from patients implicate a defect in activation-induced ectodomain-cleavage. This latter observation may serve as a paradigm for additional as yet undiscovered mutations in other members of the TNF receptor family. Sequencing studies of new Clinical Center referrals have brought the total number of disease-associated mutations to 13, and careful clinical observations of these families are likely to deepen our understanding of the role of TNF pathways in human disease. Our data indicate that TNF receptor mutations are not restricted to the Irish and Scottish populations, though there is an unexplained enrichment for these ethnicities among affected families. During the next year, our objectives will be:1) continuing mutational and genotype-phenotype studies of selected patients and families referred to the NIH Clinical Center; 2) developing a blinded, placebo-controlled crossover study to evaluate the efficacy of etanercept in TRAPS more systematically; 3) collaborative studies with the Lymphocyte Cell Biology Section of ARB to study signaling in cells transfected with TNFR1 bearing disease- associated mutations; and 4) developing a TRAPS knockin mouse.

背景主要继承的周期性发烧综合征是一组 疾病的特征是发烧发作带有浆膜，滑膜， 结膜或皮肤炎症；随着时间的流逝，一些患者也 发展系统性AA淀粉样变性。这些条件可以区分 临床上隐藏的家族性地中海热 （fmf）由于他们的延长（大于1周）的攻击， 周围水肿/结膜炎，迁移红斑和差 对秋水仙碱的治疗反应。在我们发现一个共同点之前 遗传病因，主要遗传的周期性发烧综合征 包含了几个诊断实体，包括家族性的Hibernian 发烧（FHF），良性常染色体显性家族周期性发烧和 常染色体显性周期性热和淀粉样变性。在1998年初 阿德莱德的合作者建立了与染色体的遗传联系 12p13用于良性常染色体显性家族周期性发烧，并且 此后不久，伦敦的一名合作者建立了与 FHF的同一区域。目前研究的目的是1999年 报告期我们的目标是建立一个位置克隆 最终确定周期性发烧基因的项目 12p13染色体。由于我们能够在 时间比预期的要短，我们建立了其他目标：a） 研究该基因的突变如何引起疾病； b）执行 对选定转诊至临床中心的突变筛查，以及 研究基因型 - 表型关系； c）承担飞行员 关于主要遗传周期热治疗的研究 综合征。引起基因的最后一年识别 主要继承的周期性发烧：从NIH中汇集血统 和我们的合作者，我们能够定义大约8厘米 包括基因的候选间隔（TNFRSF1A，以前为TNFR1） 编码55 kDa肿瘤坏死因子（TNF）受体以及 其他几个合理的候选人。该TNF受体蛋白是 被称为TNFR1，P55和CD120A。通过基因组测序，我们 在7个不同家族的TNFRSF1A中鉴定了6种不同的突变 具有主要继承的周期性发烧。 6个突变中的五个 涉及将直接破坏的半胱氨酸中的错义替代 细胞外二硫键，而第六个突变与 这些半胱氨酸之一，可能间接抑制二硫键。 有突变的家庭不仅限于爱尔兰或苏格兰 祖先。我们提出了首字母缩略词陷阱（TNF受体相关 周期性综合征）识别所有这些的常见发病机理 各种种族背景和临床表型的家族。事实 TNFR1成为同构二聚体可能会占主导地位 继承方式，因为即使是三个链中的一个突变 可能导致复合体的重大变化。 疾病的分子发病机理：与淋巴细胞合作 关节炎和风湿病分支的细胞生物学部分，我们 研究了这些突变可能的可能机制 引起高炎性表型。没有增加 放射性标签TNF对白细胞的结合亲和力是患者的白细胞 与对照组相比。我们还考虑了 与疾病相关的突变可能导致构型激活， 也许通过允许分子间二硫化物均匀化和 非配体非依赖性激活。这是通过研究体外测试的 由白细胞产生IL-6，TNF响应性细胞因子 患者。基线IL-6产生没有增加，剂量 - 用TNF刺激的纯化的单核细胞的响应相似 对控件，因此反对构成激活。我们确实做到了 但是，找到这些突变导致特定缺陷的证据 在激活引起的55 kDa TNF受体的脱落中。这 首先建议可能是这样的观察 27个具有4个不同突变的突变阳性个体大约 循环可溶性p55的正常水平的50％和一个明显的 攻击期间可溶性p55的增加（相对于患者） 与其他炎症条件”。白细胞的密集研究 来自3例C52F突变的患者显示静息水平升高 膜TNFR1相对于正常对照。用佛波刺激 酯（PMA）通常诱导金属蛋白酶介导的TNFR1裂解 以及细胞表面的其他几个受体。通过流式细胞仪， PMA引起的TNFR1从两个粒细胞的表面清除 相对于正常对照，单核细胞显着降低。在 对比，C52F患者的75 kDa TNF受体（P75）的清除率 与对照相媲美，因此反对广义缺陷 在受体清除率中。而且，当患者或控制白细胞时 用PMA培养，上清液中可溶性p55水平为 C52F患者比对照组低得多 可溶性P75在患者和对照上清液中相似。拍摄 这些数据一起表明一个重要的生理 C52F突变的结果是TNFR1的裂解受损 胞外域，从而通过重复来颠覆正常的稳态 通过细胞结合的受体刺激，并导致减少 潜在拮抗循环p55的水平。陷阱是第一个 人类疾病被证明是由裂解中的缺陷引起的 细胞因子受体。突变筛查和基因型 - 表型 研究：在我们的初步报告之后描述了前六个 tnfrsf1a突变，我们筛选了更多的家庭 评估周期性发烧。通过这些努力，我们确定了 另外7个突变，所有突变都会影响细胞外域 p55 TNF受体。 7个中有六个是错义突变，2 影响与形成二硫键形成有关的半胱氨酸。第七 突变是产生剪接受体的单个基础替代 内含子2中的位点，导致在 细胞外域。最初通过基因组检测到该突变 测序以及对剪接的影响已通过cDNA证实 RT-PCR产品的测序。值得注意的是，在16个 我们发现TNFRSF1A突变的家庭，有13个 不同的突变。在2中看到了上述剪接突变 在3个家庭中观察到家庭，以及T50M突变。但是，通过 微卫星单倍型分析，甚至这些分析也代表了独立的 突变事件。这与FMF形成鲜明对比，其中有几个 创始人突变/单倍型占多数载体 几个人群中的染色体。尽管如此，尽管 在各个种族中看到陷阱的事实，完全有60％ 我们的家人是苏格兰或爱尔兰血统，提高了可能性 在爱尔兰人和苏格兰的第二个基因座的允许基因型 人群。仔细观察TNFRSF1A突变的家庭 揭示了比描述的要广泛得多的临床光谱 原型FHF。系统性AA淀粉样变性已记录在 具有C30R，C33Y，C33G，C52F和C88Y突变的家庭。皮疹 与一名成员中观察到与皮肌炎中发现的相似之处 具有C30S突变的家庭。与已知的生物学一致 TNF在促进厌食和恶病质上的影响，经常捕获患者 在发热攻击中显示出重大减肥，一个 R92Q突变的患者已明显浪费和脂肪肉芽。 考虑到中枢神经系统中TNF受体的表达， 以及TNF转基因小鼠中神经系统异常的存在，我们 正在积极研究神经系统症状的潜力 陷阱。治疗研究：在过去的六个月中，我们已经开始 重组P75 TNFR的使用试验研究：FC融合蛋白 （Etanerce）在陷阱的处理中。期望这个代理人 减少TNF依赖性炎症的激活，并可能弥补 观察到的循环可溶性p55的缺陷。总共7个陷阱 患者已接受依那耐治疗； 3具有C52F突变，2 具有C33G突变，1个具有C30S和T50M突变。 该药物在所有7例患者中都富有耐受性。临床 在最佳研究病例中的反应，患有C52F突变的患者， 一直很戏剧性，其余的6 一直很有利。结论和有意义的一年我们 大大增加了我们对主要继承的理解 周期性发烧综合征。首先，我们发现这些疾病是 由TNFRSF1A突变引起的，该基因编码55 kDa TNF的受体。我们提出了新的首字母缩写陷阱（TNF 与受体相关的周期综合征），包括所有突变阳性 患者，无论他们的种族背景如何 它们周期性炎症的特征。六个首字母中的五个 突变直接破坏内部内部二硫键 细胞外结构域和白细胞的功能研究 患者暗示了激活诱导的外生域切解中的缺陷。 后一个观察结果可能是额外的范式 TNF受体家族其他成员中未发现的突变。 新临床中心推荐的测序研究带来了 疾病相关突变的总数为13，并且谨慎 这些家庭的临床观察可能会加深我们 了解TNF途径在人类疾病中的作用。我们的数据 表明TNF受体突变不限于爱尔兰 和苏格兰人口，尽管有无法解释的 受影响家庭中的这些种族。在第二年，我们 目标将是：1）继续突变和基因型 - 表型 参考NIH临床的选定患者和家庭的研究 中心; 2）开发一项盲，安慰剂控制的跨界研究 更系统地评估Etanercept在陷阱中的功效； 3） 与ARB的淋巴细胞细胞生物学部分合作研究 研究用TNFR1轴承疾病转染的细胞中的信号传导 - 相关突变； 4）开发一个陷阱小鼠。