Genetics Of The Dominantly Inherited Periodic Fever Synd

显性遗传性周期性发热综合征的遗传学

• 批准号: 6542715
• 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/6542715
• 关键词: autosomal dominant trait; biological response modifiers; clinical trials; cytokine receptors; drug screening /evaluation; family genetics; gene expression; gene mutation; genetic disorder; genetic mapping; genetically modified animals; human subject; human therapy evaluation; hyperthermia; immunosuppressive; laboratory mouse; linkage mapping; medical outreach /case finding; nonsteroidal antiinflammatory agent; patient oriented research; recombinant proteins; syndrome; tumor necrosis factor alpha; autosomal dominant trait; biological response modifiers; clinical trials; cytokine receptors; drug screening /evaluation; family genetics; gene expression; gene mutation; genetic disorder; genetic mapping; genetically modified animals; human subject; human therapy evaluation; hyperthermia; immunosuppressive; laboratory mouse; linkage mapping; medical outreach /case finding; nonsteroidal antiinflammatory agent; patient oriented research; recombinant proteins; syndrome; tumor necrosis factor alpha

Background In 1999 we discovered mutations in the gene (TNFRSF1A) encoding the 55 kDa tumor necrosis factor (TNF) alpha receptor in seven families with dominantly inherited periodic fever and inflammation. In addition to their fever and serosal inflammation, these families shared several distinguishing clinical features, including prolonged (greater than one week) attacks, a characteristic migratory erythematous rash with associated myalgia, conjunctival involvement, and a more marked therapeutic response to corticosteroids than to colchicine. This syndrome had first been described in a large Irish family, and had therefore been termed "familial Hibernian fever," but upon finding mutations not only in Irish families but in other nationalities, we suggested the more ethnically neutral name "TNF receptor-associated periodic syndrome (TRAPS)." All of the mutations we identified were missense mutations in the extracellular cysteine-rich domains of the TNF receptor, with five disrupting highly conserved disulfide bonds. For the C52F mutation, we demonstrated a defect in activation-induced ectodomain shedding of the TNF receptor, which may interfere with normal homeostasis in inflammation. During the 2000 reporting period we: (1) described eight additional mutations, including one splice mutation, in TNFRSF1A among newly referred patients. Two of these substitutions, R92Q and P46L, were found in about 1% of control chromosomes, but at even higher frequencies among periodic fever patients; (2) demonstrated receptor shedding defects for five additional mutations, but not for R92Q; (3) identified a common intragenic haplotype for apparently unrelated patients with the R92Q mutation, but not for T50M; (4) provided preliminary data that the frequency of R92Q is increased among patients in an early arthritis clinic; (5) performed linkage studies suggesting further genetic heterogeneity among the dominantly inherited periodic fevers; (6) initiated a treatment protocol to evaluate the efficacy of the anti-TNF agent etanercept in TRAPS; and (7) began constructing targeting vectors to develop TRAPS knockin mice. Results of the Last Year Mutational studies: Although we have identified a number of new cases with already known mutations, we have found only one new TNFRSF1A mutation (C43S) among the last year's referrals. This brings to 17 the total number of known mutations. All are in the extracellular domain of the receptor; one is a splicing mutation, nine are missense substitutions of cysteines, and the remainder are missense mutations at other residues. Genotype-phenotype studies performed before the discovery of C43S indicated a statistically insignificant increase in penetrance for cysteine substitutions (54/59 vs. 46/58) over other mutations. Based on our own patients and those reported in the literature, we found that the frequency of amyloidosis in TRAPS is approximately 14%, with 13 of the 14 cases occurring in patients with cysteine substitutions. In a study of 90 sporadic cases of periodic fever, complete genomic sequencing of the exons and splice junctions identified no additional mutations. A followup of our earlier studies of R92Q in early arthritis identified a total of 7 of 135 patients who bore this mutation. Etanercept study: We have now reached our accrual limit of 15 for Protocol 00-AR-0112, to examine systematically the use of etanercept in TRAPS. The trial consists of a 3 month observation period on "standard therapy" (for most patients, intermittent corticosteroids), 3 months on etanercept twice weekly (25 mg SC for adults, 0.4 mg/kg for children), 3 months on etanercept 3 times a week if there has not been a complete response to twice weekly (otherwise, a continuation of etanercept twice weekly), and a 3 month washout period. Patients are monitored for the number and severity of attacks, baseline and attack-associated levels of a number of inflammatory parameters, and the need for other medications (analgesics, corticosteroids, etc.). Of the 15 patients enrolled, 10 have the T50M mutation, 2 have H22Y, 1 has P46L, 1 has R92Q, and 1 has C33G. None have evidence of systemic amyloidosis. There have been two withdrawals: the R92Q patient withdrew for lack of efficacy, and the C33G patient was withdrawn in the first quarter because of inability to undergo periodic blood testing. Of the remaining 13, all have required a dose escalation in the third quarter. Ten have completed all four quarters, and 3 are in the washout phase. Of the 10 who have completed the study, all have had a dose-dependent decrease in their attack score and their use of other medications, which rebounded during the washout phase. Development of TRAPS knockin mice: We now have chimeric mice with the T50M mutation, and these are being tested for germline transmission. Embryonic stem cells with the C33Y and C52F mutations are currently being prepared for blastocyst injection. Development of an in vitro system to study the function of mutant forms of TNFRSF1A: We have developed expression vectors for wild-type TNFRSF1A, and for the H22Y, C30S, C33G, T50M, C52F, C88R, and R92Q mutations. Using biotinylated TNF and flow cytometry, we have found that R92Q receptors bind TNF as well as the wild type, that TNF binding to H22Y and C30S mutants is decreased but not abolished, and that TNF binding to the other 4 mutants (C33G, T50M, C52F, and C88R) is totally ablated. Experiments are in progress to examine receptor pre-ligand assembly by fluorescence energy transfer (FRET), protein size and quantity by Western analysis, signaling through the NF kappa B, jun kinase, and caspase pathways, and activation-induced receptor shedding. Conclusions and Significance The studies of the last year provide additional details in the characterization of TRAPS. Among the mutational studies, the most significant findings are the association of cysteine mutations with an increased risk of systemic amyloidosis, and the increased frequency of the R92Q mutation with early arthritis. The etanercept treatment protocol is now near completion, and it appears to confirm our earlier pilot experience that this agent reduces the frequency and severity of TRAPS attacks. The data on transfected mutant receptors strongly suggest that there may be additional mechanisms of inflammation in TRAPS other than the failure of activation-induced receptor cleavage. The observation that C33G, T50M, C52F, and C88R mutants do not bind TNF might suggest that these mutations lead to shunting of TNF signaling to p75 receptors, which lack a death domain and therefore cannot signal apoptosis. Thus, as appears to be the case for other periodic fevers, TRAPS may in part be caused by a failure of apoptosis in a subset of leukocytes involved in the early steps of the inflammatory cascade. During the next year, our objectives will be: (1) to continue mutational and genotype-phenotype studies of selected patients and families referred to the NIH Clinical Center; (2) to examine the role of TNFRSF1A sequence variants in a range of inflammatory conditions; (3) to complete our clinical trial of etanercept in TRAPS, and possibly to initiate a followup study comparing etanercept with another TNF inhibitor; (4) to begin phenotypic, biochemical, and cellular studies of T50M TRAPS knockin mice; and (5) to continue in vitro functional assays in transfected cell lines.

背景在1999年，我们发现了编码55 kDa肿瘤坏死因子（TNF）α受体的基因（TNFRSF1A）中的突变。除了发烧和浆膜炎症外，这些家族还具有多种显着的临床特征，包括长时间（大于一周）的攻击，一种具有特征性的迁移红斑皮疹，与肌痛相关，结膜参与以及对皮质类固醇的治疗反应更明显。该综合征首先在一个大型爱尔兰家庭中被描述，因此被称为“家族性的Hibernian热”，但不仅在爱尔兰家庭中发现突变，而且在其他民族中发现突变后，我们建议更具种族中性的名称“ TNF受体相关的周期性综合征（陷阱））。我们确定的所有突变都是TNF受体的细胞外半胱氨酸域中的错义突变，五个破坏了高度保守的二硫键。对于C52F突变，我们证明了TNF受体激活诱导的外域脱落的缺陷，这可能会干扰炎症中正常的稳态。 在2000年的报告期间，我们：（1）在新推荐的患者中描述了TNFRSF1A中的八个其他突变，包括一个剪接突变。在大约1％的对照染色体中发现了其中两个替代，R92Q和P46L，但在周期性发烧患者中的频率更高。 （2）证明了另外五个突变的受体脱落缺陷，但没有R92Q； （3）确定了显然无关的R92Q突变患者的常见基因内单倍型，但对于T50M而言却没有； （4）提供了初步数据，即早期关节炎诊所患者中R92Q的频率增加； （5）进行了连锁研究，表明主要遗传的周期性发动机之间的进一步遗传异质性； （6）启动了一种治疗方案，以评估抗TNF剂etanercept在陷阱中的功效； （7）开始构建靶向载体以发展陷阱敲击蛋白小鼠。 去年突变研究的结果：尽管我们已经确定了许多已知突变的新病例，但在去年的推荐中，我们只发现了一个新的TNFRSF1A突变（C43S）。这使已知突变的总数达到17。所有这些都位于受体的细胞外结构域中；一个是剪接突变，九个是半胱氨酸的错义取代，其余的是其他残基的错义突变。在发现C43S之前进行的基因型 - 表型研究表明，与其他突变相比，半胱氨酸取代（54/59 vs. 46/58）在统计学上微不足道的渗透率增加。根据我们自己的患者和文献报道的患者，我们发现陷阱中淀粉样变性的频率约为14％，半胱氨酸替代患者中有14例中有13例。在对90例周期性发烧的零星病例的研究中，外显子和剪接连接的完全基因组测序尚未鉴定出其他突变。我们早期对R92Q早期关节炎的研究的随访确定了135例患有这种突变的患者中的7例。 Etanercept研究：对于00AR-0112方案，我们现在已经达到了15的应计限制，以系统地检查eTanercept在陷阱中的使用。 The trial consists of a 3 month observation period on "standard therapy" (for most patients, intermittent corticosteroids), 3 months on etanercept twice weekly (25 mg SC for adults, 0.4 mg/kg for children), 3 months on etanercept 3 times a week if there has not been a complete response to twice weekly (otherwise, a continuation of etanercept twice weekly), and a 3 month washout period.监测患者的攻击，基线和攻击相关的多种炎症参数水平，以及对其他药物（镇痛药，皮质类固醇等）的需求。在入学的15例患者中，有10例具有T50M突变，其中2例H22Y，1例患有P46L，1例具有R92Q，1例具有C33G。没有人有全身性淀粉样变性的证据。有两次戒断：R92Q患者因缺乏疗效而撤回了，并且C33G患者在第一季度撤回，因为无法进行周期性的血液检查。在其余的13个中，所有人都需要在第三季度升级。十个已经完成了所有四个季度，其中3个处于冲洗阶段。在完成这项研究的10个中，所有人的攻击评分和对其他药物的使用都依赖于剂量，这些药物在冲洗阶段反弹。 陷阱蛋白小鼠的发育：我们现在具有带有T50M突变的嵌合小鼠，并且正在测试生殖线传播。目前正在准备胚泡注射C33Y和C52F突变的胚胎干细胞。 开发一种体外系统来研究TNFRSF1A突变形式的功能：我们已经开发了野生型TNFRSF1A的表达矢量，以及H22Y，C30S，C33G，T50M，T50M，C52F，C52F，C88R和R92Q突变。使用生物素化的TNF和流式细胞仪，我们发现R92Q受体结合了TNF和野生型，TNF与H22Y和C30S突变体的结合被减少但未被废除，并且TNF与其他4个突变体（C33G，T50m，T50m，C52F和C888）的结合。正在进行实验，以通过荧光能量转移（FRET），蛋白质的大小和数量来检查受体前配体组装，通过西方分析，通过NF Kappa B，Jun激酶和caspase途径进行信号，以及激活诱导的受体脱落。结论和意义上一年的研究为陷阱表征提供了更多细节。在突变研究中，最重要的发现是半胱氨酸突变与全身性淀粉样变性的风险增加以及早期关节炎的R92Q突变频率的增加。 Etanercept治疗方案现已接近完成，它似乎证实了我们较早的试点经验，即该药物降低了陷阱攻击的频率和严重性。转染突变受体的数据强烈表明，除了激活诱导的受体裂解失败以外，陷阱中可能还存在其他炎症机制。 C33G，T50M，C52F和C88R突变体不结合TNF的观察结果可能表明这些突变导致TNF信号传导与P75受体分流，后者缺乏死亡结构域，因此无法信号凋亡。因此，与其他周期性发动机似乎是这种情况，陷阱可能部分是由于炎症级联反应早期涉及的白细胞中凋亡失败引起的。 在第二年，我们的目标将是：（1）继续对NIH临床中心的选定患者和家庭进行突变和基因型 - 表型研究； （2）检查TNFRSF1A序列变体在一系列炎症条件下的作用； （3）完成我们对陷阱中依那耐酸的临床试验，并可能启动一项随访研究，将Etanercept与另一个TNF抑制剂进行比较； （4）开始进行T50M陷阱蛋白小鼠的表型，生化和细胞研究； （5）继续在转染的细胞系中继续体外功能测定。