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: 6823106
• 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/6823106
• 关键词: antiinflammatory agents; autosomal dominant trait; biological response modifiers; clinical trials; cytokine receptors; drug screening /evaluation; family genetics; gene expression; gene mutation; gene targeting; genetic disorder; genetically modified animals; human subject; human therapy evaluation; hyperthermia; immunosuppressive; interleukin 1; laboratory mouse; linkage mapping; medical outreach /case finding; nonsteroidal antiinflammatory agent; patient oriented research; recombinant proteins; syndrome; tumor necrosis factor alpha

Background In 1999 our laboratory led an international consortium in reporting that mutations in TNFRSF1A, the gene encoding the 55 kDa tumor necrosis factor (TNF) receptor, cause a dominantly inherited syndrome of fever and inflammation, and proposed the name "TNF receptor-associated periodic syndrome" (TRAPS) for this condition. We demonstrated 6 mutations in the extracellular domain of the TNFRSF1A protein, 5 of which were missense substitutions at cysteine residues, resulting in the disruption of highly conserved disulfide bonds. The 7 original families included the Irish-Scottish family prototypic for "familial Hibernian fever," as well as families from other ethnic groups. In 3 C52F patients, we found a defect in the activation-induced shedding of the p55 (but not p75) TNF receptor, possibly leading to impaired homeostasis in the inflammatory response. During the 4 years leading to the present reporting period, we have identified new TRAPS mutations, studied genotype-phenotype correlations, and established p55 receptor shedding defects for most, but not all, mutations tested. We also conducted an open-label, dose-escalation study of the TNFR p75:Fc fusion protein, etanercept, in 15 TRAPS patients. There were highly significant dose-dependent improvements in a TRAPS attack score while on etanercept. Finally, we have developed TRAPS knockin mice for further mechanistic studies. During the immediately antecedent reporting period, we also identified de novo dominant mutations in a second gene, CIAS1 (also known as NALP3 or PYPAF1), that cause a distinct disorder known as neonatal onset multisystem inflammatory disease (NOMID) or chronic infantile neurologic cutaneous and arthropathy (CINCA) syndrome. Consistent with the recently discovered role of cryopyrin, the protein product of CIAS1, in IL-1 regulation, we found evidence for increased monocyte IL-1beta in a mutation-positive patient compared with normal controls. Results of the Last Year Clinical observations on TRAPS: During the last year we analyzed the biochemical markers of inflammation for the TRAPS-etanercept protocol, which had closed shortly before the beginning of the present reporting period. We measured the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and serum amyloid A (SAA) during 3-month baseline, low dose (2x/wk) and high dose (3x/wk) etanercept, and washout periods, both between and during any TRAPS attacks that the patients experienced. In general, etanercept was associated with significant reductions in all 3 acute phase reactants during symptomatic and asymptomatic periods. CRPs remained above the upper limit of normal even during the asymptomatic periods with both low- and high-dose etanercept administration. There were significant increases in acute phase reactants following discontinuation of etanercept. TRAPS knockin mice: Using standard techniques of homologous recombination in embryonic stem cells, followed by in vitro selection and blastocyst injection, we have developed two different types of Tnfrsf1a knockin mice. The first has the substitution of methionine for threonine at residue 50 (T50M), and represents one of the more common and severe mutations associated with human TRAPS. The second has the substitution of tyrosine for cysteine at residue 33 (C33Y), which is the mutation in the original Irish-Scottish "familial Hibernian fever" family. Homozygous animals for T50M (derived from 2 different ES clones) and C33Y (1 clone) have been generated. These strains have subsequently been crossed with a Cre recombinase strain, permitting the excision of the neomycin resistance cassette flanked by lox p sites. These lines are currently being backcrossed onto the C57BL6 and 129Sv backgrounds. On the mixed C57BL6/129 background, we have confirmed the respective knockin mutations by DNA sequencing. RT-PCR from both T50M and C33Y mice demonstrated Tnfrsf1a transcripts of the appropriate size. Immunoprecipitation and Western blotting with anti-mouse Tnfrsf1a showed intact 55 kDa receptor in leukocytes from homozygous T50M and C33Y mice. More extensive phenotypic and functional studies have been carried out on the T50M mice. By flow cytometry, T50M homozygotes showed detectable, but reduced, levels of leukocyte cell surface Tnfrsf1a. T50M heterozygotes and homozygoes did not spontaneously develop fevers. Homozygotes showed resistance to the lethal effects of bacterial lipopolysaccharide (LPS) coinjected with D-galactosamine, similar to that seen with Tnfrsf1a knockout mice. The T50M knockin mice were also resistant to the thermal effects of low-dose LPS injection. In addition, homozygous T50M mice showed detectable but diminished levels of soluble serum Tnfrsf1a following low-dose LPS injection, with heterozygous littermates exhibiting an intermediate phenotype. These data have led us to hypothesize that the mutant T50M Tnfrsf1a receptor may not reach the cell surface as readily as wild-type receptor, and that, either because of this or as an additional defect, there is impaired TNF signaling through this receptor. Genetic studies in NOMID: We have analyzed an additional 8 patients with clinical NOMID for CIAS1 mutations. We found mutations in only 4, bringing our total to 10 mutation-positive patients out of 21, further strengthening the case for genetic heterogeneity. Two of the new mutations, F523C and G326E, are novel. Extending our previous finding of increased IL-1beta production in a patient with the D303N CIAS1 mutation, we found increased levels of IL-1beta precursor regardless of whether a CIAS1 mutation was present. Given the existence of 13 other human proteins with a domain structure similar to cryopyrin (sometimes denoted NALP or PYPAF proteins), we screened our mutation-negative patients for mutations in several related genes with similar tissue expression profiles. These included NALP1/DEFCAP, NALP2/PYPAF2, NALP4/PYPAF4, NALP6/PYPAF5, NALP12/PYPAF7. We also screened ASC, which encodes a protein that interacts with cryopyrin to regulate apoptosis and proinflammatory caspases, and the gene encoding the IL-1 receptor antagonist. We have identified several missense substitutions in these molecules, and we are currently screening controls and evaluating the functional consequences of these variants. Conclusions and Significance Taken together, our data confirm a major role for abnormalities in cytokine signaling in the dominantly inherited periodic fevers. TNF and IL-1 are the 2 major pyrogenic cytokines in man, and we have now found pathogenic mutations in both signaling pathways. The clinical importance of this insight is emphasized by the success of the TNF-inhibitor etanercept in controlling the symptoms and acute-phase response in TRAPS, although the optimal regimen required to prevent systemic amyloidosis remains to be established. Somewhat paradoxically, laboratory studies of the T50M TRAPS knockin mouse suggest that TNFRSF1A mutations may actually lead to decreased signaling through the p55 receptor, perhaps indicating a role for increased signaling through the p75 TNF receptor in TRAPS. The finding of mutations in cryopyrin, a newly recognized IL-1 regulator, in NOMID raises the hope that IL-1 inhibition may have an impact in this condition similar to the clinical benefit of etanercept in TRAPS. During the next year, our objectives will be: 1) to continue mutational studies of genes in the TNF and IL-1 pathways in patients with uncharacterized inflammatory disorders; 2) to continue our physiologic studies of TRAPS knockin mice; 3) in collaboration with Dr. Goldbach-Mansky, to conduct therapeutic studies of anakinra, the IL-1 receptor antagonist, in NOMID; and 4) to study gene expression profiles of patients with TRAPS and NOMID during and between attacks.

背景 1999年，我们的实验室领导了一个国际联盟，报道了TNFRSF1A的突变，该基因编码了55 kDa肿瘤坏死因子（TNF）受体，引起了发烧和炎症的主要遗传综合征，并提出了“ TNF受体搭配的周期性综合征”的名称（TRAPS）（TRAPP）（TRAPP）。我们在TNFRSF1A蛋白的细胞外结构域中证明了6个突变，其中5种是半胱氨酸残基的错义取代，导致破坏高度保守的二硫键键。这7个原始家庭包括爱尔兰 - 苏格兰家庭的原型“家族性冬眠”，以及其他种族的家庭。在3例C52F患者中，我们发现p55（而不是p75）TNF受体的激活诱导的脱落缺陷，这可能导致炎症反应中的稳态受损。 在导致当前报告期的4年中，我们已经确定了新的陷阱突变，研究了基因型 - 表型相关性，并确定了大多数（但不是全部）测试的大多数（但不是全部）的P55受体脱落缺陷。我们还对15例陷阱患者进行了TNFR P​​75：FC融合蛋白Etanercept的开放标签，剂量降低研究。在Etanercept上，陷阱攻击评分的剂量依赖性依赖性改善。最后，我们开发了陷阱蛋白小鼠以进行进一步的机械研究。 在立即的先进报告期间，我们还确定了第二个基因CIAS1（也称为NALP3或PYPAF1）中的从头显着突变，该突变引起了一种独特的疾病，称为新生儿发作多系统炎症性疾病（NOMID）或慢性婴儿神经学性皮肤病和促进性皮肤病（Cinca）（Cinca）（Cinca）。与在IL-1调节中的Cryopyrin最近发现的Cryopyrin的作用一致，我们发现与正常对照组相比，突变阳性患者中单核细胞IL-1BETA增加的证据。 去年的结果 陷阱上的临床观察结果：在去年，我们分析了陷阱 - 伊蒂普方案的炎症的生化标记，该标志在本报告期开始之前不久就关闭了。我们在3个月的基线期间测量了红细胞沉积速率（ESR），C反应蛋白（CRP）和血清淀粉样蛋白A（SAA），低剂量（2x/wk）和高剂量（3x/wk）etanercept，以及在任何陷阱的患者之间，以及在任何陷阱的患者之间和过程中都有经验丰富的患者。通常，在有症状的和无症状的时期，Etanercept与所有3个急性相应物的大幅降低有关。即使在无症状和高剂量依然酸酯给药的无症状时期，CRP仍保持在正常的上限之上。依然切齿中停止急性相应物的急性相应物显着增加。 陷阱敲击蛋白小鼠：使用胚胎干细胞中同源重组的标准技术，然后进行体外选择和胚泡注射，我们开发了两种不同类型的TNFRSF1A敲击蛋白小鼠。第一个在残基50（T50M）处取代蛋氨酸代表苏氨酸，代表与人陷阱相关的最常见和严重的突变之一。第二个是在残基33（C33Y）处取代酪氨酸代替半胱氨酸，这是原始的爱尔兰 - 苏格兰“家族性霍伯利亚热”家庭中的突变。 T50M（源自2种不同的ES克隆）和C33Y（1克隆）的纯合动物已产生。随后将这些菌株与CRE重组酶菌株交叉，允许切除新霉素的耐药性盒，侧面是Lox P位点。这些线路目前正在将其回到C57BL6和129SV背景上。在混合的C57BL6/129背景下，我们通过DNA测序证实了各自的敲除突变。来自T50M和C33Y小鼠的RT-PCR显示了适当尺寸的TNFRSF1A转录本。抗小鼠TNFRSF1A的免疫沉淀和蛋白质印迹显示出来自纯合T50M和C33Y小鼠的白细胞中的55 kDa受体。 在T50M小鼠上已经进行了更广泛的表型和功能研究。通过流式细胞仪，T50M纯合子表现出可检测但降低的白细胞细胞表面TNFRSF1A的水平。 T50M杂合子和纯合子并未自发发展发烧。纯合子表现出与D-半乳糖胺共同作用的细菌脂多糖（LPS）的致命作用的抗性，类似于TNFRSF1A敲除小鼠的致命作用。 T50M敲击蛋白小鼠也对低剂量LPS​​注射的热作用有抵抗力。此外，低剂量LPS​​注射后，纯合T50M小鼠显示出可检测但降低的可溶性血清TNFRSF1A，杂合的同窝仔表现出中间表型。这些数据使我们假设突变体T50M TNFRSF1A受体可能不会像野生型受体那样容易到达细胞表面，并且由于这种受体，或者是由于这种受体的额外缺陷，通过该受体会受损。 NOMID中的遗传研究：我们分析了另外8例CIAS1突变临床Nomid的患者。我们发现突变仅4个，使我们的总数达到21名突变阳性患者，进一步加强了遗传异质性的案例。新的两个突变F523C和G326E是新颖的。为了扩大D303N CIAS1突变患者IL-1BETA产生增加的发现，我们发现IL-1Beta前体的水平升高，无论是否存在CIAS1突变。鉴于存在类似于冷冻蛋白（有时为NALP或PYPAF蛋白）的域结构的13种其他人蛋白，我们在几种具有相似组织表达谱的几个相关基因中筛选了突变阴性患者的突变患者。其中包括NALP1/DEFCAP，NALP2/PYPAF2，NALP4/PYPAF4，NALP6/PYPAF5，NALP12/PYPAF7。我们还筛选了ASC，该ASC编码一种与冷冻蛋白相互作用以调节凋亡和促炎性胱天蛋白酶的蛋白质，以及编码IL-1受体拮抗剂的基因。我们已经确定了这些分子中的几个错义取代，目前正在筛选控制并评估这些变体的功能后果。 结论和意义 综上所述，我们的数据证实了主要遗传的周期性发烧中细胞因子信号传导异常的主要作用。 TNF和IL-1是MAN中的2个主要的热原细胞因子，我们现在在两个信号通路中都发现了致病性突变。尽管TNF抑制剂Etanercept的成功控制了陷阱中的症状和急性期反应，但这种见解的临床重要性是由预防全身性淀粉样变性的最佳方案来确定的。有点自相矛盾的是，对T50M陷阱的实验室研究表明，TNFRSF1A突变实际上可能导致通过P55受体的信号下降，这可能表明通过捕集圈中P75 TNF受体的信号增加起作用。新认识的IL-1调节剂中冷冻蛋白突变的发现提出了希望在这种情况下IL-1抑制作用可能会产生类似于Etanercept在陷阱中的临床益处的影响。 在明年，我们的目标将是：1）继续对未表征炎症性疾病的患者进行TNF和IL-1途径中基因的突变研究； 2）继续我们对陷阱蛋白小鼠的生理研究； 3）与Goldbach-Mansky博士合作，在Nomid中对IL-1受体拮抗剂Anakinra进行治疗研究； 4）研究在攻击期间和两次攻击期间和新虫的患者的基因表达谱。