Genetics, Pathophysiology, and Treatment of Recessive Autoinflammatory Diseases

隐性自身炎症性疾病的遗传学、病理生理学和治疗

• 批准号: 8948387
• 负责人: Daniel Kastner
• 金额: $ 101.82万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 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/8948387
• 关键词: Acute; Acute-Phase Proteins; Affect; Affinity; Amino Acids; Anemia; Animal Model; Animals; Arabs; Atrophic; Autoimmune Process; B-Lymphocytes; Biochemical; Biological Assay; Biology; Biopsy; Blood; Blood Platelets; Blood Vessels; Bone Marrow; Brain; Brain Ischemia; Brothers; Candidate Disease Gene; Caucasians; Caucasoid Race; Cell Line; Cell Lineage; Cell Nucleus; Cell physiology; Cells; Child; Chromosomes, Human, Pair 22; Chromosomes, Human, Pair 3; Clinical; Coculture Techniques; Cytoplasm; DNA; Data; Databases; Deaminase; Defect; Deoxyadenosines; Dermis; Development; Developmental Delay Disorders; Diagnosis; Disease; Drug Kinetics; Endothelial Cells; Enzymes; Event; Exanthema; Exons; Failure; Family; Fever; Fresh Frozen Plasmas; Functional disorder; Gene Frequency; Genes; Genetic; Genomics; Goals; Growth Factor; Hair Cells; Hepatosplenomegaly; Homologous Gene; Human; Hydrocephalus; Immunoglobulin M; Immunohistochemistry; Immunologic Deficiency Syndromes; Inflammation; Inflammation Process; Inflammatory; Inflammatory Infiltrate; Infusion procedures; Inherited; Inosine; Interferons; Interleukin-6; Intermittent Pyrexia; Intracranial Hemorrhages; Investigation; Ischemic Stroke; Journals; Knock-out; Laboratories; Labyrinth; Lead; Lesion; Leukocytes; Literature; Magnetic Resonance Imaging; Medicine; Missense Mutation; Mitochondria; Modeling; Monitor; Mus; Muscle hypotonia; Musculoskeletal; Mutation; Myelogenous; Myeloid Cells; National Heart, Lung, and Blood Institute; Natural History; Nature; Neutropenia; New England; Optic Nerve; Orthologous Gene; Paper; Parents; Pathologic Nystagmus; Patients; Peripheral; Phenotype; Polyarteritis Nodosa; Portal Hypertension; Protein Biosynthesis; Proteins; Publications; Publishing; Reaction; Recurrence; Relative (related person); Reporting; Role; Sampling; Sensorineural Hearing Loss; Serum; Severe Combined Immunodeficiency; Sister; Skin; Staining method; Stains; Stroke; Symptoms; Syndrome; T-Lymphocyte; TNF gene; Tail; Transfer RNA; Transfer RNA Aminoacylation; Transferase; Tumor Necrosis Factor-alpha; U937 Cells; United States National Institutes of Health; Untranslated Regions; Vascular Diseases; Vasculitis; Warfarin; Zebrafish; adenosine deaminase; adenosine deaminase deficiency; base; cytokine; early onset; exome; exome sequencing; gastrointestinal symptom; human TNF protein; improved; inhibitor/antagonist; knockin animal; knockout animal; lateral line; loss of function mutation; macrophage; monocyte; mutant; neutrophil; novel; null mutation; paralogous gene; protein function; prototype; screening; small hairpin RNA

During the current reporting period we have focused on 2 new diseases identified through whole-exome sequencing. We discovered the first, denoted the deficiency of adenosine deaminase 2 (DADA2), during the previous reporting period. During the past year we completed our initial characterization of DADA2 and published our findings in the New England Journal of Medicine. The second disorder, caused by mutations in TRNT1, was discovered in our laboratory during the last six months, and is now under active investigation. Both projects are summarized below. Deficiency of ADA2, a New Autoinflammatory Disease Beginning about 10 years ago, we saw a total of 5 patients at the NIH Clinical Center with intermittent fevers, recurrent lacunar strokes, elevated acute phase reactants, livedoid rash, hepatosplenomegaly, and hypogammaglobulinemia. In 4 patients skin biopsies of livedoid lesions showed an inflammatory infiltrate of neutrophils, macrophages, and T lymphocytes; in one patient necrotizing vasculitis was present in the deep dermis. Magnetic resonance imaging showed evidence for small vessel acute brain ischemia and old ischemic strokes in the deep nuclei of the brain. Several stroke events were hemorrhagic or underwent hemorrhagic transformation, although the interpretation is clouded by the concomitant use of anti-platelet agents and/or warfarin. Four patients had hepatosplenomegaly, and one had documented portal hypertension. IgM levels were consistently low in all 5. We performed whole-exome sequencing in the first 3 patients and their unaffected parents, filtering the data under both recessive and de novo dominant models. A single common candidate gene was identified only under the recessive model. We found compound heterozygous predicted deleterious mutations in CECR1 (chromosome 22), encoding adenosine deaminase type 2 (ADA2). Two of the patients shared the p.Tyr453Cys mutation; one patient had a 28 kb genomic deletion that included the 5 prime UTR and exon 1 of CECR1. Among the 3 patients we identified a total of 5 CECR1 mutations. We then sequenced CECR1 in the 2 remaining NIH patients and a patient from the UK with a similar phenotype. All 3 were compound heterozygous for CECR1 mutations, including 3 novel mutations. We subsequently performed CECR1 candidate gene sequencing on 3 Turkish patients, 2 brothers who carried the diagnosis of polyarteritis nodosum (PAN) and a third patient with necrotizing small vessel vasculitis. All 3 were homozygous for the p.Gly47Arg mutation. Among these 9 patients we identified 9 predicted deleterious ADA2 mutations, including 2 null mutations. Mutations in the related protein ADA1 cause severe combined immunodeficiency disease (SCID) because of the failure to catalyze the conversion of adenosine (and deoxyadenosine) to inosine (and deoxyinosine), and the consequent accumulation of toxic metabolites. ADA2 also catalyzes this reaction, but with a lower affinity for adenosine. Using enzyme assays that distinguish ADA1 from ADA2 activity, we found that patients had nearly absent ADA2 activity, but normal ADA1 activity, in the blood. Direct assays of adenosine/deoxyadenosine metabolites were negative. Assays of serum cytokines, peripheral T cell function, and peripheral B cells demonstrated modest defects in the B cell compartment. Although there is no clear CECR1 ortholog in the mouse, there are 2 paralogs in the zebrafish. Morpholino knockdown of one of the zebrafish paralogs (cecr1b) caused intracranial hemorrhages and neutropenia, phenotypes that were rescued by wild type but not mutant human CECR1. These data, in conjunction with data from the literature indicating that ADA2 may be the prototype for a family of adenosine deaminase-derived growth factors, prompted a closer examination of the role of ADA2 in endothelial and leukocyte development in patients. We found that ADA2 is not produced in human endothelial cells. From the literature it is known that ADA2 is expressed in myeloid cells. Immunohistochemistry of skin and brain biopsies from patients demonstrates both endothelial damage and activation; staining for IL-1beta, TNF-alpha, and iNOS indicates inflammation. Using shRNA to silence the expression of ADA2 in myeloid U937 cells, and studies of patient monocytes stimulated with defined growth factors, we found that ADA2 deficiency is associated with a skewing of monocytes to the more inflammatory M1 subpopulation. When shRNA-treated U937 cells, or untreated patient monocytes, were cocultured with endothelial cell layers, they induced damage, relative to control U937 cells or healthy control monocytes. Thus, loss-of-function mutations in CECR1 cause a spectrum of vascular and inflammatory phenotypes ranging from early-onset recurrent stroke to systemic vasculopathy and/or vasculitis. Together, zebrafish and patient data indicate that ADA2 deficiency may diminish endothelial integrity while polarizing macrophage/monocyte subsets towards pro-inflammatory cells, establishing a vicious circle of vasculopathy and inflammation. Since the publication of our initial paper, we have continued to monitor the natural history of this disorder, and have identified 8 additional patients. There are now a total of 15 documented missense CECR1 mutations and 1 genomic deletion. Based on encouraging data from Israeli PAN patients with CECR1 mutations, some of our patients are now receiving TNF inhibitors. We are also evaluating the pharmacokinetics of ADA2 blood levels in patients who have received infusions of fresh frozen plasma, we are beginning to screen other vasculitis patients for CECR1 mutations, and we are establishing zebrafish cecr1 knockout lines for mechanistic studies. TRNT1-Associated Periodic Fever Syndrome Through whole-exome sequencing and candidate gene screening, we identified 5 children from 4 unrelated families with unexplained autoinflammatory disease and shared mutations in one common gene. All patients carried missense recessive mutations in TRNT1 (tRNA nucleotidyl transferase, CCA-adding, 1), encoded on chromosome 3. Two affected sisters from a Saudi Arabian consanguineous family were homozygous for a p.His215Arg missense mutation, while the other 3 children were compound heterozygous for a missense mutation, p.Ile223Thr or p.Arg99Trp, and one shared mutation, p.Asp163Val. p.His215Arg was not found in any public database nor in 1061 Arab control DNA samples. Among the 3 Caucasian mutations, p.Arg99Trp was novel whereas p.Ile223Thr and p.Asp163Val were found at a very low allele frequency (<0.001) in the NHLBI exome database. All mutations affect highly conserved amino acid residues and are predicted to be damaging to protein function. The TRNT1 protein adds CCA to the 3 prime end of all transfer RNAs both in the cytoplasm and mitochondria, which is critical for tRNA aminoacylation, protein synthesis, and tRNA degradation. Given the essential nature of this enzyme, disease-associated mutations are likely to be hypomorphs. All patients had recurrent episodes of fever and gastrointestinal symptoms with multisystem features that included developmental delay, nystagmus, hypotonia, optic nerve atrophy, sensorineural hearing loss, dysmorphic features, musculoskeletal symptoms, and B cell immunodeficiency. Flow cytometric studies suggested a maturation defect of the B cell lineage in the bone marrow. Preliminary data from cytokine analyses in two patients have shown elevated levels of the proinflammatory cytokines interleukin 6 and type 1 interferon. Knockdown of the zebrafish TRNT1 homolog caused hydrocephaly, defects in tail development, anemia, and a reduction in the number of hair cells present in the lateral line, which functions similarly to the human inner ear. Current studies focus on the relationship between defective tRNA processing and inflammation.

在当前报告期内，我们重点关注通过全外显子组测序发现的两种新疾病。我们在上一个报告期间发现了第一个，即腺苷脱氨酶 2 (DADA2) 的缺乏。 去年，我们完成了 DADA2 的初步表征，并在《新英格兰医学杂志》上发表了我们的研究结果。第二种疾病是由 TRNT1 突变引起的，是我们实验室在过去六个月中发现的，目前正在积极研究中。这两个项目总结如下。 ADA2 缺乏症，一种新的自身炎症性疾病 从大约 10 年前开始，我们在 NIH 临床中心总共看到了 5 名患有间歇性发热、反复腔隙性中风、急性期反应物升高、青斑皮疹、肝脾肿大和低丙种球蛋白血症的患者。 4 名患者的青斑样病变皮肤活检显示中性粒细胞、巨噬细胞和 T 淋巴细胞炎症浸润；一名患者真皮深层出现坏死性血管炎。磁共振成像显示大脑深部核团存在小血管急性脑缺血和陈旧性缺血性中风的证据。一些中风事件是出血性的或经历了出血性转化，尽管抗血小板药物和/或华法林的同时使用使解释变得模糊。 4 名患者出现肝脾肿大，1 名患者出现门静脉高压。所有 5 种患者的 IgM 水平始终较低。 我们对前 3 名患者及其未受影响的父母进行了全外显子组测序，在隐性和从头显性模型下过滤数据。仅在隐性模型下才鉴定出单个共同候选基因。我们发现编码 2 型腺苷脱氨酶 (ADA2) 的 CECR1（22 号染色体）中存在预测的复合杂合有害突变。其中两名患者具有 p.Tyr453Cys 突变；一名患者存在 28 kb 基因组缺失，其中包括 CECR1 的 5 Prime UTR 和外显子 1。在这 3 名患者中，我们总共发现了 5 个 CECR1 突变。然后，我们对剩下的 2 名 NIH 患者和一名来自英国的具有相似表型的患者进行了 CECR1 测序。所有 3 例均为 CECR1 突变复合杂合子，其中包括 3 个新突变。随后，我们对 3 名土耳其患者、2 名诊断为结节性多动脉炎 (PAN) 的兄弟和另一名患有坏死性小血管炎的患者进行了 CECR1 候选基因测序。所有 3 个均为 p.Gly47Arg 突变纯合子。在这 9 名患者中，我们鉴定了 9 种预测的有害 ADA2 突变，其中包括 2 种无效突变。 相关蛋白 ADA1 的突变会导致严重的联合免疫缺陷病 (SCID)，因为无法催化腺苷（和脱氧腺苷）转化为肌苷（和脱氧肌苷），并随之积累有毒代谢物。 ADA2 也催化该反应，但对腺苷的亲和力较低。使用区分 ADA1 和 ADA2 活性的酶测定，我们发现患者血液中几乎没有 ADA2 活性，但 ADA1 活性正常。腺苷/脱氧腺苷代谢物的直接测定结果为阴性。血清细胞因子、外周 T 细胞功能和外周 B 细胞的检测显示 B 细胞区室存在适度缺陷。 虽然小鼠中没有明确的 CECR1 直系同源物，但斑马鱼中有 2 个旁系同源物。斑马鱼旁系同源物之一 (cecr1b) 的吗啡啉敲低会导致颅内出血和中性粒细胞减少症，这些表型可以被野生型而非突变型人类 CECR1 挽救。这些数据与表明 ADA2 可能是腺苷脱氨酶衍生生长因子家族原型的文献数据相结合，促使人们更仔细地研究 ADA2 在患者内皮细胞和白细胞发育中的作用。我们发现 ADA2 不是在人内皮细胞中产生的。从文献中已知ADA2在骨髓细胞中表达。患者皮肤和脑活检的免疫组织化学显示内皮损伤和激活； IL-1β、TNF-α 和 iNOS 染色表明存在炎症。使用 shRNA 沉默髓系 U937 细胞中 ADA2 的表达，并对用特定生长因子刺激的患者单核细胞进行研究，我们发现 ADA2 缺陷与单核细胞偏向炎症性更强的 M1 亚群相关。当 shRNA 处理的 U937 细胞或未经处理的患者单核细胞与内皮细胞层共培养时，相对于对照 U937 细胞或健康对照单核细胞，它们会引起损伤。 因此，CECR1 的功能丧失突变会导致一系列血管和炎症表型，从早发复发性中风到全身性血管病变和/或血管炎。斑马鱼和患者数据共同表明，ADA2 缺陷可能会降低内皮完整性，同时使巨噬细胞/单核细胞亚群向促炎细胞极化，从而形成血管病变和炎症的恶性循环。 自从我们的第一篇论文发表以来，我们继续监测这种疾病的自然病程，并确定了另外 8 名患者。目前总共记录了 15 个 CECR1 错义突变和 1 个基因组缺失。根据来自患有 CECR1 突变的以色列 PAN 患者的令人鼓舞的数据，我们的一些患者现在正在接受 TNF 抑制剂治疗。我们还在评估接受新鲜冷冻血浆输注的患者的 ADA2 血液水平的药代动力学，我们开始筛查其他血管炎患者的 CECR1 突变，并且我们正在建立斑马鱼 cecr1 敲除系用于机制研究。 TRNT1-相关周期性发热综合征 通过全外显子组测序和候选基因筛查，我们鉴定出来自 4 个不相关家庭的 5 名儿童患有不明原因的自身炎症性疾病，并且存在一个共同基因的突变。所有患者均携带编码在 3 号染色体上的 TRNT1（tRNA 核苷酸转移酶，CCA 添加，1）错义隐性突变。来自沙特阿拉伯近亲家庭的两名受影响的姐妹为 p.His215Arg 错义突变纯合子，而其他 3 名儿童为 p.Ile223Thr 错义突变复合杂合子 p.Arg99Trp 和一个共享突变 p.Asp163Val。 p.His215Arg 在任何公共数据库和 1061 个阿拉伯对照 DNA 样本中均未发现。在 3 个白种人突变中，p.Arg99Trp 是新突变，而 p.Ile223Thr 和 p.Asp163Val 在 NHLBI 外显子组数据库中的等位基因频率非常低 (<0.001)。所有突变都会影响高度保守的氨基酸残基，预计会损害蛋白质功能。 TRNT1 蛋白将 CCA 添加到细胞质和线粒体中所有转移 RNA 的 3 末端，这对于 tRNA 氨酰化、蛋白质合成和 tRNA 降解至关重要。考虑到这种酶的本质，与疾病相关的突变很可能是亚型。 所有患者均出现反复发作的发热和胃肠道症状，并伴有多系统特征，包括发育迟缓、眼球震颤、张力减退、视神经萎缩、感音神经性听力损失、畸形特征、肌肉骨骼症状和 B 细胞免疫缺陷。流式细胞术研究表明骨髓中 B 细胞谱系存在成熟缺陷。两名患者细胞因子分析的初步数据显示促炎细胞因子白细胞介素 6 和 1 型干扰素水平升高。斑马鱼 TRNT1 同源物的敲除会导致脑积水、尾部发育缺陷、贫血以及侧线毛细胞数量减少，侧线毛细胞的功能与人类内耳相似。 目前的研究重点是 tRNA 加工缺陷与炎症之间的关系。