FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

范可尼贫血：基因型-表型相关性

• 批准号: 10267077
• 负责人: settara chandrasekharappa
• 金额: $ 86.81万
• 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/10267077
• 关键词: Accounting; Affect; Age; Aldehydes; Alleles; Amino Acids; Anemia; Asia; Back; Blood; Blood Cell Count; Blood Platelets; Breast Cancer Risk Factor; Cell Count; Characteristics; Clinical Management; Clinical Treatment; Codon Nucleotides; Collaborations; Complement; Complex; Congenital Abnormality; DNA; DNA Crosslinking Agent; DNA Interstrand Crosslinking; DNA Repair; DNA Repair Pathway; Data; Development; Diagnosis; Diamond-Blackfan anemia; Disease; Dyskeratosis Congenita; Embryo; Enrollment; Enzymes; Erythrocytes; Evaluation; Exhibits; Exons; FANCB; FANCB gene; FANCG gene; Family; Family member; Family-Based Registry; Fanconi Anemia Complementation Group A Protein; Fanconi' s Anemia; Female; Fishes; Gene Mutation; General Population; Generations; Genes; Genetic; Genetic Heterogeneity; Genome; Genomics; Genotype; Goals; Haplotypes; Head and Neck Squamous Cell Carcinoma; Hematological Disease; Hematopoietic; Heterozygote; Human; India; Individual; Inherited; International; Journals; Knock-out; Larva; Lasers; Length; Leukocytes; Life; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Manuscripts; Measures; Metabolism; Methodology; Modeling; Molecular Diagnosis; Monoubiquitination; Mosaicism; Mutation; Myeloid Leukemia; Outcome; Pancytopenia; Pathogenicity; Patients; Phenotype; Predisposition; Process; Proteins; Protocols documentation; Publishing; RNA; RNA Splicing; RNA analysis; Registries; Reporting; Residual state; Role; Series; Severities; TP53 gene; Techniques; Technology; Thrombosis; Time; Tissues; Ubiquitination; United States National Institutes of Health; Universities; Variant; Venous; Zebrafish; aldehyde dehydrogenases; bone marrow failure syndrome; causal variant; cohort; early onset; erythritol anhydride; flexibility; founder mutation; genome database; insertion/deletion mutation; knockout gene; male; mutant; mutation screening; next generation sequencing; patient population; screening; screening panel; sex; tumor

Once diagnosed with Fanconi anemia (FA), identification of the causative gene and the mutations is an arduous task. FA genes are large, with multiple exons, and harbor a wide spectrum of compound heterozygous mutations spread throughout the gene including large genomic deletions. More FA genes are being identified requiring the screening methodologies to be flexible to accommodate newly identified FA genes. Thus, molecular diagnosis of a large number of families enrolled in the International Fanconi Anemia Registry (IFAR) remained unknown. Though FA patients can carry mutations in any of the 22 known genes, FANCA (64%), FANCC (12%), and FANCG (8%) are the three most commonly observed disease-causing genes among FA patients, accounting for 84% of cases worldwide. Our current efforts are focused on employing nextgen sequencing technologies to sequence a large (23 Mb) region of the genome, targeting the entire length of all FA and FA-related DNA-repair pathway genes. The genes being sequenced, also include those encoding ALDH and ADH and other enzymes involved in the generation and metabolism of aldehydes, which are key endogenous DNA crosslinking agents. A collaborative study initiated with our colleagues at NCI/NIH, now extends our disease-causing gene/mutation screening efforts to other inherited bone marrow failure syndromes (IBMFS), dyskeratosis congenita (DC) and Diamond-Blackfan anemia (DBA), in addition to FA. Thus, our mutation screening panel now includes 150 genes. In addition to sequence variants, we are now able to detect deletions/duplications and determine their precise boundaries from the nextgen sequence reads, as well. So far, through our collaboration with the Rockefeller University, we have identified biallelic mutations in 572 IFAR families diagnosed with FA and now extending similar efforts towards 100 individuals enrolled at NCI/NIH as a part of IBMFS studies. A few highlights from this and other efforts are described below. We characterized both mutations in 159 FANCA patients and (published in Human Mutation, 2018). The genetic heterogeneity was apparent from the observation that all but seven families harbored distinct combinations of two mutations. We found that certain mutations predicted to encode missense changes are, indeed, causing aberrant splicing, instead. We are now finding that genetic heterogeneity, a wide spectrum of pathogenic variants including large deletions, missense, nonsense, indel and splice aberrations, is prevalent across other FA genes as well, and underscores the need for RNA analysis to be an integral part of the molecular diagnosis of FA. We also observed that deletions extending beyond the 5 end and codon 1 missense variants in FANCA are, indeed, null alleles as they do not express the encoded RNA or protein, respectively. FA due to X-linked FANCB mutations are rare and account for only 2% of all FA patients. We characterized 19 patients from 16 families with FANCB mutations. No two patients carried the same FANCB mutation. Those with FANCB deletion or truncation demonstrated earlier than average onset of bone marrow failure, and more severe congenital abnormalities compared to a large series of FA individuals in the published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity was associated with the extent of residual FANCD2 monoubiquitination activity. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination showed earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our report revealing a genotype-phenotype correlation within FA-B complementation group of FA is now published in the journal, Blood. Unlike FA-A and FA-B, groups with high genetic heterogeneity, we found a founder mutation in a very rare group, FAL, representing only 0.2% of the patient population. The causative variant, predicted to be synonymous, K364K, however, causes aberrant splicing eliminating the exon 13 carrying the variant and thus removing 24 amino acids from the encoded protein. This variant caused FA in 12 patients from India and the carriers share a common haplotype from an ancestral allele that dates back 2700 years. The public genome databases reveal the variant to be isolated to South Asia. A manuscript describing these findings is now published in the journal Human Mutation. Similar to the FA-A, FA-B, and FA-L groups, studies on FA patients from FA-D2, FA-E, and FA-J groups are now underway. We have generated and characterized knockouts of 17 FA genes in zebrafish (published in PLOS Genetics 2019). Recently, we characterized the consequences of deficiency of two FA-associated proteins, faap100 and faap24, in zebrafish. Both proteins are components of the FA core complex that facilitates ubiquitination of FA-I and FA-D2 proteins. To date, mutations in these genes have not been reported in FA patients. The faap100 nulls exhibited complete conversion of female-to-male sex-reversal, a characteristic of all FA gene mutants, whereas the faap24 mutants did not. The sensitivity of the embryos for DNA crosslinking agent, diepoxybutane (DEB), was very much apparent for faap100 but not for the faap24. Overall, our data show that deficiency of faap100 results in phenotypes consistent with other FA gene knockouts in zebrafish. On the contrary, deficiency of faap24 did not show similar phenotypes. We are pursuing efforts to develop zebrafish mutants as a model to study the FA disease process, particularly, hematopoietic disease and cancer predisposition. We evaluated differential blood cell counts for two mutant lines, fanca and fanco: we find that, as they age, the nulls exhibit decreasing cell counts for all lineages (thrombocytes, red blood cells, and white blood cells). The number and function of thrombocytes were evaluated by measuring time to occlusion (TTO), either venous or arterial laser thrombosis on 5 dpf larvae that were treated with DEB. The treated larvae showed prolongation of TTO compared to untreated larvae indicating inefficient thrombosis in mutants. Similarly, we have generated double knockouts (an FA gene along with TP53) and we are characterizing the tumors that develop in these fish.

一旦诊断出范可尼贫血（FA），识别致病基因和突变是一项艰巨的任务。 FA 基因很大，具有多个外显子，并且具有广泛的复合杂合突变，分布在整个基因中，包括大量基因组缺失。更多 FA 基因正在被鉴定，这就要求筛选方法能够灵活地适应新鉴定的 FA 基因。因此，国际范可尼贫血登记处（IFAR）登记的大量家庭的分子诊断仍然未知。尽管 FA 患者可携带 22 种已知基因中任何一种的突变，但 FANCA (64%)、FANCC (12%) 和 FANCG (8%) 是 FA 患者中最常见的三种致病基因，占全球病例的 84%。 我们目前的工作重点是采用下一代测序技术对基因组的大区域 (23 Mb) 进行测序，针对所有 FA 和 FA 相关 DNA 修复途径基因的整个长度。正在测序的基因还包括编码 ALDH 和 ADH 以及参与醛类生成和代谢的其他酶的基因，醛类是关键的内源性 DNA 交联剂。我们与 NCI/NIH 的同事发起的一项合作研究，现在将我们的致病基因/突变筛查工作扩展到除 FA 之外的其他遗传性骨髓衰竭综合征 (IBMFS)、先天性角化不良 (DC) 和 Diamond-Blackfan 贫血 (DBA)。因此，我们的突变筛选小组现在包括 150 个基因。除了序列变异之外，我们现在还能够检测缺失/重复，并从下一代序列读取中确定它们的精确边界。到目前为止，通过与洛克菲勒大学的合作，我们已经在 572 个诊断为 FA 的 IFAR 家族中发现了双等位基因突变，并且现在将类似的工作扩展到 NCI/NIH 登记的 100 名个人，作为 IBMFS 研究的一部分。下面描述了这项工作和其他工作的一些亮点。 我们对 159 名 FANCA 患者的这两种突变进行了表征（发表在《Human Mutation》，2018 年）。通过观察发现，除了七个家族之外，所有家族都含有两种突变的不同组合，遗传异质性是显而易见的。我们发现，某些预测编码错义变化的突变实际上会导致异常剪接。我们现在发现，遗传异质性，即广泛的致病变异，包括大缺失、错义、无义、插入缺失和剪接畸变，在其他 FA 基因中也很普遍，这强调了 RNA 分析需要成为 FA 分子诊断的一个组成部分。 我们还观察到，FANCA 中超出 5 末端和密码子 1 错义变体的缺失实际上是无效等位基因，因为它们分别不表达编码的 RNA 或蛋白质。 由 X 连锁 FANCB 突变引起的 FA 很罕见，仅占所有 FA 患者的 2%。我们 对来自 16 个家族的 19 名患有 FANCB 突变的患者进行了特征分析。没有两名患者携带相同的 FANCB 突变。与已发表报告中的大量 FA 个体相比，FANCB 缺失或截短的患者出现骨髓衰竭的时间早于平均水平，且先天性异常更为严重。这反映了 FANCB 蛋白在 FANCD2 单泛素化酶激活中不可或缺的作用，这是修复 DNA 链间交联的重要步骤。对于 FANCB 错义变体，更多不同的严重程度与残余 FANCD2 单泛素化活性的程度相关。携带 FANCD2 单泛素化显着降低的错义变异的个体表现出较早的血液疾病发病和较短的生存期。相反，FANCD2 单泛素化接近正常的变异与更有利的结果相关。我们的报告揭示了 FA 的 FA-B 互补组内基因型-表型相关性，现已发表在《Blood》杂志上。 与具有高度遗传异质性的 FA-A 和 FA-B 群体不同，我们在 非常罕见的群体，FAL，仅占患者群体的 0.2%。致病变异， 预计 K364K 是同义的，然而，它会导致异常剪接，消除外显子 13 携带变体，从而从编码的蛋白质中去除 24 个氨基酸。这个变体 导致 12 名印度患者出现 FA，携带者拥有来自祖先的共同单倍型 等位基因的历史可以追溯到 2700 年前。公共基因组数据库显示该变种是在南亚分离出来的。描述这些发现的手稿现已发表在《人类突变》杂志上。与 FA-A、FA-B 和 FA-L 组类似，针对 FA-D2、FA-E 和 FA-J 组 FA 患者的研究目前正在进行中。 我们已经在斑马鱼中生成并表征了 17 个 FA 基因的敲除（发表于 PLOS Genetics 2019）。最近，我们描述了斑马鱼中两种 FA 相关蛋白 faap100 和 faap24 缺乏的后果。这两种蛋白都是 FA 核心复合物的组成部分，促进 FA-I 和 FA-D2 蛋白的泛素化。迄今为止，尚未报道 FA 患者中这些基因发生突变。 faap100 nulls 表现出雌性向雄性性逆转的完全转变，这是所有 FA 基因突变体的特征，而 faap24 突变体则没有。胚胎对 DNA 交联剂二环氧丁烷 (DEB) 的敏感性对于 faap100 非常明显，但对于 faap24 则不然。总体而言，我们的数据表明 faap100 缺陷导致斑马鱼的表型与其他 FA 基因敲除一致。相反，faap24缺陷则没有表现出类似的表型。 我们正在努力开发斑马鱼突变体作为研究 FA 疾病过程的模型，特别是造血系统疾病和癌症易感性。我们评估了 Fanca 和 fanco 这两个突变系的血细胞计数差异：我们发现，随着年龄的增长，空细胞显示出所有谱系（血小板、红细胞和白细胞）的细胞计数减少。通过测量用 DEB 处理的 5 dpf 幼虫上静脉或动脉激光血栓形成的闭塞时间 (TTO) 来评估血小板的数量和功能。与未处理的幼虫相比，经过处理的幼虫表现出 TTO 延长，表明突变体中血栓形成效率低下。同样，我们已经产生了双敲除（FA 基因和 TP53），并且我们正在表征这些鱼中形成的肿瘤。