FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

范可尼贫血：基因型-表型相关性

• 批准号: 10691101
• 负责人: settara chandrasekharappa
• 金额: $ 94.73万
• 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/10691101
• 关键词: Acute Myelocytic Leukemia; Affect; Age; Age of Onset; Alcohols; Aldehydes; Alleles; Anemia; Aplastic Anemia; Appearance; Asian population; Biological Assay; Bone marrow failure; Breast Cancer Risk Factor; Candidate Disease Gene; Carbon; Cell Line; Cells; Cessation of life; Chromosomes; Clinical; Clinical Management; Clinical Treatment; Complement; Complementary DNA; Congenital Abnormality; DNA; DNA Crosslinking Agent; Defect; Development; Diagnosis; Disease; Dysmyelopoietic Syndromes; Enzymes; Evaluation; Event; Exhibits; FANCB; Family; Family member; Fanconi Anemia Complementation Group A Protein; Fanconi' s Anemia; Fishes; Frequencies; Gene Mutation; Gene Targeting; Generations; Genes; Genetic Variation; Genomics; Genotype; Goals; Growth; Head and Neck Squamous Cell Carcinoma; Hematological Disease; Hematology; Hematopoietic; Heterogeneity; Heterozygote; India; Individual; Inherited; Knock-out; Lead; Length; Life; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Messenger RNA; Metabolism; Methodology; Mitotic Recombination; Modeling; Molecular Diagnosis; Mosaicism; Mutation; Nature; Pancytopenia; Pathogenicity; Patient-Focused Outcomes; Patients; Phenotype; Population; Predisposition; Privatization; Process; Proteins; Protocols documentation; Publishing; RNA; RNA Splicing; Rare Diseases; Reporting; Residual state; Risk; SNP array; Severities; Siblings; Solid Neoplasm; South Asian; TP53 gene; Techniques; Technology; Thrombosis; Tissues; Transcript; Tumor Pathology; United States National Institutes of Health; Universities; Variant; Zebrafish; acetaldehyde dehydrogenase; aged; aldehyde dehydrogenases; attenuation; bone marrow failure syndrome; cancer predisposition; cohort; comparative; deep sequencing; density; disease-causing mutation; founder mutation; genetic variant; insertion/deletion mutation; knockout gene; mutant; protein function; tumor

Our current efforts are focused on employing NextGen technologies to sequence 152 genes, targeting the entire length of all FA and other inherited bone marrow failure syndromes (IBMFS) genes (59). ADH/ALDH (27) and 1-Carbon metabolism (47) genes are also being sequenced as they encode enzymes involved in the generation and metabolism of aldehydes, which are key endogenous DNA crosslinking agents. 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. For identification of large-size genomic changes including isodisomy caused by mitotic recombination in patients displaying somatic mosaicism, we employ high-density (1M) SNP arrays. For elucidating the nature of cDNA products generated by aberrant splicing, we use PacBio sequencing technology. We are also pursuing efforts to develop zebrafish mutants as a model to study the FA disease process, particularly hematopoietic disease and cancer predisposition. In the recent years, we have reported the molecular diagnosis of 159 patients with mutations in FANCA which revealed that all but seven families harbored a distinct pair of mutations, thus defining the genotypic heterogeneity among FANCA patients. We also identified and characterized a FANCL founder mutation specific to the South Asian population that originated 2700 years ago. The FA-L group represents only 0.4% of the FA population worldwide but it is the third most common group in India. We have also published two studies associating genetic variation to disease presentation in FANCB patients: 1) The case of a FANCB patient that presented with a milder disease, and for which we determined that the patients outcome was a consequence of a large intragenic duplication in FANCB that was unstable and reverted, resulting in mosaic expression. 2) The characterization of disease-causing mutations and their effect on the encoded RNA and protein function for 19 X-linked FANCB patients from 16 families, associating the severity to the type of gene variant and the residual activity of mutant protein. We have reported the hypomorphic nature of a FANCA variant c.4199G>A/p.R1400H. We now find another instance where the nature of the disease-causing pathogenic variant resulted in milder disease. This involved carriers of the FANCA variant c.3624C>T. This variant is predicted to be synonymous (p.S1208S), but affects splicing leading to a pathogenic four bp deletion (p.S1208Ifs*38). The hematologic onset in six patients carrying this variant was found to be much later than FA patients in general (median age of 22.5 vs seven years). Deep sequencing the transcript region containing the aberrant splicing event revealed that about 6-10% of transcripts carried the canonical splice product, resulting in functional protein, thus explaining the milder phenotype of these patients. Functional assays confirmed residual function of FANCA in cell lines from these patients. Similar to the FA-A, FA-B, and FA-L groups, studies on FA patients from FA-D2, FA-E, and FA-F groups are now underway. In general, mutations in each group are private, reflecting allelic heterogeneity. However, we do find recurring variants in FA groups, such as FANCF, in which we identified two variants with comparatively increased frequency, c.230_252del23 and c.484_485delCT, representing 33% and 30% of FANCF families, respectively, and identified c.230_252del23 as a founder variant of recent origin. We also find a variant that is present in 17/26 (65%) FANCJ families. We identified evidence of somatic mosaicism in 32 individuals from 30 families. The advent of a de novo compensatory variant or the reversal of a pathogenic variant was observed in five and 13 individuals, respectively. Isodisomy of an FA gene resulting from mitotic recombination was exhibited by 15 individuals. In two families there were siblings that each exhibited mosaicism via different mechanisms. The isodisomy extended to the chromosome terminus in each case, influencing the allelic expression in two different ways: six cases exhibited the breakpoint junction within the FA gene, between two biallelic pathogenic variants, resulting in the replacement of the pathogenic variant by a wildtype sequence; nine cases exhibited isodisomy of the entire FA gene, resulting in a loss of one variant and duplication of the second. Of the nine cases with isodisomy of the entire gene we performed functional analysis on 5/8 variants, as one variant occurred in two cases, and in each case the duplicated variant exhibited hypomorphic function. Neither isodisomy event appear to influence the age at onset of hematologic disease, but the cases with intragenic isodisomy junction showed longer survival, indicating that the loss of variant and subsequent expansion of modified cells promoted a positive effect on the hematologic disease course. We are also pursuing efforts to develop zebrafish mutants as a model to study the FA disease process, particularly, hematopoietic disease and cancer predisposition. We have generated and characterized knockouts of 17 FA genes in zebrafish. We demonstrated that deficiency of faap100, an FA-candidate gene, results in phenotypes consistent with other FA gene knockouts in zebrafish. Similar phenotypes were apparent in zebrafish mutants for slx4ip protein, suggesting that SLX4IP could also be an FA-candidate gene. We demonstrated pancytopenia and thrombosis defects in zebrafish mutants with inactivation of the FANCA or FANCO gene, resembling aplastic anemia associated with FA. We also are making an effort to explore tumor development in FA gene mutants. As none of the FA gene homozygous knockouts showed reduced survival or indications of tumor development, we introduced TP53 knockout allele (7bp indel) in to FANCI or FANCP knockout lines to generate double knockouts (FANCI-TP53 and FANCP-TP53). As the fish aged, we evaluated the pathology of the tumor development after the appearance of abnormal growth (or natural death). In addition, we checked all the fish as they reached 7mpf. An associated loss of TP53 led to the development of tumors. Characterization of these tumors is underway.

我们目前的工作重点是利用 NextGen 技术对 152 个基因进行测序，针对所有 FA 和其他遗传性骨髓衰竭综合征 (IBMFS) 基因的全长 (59)。 ADH/ALDH (27) 和 1-碳代谢 (47) 基因也正在进行测序，因为它们编码参与醛类生成和代谢的酶，醛类是关键的内源性 DNA 交联剂。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。为了识别大尺寸基因组变化，包括表现出体细胞嵌合体的患者中由有丝分裂重组引起的等二体性，我们采用高密度 (1M) SNP 阵列。为了阐明异常剪接产生的 cDNA 产物的性质，我们使用 PacBio 测序技术。我们还致力于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。 近年来，我们报道了 159 例 FANCA 突变患者的分子诊断，结果显示，除了 7 个家族外，所有家族都携带一对不同的突变，从而确定了 FANCA 患者的基因型异质性。我们还鉴定并鉴定了起源于 2700 年前的南亚人群特有的 FANCL 创始人突变。 FA-L 群体仅占全球 FA 人口的 0.4%，但在印度却是第三大常见群体。我们还发表了两项将 FANCB 患者的遗传变异与疾病表现相关联的研究：1) 一名 FANCB 患者的病例表现出较轻的疾病，我们确定该患者的结果是 FANCB 中大量基因内重复的结果，该重复不稳定并可恢复，从而导致嵌合表达。 2) 来自16个家族的19名X连锁FANCB患者的致病突变特征及其对编码RNA和蛋白质功能的影响，将严重程度与基因变异类型和突变蛋白的残留活性相关联。 我们报道了 FANCA 变体 c.4199G>A/p.R1400H 的亚等位性。我们现在发现了另一个例子，其中致病性变异的性质导致了较轻的疾病。这涉及 FANCA 变体 c.3624C>T 的携带者。该变体预计是同义的 (p.S1208S)，但会影响剪接，导致致病性 4 bp 缺失 (p.S1208Ifs*38)。研究发现，6 名携带该变异的患者的血液学发病时间比一般 FA 患者要晚得多（中位年龄为 22.5 岁，中位年龄为 7 岁）。对包含异常剪接事件的转录物区域进行深度测序发现，约 6-10% 的转录物携带规范剪接产物，产生功能性蛋白质，从而解释了这些患者的较轻表型。功能测定证实了 FANCA 在这些患者细胞系中的残留功能。 与 FA-A、FA-B 和 FA-L 组类似，针对 FA-D2、FA-E 和 FA-F 组 FA 患者的研究目前正在进行中。一般来说，每个群体的突变都是私人的，反映了等位基因的异质性。然而，我们确实在 FA 组中发现了重复出现的变体，例如 FANCF，其中我们识别了频率相对较高的两个变体 c.230_252del23 和 c.484_485delCT，分别代表 FANCF 家族的 33% 和 30%，并将 c.230_252del23 确定为最近起源的创始人变体。我们还发现 17/26 (65%) FANCJ 家族中存在一种变异。 我们在 30 个家庭的 32 名个体中发现了体细胞嵌合的证据。分别在 5 名和 13 名个体中观察到新的补偿性变异的出现或致病性变异的逆转。 15 个个体表现出由有丝分裂重组产生的 FA 基因的同二体性。在两个家庭中，都有兄弟姐妹通过不同的机制表现出嵌合体。在每个病例中，等二体性延伸至染色体末端，以两种不同的方式影响等位基因表达：6个病例在两个双等位基因致病变异之间的FA基因内表现出断点连接，导致致病变异被野生型序列取代；九个病例表现出整个 FA 基因的二倍体，导致一种变异的丢失和另一种变异的重复。在整个基因同二体的九个病例中，我们对 5/8 个变体进行了功能分析，因为两个病例中出现了一个变体，并且在每种情况下重复的变体都表现出亚等位功能。两种二倍体事件似乎都不影响血液疾病的发病年龄，但具有基因内二倍体连接的病例显示出更长的生存期，表明变异的丢失和随后修饰细胞的扩增促进了对血液病病程的积极影响。 我们还致力于开发斑马鱼突变体作为模型来研究 FA 疾病过程，特别是造血系统疾病和癌症易感性。我们已经在斑马鱼中生成并敲除 17 个 FA 基因。我们证明，FA 候选基因 faap100 的缺陷会导致斑马鱼中与其他 FA 基因敲除一致的表型。类似的表型在 slx4ip 蛋白的斑马鱼突变体中也很明显，这表明 SLX4IP 也可能是 FA 候选基因。我们证明了 FANCA 或 FANCO 基因失活的斑马鱼突变体存在全血细胞减少症和血栓形成缺陷，类似于与 FA 相关的再生障碍性贫血。我们还致力于探索 FA 基因突变体的肿瘤发展。由于 FA 基因纯合敲除均未显示存活率降低或肿瘤发展迹象，因此我们将 TP53 敲除等位基因（7bp 插入缺失）引入 FANCI 或 FANCP 敲除系中，以产生双敲除（FANCI-TP53 和 FANCP-TP53）。随着鱼的衰老，我们在出现异常生长（或自然死亡）后评估肿瘤发展的病理学。此外，我们检查了所有达到 7mpf 的鱼。 TP53 的相关缺失导致肿瘤的发生。这些肿瘤的表征正在进行中。