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; 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.

我们目前的努力集中在采用NextGen技术来对152个基因进行序列，以针对所有FA和其他遗传性骨髓衰竭综合征（IBMFS）基因的整个长度（59）。 ADH/ALDH（27）和1-碳代谢（47）基因也被测序，因为它们编码参与醛的产生和代谢的酶，这是关键的内源性DNA交联药物。除了序列变体外，我们现在还能够检测缺失/重复，并从NextGen序列读取中确定它们的精确边界。为了鉴定大型基因组变化，包括表现出躯体镶嵌的患者的有丝分裂重组引起的异构切除术，我们采用了高密度（1M）SNP阵列。为了阐明异常剪接产生的cDNA产品的性质，我们使用PACBIO测序技术。我们还努力开发斑马鱼突变体，作为研究FA疾病过程的模型，尤其是造血疾病和癌症的易感性。 近年来，我们报道了FANCA中159例突变患者的分子诊断，该患者表明，除7个家庭外，所有家族都具有明显的突变，从而定义了FANCA患者的基因型异质性。我们还确定并表征了由2700年前的南亚人口特有的FANCL创始人突变。 FA-L组仅占全球足总人口的0.4％，但它是印度第三大常见的群体。我们还发表了两项研究，将遗传变异与范围患者的疾病呈递相关联：1）粉丝患者出现了温和疾病的情况，我们确定患者的结果是粉丝中大量内部重复性的结果，该重复是不稳定和恢复的，导致摩西式表达。 2）引起疾病的突变的表征及其对来自16个家族的19个X连接的Fancb患者对编码的RNA和蛋白质功能的影响，将严重程度与基因变异的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的造型性质。现在，我们找到了另一个实例，其中引起疾病​​的致病变异性质导致了温和的疾病。这涉及Fanca变体C.3624C> t的载体。预计该变体是同义词（P.S1208S），但会影响剪接导致致病性四个BP缺失（P.S1208ifs*38）。发现六名携带此变异的六名患者的血液学发作比一般的FA患者晚得多（中位年龄为22.5 vs 7年）。深层测序包含异常剪接事件的转录区域表明，大约6-10％的转录本载有规范的剪接产物，从而产生功能性蛋白质，从而解释了这些患者的温和表型。功能分析证实了这些患者细胞系中芬卡的残留功能。 类似于FA-A，FA-B和FA-L组，对FA-D2，FA-E和FA-F组的FA患者的研究正在进行中。通常，每个组的突变都是私有的，反映了等位基因异质性。但是，我们确实发现FA组（例如FANCF）的反复变体，其中我们确定了两个频率相对较高的变体，C.230_252DEL23和C.484_485DELCT，分别代表Fancf家族的33％和30％，并确定了C.230_252Del23的最新杂种。我们还发现了一种在17/26（65％）的FANCJ家族中存在的变体。 我们确定了来自30个家庭的32个人中体细胞镶嵌的证据。在五个和13个人中分别观察到了从头补偿性变体的出现或致病变体的逆转。 15个个体表现出了因有丝分裂重组而引起的FA基因的异构体。在两个家庭中，每个兄弟姐妹都通过不同的机制表现出镶嵌性。在每种情况下，异构切开术扩展到染色体末端，以两种不同的方式影响等位基因表达：六个病例在FA基因内表现出断点连接，在两个双重性的致病变异之间，导致通过WildType序列替换病原变异的病原变体； 9例表现出整个FA基因的异构疾病，导致一种变体的损失和第二种变体的重复。在整个基因的九个病例中，我们对5/8变体进行了功能分析，因为在两种情况下发生了一个变体，在每种情况下，重复的变体都表现出差态功能。异构疾病事件似乎都会影响血液学疾病发作时的年龄，但是结构内切开术结局的病例显示出更长的生存期，表明变异的丧失和随后的修饰细胞的扩张促进了对血液学疾病病程的积极影响。 我们还在努力开发斑马鱼突变体，作为研究FA疾病过程的模型，特别是造血疾病和癌症的易感性。我们在斑马鱼中产生了17个FA基因的敲除。我们证明了FAAP100（一种FA候选基因）的缺乏导致表型与斑马鱼中其他FA基因敲除一致。在斑马鱼突变体中，SLX4IP蛋白的表型也很明显，这表明SLX4IP也可能是FA候选基因。我们证明了斑马鱼突变体中的全细胞减少症和血栓形成缺陷，而Fanca或Fanco基因失活，类似于与FA相关的常层性贫血。我们还在努力探索FA基因突变体的肿瘤发展。由于FA基因纯合子敲除均未显示出肿瘤发育的存活率降低或迹象，因此我们将TP53敲除等位基因（7BP Indel）引入了Fanci或FANCP敲除线，以产生双重敲除（Fanci-TP53和FANCP-TP53）。随着鱼的年龄，我们评估了异常生长（或自然死亡）后肿瘤发育的病理。此外，我们检查了所有的鱼，它们达到7MPF。 TP53的相关损失导致肿瘤的发展。这些肿瘤的表征正在进行中。