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，目标是所有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）基因也在测序中，因为它们编码醛的产生和代谢酶，醛是关键的内源性DNA交联剂。除了序列变异外，我们现在还能够检测到缺失/重复，并从下一代序列读取中确定它们的精确边界。为了鉴定显示体细胞嵌合体的患者有丝分裂重组引起的大尺寸基因组变化，包括同位体，我们采用高密度（1M） SNP阵列。为了阐明由异常剪接产生的cDNA产物的性质，我们使用PacBio测序技术。我们也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易感性。