FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

范可尼贫血：基因型-表型相关性

• 批准号: 9571134
• 负责人: settara chandrasekharappa
• 金额: $ 78.54万
• 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/9571134
• 关键词: Adopted; Affect; Age; Alleles; BRCA2 gene; Back; Blood Cells; Cell Fraction; Clinical Management; Clinical Treatment; Collaborations; Complement; Congenital Abnormality; Copy Number Polymorphism; DNA; DNA Repair Pathway; Diagnosis; Disease; Enrollment; Evaluation; Exons; FANCB gene; FANCG gene; Family; Family-Based Registry; Fanconi Anemia Complementation Group A Protein; Fanconi' s Anemia; Fibroblasts; Frequencies; Gene Mutation; General Population; Genes; Genome; Genomic DNA; Genomics; Genotype; Goals; Head and Neck Squamous Cell Carcinoma; Hematopoietic; Heterozygote; Hybridization Array; Inherited; International; Life; Link; Malignant neoplasm of pancreas; Manuscripts; Massive Parallel Sequencing; Methodology; Molecular Diagnosis; Mosaicism; Mutation; Myeloid Leukemia; Pancytopenia; Patients; Phenotype; Predisposition; Preparation; Promoter Regions; Proteins; Protocols documentation; Publications; Publishing; RNA Splicing; RNA analysis; Rare Diseases; Registries; Reporting; Risk; Role; SNP array; Siblings; Techniques; Technology; Testing; Time; Tissues; United States National Institutes of Health; Universities; Variant; Zebrafish; blood fractionation; cohort; comparative genomic hybridization; disease-causing mutation; genetic variant; insertion/deletion mutation; malignant breast neoplasm; null mutation; repaired

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. Thus, molecular diagnosis of a large number of families enrolled in the International Fanconi Anemia Registry (IFAR) remained unknown. Within the last two years, there were reports of five new FA genes being identified, thus bringing the number of known FA genes to 21. Though FA patients can carry mutations in any of the 21 known genes, about two-thirds are affected by mutations in FANCA. Our current efforts are focused on employing massively parallel sequencing technologies to sequence large (2-3 Mb) regions of the genome, targeting all FA and FA-related DNA-repair pathway genes. We also adopt Comparative Genome Hybridization arrays (aCGH), and SNP arrays to explore large-size copy number variants in a similar set of genes. So far, through our collaboration with the Rockefeller University, we have identified bi-allelic mutations in 500 IFAR families, and these include 313 FANCA, 16 FANCB, 54 FANCC, 15 FANCD1, 21 FANCD2, seven FANCE, 15 FANCF, 23 FANCG, 11 FANCI, 15 FANCJ, five FANCL, four FANCN, three FANCP and one FANCT. A few highlights from this effort include: 1) identification of 16 patients with an X-linked FANCB mutation, a very rare FA group, and in four patients, the mutation was de novo; 2) Identification of rare disease-associated deletions in 148 families including 131 FANCA, 8 FANCC, 5 FANCD2, one FANCJ, one FANCI, and 2 FANCB using aCGH; 3) determination of the precise breakpoints for 90 deletions, and discovered the mechanisms leading to deletions; 4) found that fifty-five FANCA deletions overlapped exon 1 and extended beyond the 5 gene terminus, eliminating a putative promoter region and, likely expression. This was confirmed by RNA analysis. A manuscript describing characterization of both mutations in 160 FANCA patients in under revision. For the past two years, as a part of our detailed molecular diagnosis, we have been exploring causes and consequences of mosaicism in FA families. It is estimated that 20% of FA patients may display somatic mosaicism, a scenario where a fraction of cells from hematopoietic lineages may have lost, or repaired, one of the inherited mutations. This phenomenon results in a functional allele in the fraction of blood cells with reverse mosaicism (RM), and may often provide protection from hematopoietic diseases. Somatic mosaicism in a patient is evident when his/her blood cells were subjected to DNA breakage test at the time of diagnosis. We completed our study of three siblings in a family with mutations in FANCG displaying RM, each displaying a different mechanism that resulted in RM, and a manuscript describing these findings is under preparation. We also completed another study where a FANCB patient displayed RM. The patient harbored a 10kb intragenic duplication in FANCB. This duplication was unstable and reverted back to wild type in patient cells from peripheral blood and also, to some extent, from fibroblasts. A manuscript describing the mosaicism displayed by the FANCB patient is under revision. In an effort to evaluate the frequency of FA gene variants in patients diagnosed with HNSCC under the age of 50, we sequenced genomic DNA of 492 patients for variations in all FA genes. The findings from this study were published this year. In addition, we have generated zebrafish carrying mutations in each of the 19 FA genes. The initial characterization of FA null mutations in zebrafish is now complete and a manuscript describing these efforts is being prepared for publication.

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。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。过去两年内，有报道称发现了 5 个新的 FA 基因，从而使已知 FA 基因的数量达到 21 个。尽管 FA 患者可能携带 21 个已知基因中任何一个的突变，但大约三分之二的患者受到 FANCA 突变的影响。我们目前的工作重点是采用大规模并行测序技术对基因组的大区域 (2-3 Mb) 进行测序，针对所有 FA 和 FA 相关的 DNA 修复途径基因。我们还采用比较基因组杂交阵列 (aCGH) 和 SNP 阵列来探索一组相似基因中的大尺寸拷贝数变异。 到目前为止，通过与洛克菲勒大学的合作，我们已经在500个IFAR家族中鉴定出双等位基因突变，其中包括313个FANCA、16个FANCB、54个FANCC、15个FANCD1、21个FANCD2、7个FANCE、15个FANCF、23个FANCG、11个FANCI、15个FANCJ、5个FANCL、4个FANCN、3个FANCP 和一个 FANCT。 这项工作的一些亮点包括：1) 鉴定出 16 名具有 X 连锁 FANCB 突变的患者，这是一个非常罕见的 FA 组，其中 4 名患者的突变是从头发生的； 2）使用aCGH鉴定148个家系中罕见疾病相关的缺失，包括131个FANCA、8个FANCC、5个FANCD2、1个FANCJ、1个FANCI和2个FANCB； 3）确定了90个缺失的精确断点，并发现了导致缺失的机制； 4) 发现 55 个 FANCA 缺失与外显子 1 重叠并延伸到 5 基因末端之外，消除了推定的启动子区域和可能的表达。 RNA分析证实了这一点。 一份描述 160 名 FANCA 患者两种突变特征的手稿正在修订中。 在过去的两年里，作为我们详细分子诊断的一部分，我们一直在探索 FA 家族嵌合体的原因和后果。据估计，20% 的 FA 患者可能表现出体细胞镶嵌现象，在这种情况下，造血谱系的一小部分细胞可能丢失或修复了一种遗传突变。这种现象会在具有反向嵌合体（RM）的血细胞部分中产生功能性等位基因，并且通常可以提供针对造血系统疾病的保护。当诊断时对患者的血细胞进行 DNA 断裂测试时，患者的体细胞嵌合现象很明显。我们完成了对一个家庭中三个兄弟姐妹的研究，该家庭中的三个兄弟姐妹患有显示 RM 的 FANCG 突变，每个兄弟姐妹都显示出导致 RM 的不同机制，描述这些发现的手稿正在准备中。 我们还完成了另一项研究，其中一名 FANCB 患者表现出 RM。该患者的 FANCB 中存在 10kb 的基因内重复。这种复制是不稳定的，并且在来自外周血以及在某种程度上来自成纤维细胞的患者细胞中恢复为野生型。一份描述 FANCB 患者表现出的嵌合现象的手稿正在修订中。 为了评估 50 岁以下诊断为 HNSCC 的患者中 FA 基因变异的频率，我们对 492 名患者的基因组 DNA 进行了测序，以了解所有 FA 基因的变异。这项研究的结果于今年发表。此外，我们还培育出了 19 个 FA 基因均携带突变的斑马鱼。斑马鱼 FA 无效突变的初步表征现已完成，描述这些工作的手稿正在准备出版。