FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

范可尼贫血：基因型-表型相关性

• 批准号: 8750654
• 负责人: elaine ostrander
• 金额: $ 76.67万
• 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/8750654
• 关键词: Affect; BRCA2 gene; Biological Assay; Cataloging; Catalogs; Clinical Management; Clinical Treatment; Cloning; Code; Congenital Abnormality; Custom; DNA; DNA Repair Pathway; DNA Sequence; Detection; Diagnosis; Dideoxy Chain Termination DNA Sequencing; Disease; Elements; Enrollment; Ensure; Evaluation; Exons; FANCG gene; Family; Family member; Fanconi' s Anemia; Future; Gene Mutation; General Population; Genes; Genome; Genomics; Genotype; Goals; Haplotypes; Head and Neck Cancer; Heterozygote; Hybridization Array; Individual; International; Introns; Length; Life; Malignant Neoplasms; Malignant neoplasm of pancreas; Mediating; Methodology; Methods; Molecular Diagnosis; Mutation; Myeloid Leukemia; Pancytopenia; Pathogenicity; Patients; Phenotype; Predisposition; Process; Proteins; Protocols documentation; RNA Splicing; Reading; Registries; Risk; Role; Sampling; Techniques; Technology; Transcript; Universities; Variant; cohort; comparative genomic hybridization; design; disease-causing mutation; genome sequencing; insertion/deletion mutation; malignant breast neoplasm; next generation sequencing; screening

Once diagnosed with Fanconi anemia (FA), identification of the causative gene and the mutations is an arduous task. The conventional screening process is a sequential, multi-step approach and, thus, is inefficient and expensive to perform. 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 nearly half of the 800 families enrolled in the International Fanconi Anemia Registry (IFAR) remained unknown. We employed the massively parallel sequencing technologies to sequence large (2Mb) regions of the genome representing all FA and related DNA-repair pathway genes. We designed Comparative Genome Hybridization arrays (aCGH) arrays to explore large-size copy number variants in the same set of genes. We also employed RNAseq technologies for determining the pathogenicity of unsuspecting variants resulting in aberrant splicing. The use of complementary technologies allowed for successful identification of mutations in FA genes in 43 individuals: FANCA (17), FANCB (4), FANCC (5), FANCD1 (1), FANCE (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (4) and FANCL (3). The strategy we employed was an effective approach to identify variations underlying a highly genetically heterogeneous disorder such as FA, and ensures a timely and efficient molecular diagnosis of future enrollees. Though FA patients can carry mutations in any of the 16 known genes, about two-thirds are affected by mutations in FANCA gene. Thus, for all FA individuals checking for FANCA mutations may serve as an efficient initial step. Earlier, we had used Sanger sequencing method to sequence FANCA coding region and splice junctions in DNA from 195 FA patients. This year, we explored a next-generation sequencing methodology, Truseq custom amplicon, for screening all the 43 FANCA exons along with 100bp of the adjacent regions in DNA from 58 patients. Sixty seven custom amplicons (200 bp in length) were designed, and they targeted a total of 14,642 bp that covered nearly the entire length of the RefSeq FANCA transcript (6090 of the 6191bp). Upon capturing, sequencing and aligning to the reference genome, the sequence depth ranged widely from 204 - 6215 (median 2762) except for four exons (1, 6-7, 15) where the depth was much less and ranged from 20 - 78. Of the 58 DNA sequenced, we found two FANCA mutations for 24, one in 24, and none in ten. The Truseq custom amplicon allows for an efficient evaluation of sequence variations in a large number of DNA samples at once, and the read depth (100s-1000s fold) should allow for detection of variants present in a small proportion of patient DNA. Deletions contribute to a substantial proportion of mutations in FANCA. As part of a comprehensive effort to identify all the disease-causing mutations for patients enrolled IFAR, we analyzed 202 FA families for deletion and insertion mutations using high throughput methods including Comparative Genome Hybridization arrays (aCGH). The arrays contained 135,000 50mer probes, spaced an average interval of 37bp, spanning up to 200kb upstream and downstream of the 15 known FA genes and 12 other functionally relevant genes. We found deletions in 98 families consisting of 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. The precise boundaries identified by aCGH enabled design of PCR assays across the deleted regions, followed by cloning and sequencing across the breakpoints. Fifty-two FANCA deletion ends, and one FANCC deletion end were found to extend beyond the gene boundaries, potentially affecting neighboring genes. Eighty percent of the FANCA deletion breakpoints are Alu-Alu mediated, predominantly by AluY elements. Individual Alu hotspots were identified in introns 21, 17 and 5. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry, and all are of recent origin. We are now employing MLPA for detection of deletions in FANCA exons for patient DNA samples in smaller quantities and thus insufficient for CGH analysis. Detailed characterization of deletions is critical for a better understanding of the FA phenotypes. In summary, our sequencing and arrayCGH efforts have resulted in identifying a spectrum of FA gene mutations for over 230 patients. Our goal is to comprehensively catalog mutations in all patients enrolled in the IFAR.

一旦被诊断为范可尼贫血(FA)，识别致病基因和突变是一项艰巨的任务。传统的筛选过程是顺序的、多步骤的方法，因此执行起来效率低且成本高。FA基因很大，有多个外显子，并且含有广泛分布在整个基因中的复合杂合性突变，包括大的基因组缺失。因此，在国际Fanconi贫血登记中心(IFAR)登记的800个家庭中，近一半家庭的分子诊断仍然未知。 我们使用大规模并行测序技术对代表所有FA和相关DNA修复途径基因的基因组大(2Mb)区域进行了测序。我们设计了比较基因组杂交阵列(ACGH)阵列，以探索同一组基因中的大拷贝数变异。我们还使用了RNAseq技术来确定导致异常剪接的毫无戒备的变异的致病性。利用辅助技术成功鉴定了43名个体的FA基因突变：FANCA(17人)、FANCB(4人)、FANCC(5人)、FANCD1(1人)、FANCE(1人)、FANCD2(3人)、FANCF(2人)、FANCG(2人)、FANCI(1人)、FANCJ(4人)和FANCL(3人)。我们采用的策略是一种有效的方法来识别诸如FA等高度遗传异质性疾病的潜在变异，并确保对未来的参与者进行及时和有效的分子诊断。 虽然FA患者可以携带16个已知基因中的任何一个突变，但约三分之二受到FANCA基因突变的影响。因此，对于所有FA个体来说，检查FANCA突变可能是一个有效的初始步骤。早些时候，我们已经使用Sanger测序法对195例FA患者DNA中的FANCA编码区和剪接连接进行了测序。今年，我们探索了一种新一代测序方法，Truseq定制扩增，用于筛查58名患者DNA中所有43个FANCA外显子以及100个碱基的相邻区域。共设计了67个定制扩增片段(长度为200个碱基)，它们针对的总长度为14,642bp，几乎覆盖了RefSeq FANCA转录本的整个长度(6191个碱基对中的6090个)。在捕获、测序和与参考基因组比对后，除4个外显子(1，6-7，15)外，序列深度在204-6215(中位数2762)之间变化很大，其深度要小得多，范围为20-78。在测序的58个DNA中，我们发现了24个FANCA突变中的两个，24个中有一个突变，10个中没有突变。Truseq定制扩增子允许一次对大量DNA样本中的序列变异进行有效评估，并且读取深度(100s-1000s倍数)应该允许检测在一小部分患者DNA中存在的变异。 缺失导致了相当大比例的FANCA突变。作为为参加IFAR的患者确定所有致病突变的全面努力的一部分，我们使用包括比较基因组杂交阵列(ACGH)在内的高通量方法分析了202个FA家系的缺失和插入突变。这些阵列包含135,000个50,000个探针，平均间隔为37bp，跨越15个已知FA基因和12个其他功能相关基因的上下游200kb。我们在98个家系中发现了缺失，包括88个FANCA、7个FANCC、2个FANCD2和1个FANCB。通过CGH确定的精确边界使得能够设计跨缺失区域的聚合酶链式反应分析，随后跨断点进行克隆和测序。52个FANCA缺失末端和1个FANCC缺失末端被发现延伸到基因边界之外，潜在地影响邻近基因。80%的FANCA缺失断裂点是由Alu-Alu介导的，主要由AluY元件介导。在21号、17号和5号内含子上发现了单个Alu热点。对4个FANCA缺失家系的单倍型分析表明，其中3个有共同的祖先，而且都是最近起源的。我们现在使用MLPA来检测患者DNA样本中FANCA外显子的缺失，数量较少，因此不足以进行CGH分析。缺失的详细特征对于更好地理解FA表型至关重要。 总而言之，我们的测序和阵列CGH努力已经为230多名患者鉴定了FA基因突变谱。我们的目标是全面记录所有参加IFAR的患者的突变情况。