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.

一旦被诊断出患有法科尼贫血（FA），病因基因和突变是一项艰巨的任务。常规的筛选过程是一种顺序的多步进方法，因此执行效率低下且昂贵。 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，包括大型基因组缺失。因此，在国际法科尼贫血注册中（IFAR）中招收的800个家庭中，将近一半的分子诊断尚不清楚。 我们采用了大量平行的测序技术来序列，代表所有FA和相关的DNA修复途径基因的大型（2MB）区域。我们设计了比较基因组杂交阵列（ACGH）阵列，以探索同一基因集中的大尺寸拷贝数变体。 我们还采用了RNASEQ技术来确定导致异常剪接的毫无戒心变体的致病性。使用补充技术允许成功鉴定43个个体中FA基因突变：Fanca（17），Fancb（4），Fancc（5），Fancd1（1），Fance（1），Fancd2（1），Fancd2（3），Fancf（ 2），Fancg（2），Fanci（1），Fancj（4）和Fancl（3）。 我们采用的策略是一种有效的方法，可以识别出高度遗传异质性疾病（例如FA）的基础的变异，并确保对未来参与者的及时有效分子诊断。 尽管FA患者可以在16个已知基因中的任何一个中携带突变，但大约三分之二的人受Fanca基因突变的影响。因此，对于所有FA个人检查FANCA突变的人都可以作为有效的初始步骤。早些时候，我们使用了Sanger测序方法来对195名FA患者的DNA中的Fanca编码区和剪接连接序列进行序列。今年，我们探索了下一代测序方法Truseq Custom Amplicon，用于筛选所有43个FANCA外显子，以及来自58名患者DNA的100bp邻近区域。设计了67个定制扩增子（长度为200 bp），它们的目标是14,642 bp，几乎涵盖了RefSeq Fanca转录本的整个长度（6191BP的6090）。捕获，测序和对齐参考基因组后，序列深度的范围在204-6215（中位2762）范围内，除了四个外显子（1、6-7、15）之外，深度较小，范围较小，范围为20-78。在测序的58个DNA中，我们发现了两个Fanca突变为24，一个24分之一，十个中没有。 TRUSEQ自定义扩增子可以同时对大量DNA样品中的序列变化进行有效评估，并且读取深度（100S-1000S折叠）应允许检测以较小比例的患者DNA中存在的变体。 删除造成了大量的FANCA突变。作为确定IFAR入选患者的所有引起疾病的突变的全面努力的一部分，我们使用高吞吐量方法（包括比较基因组杂交阵列（ACGH））分析了202个FA家族的缺失和插入突变。阵列包含135,000个50mer探针，间隔为37bp的平均间隔，跨越了15个已知的FA基因的上游和下游200KB，其他12个其他与功能相关的基因的跨度为200kb。 我们发现了98个家庭中的删除，包括88个Fanca，7 Fancc，2个FANCD2和一个Fancb家庭。通过ACGH启用了跨删除区域的PCR测定的设计确定的精确边界，然后在断点上克隆和测序。发现52个FANCA缺失末端，发现一个FANCC缺失端超出了基因边界，可能会影响邻近的基因。百分之八十的FANCA缺失断点是Alu-Alu介导的，主要是由Aluy元素介导的。在内含子21、17和5中确定了单个的Alu热点。定义多个家庭共享的四个FANCA缺失的单倍型表明，三个是共同的血统，所有这些都具有最近的起源。现在，我们使用MLPA来检测FANCA外显子中的缺失，以较少数量的患者DNA样品，因此不足以进行CGH分析。缺失的详细表征对于更好地理解FA表型至关重要。 总而言之，我们的测序和ARRAYCGH努力导致鉴定了230多名患者的FA基因突变。我们的目标是全面地对IFAR入学的所有患者进行分类突变。