FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

范可尼贫血：基因型-表型相关性

• 批准号: 8149406
• 负责人: settara chandrasekharappa
• 金额: $ 65.98万
• 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/8149406
• 关键词: Fanconi' s Anemia; Genotype; Phenotype

Once diagnosed with Fanconi anemia (FA), identification of the mutations remains an arduous task at present. The current screening process is a sequential, multi-step approach and successful identification of mutations may be delayed or hindered at any of these steps: establishing cell lines, growing and transducing cells for complementation, and procuring an efficient transducing vector for all FA genes. FA genes are large, with multiple exons, and harbor a wide spectrum of compound heterozygous mutations spread throughout the gene. Multi-exon size genomic deletions of FA genes are also well documented, and therefore, PCR amplification of exons and Sanger sequencing may not yield both the mutations. Therefore, there is a need for an efficient approach that scans the entire length of all the FA genes, and detects wide spectrum of changes. The next-gen sequencing (NGS) allows sequencing large (megabase) regions of the genome rapidly. This enables identification of mutations, directly from DNA, with no prior requirement for establishment of cell lines and determination of the complementation group. We have targeted 13 FA and 11 additional genes that are associated with DNA repair pathways. We employed MIP (Molecular Inversion probe) selection approach for enrichment of the genomic regions of the targeted 24 genes. Essentially, probes were designed to capture 5136 regions, and each test DNA was subjected to the MIP selection. A library of the enriched material was sequenced using a sequencing instrument (Illumina GAII) in a single-end 36 bp configuration. As an initial step, we tested six DNAs, each with one (or both) previously known mutation in a different FA gene. The MIP selection and sequencing helped identify all the known mutations in the DNAs we tested, thus demonstrating that the methodology may be suitable for screening FA gene mutations. Efforts are underway to employ this methodology to test the DNA from FA individuals with no known mutations. Improvements in the selection strategies, as they emerge, will be employed for optimal utilization of the next-gen sequencing technology for screening for mutations in the known FA genes. A small number of individuals diagnosed with FA do not harbor mutations in any of the known 13 FA genes, suggesting that there may be additional gene(s) mutations in which may contribute to FA. We intend to employ the next-gen sequencing technologies for identification of yet unidentified FA genes. Multi-exon size genomic deletions of FA genes are well documented, and such deletions account for more than a quarter of the mutations in the FANCA gene. At present, deletion mutations are often difficult to discern from the next-gen sequencing data. Comparative genomic hybridization (CGH) using high-density oligo arrays allows for efficient identification of genomic deletions and duplications at a high resolution. Unlike the current methods, aCGH analysis is not limited only to the exonic regions, and therefore can identify the precise ends of a deletion. The high-density arrays accommodate the entire genomic regions of all the FA genes (and many other genes of interest as well), and thus can screen for deletions in all the FA genes at once. A CGH array was developed with 135,000 oligonucleotides, representing 37 genes that includes the 13 FA genes, and several others known to participate in a DNA repair pathway. The median spacing of probes is 14nt. DNA from FA patients was processed to incorporate a Cy5 fluorescent tag, and a reference genomic DNA was similarly processed to incorporate the Cy3 tag. Data from CGH analysis has been collected on DNA from 72 FA patients, which included several controls with known deletions. 50 DNAs assigned to the FANCA gene were analyzed and CGH arrays helped identify deletions in 27. Similarly deletions/duplications were identified in one each of the two DNAs tested from the FA-B, FA-C and FA-G groups. Two FANCA DNAs displayed two overlapping deletions, and another showed a homozygous deletion, and the precise breakpoints of both the deletions could be inferred from the data. We found a deletion in the FANCA gene in a sample with no prior assigned complementation group. Deletions/duplications in any of the FA genes can be queried in a single assay using CGH. Availability of the precise breakpoints will allow for identification of the samples with a shared common deletion interval. In addition to FA individuals, a deletion in FANCC and another in FANCD1 gene have been reported in two pancreatic cancer cell lines. We tested and found that our CGH arrays can identify such deletions from the xenograft of the tumor and the tumor cell line. Thus a combination of aCGH and next-gen sequencing technologies can be employed as a comprehensive screening approach for scanning all the FA genes in individuals diagnosed with FA. In addition, this offers an opportunity to explore the role of FA genes in pancreatic and other cancers.

一旦被诊断为范可尼贫血（FA），目前鉴定突变仍然是一项艰巨的任务。目前的筛选过程是一个连续的，多步骤的方法和突变的成功鉴定可能会延迟或阻碍在这些步骤中的任何一个：建立细胞系，生长和转导细胞的互补，并获得一个有效的转导载体的所有FA基因。FA基因很大，具有多个外显子，并且在整个基因中具有广泛的复合杂合突变。FA基因的多外显子大小的基因组缺失也被充分记录，因此，外显子的PCR扩增和桑格测序可能不会产生两种突变。因此，需要一种有效的方法，扫描所有FA基因的整个长度，并检测广谱的变化。 下一代测序（NGS）允许快速对基因组的大（兆碱基）区域进行测序。 这使得能够直接从DNA鉴定突变，而不需要预先建立细胞系和确定互补组。 我们已经靶向了13个FA和11个与DNA修复途径相关的额外基因。我们采用MIP（分子倒置探针）选择方法富集靶向的24个基因的基因组区域。基本上，探针被设计为捕获5136个区域，并且每个测试DNA经受MIP选择。使用测序仪器（Illumina GAII）以单端36 bp构型对富集材料的文库进行测序。作为第一步，我们测试了六个DNA，每个DNA在不同的FA基因中具有一个（或两个）先前已知的突变。 MIP选择和测序有助于鉴定我们测试的DNA中的所有已知突变，从而证明该方法可能适用于筛选FA基因突变。目前正在努力采用这种方法来测试来自没有已知突变的FA个体的DNA。选择策略的改进，因为他们出现，将用于最佳利用下一代测序技术筛选已知的FA基因的突变。少数被诊断患有FA的个体在已知的13个FA基因中的任何一个中都没有突变，这表明可能存在可能导致FA的其他基因突变。我们打算采用下一代测序技术来鉴定尚未鉴定的FA基因。 FA基因的多外显子大小的基因组缺失是有据可查的，并且这种缺失占FANCA基因突变的四分之一以上。目前，缺失突变通常难以从下一代测序数据中辨别出来。使用高密度寡核苷酸阵列的比较基因组杂交（CGH）可以高分辨率有效识别基因组缺失和重复。与目前的方法不同，aCGH分析不仅限于外显子区域，因此可以识别缺失的精确末端。高密度阵列容纳了所有FA基因（以及许多其他基因）的整个基因组区域。 感兴趣），因此可以在所有FA基因中筛选缺失， 一次CGH阵列用135，000个寡核苷酸开发，代表37个基因，包括13个FA基因和已知参与DNA修复途径的其他几个基因。探针的中位间距为14 nt。对来自FA患者的DNA进行处理以掺入Cy 5荧光标签，并且对参考基因组DNA进行类似的处理以掺入Cy 3标签。从CGH分析的数据收集了72例FA患者的DNA，其中包括几个已知缺失的对照。对50个FANCA基因的DNA进行了分析，CGH阵列有助于识别 删除27。类似地，在一个基因组中鉴定出缺失/重复。 来自FA-B、FA-C和FA-G组的两种DNA中的每一种。两 FANCA DNA显示两个重叠缺失，另一个显示一个重叠缺失。 纯合缺失，两种缺失的精确断点可以 从数据中推断出来。 我们发现FANCA基因缺失， 没有预先指定互补组的样本。 任何FA基因中的缺失/重复可以在 使用CGH的单次测定。精确断点的可用性将允许识别具有共享共同缺失间隔的样品。除FA个体外，在两个胰腺癌细胞系中还报道了FANCC和FANCD 1基因的缺失。我们测试并发现，我们的CGH阵列可以从肿瘤和肿瘤细胞系的异种移植物中鉴定出这种缺失。因此，aCGH和下一代测序技术的组合可以用作扫描被诊断患有FA的个体中的所有FA基因的综合筛查方法。此外，这为探索FA基因在胰腺癌和其他癌症中的作用提供了机会。