FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

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）技术允许快速测序基因组的大（兆）区域。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个与下一代测序的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）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选择和测序有助于识别我们测试的DNA中的所有已知突变。然后，我们选择了12个没有指定组的额外DNA，因此没有已知的突变。我们能够在其中的11个中找到基因和失活突变，从而允许在没有互补组的先验知识的情况下识别突变。一个样本没有任何FA基因突变，这可能属于少数被诊断患有FA的个体，但互补试验无法将其分配到任何已知的组中，这表明可能还有其他FA基因有待发现。对这些样品应用全外显子组测序应有助于鉴定额外的FA基因。 虽然FA患者可以携带15种已知FA基因中的任何一种突变，但大约三分之二的患者受到单一基因FANCA突变的影响。 因此，对于没有指定互补组的所有FA个体，通过桑格测序方法检查FANCA突变将作为有效的初始步骤。我们对88名此类患者的DNA进行了测序，正如预期的那样，我们发现58/88携带FANCA基因突变。 非FANCA个体可以进行下一代测序，事实上，选择用于下一代测序的12个样本中有8个来自非FANCA组，并且发现每个样本在不同的FA基因中含有突变。除了这88名患者，我们还对另外110名FA个体进行了测序，这些个体通过互补试验被分配到FANCA组，但其中一种或两种致病突变尚不清楚。对200例携带FANCA基因突变的FA个体进行测序，发现了几个新的突变（39个点，14个indel和18个剪接）。 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，很难从下一代测序数据中辨别缺失。使用高分辨率、高密度寡核苷酸阵列的比较基因组杂交（CGH）允许对基因组缺失和重复进行高效鉴定和精确作图。CGH阵列用135，000个寡核苷酸开发，代表包括所有FA基因的37个基因，以及已知参与DNA修复途径的其他几个基因。这些数组帮助识别了几个缺失。意识到很大比例的缺失超出了基因，我们设计了另一个CGH阵列，可以查询基因两侧高达200 kb的基因组区域。我们现在已经确定了近65个FANCA缺失的精确边界，其中28个延伸到基因区域之外。我们在7例患者中发现FANCC基因缺失，其中5例具有相同的缺失，FANCB和FANCD 2缺失各1例。对于更大的缺失，我们使用高密度SNP芯片，这些芯片扫描整个基因组区域，并揭示除了缺失和重复之外的任何其他染色体变异 下一代测序，CGH和SNP阵列技术，沿着桑格测序，将使我们实现我们的目标，即确定我们收集的所有FA样本中的致病突变。 除了FA个体之外，这些技术还可用于探索FA基因在胰腺癌和其他癌症中的作用。

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) technologies allow 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 for next-gen sequencing. 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). 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. We then chose 12 additional DNAs with no assigned group and thus no known mutations. We were able to find the gene and the inactivating mutations in 11 of them, and thus allowing identification of the mutations without a prior knowledge of the complementation group. One sample did not harbor mutations in any of the FA genes, and this may belong to a small number of individuals diagnosed with FA but the complementation test could not assign them to any known group, suggesting that there may be additional FA gene(s) to be discovered. Application of whole exome sequencing on these samples should help identify additional FA gene(s). Though FA patients can carry mutations in any of the 15 known FA genes, about two-thirds are affected by mutation in a single gene, FANCA. Thus, for all FA individuals with no assigned complementation group, checking for FANCA mutations by Sanger sequencing method will serve as an efficient initial step. We sequenced DNA from 88 such patients, and as anticipated, we found 58/88 to carry mutations in the FANCA gene. The non-FANCA individuals can be subjected to next-gen sequencing, and in fact, eight of the twelve samples chosen for next-gen sequencing were from the non-FANCA group, and found that each harbored mutations in a distinct FA gene. In addition to the 88 patients, we sequenced another 110 FA individuals who have been assigned to the FANCA group by complementation test, but either one or both of the disease-causing mutations were not yet known. Sequencing of 200 FA individuals carrying mutations in FANCA gene has allowed us to discover several novel (39 point, 14 indel and 18 splice) mutations. 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, it is difficult to discern deletions from the next-gen sequencing data. Comparative genomic hybridization (CGH) using high-resolution, high-density oligo arrays allows for efficient identification and precise mapping of genomic deletions and duplications. A CGH array was developed with 135,000 oligonucleotides, representing 37 genes that included all the FA genes, and several others known to participate in a DNA repair pathway. The arrays helped identify several deletions. Realizing that a good proportion of deletions go beyond the gene, we have designed another CGH array that queries the genomic regions up to 200kb on either side of the gene. We have now determined the precise boundaries for nearly 65 FANCA deletions with 28 extending beyond the gene region. We found FANCC gene deletion in seven patients with five sharing the same deletion, and FANCB and FANCD2 deletions in one sample each. For even larger deletions, we employ high-density SNP chips, and these chips scan the entire genomic region and reveal any other chromosomal variations, in addition to deletions and duplications The next-gen sequencing, CGH and SNP array technologies, along with the Sanger sequencing, will take us towards our goal of determining both the disease-causing mutations in all the FA samples in our collection. In addition to FA individuals, these technologies can be employed to explore the role of FA genes in pancreatic and other cancers.