FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

一旦被诊断患有范可尼贫血（FA），鉴定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方法，因此效率低且执行起来昂贵。FA基因是大的，具有多个外显子，并且具有广泛的复合杂合突变，其分布在整个基因中，包括大的基因组缺失。因此，在国际范可尼贫血登记处（IFAR）登记的800个家庭中，近一半的家庭的分子诊断仍然未知。 虽然FA患者可以携带16种已知基因中的任何一种突变，但约三分之二的患者受到FANCA基因突变的影响。因此，对于所有FA个体，检查FANCA突变可以作为有效的初始步骤。早些时候，我们使用桑格测序和Truseq定制扩增子测序方法对250名FA患者DNA中的FANCA编码区和剪接点进行测序。我们采用大规模平行测序技术对基因组的大（2 Mb）区域进行测序，靶向所有FA和相关DNA修复途径基因。我们设计了比较基因组杂交阵列（aCGH）来探索同一组基因中的大尺寸拷贝数变异。我们还采用了高desnisty SNP阵列的基因组，特别是在FA基因的区域的拷贝数变化的评估。利用互补技术，今年成功鉴定了37个家庭的FA基因双等位基因突变：FANCA（18），FANCB（2），FANCC（2），FANCD 1（2），FANCD 2（6），FANCF（1），FANCG（1），FANCI（1），FANCJ（2）和FANCN（2）。 我们采用的策略是一种有效的方法，以确定潜在的高度遗传异质性疾病，如FA的变化，并确保及时和有效的分子诊断未来的登记。 对于FA先证者，其父母的DNA是可用的，我们已经研究了突变的父母遗传。这项分析，结合SNP/STRP数据，提供了证据，单亲二体性（UDP）的原因，在三个FA先证者的FA。对亲本DNA的突变分析也有助于鉴定4名患者的体细胞嵌合现象，这是由于从血液中制备的DNA（或从淋巴细胞建立的LCL细胞系）中缺乏两种突变之一。血细胞中的体细胞嵌合现象通常导致对造血系统疾病的保护。虽然这似乎是一个有益的变化，但这种保护可能不会延伸到他们患癌症的倾向。我们现在正在探索导致体细胞镶嵌现象的机制。 头颈部癌（HNSCC）是FA患者中最常见的实体恶性肿瘤之一。高达25%的FA患者的唯一表现为实体和血液恶性肿瘤。由于这些表现是非典型的，这些患者并不总是进行FA检测。此类癌症的标准治疗通常涉及包括放疗和化疗的治疗方式，已知这些治疗会导致FA患者出现危及生命的并发症。因此，不知道诊断为HNSCC的患者的FA状态可能具有严重的临床后果。我们正在探索未确诊的FA在450例年轻成人头颈部（H&N）癌症样本的程度。我们已经完成了16个已知的FA基因和24个与头颈癌相关的其他基因的测序，我们正在分析致病变异的数据。

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. 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 used Sanger sequencing and Truseq custom amplicon sequencing methods to sequence FANCA coding region and splice junctions in DNA from 250 FA patients. We employed massively parallel sequencing technologies to sequence large (2Mb) regions of the genome, targeting all FA and related DNA-repair pathway genes. We designed Comparative Genome Hybridization arrays (aCGH) to explore large-size copy number variants in the same set of genes. We also employ high-desnisty SNP arrays for evaluation of copy number changes in the genome and particularly in the regions of FA genes. The use of complementary technologies allowed for successful identification of biallelic mutations in FA genes in 37 families this year: FANCA (18), FANCB (2), FANCC (2), FANCD1 (2), FANCD2 (6), FANCF (1), FANCG (1), FANCI (1), FANCJ (2) and FANCN (2). 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. For FA probands whose parental DNA are available, we have studied the parental inheritance of mutations. This analysis, in combination with SNP/STRP data, has provided evidence for uniparentaldisomy (UDP) as the cause of FA in three FA probands. Analysis of parental DNA for mutations has also helped identify somatic mosaicism in four patients, which results from the absence of one of the two mutations in their DNA prepared from blood (or LCL cell lines established from lymphocytes). Somatic mosaicism in blood cells often leads to protection from hematopoietic diseases. Though this appears to be a beneficial change, this protection may not extend to their propensity to develop cancers. We are now exploring the mechanism that leads to somatic mosaicism. Head and Neck cancer (HNSCC) is one of the most common solid malignancies in patients with FA. Up to 25% of patients with FA have solid and hematological malignancies as their sole presenting manifestation. Since these presentations are atypical, these patients are not always tested for FA. The standard of care for such cancers often involves treatment modalities that include both radiation and chemotherapy and these treatments are known to cause life-threatening complications in FA patients. Thus, not knowing the FA status of a patient diagnosed with HNSCC can have severe clinical consequences. We are exploring the extent of undiagnosed FA in a sample of 450 young adults with Head and Neck (H&N) cancer. We have completed sequencing 16 known FA genes and 24 other genes linked to head and neck cancer, and we are in the process of analyzing the data for pathogenic variants.