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。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）登记的大量家庭的分子诊断仍然未知。在过去的两年中，有报道称发现了5个新的FA基因，从而使已知的FA基因数量达到21个。虽然FA患者可以携带21种已知基因中的任何一种突变，但大约三分之二的患者受到FANCA突变的影响。我们目前的努力集中在采用大规模平行测序技术来测序基因组的大区域（2-3 Mb），靶向所有FA和FA相关的DNA修复途径基因。我们还采用比较基因组杂交阵列（aCGH）和SNP阵列来探索类似基因组中的大拷贝数变异。 到目前为止，通过与洛克菲勒大学的合作，我们已经在500个IFAR家族中鉴定出双等位基因突变，其中包括313个FANCA，16个FANCB，54个FANCC，15个FANCD 1，21个FANCD 2，7个FANCE，15个FANCF，23个FANCG，11个FANCI，15个FANCJ，5个FANCL，4个FANCN，3个FANCP和1个FANCT。 这项工作的几个亮点包括：1）鉴定了16例X连锁FANCB突变患者，这是一个非常罕见的FA组，其中4例患者的突变是从头开始的; 2）使用aCGH鉴定了148个家族中的罕见疾病相关缺失，包括131个FANCA，8个FANCC，5个FANCD 2，1个FANCJ，1个FANCI和2个FANCB; 4）发现55个FANCA缺失与外显子1重叠，并延伸到5个基因末端以外，消除了一个推定的启动子区域，可能表达。RNA分析证实了这一点。 描述160例FANCA患者两种突变特征的手稿正在修订中。 在过去的两年里，作为我们详细的分子诊断的一部分，我们一直在探索FA家族中镶嵌现象的原因和后果。据估计，20%的FA患者可能会显示体细胞嵌合现象，一种情况是来自造血谱系的一部分细胞可能已经丢失或修复了遗传突变之一。这种现象导致在具有反向镶嵌（RM）的血细胞部分中的功能等位基因，并且通常可以提供对造血疾病的保护。当患者的血细胞在诊断时进行DNA断裂试验时，患者的体细胞嵌合现象是明显的。我们完成了对一个FANCG突变家族中三个兄弟姐妹的研究，每个兄弟姐妹都显示了导致RM的不同机制，描述这些发现的手稿正在准备中。 我们还完成了另一项研究，其中一名FANCB患者显示RM。该患者在FANCB中存在10 kb的基因内重复。这种复制是不稳定的，并且在来自外周血的患者细胞中以及在一定程度上来自成纤维细胞的患者细胞中恢复为野生型。一份描述FANCB患者表现出的镶嵌现象的手稿正在修订中。 为了评估50岁以下HNSCC患者FA基因变异的频率，我们对492例患者的基因组DNA进行了测序，以确定所有FA基因的变异。这项研究的结果于今年发表。此外，我们已经产生了19个FA基因中每一个都携带突变的斑马鱼。斑马鱼中FA无效突变的初步表征现已完成，描述这些努力的手稿正在准备出版。

Once diagnosed with Fanconi anemia (FA), identification of the causative gene and the mutations is an arduous task. 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 a large number of families enrolled in the International Fanconi Anemia Registry (IFAR) remained unknown. Within the last two years, there were reports of five new FA genes being identified, thus bringing the number of known FA genes to 21. Though FA patients can carry mutations in any of the 21 known genes, about two-thirds are affected by mutations in FANCA. Our current efforts are focused on employing massively parallel sequencing technologies to sequence large (2-3 Mb) regions of the genome, targeting all FA and FA-related DNA-repair pathway genes. We also adopt Comparative Genome Hybridization arrays (aCGH), and SNP arrays to explore large-size copy number variants in a similar set of genes. So far, through our collaboration with the Rockefeller University, we have identified bi-allelic mutations in 500 IFAR families, and these include 313 FANCA, 16 FANCB, 54 FANCC, 15 FANCD1, 21 FANCD2, seven FANCE, 15 FANCF, 23 FANCG, 11 FANCI, 15 FANCJ, five FANCL, four FANCN, three FANCP and one FANCT. A few highlights from this effort include: 1) identification of 16 patients with an X-linked FANCB mutation, a very rare FA group, and in four patients, the mutation was de novo; 2) Identification of rare disease-associated deletions in 148 families including 131 FANCA, 8 FANCC, 5 FANCD2, one FANCJ, one FANCI, and 2 FANCB using aCGH; 3) determination of the precise breakpoints for 90 deletions, and discovered the mechanisms leading to deletions; 4) found that fifty-five FANCA deletions overlapped exon 1 and extended beyond the 5 gene terminus, eliminating a putative promoter region and, likely expression. This was confirmed by RNA analysis. A manuscript describing characterization of both mutations in 160 FANCA patients in under revision. For the past two years, as a part of our detailed molecular diagnosis, we have been exploring causes and consequences of mosaicism in FA families. It is estimated that 20% of FA patients may display somatic mosaicism, a scenario where a fraction of cells from hematopoietic lineages may have lost, or repaired, one of the inherited mutations. This phenomenon results in a functional allele in the fraction of blood cells with reverse mosaicism (RM), and may often provide protection from hematopoietic diseases. Somatic mosaicism in a patient is evident when his/her blood cells were subjected to DNA breakage test at the time of diagnosis. We completed our study of three siblings in a family with mutations in FANCG displaying RM, each displaying a different mechanism that resulted in RM, and a manuscript describing these findings is under preparation. We also completed another study where a FANCB patient displayed RM. The patient harbored a 10kb intragenic duplication in FANCB. This duplication was unstable and reverted back to wild type in patient cells from peripheral blood and also, to some extent, from fibroblasts. A manuscript describing the mosaicism displayed by the FANCB patient is under revision. In an effort to evaluate the frequency of FA gene variants in patients diagnosed with HNSCC under the age of 50, we sequenced genomic DNA of 492 patients for variations in all FA genes. The findings from this study were published this year. In addition, we have generated zebrafish carrying mutations in each of the 19 FA genes. The initial characterization of FA null mutations in zebrafish is now complete and a manuscript describing these efforts is being prepared for publication.