FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

一旦被诊断为范可尼贫血(FA)，识别致病基因和突变是一项艰巨的任务。FA基因很大，有多个外显子，并且含有广泛分布在整个基因中的复合杂合性突变，包括大的基因组缺失。越来越多的FA基因正在被发现，这就要求筛选方法要灵活，以适应新发现的FA基因。因此，国际范科尼贫血登记处(IFAR)登记的大量家庭的分子诊断仍然未知。虽然FA患者可以携带22个已知基因中的任何一个突变，但FANCA(%)、FANCC(12%)和FANCG(8%)是FA患者中最常见的三个致病基因，占全球病例的84%。 我们目前的工作集中在使用NextGen测序技术对基因组的一个大(2-3Mb)区域进行测序，目标是所有FA和FA相关的DNA修复途径基因的全长。虽然我们采用了比较基因组杂交阵列(ACGH)和SNP阵列来探索一组相似基因中的拷贝数变异，但我们现在能够从序列读取中检测缺失/复制并确定它们的精确边界。我们与NCI/NIH的同事共同发起的一项研究，现在将我们的致病基因/突变筛查工作扩展到其他遗传性骨髓衰竭综合征(IBMFS)、先天性角化不良(DC)和钻石-布莱克凡贫血(DBA)，以及FA。此外，我们正在探索疾病修饰基因/变体，这些基因主要是编码ALDH和ADH的基因，以及参与醛的生成和代谢的其他酶，醛是关键的内源DNA交联剂。因此，我们的突变筛查小组现在包括150个基因。到目前为止，通过我们与洛克菲勒大学的合作，我们已经在&gt；500个被诊断为FA的IFAR家庭中确定了双等位基因突变，现在将类似的努力扩展到NCI/NIH登记的IBMFS家庭。下面将介绍这项工作的一些亮点。 1)2018年《人类突变》发表了一篇手稿，描述了159名FANCA患者的这两种突变的特征。观察到，除了7个家系外，所有的家系都有两个突变的不同组合，从这一点来看，遗传异质性很明显。我们发现，某些被预测为编码错义变化的突变确实会导致异常剪接。我们现在发现，遗传异质性、广泛的变异(致病变异包括大的缺失、错义、无义、插入和剪接异常)以及作为分子诊断不可或缺的一部分的RNA分析的需要也延伸到其他FA基因。我们还观察到，FANCA中超出5end和密码子1错义变体的缺失确实是零等位基因，因为它们分别不表达编码的RNA或蛋白质。 2)由X连锁FANCB突变引起的FA非常罕见，仅占FA患者的2%。我们对来自16个家系的19名FANCB突变患者进行了特征分析。没有两名患者携带相同的FANCB突变。在已发表的报告中，与大量FA个体相比，FANCB缺失或截短者表现出比平均发病时间更早的骨髓衰竭，以及更严重的先天性异常。这反映了FANCB蛋白在FANCD2单素化的酶激活过程中不可或缺的作用，FANCD2单素化是DNA链间交联修复的关键步骤。对于FANCB错义变异体，更多的变量严重程度与残留的FANCD2单素化活性的程度有关。携带FANCD2单泛素化显著降低的错义变异的个体表现出更早的血液病发病和更短的生存时间。相反，FANCD2单素化接近正常的变异体与更有利的结果相关。我们的报告揭示了FA的FA-B互补组中的基因型-表型相关性，该报告已准备好提交给《血液》杂志。 3)与遗传异质性较高的FA-A和FA-B组不同，我们在一个非常罕见的FA-L组中发现了创始人突变，仅占患者总数的0.2%。然而，预计同义的致病变异体K364K会导致异常剪接，消除携带该变异体的外显子13，从而从编码的蛋白质中移除24个氨基酸。这种变异导致了来自印度的12名患者发生FA，携带者共享一个可以追溯到2700年前的祖先等位基因的共同单倍型。公开的基因组数据库显示，该变异将被分离到南亚。《人类突变》杂志对描述这些发现的手稿进行了审查，现在已经提交了修改后的手稿。与FA-A和FA-B组类似，目前正在对FA-D2、FA-E和FA-J组的FA患者进行研究。 4)我们已经克隆并鉴定了斑马鱼的17个FA基因和两个FA相关蛋白基因(faap100和faap24)中的每一个都存在突变。虽然零突变体的活力没有受到影响，但胚胎对DNA交联剂二环氧丁烷(DEB)的敏感性非常明显。这些空体表现出完全或接近完全的雌雄反转表型，这似乎是所有FA基因突变体的特征。这些斑马鱼突变体的特征发表在本财年的《公共科学图书馆·遗传学》上。FAAP蛋白尚未被认为是FA的致病基因，然而，对斑马鱼faap100突变体的鉴定表明，它们表现出与其他FA基因突变体相似的特征，因此可能在不久的将来被鉴定为真正的FA基因。我们正在努力开发这些突变株作为研究FA疾病过程的模型，特别是血液病和癌症易感性。

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. More FA genes are being identified requiring the screening methodologies to be flexible to accommodate newly identified FA genes. Thus, molecular diagnosis of a large number of families enrolled in the International Fanconi Anemia Registry (IFAR) remained unknown. Though FA patients can carry mutations in any of the 22 known genes, FANCA (64%), FANCC (12%), and FANCG (8%) are the three most commonly observed disease-causing genes among FA patients, accounting for 84% of cases worldwide. Our current efforts are focused on employing nextgen sequencing technologies to sequence a large (2-3 Mb) region of the genome, targeting the entire length of all FA and FA-related DNA-repair pathway genes. Although we had adopted Comparative Genome Hybridization arrays (aCGH), and SNP arrays to explore copy number variants in a similar set of genes, we are now able to detect deletions/duplications and determine their precise boundaries from the sequence reads. A collaborative study initiated with our colleagues at NCI/NIH, now extends our disease-causing gene/mutation screening efforts to other inherited bone marrow failure syndromes (IBMFS), dyskeratosis congenita (DC) and Diamond-Blackfan anemia (DBA), in addition to FA. In addition, we are exploring disease-modifying genes/variants, and these are primarily genes encoding ALDH and ADH and other enzymes involved in the generation and metabolism of aldehydes, which are key endogenous DNA crosslinking agents. Thus, our mutation screening panel now includes 150 genes. So far, through our collaboration with the Rockefeller University, we have identified bi-allelic mutations in >500 IFAR families diagnosed with FA and now extending similar efforts towards IBMFS families enrolled at NCI/NIH. A few highlights from this effort are described below. 1) A manuscript describing characterization of both mutations in 159 FANCA patients was published in 2018 in Human Mutation. The genetic heterogeneity was apparent from the observation that all but seven families harbored distinct combinations of two mutations. We found that certain mutations predicted to encode missense changes are indeed causing aberrant splicing instead. We are now finding that genetic heterogeneity, a wide spectrum of variants (the pathogenic variants include large deletions, missense, nonsense, indel and splice aberrations), and need for RNA analysis as an integral part of molecular diagnosis extend to other FA genes as well. We also observed that deletions extending beyond the 5end and codon 1 missense variants in FANCA are indeed null alleles as they do not express the encoded RNA or protein, respectively. 2) FA due to X-linked FANCB mutations are rare and accounts for only 2% of all FA patients. We characterized 19 patients from 16 families with FANCB mutations. No two patients carried the same FANCB mutation. Those with FANCB deletion or truncation demonstrated earlier than average onset of bone marrow failure, and more severe congenital abnormalities compared to a large series of FA individuals in the published reports. This reflects the indispensable role of FANCB protein in the enzymatic activation of FANCD2 monoubiquitination, an essential step in the repair of DNA interstrand crosslinks. For FANCB missense variants, more variable severity was associated with the extent of residual FANCD2 monoubiquitination activity. Individuals carrying missense variants with drastically reduced FANCD2 monoubiquitination showed earlier onset of hematologic disease and shorter survival. Conversely, variants with near-normal FANCD2 monoubiquitination were associated with more favorable outcome. Our report revealing a genotype-phenotype correlation within FA-B complementation group of FA is ready for submission to the journal, Blood. 3) Unlike FA-A and FA-B groups with high genetic heterogeneity, we found a founder mutation in a very rare group, FA-L, representing only 0.2% of the patient population. The causative variant, predicted to be synonymous, K364K, however, causes aberrant splicing eliminating the exon 13 carrying the variant and thus removing 24 amino acids from the encoded protein. This variant caused FA in 12 patients from India and the carriers share a common haplotype from an ancestral allele that dates back 2700 years. The public genome databases reveal the variant to be isolated to South Asia. A manuscript describing these findings was reviewed by the journal, Human Mutation, and a revised manuscript has now been submitted. Similar to the FA-A and FA-B groups, studies on FA patients from FA-D2, FA-E and FA-J groups are now underway. 4) We have generated and characterized zebrafish carrying mutations in each of the 17 FA, and two FA-associated protein (faap100 and faap24) genes. Although viability of the null mutants was not affected, the sensitivity of the embryos for DNA crosslinking agent, diepoxybutane (DEB), was very much apparent. The nulls showed complete or nearly-complete female-to-male sex-reversal phenotype, which appears to be characteristic of all FA gene mutants. A characterization of these zebrafish mutants was published this fiscal year in PLoS Genetics. FAAP proteins have not yet been recognized as FA-causing genes, however, characterization of zebrafish mutants for faap100, reveal that they display similar features like any other FA gene mutant, and thus could be identified as an authentic FA gene in the near future. We are pursuing efforts to develop these mutants as a model to study FA disease process, particularly, hematopoietic disease and cancer predisposition.