FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

点击翻译按钮获取中文摘要

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 (23 Mb) region of the genome, targeting the entire length of all FA and FA-related DNA-repair pathway genes. The genes being sequenced, also include those encoding ALDH and ADH and other enzymes involved in the generation and metabolism of aldehydes, which are key endogenous DNA crosslinking agents. 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. Thus, our mutation screening panel now includes 150 genes. In addition to sequence variants, we are now able to detect deletions/duplications and determine their precise boundaries from the nextgen sequence reads, as well. So far, through our collaboration with the Rockefeller University, we have identified biallelic mutations in 572 IFAR families diagnosed with FA and now extending similar efforts towards 100 individuals enrolled at NCI/NIH as a part of IBMFS studies. A few highlights from this and other efforts are described below. We characterized both mutations in 159 FANCA patients and (published in Human Mutation, 2018). 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 pathogenic variants including large deletions, missense, nonsense, indel and splice aberrations, is prevalent across other FA genes as well, and underscores the need for RNA analysis to be an integral part of the molecular diagnosis of FA. We also observed that deletions extending beyond the 5 end and codon 1 missense variants in FANCA are, indeed, null alleles as they do not express the encoded RNA or protein, respectively. FA due to X-linked FANCB mutations are rare and account 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 now published in the journal, Blood. Unlike FA-A and FA-B, groups with high genetic heterogeneity, we found a founder mutation in a very rare group, FAL, 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 is now published in the journal Human Mutation. Similar to the FA-A, FA-B, and FA-L groups, studies on FA patients from FA-D2, FA-E, and FA-J groups are now underway. We have generated and characterized knockouts of 17 FA genes in zebrafish (published in PLOS Genetics 2019). Recently, we characterized the consequences of deficiency of two FA-associated proteins, faap100 and faap24, in zebrafish. Both proteins are components of the FA core complex that facilitates ubiquitination of FA-I and FA-D2 proteins. To date, mutations in these genes have not been reported in FA patients. The faap100 nulls exhibited complete conversion of female-to-male sex-reversal, a characteristic of all FA gene mutants, whereas the faap24 mutants did not. The sensitivity of the embryos for DNA crosslinking agent, diepoxybutane (DEB), was very much apparent for faap100 but not for the faap24. Overall, our data show that deficiency of faap100 results in phenotypes consistent with other FA gene knockouts in zebrafish. On the contrary, deficiency of faap24 did not show similar phenotypes. We are pursuing efforts to develop zebrafish mutants as a model to study the FA disease process, particularly, hematopoietic disease and cancer predisposition. We evaluated differential blood cell counts for two mutant lines, fanca and fanco: we find that, as they age, the nulls exhibit decreasing cell counts for all lineages (thrombocytes, red blood cells, and white blood cells). The number and function of thrombocytes were evaluated by measuring time to occlusion (TTO), either venous or arterial laser thrombosis on 5 dpf larvae that were treated with DEB. The treated larvae showed prolongation of TTO compared to untreated larvae indicating inefficient thrombosis in mutants. Similarly, we have generated double knockouts (an FA gene along with TP53) and we are characterizing the tumors that develop in these fish.