FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

National Human Genome Research Institute

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Abstract

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.