FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

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Our current efforts are focused on employing NextGen technologies to identify genetic variations that cause Fanconi anemia and other inherited bone marrow failure disorders and influence the various phenotypes associated with the disease. We designed a custom capture gene panel consisting of 152 genes that targets the entire length of genes associated with inherited bone marrow failure syndromes (IBMFS), including all FA genes (59). ADH/ALDH (27) and 1-Carbon metabolism (47) genes are also included as they encode enzymes involved in the generation and metabolism of aldehydes, which are key endogenous DNA crosslinking agents. In addition to sequence variants, the targeted gene panel allows us to detect deletions/duplications and determine their precise boundaries from the sequence reads. For identification of large-size genomic changes including isodisomy caused by mitotic recombination in patients displaying somatic mosaicism, we employ high-density (1M) SNP arrays. For elucidating the nature of cDNA products generated by aberrant splicing, we use PacBio sequencing technology. We are also pursuing efforts to develop zebrafish mutants as a model to study the FA disease process, particularly hematopoietic disease and cancer predisposition. In recent years, we have reported the molecular diagnosis of 159 patients with pathogenic variations in FANCA, identified and characterized a FANCL founder mutation specific to the South Asian population that originated 2700 years ago, and discerned genotype-phenotype correlations for 19 X-linked FANCB patients from 16 families. Similar to the FA-A, FA-B, and FA-L groups, studies on FA patients from FA-D2, FA-E, FA-F, and FA-J groups are now underway. The FA-D2, FA-E, FA-F, and FA-J groups represent 1%-4% of FA families, and we have characterized 31, 12, 18, and 26 families, respectively. Functional evaluation of missense variants to confirm pathogenicity is underway. Previously, we had reported the hypomorphic nature of the FANCA variant c.4199G>A/p.R1400H, and we are now ready to report another instance where the nature of the disease-causing pathogenic variant resulted in milder disease. The report describes the FANCA variant c.3624C>T, which is predicted to be synonymous (p.S1208S) but affects splicing of some RNA transcripts leading to a pathogenic four bp deletion. Individuals with FA are susceptible to develop somatic mosaicism in their hematopoietic lineages. We have reported the case of a FANCB patient harboring a large intragenic duplication in FANCB that was unstable and reverted, resulting in mosaic expression and milder phenotype. In addition, we have identified and defined the mechanism leading to mosaicism in 32 individuals from 30 families, which we are currently preparing for publication. Also, I was a coauthor on a recent report detailing the patterns of mosaicism observed through clinical deep sequencing of disease-related genes in one million individuals (Truty et al., American Journal of Human Genetics 2023). We are also pursuing efforts to develop zebrafish mutants as a model to study the FA disease process, particularly hematopoietic disease and cancer predisposition. We have generated and characterized knockouts of 17 FA genes in zebrafish. In addition, we demonstrated that deficiency of faap100, an FA-candidate gene, results in phenotypes consistent with other FA gene knockouts in zebrafish. Similar phenotypes were apparent in zebrafish mutants with deficiency of slx4ip protein, suggesting that SLX4IP could also be an FA-candidate gene. Both reports describing these potential FA candidate genes are underway. However, deficiency of faap24, an FA-pathway gene, did not result in phenotypes consistent with other FA gene knockouts. Earlier, we demonstrated pancytopenia and thrombosis defects in zebrafish mutants with inactivation of the FANCA or FANCO gene, resembling aplastic anemia associated with FA. Several other FA-gene mutant lines are being evaluated for pancytopenia by measuring changes in blood cell counts. In addition, FANCA deficient transgenic zebrafish lines expressing cd41-GFP and gata1-RFP are being generated to evaluate hematopoietic lineage-specific changes. FA individuals are at increased risk (700-fold) of developing squamous cell carcinoma of the head and neck (HNSCC). We were co-authors with our collaborators from the Rockefeller University on a recently published report describing the genomic signature of cancer with FA DNA repair pathway deficiency (Weber et al., Nature 2022). Cancer with FA deficiency was characterized by the presence of a large number of structural variants, often accompanied by the loss of TP53. This study showed that the genomic instability observed in sporadic HNSCC may be due to the inability of the FA pathway to repair DNA damage caused by the increased burden of interstrand crosslinking. This report is a major step in understanding HNSCC and efforts are underway to develop methodologies to improve early detection of HNSCC.