FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

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Our current efforts are focused on employing NextGen technologies to sequence 152 genes, targeting the entire length of all FA and other inherited bone marrow failure syndromes (IBMFS) genes (59). ADH/ALDH (27) and 1-Carbon metabolism (47) genes are also being sequenced as they encode enzymes involved in the generation and metabolism of aldehydes, which are key endogenous DNA crosslinking agents. 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. 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 the recent years, we have reported the molecular diagnosis of 159 patients with mutations in FANCA which revealed that all but seven families harbored a distinct pair of mutations, thus defining the genotypic heterogeneity among FANCA patients. We also identified and characterized a FANCL founder mutation specific to the South Asian population that originated 2700 years ago. The FA-L group represents only 0.4% of the FA population worldwide but it is the third most common group in India. We have also published two studies associating genetic variation to disease presentation in FANCB patients: 1) The case of a FANCB patient that presented with a milder disease, and for which we determined that the patients outcome was a consequence of a large intragenic duplication in FANCB that was unstable and reverted, resulting in mosaic expression. 2) The characterization of disease-causing mutations and their effect on the encoded RNA and protein function for 19 X-linked FANCB patients from 16 families, associating the severity to the type of gene variant and the residual activity of mutant protein. We have reported the hypomorphic nature of a FANCA variant c.4199G>A/p.R1400H. We now find another instance where the nature of the disease-causing pathogenic variant resulted in milder disease. This involved carriers of the FANCA variant c.3624C>T. This variant is predicted to be synonymous (p.S1208S), but affects splicing leading to a pathogenic four bp deletion (p.S1208Ifs*38). The hematologic onset in six patients carrying this variant was found to be much later than FA patients in general (median age of 22.5 vs seven years). Deep sequencing the transcript region containing the aberrant splicing event revealed that about 6-10% of transcripts carried the canonical splice product, resulting in functional protein, thus explaining the milder phenotype of these patients. Functional assays confirmed residual function of FANCA in cell lines from these patients. Similar to the FA-A, FA-B, and FA-L groups, studies on FA patients from FA-D2, FA-E, and FA-F groups are now underway. In general, mutations in each group are private, reflecting allelic heterogeneity. However, we do find recurring variants in FA groups, such as FANCF, in which we identified two variants with comparatively increased frequency, c.230_252del23 and c.484_485delCT, representing 33% and 30% of FANCF families, respectively, and identified c.230_252del23 as a founder variant of recent origin. We also find a variant that is present in 17/26 (65%) FANCJ families. We identified evidence of somatic mosaicism in 32 individuals from 30 families. The advent of a de novo compensatory variant or the reversal of a pathogenic variant was observed in five and 13 individuals, respectively. Isodisomy of an FA gene resulting from mitotic recombination was exhibited by 15 individuals. In two families there were siblings that each exhibited mosaicism via different mechanisms. The isodisomy extended to the chromosome terminus in each case, influencing the allelic expression in two different ways: six cases exhibited the breakpoint junction within the FA gene, between two biallelic pathogenic variants, resulting in the replacement of the pathogenic variant by a wildtype sequence; nine cases exhibited isodisomy of the entire FA gene, resulting in a loss of one variant and duplication of the second. Of the nine cases with isodisomy of the entire gene we performed functional analysis on 5/8 variants, as one variant occurred in two cases, and in each case the duplicated variant exhibited hypomorphic function. Neither isodisomy event appear to influence the age at onset of hematologic disease, but the cases with intragenic isodisomy junction showed longer survival, indicating that the loss of variant and subsequent expansion of modified cells promoted a positive effect on the hematologic disease course. 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. 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 for slx4ip protein, suggesting that SLX4IP could also be an FA-candidate gene. We demonstrated pancytopenia and thrombosis defects in zebrafish mutants with inactivation of the FANCA or FANCO gene, resembling aplastic anemia associated with FA. We also are making an effort to explore tumor development in FA gene mutants. As none of the FA gene homozygous knockouts showed reduced survival or indications of tumor development, we introduced TP53 knockout allele (7bp indel) in to FANCI or FANCP knockout lines to generate double knockouts (FANCI-TP53 and FANCP-TP53). As the fish aged, we evaluated the pathology of the tumor development after the appearance of abnormal growth (or natural death). In addition, we checked all the fish as they reached 7mpf. An associated loss of TP53 led to the development of tumors. Characterization of these tumors is underway.