FANCONI ANEMIA:GENOTYPE-PHENOTYPE CORRELATIONS

National Human Genome Research Institute

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Abstract

Our current efforts are focused on employing nextgen sequencing 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, we employ high-density (1M) SNP arrays. 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. 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 the 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. For the six patients carrying homozygous del23 variant, the age of hematological onset was between 2-6 years, 5/6 patients developed aplastic anemia, but none had congenital abnormalities except for one individual with hypotonia. The patients with the delCT variant, however, were predominantly compound heterozygous carriers, and do not display common clinical presentations probably due to influence of the 2nd allele. We find two instances where the nature of the disease-causing pathogenic variant resulted in milder disease. In one case, siblings were not diagnosed with FA until they developed esophageal cancer at age 51 and showed adverse reaction to the conventional treatment. Their unusual delay in FA diagnosis was due to the hypomorphic nature of one of their disease-causing FANCA variants, c.4199G>A/p.R1400H. The second instance 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. 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. We evaluated differential blood cell counts for two mutant lines, fanca and fanco: finding that, as they age, the nulls exhibit decreasing cell counts for all lineages (thrombocytes, red blood cells, and white blood cells), resembling aplastic anemia associated with FA. 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, which was corrected by the expression of mRNA for the missing FA gene, fanca or fanco. We will expand our studies to other FA mutants. We will also introduce cd41:eGFP and gata1:dsRed transgenic alleles in our FA mutants to trace changes in specific blood cell lineages early in development, and to explore the associated transcriptome changes.