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. Thus, molecular diagnosis of a large number of families enrolled in the International Fanconi Anemia Registry (IFAR) remained unknown. Within the last two years, there were reports of five new FA genes being identified, thus bringing the number of known FA genes to 21. Though FA patients can carry mutations in any of the 21 known genes, about two-thirds are affected by mutations in FANCA. Our current efforts are focused on employing massively parallel sequencing technologies to sequence large (2-3 Mb) regions of the genome, targeting all FA and FA-related DNA-repair pathway genes. We also adopt Comparative Genome Hybridization arrays (aCGH), and SNP arrays to explore large-size copy number variants in a similar set of genes. So far, through our collaboration with the Rockefeller University, we have identified bi-allelic mutations in 500 IFAR families, and these include 313 FANCA, 16 FANCB, 54 FANCC, 15 FANCD1, 21 FANCD2, seven FANCE, 15 FANCF, 23 FANCG, 11 FANCI, 15 FANCJ, five FANCL, four FANCN, three FANCP and one FANCT. A few highlights from this effort include: 1) identification of 16 patients with an X-linked FANCB mutation, a very rare FA group, and in four patients, the mutation was de novo; 2) Identification of rare disease-associated deletions in 148 families including 131 FANCA, 8 FANCC, 5 FANCD2, one FANCJ, one FANCI, and 2 FANCB using aCGH; 3) determination of the precise breakpoints for 90 deletions, and discovered the mechanisms leading to deletions; 4) found that fifty-five FANCA deletions overlapped exon 1 and extended beyond the 5 gene terminus, eliminating a putative promoter region and, likely expression. This was confirmed by RNA analysis. A manuscript describing characterization of both mutations in 160 FANCA patients in under revision. For the past two years, as a part of our detailed molecular diagnosis, we have been exploring causes and consequences of mosaicism in FA families. It is estimated that 20% of FA patients may display somatic mosaicism, a scenario where a fraction of cells from hematopoietic lineages may have lost, or repaired, one of the inherited mutations. This phenomenon results in a functional allele in the fraction of blood cells with reverse mosaicism (RM), and may often provide protection from hematopoietic diseases. Somatic mosaicism in a patient is evident when his/her blood cells were subjected to DNA breakage test at the time of diagnosis. We completed our study of three siblings in a family with mutations in FANCG displaying RM, each displaying a different mechanism that resulted in RM, and a manuscript describing these findings is under preparation. We also completed another study where a FANCB patient displayed RM. The patient harbored a 10kb intragenic duplication in FANCB. This duplication was unstable and reverted back to wild type in patient cells from peripheral blood and also, to some extent, from fibroblasts. A manuscript describing the mosaicism displayed by the FANCB patient is under revision. In an effort to evaluate the frequency of FA gene variants in patients diagnosed with HNSCC under the age of 50, we sequenced genomic DNA of 492 patients for variations in all FA genes. The findings from this study were published this year. In addition, we have generated zebrafish carrying mutations in each of the 19 FA genes. The initial characterization of FA null mutations in zebrafish is now complete and a manuscript describing these efforts is being prepared for publication.
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