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. This year, there were reports of three new FA genes being identified, thus bringing the number of known FA genes to 19. Though FA patients can carry mutations in any of the 19 known genes, about two-thirds are affected by mutations in FANCA. Earlier, we did screen for mutations in FANCA, and identified both the mutations in 160 patients. However, 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. Recently, we redesigned our sequence target to include the three new FA genes. We also redesigned Comparative Genome Hybridization arrays (aCGH) to explore large-size copy number variants in the same set of genes. In the past year, we have processed DNA from 49 FA families for molecular diagnosis. Follow up on the parental inheritance of patients with homozygous mutations, along with SNP/STRP genotype data of the trio, has provided evidence for uniparental disomy (UPD) as the cause of FA in four FA patients. Discovery of UPD as a cause of FA in a patient indicates that the risk of another child diagnosed with FA in this family is reduced from 25% to nearly zero. This observation has consequences in genetic counseling of the families. It is estimated that 25% 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 identified three siblings in a family with mutations in FANCG displaying RM. The germ-line genotype of the lymphoblast cell lines (LCL) of the 3 affected siblings (FA1-FA3) indicated likely mechanisms accounting for RM. The maternal variant c.1158dupC in exon 10 was absent in the proband (FA1), likely from a gene conversion event correcting the mutant allele. FA2 and FA3 carried a paternally inherited mutation, c.1771dupC, in exon 14 and each carried a de novo variant: c.1729delC in exon 13 of FA2, and c.1761-1G>A in intron 13 of FA3.. cDNA analysis revealed that the somatic de novo variants each corrected the defect caused by the paternal mutation by restoring the reading frame except for altering amino acids encoding 577-589 and 587-589 in FA2 and FA3 respectively. The siblings did not develop bone marrow disease, probably as a consequence of RM, but all three developed cancer in their later years. FA patients are at increased risk (nearly 800 fold) to develop solid tumors. Head and Neck cancer (HNSCC) is one of the most common solid malignancies in patients with FA. Interestingly, up to 25% of patients with FA have solid and hematological malignancies as their sole presenting manifestation. Conventional cancer treatment is not well tolerated by patients with undiagnosed underlying FA disease. We initiated an effort to evaluate the FA gene mutation status in early onset HNSCC patients. For 417 patients (<50 years old) with HNSCC, germline DNA sequencing of 16 FA genes was performed. Filtering criteria were implemented to identify rare variants with the potential to alter function. The variants were then classified as: 1) previously reported in FA disease databases (FA Mutation database, and Breast cancer mutation databases), and 2) predicted to be damaging by SIFT, Polyphen2, and CADD. There were 15 patients (only two under age 40) carrying two rare variants in an FA gene. Of these, two patients carried a FA gene variant that had been previously reported in a FA disease database. Overall, 8% of patients had at least one FA gene variant previously reported in FA disease databases, and 27% of patients had at least on FA gene variant predicted to be damaging. However, there was no association between age at presentation and variant frequency overall. Further evaluation of FA gene variant frequency in cases vs population is ongoing.
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