Once diagnosed with Fanconi anemia (FA), identification of the causative gene and the mutations is an arduous task. The conventional screening process is a sequential, multi-step approach and, thus, is inefficient and expensive to perform. 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 nearly half of the 800 families enrolled in the International Fanconi Anemia Registry (IFAR) remained unknown. Though FA patients can carry mutations in any of the 16 known genes, about two-thirds are affected by mutations in FANCA gene. Thus, for all FA individuals checking for FANCA mutations may serve as an efficient initial step. Earlier, we used Sanger sequencing and Truseq custom amplicon sequencing methods to sequence FANCA coding region and splice junctions in DNA from 250 FA patients. We employed massively parallel sequencing technologies to sequence large (2Mb) regions of the genome, targeting all FA and related DNA-repair pathway genes. We designed Comparative Genome Hybridization arrays (aCGH) to explore large-size copy number variants in the same set of genes. We also employ high-desnisty SNP arrays for evaluation of copy number changes in the genome and particularly in the regions of FA genes. The use of complementary technologies allowed for successful identification of biallelic mutations in FA genes in 37 families this year: FANCA (18), FANCB (2), FANCC (2), FANCD1 (2), FANCD2 (6), FANCF (1), FANCG (1), FANCI (1), FANCJ (2) and FANCN (2). The strategy we employed was an effective approach to identify variations underlying a highly genetically heterogeneous disorder such as FA, and ensures a timely and efficient molecular diagnosis of future enrollees. For FA probands whose parental DNA are available, we have studied the parental inheritance of mutations. This analysis, in combination with SNP/STRP data, has provided evidence for uniparentaldisomy (UDP) as the cause of FA in three FA probands. Analysis of parental DNA for mutations has also helped identify somatic mosaicism in four patients, which results from the absence of one of the two mutations in their DNA prepared from blood (or LCL cell lines established from lymphocytes). Somatic mosaicism in blood cells often leads to protection from hematopoietic diseases. Though this appears to be a beneficial change, this protection may not extend to their propensity to develop cancers. We are now exploring the mechanism that leads to somatic mosaicism. Head and Neck cancer (HNSCC) is one of the most common solid malignancies in patients with FA. Up to 25% of patients with FA have solid and hematological malignancies as their sole presenting manifestation. Since these presentations are atypical, these patients are not always tested for FA. The standard of care for such cancers often involves treatment modalities that include both radiation and chemotherapy and these treatments are known to cause life-threatening complications in FA patients. Thus, not knowing the FA status of a patient diagnosed with HNSCC can have severe clinical consequences. We are exploring the extent of undiagnosed FA in a sample of 450 young adults with Head and Neck (H&N) cancer. We have completed sequencing 16 known FA genes and 24 other genes linked to head and neck cancer, and we are in the process of analyzing the data for pathogenic variants.
Showing the most recent 10 out of 44 publications
