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. More FA genes are being identified, including six more added in the last three years alone, requiring the screening methodologies to be flexible to accommodate newly identified FA genes. Thus, molecular diagnosis of a large number of families enrolled in the International Fanconi Anemia Registry (IFAR) remained unknown. Though FA patients can carry mutations in any of the 22 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. As an extension of collaborative studies with our colleagues at NCI/NIH, our gene/mutation screening efforts are now extended to study other inherited bone marrow failure syndromes (IBMFS), dyskeratosis congenita (DC) and Diamond-Blackfan anemia (DBA), in addition to FA. 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 are described below: 1) A manuscript describing characterization of both mutations in 159 FANCA patients was published in 2018. 2) Identification of 19 patients with an X-linked FANCB mutation, a very rare FA group, and in four patients, the mutation was de novo. Deep sequencing using Miseq of the amplicons with the mutation were performed to see if mother carried the mutation at a low frequency, which are often undetected by Sanger sequencing. Maternal DNA from none of the four families showed evidence for low frequency, and thus, these are indeed de novo variants. We have performed functional evaluation of missense variants, and the residual FANCB protein function of the missense variants appear to reflect the age of onset of bone marrow disease and survival of the patients. These studies are being presented at the upcoming the Fanconi aemia and American Society of Hematology annual meetings, and a manuscript is being prepared for publication. 3) 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. Determination of the precise breakpoints for 90 deletions, and discovered the mechanisms leading to deletions. Our current methodologies allow us to determine the deletion breakpoints from the nextgen sequence data, eliminating the need for amplifying, cloning and sequencing the breakpoint junction regions. The breakpoints for the remaining deletions will be identified by this efficient approach. 4) A finding 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 in cell lines from a subset of patients harboring the deletions. It was also observed that the codon 1 variants in FANCA did not express the protein. Determination of the precise molecular consequence of pathogenic variants would help evaluate their influence on the clinical presentations of the patients. 5) An observation that two patients carried identical homozygous variants in FA-L, a rare group representing only 0.2% of the patient population. Recently, two other FA research labs have identified several patients from India with the same mutations, indicative of a founder mutation. Efforts are underway to complete this collaborative study on FANCL. 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 was published in 2018. 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 last year. A similar analysis of variants in DC genes has been completed for these patients. Three patients carried variants in TERF1 (TRF1), suggesting TERF1 may be a novel DC gene. In addition, we have generated zebrafish carrying mutations in each of the 17 FA, and two FA-associated protein (FFAP) genes. Among others, either complete or partial female-to-male sex-reversal phenotype was apparent for FA gene knockouts in zebrafish. A manuscript describing a detailed characterization of these zebrafish mutants was submitted and reviewed; and at present, the manuscript is being revised to accommodate the comments raised by the reviewers.
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