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. Last 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 screened over 200 patients for mutations in FANCA, and identified both the mutations in 180 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 a similar set of genes. In addition to finding FANCA families, our sequencing efforts have identified both mutations in families belonging to other FA groups: eight FANCB, 17 FANCC, two FANCD1, 13 FANCD2, two FANCE, four FANCF, four FANCG, six FANCI, five FANCJ, four FANCL, two FANCN, and one FANCP. In the past year, as a part of our detailed molecular diagnosis, we have been exploring causes and consequences of mosaicism in FA 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. Last year, we identified three siblings in a family with mutations in FANCG displaying RM. One sibling had reversal of the maternally inherited variant back to WT. The other two siblings had distinct de novo variants, and each corrected the defect caused by the paternal mutation by restoring the reading frame except for altering 23 and 3 amino acids, encoding residues 577-589 and 587-589, respectively. We followed up on the functional evaluation of these two altered FANCG proteins, by generating cDNA expression constructs and introducing them into a FANCG-null cell line. Both the revertant variant proteins, indeed, retained FANCG function, explaining RM and the associated delay in the appearance of hematological manifestations in the patient. Patients harboring X-linked FANCB mutations usually present with a severe clinical phenotype, resembling VACTERL syndrome with hydrocephalus. We identified a FANCB patient with much milder phenotype. The patient exhibited mosaicism detected during the diagnostic DEB test, where patient LCLs were resistant to DEB while fibroblasts were sensitive. Employing aCGH and targeted next-gen sequencing, we identified an intragenic duplication in FANCB. The 9154 bp duplication included the first coding exon, exon 3, and the flanking intronic regions (chrX:14877976-14887129). Presence of a 4 bp homology (GTAG) at both ends of the breakpoint appears to indicate the duplication may have been mediated via the alt-EJ mechanism. A ddPCR assay was developed for the junction region to evaluate the extent of the duplication. The duplication was present in 90% of the patient fibroblast cell line but in only 5% of the patient LCL. This is consistent with the DEB test, indicating that the allele with duplication has reverted back to wild-type (WT) in LCL and, to a lesser extent, in fibroblast cells. The assay also detected the duplication in DNA from the mother, albeit in only 22% of her DNA, indicating her to be a mosaic carrier for the mutation. RT-PCR of RNA from patient fibroblast cells showed the WT transcript, along with an aberrant transcript, 1 kb larger. PacBio sequencing of the larger transcript revealed the aberrant transcript had insertion of the entire exon 3, predicted to introduce a stop codon at the junction. Unlike sequence variants, duplications are difficult to define precisely and quantitate accurately. Thus we demonstrated that detailed characterization of the disease-causing variant is critical for better understanding the patient phenotype. We have initiated an effort to generate zebrafish carrying mutations in each of the 19 FA genes. Fish with mutations in each gene individually, and in combinations of genes, have been generated. Efforts to characterize the consequence of these are underway.
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