Massively parallel sequencing, ArrayCGH and RNA-Seq technologies provide a comprehensive molecular diagnosis of Fanconi anemia.
ABSTRACT Current methods for detecting mutations in Fanconi anemia (FA) suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom Molecular Inversion Probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at the Rockefeller University. DNA sequencing was complemented with custom Array Comparative Genomic Hybridization (aCGH) and RNA-Seq analysis. aCGH identified deletions/duplications in four different FA genes. RNA-Seq analysis revealed lack of allele specific expression associated with a deletion, and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq targeted capture, aCGH and RNA-Seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2) and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified two novel FANCC mutations in two patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of Fanconi anemia.
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ABSTRACT: FANCD2 is a product of one of the genes associated with Fanconi anemia (FA), a rare recessive disease characterized by bone marrow failure, skeletal malformations, developmental defects, and cancer predisposition. FANCD2 forms a complex with FANCI (ID complex) and is monoubiquitinated, which facilitates the downstream interstrand crosslink (ICL) repair steps, such as ICL unhooking and nucleolytic end resection. In the present study, we focused on the chicken FANCD2 (cFANCD2) mutant harboring the Leu234 to Arg (L234R) substitution. cFANCD2 L234R corresponds to the human FANCD2 L231R mutation identified in an FA patient. We found that cFANCD2 L234R did not complement the defective ICL repair in FANCD2-/- DT40 cells. Purified cFANCD2 L234R did not bind to chicken FANCI, and its monoubiquitination was significantly deficient, probably due to the abnormal ID complex formation. In addition, the histone chaperone activity of cFANCD2 L234R was also defective. These findings may explain some aspects of Fanconi anemia pathogenesis by a FANCD2 missense mutation.PLoS ONE 12/2014; 9(12):e114752. DOI:10.1371/journal.pone.0114752 · 3.53 Impact Factor
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ABSTRACT: Accurate and timely diagnosis of inherited bone marrow failure and inherited myelodysplastic syndromes is essential to guide clinical management. Distinguishing inherited from acquired bone marrow failure/myelodysplastic syndrome poses a significant clinical challenge. At present, diagnostic genetic testing for inherited bone marrow failure/myelodysplastic syndrome is performed gene-by-gene, guided by clinical and laboratory evaluation. We hypothesized that standard clinically-directed genetic testing misses patients with cryptic or atypical presentations of inherited bone marrow failure/myelodysplastic syndrome. In order to screen simultaneously for mutations of all classes in bone marrow failure/myelodysplastic syndrome genes, we developed and validated a panel of 85 genes for targeted capture and multiplexed massively-parallel sequencing. In patients with clinical diagnoses of Fanconi anemia, genomic analysis resolved subtype assignment, including those of patients with inconclusive complementation test results. Eight out of 71 patients with idiopathic bone marrow failure or myelodysplastic syndrome were found to harbor damaging germline mutations in GATA2, RUNX1, DKC1, or LIG4. All eight of these patients lacked classical clinical stigmata or laboratory findings of these syndromes and only four had a family history suggestive of inherited disease. These results reflect the extensive genetic heterogeneity and phenotypic complexity of bone marrow failure/myelodysplastic syndrome phenotypes. This study supports the integration of broad unbiased genetic screening into the diagnostic workup of children and young adults with bone marrow failure and myelodysplastic syndromes.Haematologica 09/2014; DOI:10.3324/haematol.2014.113456 · 5.94 Impact Factor
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ABSTRACT: Fanconi anemia (FA) is a rare recessive disease resulting from mutations in one of at least 16 different genes. Mutation types and phenotypic manifestations of FA are highly heterogeneous and influence the clinical management of the disease. We analyzed 202 FA families for large deletions, using high-resolution Comparative Genome Hybridization arrays (arrayCGH), Single Nucleotide Polymorphism arrays (SNParrays) and DNA sequencing. We found pathogenic deletions in 88 FANCA, seven FANCC, two FANCD2, and one FANCB families. We find 35% of FA families carry large deletions, accounting for 18% of all FA pathogenic variants. Cloning and sequencing across the deletion breakpoints revealed that 52 FANCA deletion ends, and one FANCC deletion end extended beyond the gene boundaries, potentially affecting neighboring genes with phenotypic consequences. Seventy-five percent of the FANCA deletions are Alu-Alu mediated, predominantly by AluY elements, and appear to be caused by Non-Allelic Homologous Recombination. Individual Alu hotspots were identified. Defining the haplotypes of four FANCA deletions shared by multiple families revealed that three share a common ancestry. Knowing the exact molecular changes that lead to the disease may be critical for a better understanding of the FA phenotype, and to gain insight into the mechanisms driving these pathogenic deletion variants.This article is protected by copyright. All rights reservedHuman Mutation 08/2014; 35(11). DOI:10.1002/humu.22680 · 5.05 Impact Factor