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: In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns-multisystemic, hepatic, or in central nervous system (CNS)-and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved.02/2014; 24(1):48-56. DOI:10.1089/nat.2013.0453
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ABSTRACT: Fanconi anemia (FA) is a severe hereditary disorder with defective DNA damage response and repair. It is characterized by phenotypes including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. Recent studies in mice have suggested that the FA proteins might counteract aldehyde-induced genotoxicity in hematopoietic stem cells. Nearly half of the Japanese population carries a dominant negative allele (rs671) of the aldehyde-catalyzing enzyme ALDH2 (acetaldehyde dehydrogenase 2), providing an opportunity to test this hypothesis in humans. We examined 64 Japanese FA patients, and found that the ALDH2 variant is associated with accelerated progression of BMF, while birth weight or the number of physical abnormalities was not affected. Moreover, malformations at some specific anatomic locations were observed more frequently in ALDH2-deficient patients. Our current data indicate that the level of ALDH2 activity impacts pathogenesis in FA, suggesting the possibility of a novel therapeutic approach.Blood 09/2013; 122(18). DOI:10.1182/blood-2013-06-507962 · 10.43 Impact Factor
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ABSTRACT: The inherited bone marrow failure syndromes (IBMFS) are a set of clinically related yet heterogeneous disorders in which at least one hematopoietic cell lineage is significantly reduced. Many of the IBMFS have notably increased cancer risks, as well as other physical findings. Highly penetrant germline mutations in key pathways, such as DNA repair, telomere biology, or ribosomal biogenesis, are causative of Fanconi anemia (FA), dyskeratosis congenita (DC), and Diamond-Blackfan anemia (DBA), respectively. Next-generation sequencing (NGS) generally refers to high-throughput, large-scale sequencing technologies and is being used more frequently to understand disease etiology. In the IBMFS, NGS has facilitated the discovery of germline mutations that cause thombocytopenia absent radii syndrome (TAR), a subset of DC and DBA, and other uncharacterized, but related, disorders. Panels of large numbers of genes are being used to molecularly characterize patients with IBMFS, such as FA and DBA. NGS is also accelerating the discovery of the genetic etiology of previously unclassified IBMFS. In this review, we will highlight recent studies that have employed NGS to ascertain the genetic etiology of IBMFS, namely, FA, DC, DBA, and TAR, and discuss the translational utility of these findings.Seminars in Hematology 10/2013; 50(4):333-47. DOI:10.1053/j.seminhematol.2013.09.002 · 2.46 Impact Factor