Massively parallel sequencing, ArrayCGH and RNA-Seq technologies provide a comprehensive molecular diagnosis of Fanconi anemia.

Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, United States
Blood (Impact Factor: 10.45). 04/2013; 121(22). DOI: 10.1182/blood-2012-12-474585
Source: PubMed


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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Available from: Arleen D Auerbach, Jul 28, 2015
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    • "Although some mutations we found are the SNPs listed in the database, the functions of these mutations remain undetermined. Moreover, some SNPs can also cause disease [14], and thus we could not exclude that these SNPs are not related to FA disease. Finally, we were unable to detect new mutations in genes implicated in DNA repair pathways in any FA patients by comparison with their parents’ sequencing data. "
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    ABSTRACT: Background Fanconi anemia (FA) is a rare inherited genetic syndrome with highly variable clinical manifestations. Fifteen genetic subtypes of FA have been identified. Traditional complementation tests for grouping studies have been used generally in FA patients and in stepwise methods to identify the FA type, which can result in incomplete genetic information from FA patients. Methods We diagnosed five pediatric patients with FA based on clinical manifestations, and we performed exome sequencing of peripheral blood specimens from these patients and their family members. The related sequencing data were then analyzed by bioinformatics, and the FANC gene mutations identified by exome sequencing were confirmed by PCR re-sequencing. Results Homozygous and compound heterozygous mutations of FANC genes were identified in all of the patients. The FA subtypes of the patients included FANCA, FANCM and FANCD2. Interestingly, four FA patients harbored multiple mutations in at least two FA genes, and some of these mutations have not been previously reported. These patients’ clinical manifestations were vastly different from each other, as were their treatment responses to androstanazol and prednisone. This finding suggests that heterozygous mutation(s) in FA genes could also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers. Interestingly, we were not able to identify de novo mutations in the genes implicated in DNA repair pathways when the sequencing data of patients were compared with those of their parents. Conclusions Our results indicate that Chinese FA patients and carriers might have higher and more complex mutation rates in FANC genes than have been conventionally recognized. Testing of the fifteen FANC genes in FA patients and their family members should be a regular clinical practice to determine the optimal care for the individual patient, to counsel the family and to obtain a better understanding of FA pathophysiology.
    Full-text · Article · May 2014 · BMC Medical Genomics
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    • "Regarding clinical trials, ISIS Pharmaceuticals has recently concluded a phase 1 trial in which the safety, tolerability, and pharmacokinetics of escalating doses of the ISIS-SMNRx drug, an ISS-N1–tar- geting MOE SSO, administered into the spinal fluid as a single injection in patients with SMA (NCT01494701) has been evaluated (Southwell et al., 2012). Moreover, a phase 2 clinical trial has been initiated in patients with infantile-onset spinal muscular atrophy (NCT01839656) (Seo et al., 2013). "
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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.
    Full-text · Article · Feb 2014
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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.
    Full-text · Article · Sep 2013 · Blood
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