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

Abstract

• Application of capturing/ sequencing, copy number, and RNA analysis technologies ensures comprehensive molecular diagnosis of Fanconi anemia.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 sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 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 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA. (Blood. 2013;121(22):e138-e148)

Identification of point mutations and large intragenic deletions in Fanconi anemia using next‐generation sequencing technology

“…Indeed, massive parallel sequencing complemented with array comparative genomic hybridization and RNA sequencing analysis allowed the identification of biallelic germline mutations in different FA families (Chandrasekharappa et al. 2013). However, it has been shown that massively parallel sequencing techniques are feasible to identify CNV in FA and other diseases (Ameziane et al.…”

Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology

“…Massive parallel sequencing approaches have already been successfully used. 21,22 In our experience, we are validating the Ion PGM TM system (Life Technologies) and have identified mutations in 6 cases, including 2 belonging to group FA-C. Since preliminary data are very encouraging, we are confident to be able to apply this procedure not only in the routine of molecular screening in FA but also for re-sequencing those cases (3 in our experience) who, having been analyzed only partially, are still without a molecular diagnosis.…”

Genetic diagnosis of Fanconi anemia. Literature review

“…Возможны несколько подходов. Первый -секвенирование экзо-ма, позволяет получить максимальный объем инфор-мации [61,62,65]. Второй подход подразумевает сек-венирование ограниченного числа интересующих и уже описанных в литературе генов (таргетное ресеквени-рование), список которых можно дополнять или мо-…”