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

• Published in: Blood Vol. 121; no. 22; pp. e138 - e148
• Main Authors: Chandrasekharappa, Settara C., Lach, Francis P., Kimble, Danielle C., Kamat, Aparna, Teer, Jamie K., Donovan, Frank X., Flynn, Elizabeth, Sen, Shurjo K., Thongthip, Supawat, Sanborn, Erica, Smogorzewska, Agata, Auerbach, Arleen D., Ostrander, Elaine A., NISC Comparative Sequencing Program
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 30.05.2013; American Society of Hematology
• Subjects: Basic-Leucine Zipper Transcription Factors - genetics; Comparative Genomic Hybridization - methods; Family Health; Fanconi Anemia - diagnosis; Fanconi Anemia - ethnology; Fanconi Anemia - genetics; Fanconi Anemia Complementation Group A Protein - genetics; Fanconi Anemia Complementation Group C Protein - genetics; Fanconi Anemia Complementation Group D2 Protein - genetics; Fanconi Anemia Complementation Group G Protein - genetics; Fanconi Anemia Complementation Group L Protein - genetics; Fanconi Anemia Complementation Group Proteins - genetics; Gene Deletion; Gene Duplication; Humans; Jews - genetics; Mutation; Red Cells, Iron, and Erythropoiesis; Sequence Analysis, RNA - methods
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2012-12-474585

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. •Application of capturing/sequencing, copy number, and RNA analysis technologies ensures comprehensive molecular diagnosis of Fanconi anemia.