Journal
GENETICS IN MEDICINE
Volume 13, Issue 3, Pages 218-229Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1097/GIM.0b013e318203cff2
Keywords
next-generation sequencing; exon-capture; molecular diagnostic testing; primary ciliary dyskinesia; clinical genetics
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Funding
- National Center of Research resources, NIH [M0O1RR00046, UL-1-RR025747]
- University Cancer Research Fund (UCRF)
- UNC Center for Genomics and Society [NHGRI 5-P50-HG004488-03]
- UNC [1-UL1-RR025747-01]
- NIH [5-U54-HL096458-06, 5-R01HL071798]
- ORDR
- NHLBI, NIH
- Deutsche Forschungsgemeinschaft (DFG) [DFG Om 6/4]
- Multidisciplinary Research Grant (MRG)
- North Carolina Biotechnology Center
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Purpose: Advances in genetic sequencing technology have the potential to enhance testing for genes associated with genetically heterogeneous clinical syndromes, such as primary ciliary dyskinesia. The objective of this study was to investigate the performance characteristics of exon-capture technology coupled with massively parallel sequencing for clinical diagnostic evaluation. Methods: We performed a pilot study of four individuals with a variety of previously identified primary ciliary dyskinesia mutations. We designed a custom array (NimbleGen) to capture 2089 exons from 79 genes associated with primary ciliary dyskinesia or ciliary function and sequenced the enriched material using the GS FLX Titanium (Roche 454) platform. Bioinformatics analysis was performed in a blinded fashion in an attempt to detect the previously identified mutations and validate the process. Results: Three of three substitution mutations and one of three small insertion/deletion mutations were readily identified using this methodology. One small insertion mutation was clearly observed after adjusting the bioinformatics handling of previously described SNPs. This process failed to detect two known mutations: one single-nucleotide insertion and a whole-exon deletion. Additional retrospective bioinformatics analysis revealed strong sequence-based evidence for the insertion but failed to detect the whole-exon deletion. Numerous other variants were also detected, which may represent potential genetic modifiers of the primary ciliary dyskinesia phenotype. Conclusions: We conclude that massively parallel sequencing has considerable potential for both research and clinical diagnostics, but further development is required before widespread adoption in a clinical setting. Genet Med 2011: 13(3): 218-229.
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