Development Of A High-throughput Resequencing Array For Pathogenic Mutation Detection Of Osteogenesis Imperfecta

Objective: Osteogenesis imperfecta(OI) is rare inherited skeletal diseases characterizedby bone fragility and low bone density. Most cases are caused by the autosomaldominant mutations such as COL1A1, COL1A2, CRTAP, LEPRE1, FKBP10. Genetictesting of the disease-causing mutations is important in this diagnostic scheme, but thelarge gene sizes make it difficult to detect efficiently and inexpensively. We aimed todevelop a technological tool to identify the nucleotide sequence of these genes rapidlyand accurately.Method: We have built up a CustomSeq Affymetrix Resequencing Array to enablehigh-throughput sequencing of5genes at the same time. The blood from14OI patientsand85normal controls were assayed. The genomic DNA was amplified usinglong-range PCR (LR-PCR). DNA fragmentation, and chip hybridization wereperformed according to Affymetrix recommendations. Hybridization signals wereanalyzed using GeneChip Sequence Analysis Software (GSEQ). This resequencingapproach was validated by capillary sequencing.Result: Resequencing array call rates is96–98%, and the agreement between microarrayand capillary sequencing was99.99%. In14OI patients,13patients were caused bypoint mutations, and all of point mutations could be detected successfully by the chip. Anovel mutation was able to be found by this technology, which was not detected in theothers. The sequence in1patient is inserted into the base, but failed to be checked outsuccessfully though the technology. In85normal controls, there were no mutationswhich were detected by the chip. Conclusion: We developed a high-throughput resequencing array that could detect thedisease-associated mutations of OI providing a potential tool to facilitate the large-scalegenetic screening for OI patients.