Objective: To identify mutations of the type I collagen genes (COL1A1 and COL1A2) in the affected with osteogenesis imperfecta (OI), to establish the spectrum of COL1A1/2 mutations in Chinese OI patients, and to provide prenatal gene diagnosis to the fetuses at high risk.
Methods: Genomic DNA was extracted from peripheral blood by the standard SDS-proteinase K-phenol/chloroform method. All the coding regions and exon/intron boundaries of COL1A1/2 were screened in 200 OI cases by conventional Sanger sequencing and targeted next-generation sequencing (NGS) on Ion Torrent-personalized genome sequencing operation (Ion PGM™). For familial cases, candidate mutations were validated in all available family members using high resolution melting analysis (HRM). In sporadic cases, only parents were examined to determine the origin of the identified mutation.Prenatal gene diagnosis was carried out by PCR direct sequencing and linkage analysis using microsatellite markers.
Results: In total, the authors identified 125 differently pathogenic mutations, including 74 in COL1A1 and 51 in COL1A2, in 158 probands, with a mutation detection rate of 79% (158/200). Among the 125 identified mutations, there were 63 novel mutations (33 in COL1A1 and 30 in COL1A2) and 13 recurrent mutations found in 46 probands (seven mutations recurring for two times, and the other six mutations recurring for more than 4 times). They performed prenatal genetic testing in 74 fetuses and found that 40 ones carried COL1A1/2 mutations identified in the corresponding probands.
Conclusions: The authors have developed a combined approach for genetic testing of OI, extended the COL1A1/2 mutation spectrum in Chinese OI patients, and confirmed gene diagnosis in a relatively large cohort of OI probands and fetuses.