The application of high-resolution melting analysis in gene mutation screening of osteogenesis imperfecta

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Objective To explore the application of high-resolution melting analysis (HRMA) in gene mutations screening of osteogenesis imperfecta (OI). Methods Clinical data of five children with OI was collected from March to December 2012 in Tianjin Hospital. Blood samples from five children with OI and their parents with a family history of OI were collected as well as normal controls. All exons and their flanking sequences of COL1A1 and COL1A2 gene were screened using PCR-HRMA and validated by the gene sequencing. Results PCR-HRMA showed abnormal results from five children in the COL1A1 gene exon 11, exon 39, exon 8 and the COL1A2 gene exon 19 screening area, respectively. Melting curves of children with OI were differences from normal controls, which showed the mutations of genes. Standard melting curve showed five children with mutations of heterozygous mutation. Sequencing analysis showed that children with COL1A1 gene mutation, c.768dupC, c.2644C> T, c.635G> A and COL1A2 gene mutation c.982G> A, c.948C>T, respectively. COL1A1 gene mutation caused a premature stop codon in children 1, 2 and clinical diagnosis with typeⅠOI. Genetic mutations in children 3, 4 with OI in alpha helix structure domain Gly alternative, and clinical diagnosis with type Ⅳ OI. Children 5 gene mutation was nonsense mutations. This variation is not the cause of OI, Which possible causes need to be researched. Conclusions PCR-HRMA has a low cost, easy operation, fast, high flux, pollution-free advantages. PCR-HRMA is a new effective method for OI mutation screening. The study found a new mutation of COL1A1 gene, c.768dupC. (Chin J Lab Med, 2015, 38: 35-39) Key words: Osteogenesis imperfecta; Collagen type Ⅰ; Collagen type Ⅱ; High resolution melting