Validation of a quantitative PCR-high-resolution melting protocol for simultaneous screening ofCOL1A1andCOL1A2point mutations and large rearrangements: Application for diagnosis of osteogenesis imperfecta

Abstract

Osteogenesis imperfecta (OI) is a connective tissue disorder mostly characterized by autosomal dominant inheritance. Over 1,100 causal mutations have been identified scattered along all exons of genes encoding type I collagen precursors, COL1A1 and COL1A2. Because of the absence of mutational hotspots, Sanger sequencing is considered the gold standard for molecular analysis even if the workload is very laborious and expensive. To overcome this issue, different prescreening methods have been proposed, including DHPLC and biochemical studies on cultured dermal fibroblasts; however, both approaches present different drawbacks. Moreover, in case of patients who screen negative for point mutations, an additional screening step for complex rearrangements is required; the added causative variants expected from this approach are about 1-2%. The aim of this study was to optimize and validate a new protocol that combines quantitative PCR (qPCR) and high-resolution melting (HRM) curve analysis to reduce time and costs for molecular diagnosis. Results of qPCR-HRM screening on 57 OI patients, validated by DHPLC-direct sequencing and multiplex ligation-dependent probe amplification (MLPA), indicate that all alterations identified with the mentioned methodologies are successfully detected by qPCR-HRM. Moreover, HRM was able to discriminate complex genotypes and homozygous variants. Finally, qPCR-HRM outperformed direct sequencing and DHPLC-MLPA in terms of rapidity and costs.

Genetic analysis of osteogenesis imperfecta in the Palestinian population: molecular screening of 49 affected families

“…The phenotypes of patients with COL1A1 and COL1A2 variants (Table ) were in line with previously reported genotype–phenotype correlations, with haploinsufficiency resulting in milder phenotypic abnormalities and pathogenic missense variants causing more severe phenotypes (Gentile et al. ; Vandersteen et al. ).…”

“…The phenotypes of patients with COL1A1 and COL1A2 variants (Table 1) were in line with previously reported genotype-phenotype correlations, with haploinsufficiency resulting in milder phenotypic abnormalities and pathogenic missense variants causing more severe phenotypes (Gentile et al 2012;Vandersteen et al 2014). However, the novel splice site variant c.3531+1G>T is associated with moderate phenotypic features, which is at odds with an earlier report that this variant resulted in mild OI (Willing et al 1994).…”

COL1‐related overlap disorder: A novel connective tissue disorder incorporating the osteogenesis imperfecta/Ehlers‐Danlos syndrome overlap

“…Among variants affecting COL1A1 , three [c.326G > A p.(Gly109Asp), c.581G > C p(Gly194Ala), c.670G > A p.(Gly224Ser)] fell within or near the procollagen N‐proteinase cleavage site, whereas two [c.1243C > T p.(Arg415*), c.2073delT p.(Gly962Valfs*74)] did not (Figure L) . The c.581G > C p(Gly194Ala) and c.2073delT p.(Gly962Valfs*74) were previously reported in OI type I patients, whereas c.1243C > T p.(Arg415*) was first reported in 1996 and has been associated with OI type I, III/IV and IV in the Leiden Online Variants Database (LOVD). The c.326G > A p.(Gly109Asp) and c.670G > A p.(Gly224Ser) were unpublished (Results S1 and S2; Figures S1 and S2).…”

“…(Gly962Valfs*74) were previously reported in OI type I patients, 17,18 whereas c.1243C > T p.(Arg415*) was first reported in 1996 19 and has been associated with OI type I, III/IV and IV in the Leiden Online Variants Database (LOVD). The c.326G > A p.(Gly109Asp) and c.670G > A p.(Gly224Ser) were unpublished (Results S1 and S2; Figures S1 and S2).…”

Ethyl methanesulfonate mutant library construction in Gossypium hirsutum L. for allotetraploid functional genomics and germplasm innovation

“…Due to the differences between wild-type and EMS mutant samples, such as gene length and the mutation content and distribution of GC base pairs, the melting curve shape due to each duplex having a characteristic melting temperature after polymerase chain reaction (PCR) amplification can show mutations at the base level (Reed and Witter, 2004;Montgomery et al, 2007). Moreover, HRM has many advantages, such as eliminating the risk of contamination during scanning, having a moderate price, and reducing processing time (Gentile et al, 2012). Therefore, this technology was used to detect EMS mutants in cotton in this study.…”