Validated high-resolution melting assays for efficient screening of DENND1A, LHCGR, FSHR, THADA, and CYP17A1 polycystic ovary syndrome-associated gene polymorphisms

Introduction: High-resolution melting (HRM) analysis is a fast, sensitive, cost-effective, post-qPCR mutation detection method. HRM analysis represents an advancement over previous DNA dissociation studies, serving to classify DNA samples based on their dissociation behaviour as they melt from dsDNA to ssDNA, utilising special dsDNA saturating fluorescent dyes such as LCGREEN, EVaGreen, and ResoLight. This study aimed to design and validate high-resolution melting assays for the screening of six polycystic ovary syndrome (PCOS) associated single-nucleotide polymorphisms, namely DENND1A (DENN/MADD domain-containing 1A) rs2479106 and rs10986105, THADA (Thyroid Adenoma-Associated Protein) rs13429458, LHCGR (Luteinising Hormone/Choriogonadotropin Receptor) rs13405728, FSHR (Follicle-Stimulating Hormone Receptor) rs6166, and CYP17A1 (Cytochrome P450 17A1) rs743572. Materials and methods: A total of forty-five peripheral blood samples obtained from the infertility clinic at Hospital Sultan Abdul Aziz Shah were collected for gDNA extraction, primers were designed, and optimised for HRM analysis to be conducted on the Light cycler release 1.5.1.62SP3 software. Results: The HRM-predicted genotypes of these target SNPs in all gDNA samples were 100% consistent with Sanger DNA sequencing results, illustrating the accuracy and efficiency of this method for high-throughput SNP genotyping. Conclusion: HRM is an efficient technique for rapidly and effectively screening specific SNPs across a large-scale population study.

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