Development of cost-effective and accurate allele-specific PCR for determination of M2/ANXA5 haplotype in recurrent pregnancy loss

Abstract

Repeated Pregnancy Loss (RPL) affects approximately 1 out of 20 pregnant women globally; it is traumatic for parents seeking parenthood with ensuing anxieties for the next pregnancy. M2/ANXA5 haplotype is a hereditary predisposition gene for thrombophilia-associated RPL; the association between M2/ANXA5 haplotype and RPL is further ascertained in a recent meta-analysis. Precision treatments have been proposed for RPL women with M2/ANXA5 haplotype. Therefore, screening for M2/ANXA5 haplotype is highly recommended as a panel of laboratory investigations for idiopathic RPL. To date, direct sequencing of PCR products is the most common method for the determination of M2/ANXA5 haplotype; this method is however tedious, expensive and time- consuming. Hereby, we demonstrate a simple and robust allele-specific PCR (AS-PCR) that detects two inherent SNPs in a single tube, which could serve as a routine genotyping tool for M2/ANXA5 haplotype. This test is rapid, only taking maximum 4 working hours to complete the analysis. Validation of the assay by 105 clinical DNA samples yields 100% concordance rate with the DNA sequencing results.