Genetic Screening for SLE: A Validated qPCR Assay for PTPN22 SNP Polymorphisms

Abstract

Objective: Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease. Multiple factors can trigger this dysregulation of the immune system, including genetic factors such as mutations in the PTPN22 gene. The objective of our study is to develop and validate a quantitative polymerase chain reaction (qPCR) assay for detecting polymorphisms in the PTPN22 gene.
Methodology: Using the PTPN22 gene and SNP rs2476601 sequence, two sets of primers were designed. The first set is for sequencing the partial gene PTPN22, and the second set is for SNP detection using qRT-PCR, focusing on the wild-type allele and two key single nucleotide polymorphisms (SNPs), SNP-G and SNP-T. The qPCR assay was designed with allele-specific probes and primers, and with RNaseP used as an endogenous control. DNA from patient and control populations was used to validate the specificity, sensitivity and reliability of the assay across various parameters.
Results: qPCR assay could discriminate wild-type from mutant alleles with good sensitivity and specificity. In SLE patient samples, the predominant mutation identified was SNP-G while the lowest level was that of SNP-T.
Scope: The developed qPCR assay has the ability to measure polymorphisms in SLE and, therefore this development opens up new vistas to investigate genetic variations conferring susceptibility to SLE as well as complications. A validated qPCR assay described here is an efficient method for studying polymorphisms of PTPN22 with a wide range of applications in genetics research and clinical diagnostics with personalized approaches.