Development of an ARMS-qPCR Strategy for the Rapid Genetic Diagnosis of Nagashima-Type Palmoplantar Keratoderma

Nagashima-type palmoplantar keratoderma, recently termed as SERPINB7/SERPINA12-palmoplantar epidermal differentiation disorders (pEDD) is the most prevalent palmoplantar keratoderma in East Asia, caused by variants in the SERPINB7 and SERPINA12. Sanger sequencing and next-generation sequencing (NGS) are the gold standard methods for detecting these variants, but their complexity and high cost limit clinical application. This study aimed to develop a rapid, cost-effective, and accurate detection strategy using the amplification refractory variant system-based real-time PCR (ARMS-qPCR) system. Eight key variants were selected: five in SERPINB7 (c.796C>T, c.522dupT, c.650_653delCTGT, c.455G>T, and c.745-553 T>G) and three in SERPINA12 (c.970_971del, c.635-7A>G, c.656A>G). Specific primers for wild-type and mutant sequences were designed based on the ARMS-PCR principle, and reaction conditions were optimized. The ARMS-qPCR strategy was applied to 103 blood samples from SERPINB7/SERPINA12-pEDD patients (n = 53) and healthy controls (n = 50). All samples were validated by Sanger sequencing or NGS, achieving 100% consistency in detecting key variants and an overall accuracy of 93.2% for disease prediction. In conclusion, our ARMS-qPCR strategy provides a rapid, cost-effective, and accurate method for detecting SERPINB7/SERPINA12-pEDD variants, demonstrating significant potential for clinical diagnosis and genetic counseling.