A PCR-RFLP method for detection of the LNPEP encoding human insulin-regulated aminopeptidase (IRAP) rs4869317 polymorphism.

ABSTRACT

Background & objectives: Insulin regulated aminopeptidase (IRAP) has been related to certain pathologies such as breast cancer, Alzheimer´s disease and septic shock. IRAP is encoded by the leucyl/cystinyl aminopeptidase (LNPEP) gene. The genetic variation in the LNPEP gene has been analyzed in relation with the mortality and vasopressin clearance in septic shock. The LNPEP rs4869317 SNP (single nucleotide polymorphism) was the most significantly associated SNP with vasopressinase activity, being TT genotype associated with increased mortality. The objective of the present study was to develop a simple method to allow a quick and affordable genotyping for the rs4869317 SNP of LNPEP gene. Methods: Blood DNA samples were obtained from randomly selected healthy volunteers (n=28). A pair of primers was designed to amplify an 834 bp region of the LNPEP gene containing the rs4869317 SNP. The two alleles (T or A) were detected by digestion of the PCR products with the PacI restriction endonuclease. This enzyme only cuts the PCR products when the adenine is present in the SNP. Results: All individuals showed RFPL (restriction fragment length polymorphism) fragments for the expected genotypes (TT, TA or AA). The methodology was validated by sequencing of the amplified DNAs from several ‘T/T’ and ‘A/A’ homozygotes and ‘T/A’ heterozygotes. The results from both methods showed agreement. Interpretation & conclusions: The PCR-RFLP is a simple and reliable method that allows a quick genotyping for the rs4869317 SNP of LNPEP gene. The study of this polymorphism could be useful in future investigations to analyze the role of genetic variants of IRAP in several physiological/pathological conditions.