Genetic variants modulate the PTGDR expression

Resumen

Background:Asthma and allergy are com-plex diseases that result from interactionbetween the genetic background and envi-ronmental factors. PTGDR, a receptor ofPGD2 (Prostaglandin D2) has beenreported as a candidate gene in asthma.The goal of this study was to determinethe contribution of different promoter hap-lotype variants toPTGDRgene expressionin cell culture.Method:The A549 cell line was trans-fected with fourPTGDRexpression plas-mids. Wild-typePTGDRcDNA clonedinto a pCMV6-entry vector was the originof thePTGDRconstructs. Using theXhoIandBglII restriction enzymes we replacedthe constitutive cytomegalovirus promoterof pCMV6-PTGDRby one of the fourpromoter sequences ofPTGDRwith thefollowingalternativehomozygous haplotypes:CTCT, CCCT, TCCT andCCCC (positions613,549,441 and197). After 24 h of transient transfection,cells were collected and total RNA wasisolated. Relative quantitative PCR ofPTGDRwas performed using SYBRGreen I Master in a Light Cycler 480instrument. Fold induction was calculatedusing the comparative Ct method. TheGAPDHgene was used as internal control.Results:We found significant differencesin thePTGDRexpression among the hap-lotype variants after transient transfectionanalysis in the A549 cell line. The haplo-type with the CCCC sequence (613C,549C,441C, and197C) showed thehighestPTGDRexpression, while theTCCT haplotype showed a lowest expres-sion.PTGDRexpression after normaliza-tion (meanSD) was expressed as foldincrements, considering the wild variantCTCT as value 1: CCCC: 1.400.08;CCCT: 1.040.12; TCCT: 0.520.04,(P<0.039) for CCCC and TCCT pairwisecomparison by analysis of variance.Conclusion:Our results suggest a differen-tialPTGDRexpression according to thepromoter haplotype combination. In previ-ous reports the CCCC genetic variant alsoexhibit a high promoter activity in reportergene assays and was associated to predis-position to asthma