Cristina Moreno Fajardo

Polymorphisms in Genes Encoding Metalloproteinase 9 and Lymphotoxin- Alpha can Influence Warfarin Treatment

Objectives: Warfarin treatment is influenced by environmental and genetic factors. The influence of polymorphisms in genes encoding metalloproteinase 9 (MMP9), lymphotoxin-alpha (LTA) andTNFSF14 (LIGHT), related to the inflammatory process of coronary artery disease, on warfarin dose and time to reach target was investigated in this study. Methods: Outpatients on warfarin treatment (n=227), 20 to 92 years, were enrolled at the Institute Dante Pazzanese of Cardiology (IDPC). Genomic DNA was obtained from peripheral whole blood to evaluate MMP9 rs17576 (Gln279Arg, A>G), LTA rs1041981 (Thr60Asn, C>A) and rs909253 (c.252T>C) and TNFSF14rs2291668 (c.147C>T) and rs344560 (Lys214Glu, G>A) polymorphisms by pyrosequencing in Q24PyroMark. Results: The patients carrying MMP9 rs17576GG genotype were more likely to require a lower warfarin weekly dose (OR: 2.73, 95% CI: 1.01-7.41, p=0.048). Also, LTA rs909253 variant was associated with a longer time to reach the target international normalized ratio (INR) (OR: 1.98, 95% CI: 1.02-3.86, p=0.043). Age was inversely correlated with the target INR (r=-0.387, p<0.001), and dose was directly correlated with time to reach target INR (r=0.244, p<0.001). Conclusion: MMP9 rs17576 variant may have an important influence on warfarin weekly dose, and that LTA rs909253 polymorphism may also influence the time to reach the target INR.

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.

Influence of SLCO1B1 and SLCO2B1 Polymorphisms on Tacrolimus Pharmacokinetics and Clinical Response

Background: Immunosuppressant such as tacrolimus have narrow therapeutic range and are often associated with increased risk of nefrotoxicity in individuals that receive this drug after renal transplantation. Variants in transporters genes have been associated with variability in plasma concentration of tacrolimus and higher risk of adverse effects. Our aim was to investigate the effect of SLCO1B1 (c.388A>G, c.521T>C) and SLCO2B1 (c.- 71T>C) variants on the efficacy and safety of tacrolimus immunosuppressive therapy in kidney transplant recipients. Methods: SLCO1B1 and SLCO2B1 polymorphisms were detected by TaqMan genotyping and were associated to tacrolimus pharmacokinetics and incidence of acute rejection or diarrhea. Results: Carriers of the allele SLCO1B1 c.388G had lower dose adjusted blood concentration (CO/D) of tacrolimus when compared to 388AA carriers, while SLCO1B1 c.521T>C had no effect. Carriers of CC genotype of SLCO2B1 c.-71T>C SNP had higher CO/D of tacrolimus when compared to TT carriers. When we consider the effect of the haplotype (c.388A>G and c.521T>C) of SLCO1B1 on tacrolimus CO/D and incidence of rejection, carriers of SLCO1B1 *1b haplotype had lower CO/D and lower incidence of rejection when compared to wild type haplotype *1a (p>0.05). Conclusions: SLCO1B1 and SLCO2B1 polymorphisms can contribute for a more safety immunossupressive treatment in kidney recipients.