Analysis of Genetic Variation in CD40 and CD40L: Relationship with mRNA Relative Expression and Soluble Proteins in Acute Coronary Syndrome.

Acute coronary syndrome (ACS) can be triggered by the presence of inflammatory factors which promote the activation of immune cells by costimulatory molecules such as CD40 and its ligand CD40L. Environmental and genetic factors are involved in the etiology of the ACS. The aim of this study was to explore the gene and protein expression associated with CD40 and CD40L genetic variants in ACS patients from the western Mexican population. A total of 620 individuals from western Mexico were recruited: 320 ACS patients and 300 individuals without a history of ischemic cardiopathy were evaluated. The genotype was determined using TaqMan SNP genotyping assays. CD40 and CD40L expressions at the mRNA level were quantified using TaqMan Gene Expression Assays. Soluble protein isoforms were measured by enzyme-linked immunosorbent assay. We did not find evidence of association between CD40 (rs1883832, rs4810485, and rs11086998) and CD40L (rs3092952 and rs3092920) genetic variants and susceptibility to ACS, although rs1883832 and rs4810485 were significantly associated with high sCD40 plasma levels. Plasma levels of sCD40L can be affected by gender and the clinical spectrum of acute coronary syndrome. Our results do not suggest a functional role of CD40 and CD40L genetic variants in ACS. However, they could reflect the inflammatory process and platelet activation in ACS patients, even when they are under pharmacological therapy. Due to the important roles of the CD40-CD40L system in the pathogenesis of ACS, longitudinal studies are required to determine if soluble levels of CD40 and CD40L could be clinically useful markers of a recurrent cardiovascular event after an ACS.

The 3'-UTR (CA)n microsatellite on CD40LG gene as a possible genetic marker for rheumatoid arthritis in Mexican population: impact on CD40LG mRNA expression.

The objective of this study was to determine the association of the CD40LG 3'-UTR (CA)n microsatellite with rheumatoid arthritis (RA) and CD40LG mRNA levels in females from western Mexico. A case-control study with 219 RA patients and 175 control subjects (CS) was conducted. Genotyping was performed by polymerase chain reaction (PCR), X 2 test was used to compare genotype and allele frequencies, and odds ratios and 95% confidence intervals were calculated to evaluate the association between RA and the microsatellite. CD40LG mRNA expression was assessed by real-time quantitative PCR. For comparisons between groups, Kruskal-Wallis or Mann-Whitney U tests for non-parametric data and ANOVA test for parametric data were performed. Among the 13 different alleles identified, CA25 was the most represented (45.4% RA and 46.3% CS). Stratification according to CA repeats as CA25 showed a tendency towards a higher frequency of >CA25 alleles in RA patients (29%) compared to CS (23.4%). There was no association between any genotype and the clinical parameters of RA patients. According to the 2-∆∆Cq method, CD40LG mRNA expression in RA patients was 4.5-fold higher compared to CS; this difference was significant when assessed by the 2-∆Cq method (p = 0.028). Compared to carriers of the CA25/CA25 genotype, CS carrying the CA25/>CA25 carriers. The 3'-UTR CD40LG (CA)n microsatellite is not a genetic marker for RA in western Mexican population; however, results suggest that it plays a role in the CD40LG mRNA expression.

Assessment of the rs4340 ACE gene polymorphism in acute coronary syndrome in a Western Mexican population.

Acute coronary syndrome (ACS) is considered one of the main causes of death worldwide. Contradictory findings concerning the impact of the angiotensin-converting enzyme (ACE) gene on cardiovascular diseases have been reported. Previous conclusions point out that the variability in results depends on ethnicity and genetic polymorphisms to determine the association of rs4340 polymorphisms of the ACE gene and ACE circulating levels in ACS. Genotyping of rs4340 polymorphisms was performed in a total of 600 individuals from Western Mexico divided into two groups: the ACS and the control group (CG). The polymorphisms were identified by polymerase chain reaction. Serum ACE concentration was determined by enzyme-linked immunosorbent assay. D/D carriers had higher ACE levels than I/I carriers (3.6 vs 2.8 ng/mL, P < 0.0021) in the CG. The D/D genotype of the rs4340 polymorphism is associated with higher ACE concentration levels; however, the polymorphism was not associated with ACS.

Aberrant expression of interleukin-10 in rheumatoid arthritis: Relationship with IL10 haplotypes and autoantibodies.

Interleukin 10 (IL-10) is an immunomodulatory cytokinethat plays a central rolein the pathogenesis of autoimmune diseases. Different studies consistently show increased IL-10 serum levels in rheumatoid arthritis (RA) and it appears to be caused by genetic variants. Three polymorphisms situated at positions -1082, -819 and -592 of IL10 gene and its major haplotypes have been associated with regulating IL10 promoter activity. In this study, we evaluated whether IL10 haplotypes are associated with mRNA expression and IL-10 serum levels as well as susceptibility to RA in a Western Mexican population. A total of 240 RA patients and 240 control subjects (CS) were included. Genotyping of IL10 polymorphisms was performed by PCR and PCR-RFLP, respectively. IL10 mRNA expression was determined by real-time PCR and IL-10 serum levels were measured using an ELISA kit. IL10 mRNA expression was 50-fold higher in RA patients than CS (p<0.001), while IL-10 serum levels did not show differences between groups. However, high IL-10 serum levels were positively related to a higherseropositivityfor rheumatoid factor (FR) and anti-CCP antibodies (p<0.05). No significant differences between the distribution of haplotype frequencies were observed between both study groups, but GCC haplotype was associated with higher IL-10 serum levels compared with the ACC and ATA haplotypes in RA patients (p<0.05). In addition, patients carrying ATA and GCC haplotypes showed higher mRNA expression than ACC (5.4-fold and 8.8-fold, respectively) and surprisingly, this trend was reversed in the controls, although it was not significant. In conclusion, our findings suggest that IL10 (GCC, ACC, and ATA) haplotypes may not be a susceptibility marker for RA in a population from Western Mexico. Nevertheless, independently of the presence of these variants, there is an aberrant overexpression of IL10 gene in RA, and it may play an important role in the pathogenesis of RA.

CD40 functional gene polymorphisms and mRNA expression in rheumatoid arthritis patients from western Mexico.

The CD40 pathway is involved in the development and pathogenesis of autoimmune diseases, including rheumatoid arthritis (RA). Two single nucleotide polymorphisms (SNPs) in the CD40 gene, rs1883832 and rs4810485, are associated with susceptibility to inflammatory and autoimmune diseases and are thought to alter CD40 expression at the mRNA and protein level. This study assessed for the first time the association of these SNPs with RA and CD40 mRNA levels in a western Mexican population. A total of 278 RA patients and 318 control subjects were included. Genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism, and CD40 mRNA expression was determined by real-time quantitative PCR. No significant differences in genotype and allele frequencies were identified between the RA patients and controls. When stratified by genotype, these SNPs were not found to be associated with the presence of autoantibodies or the clinical activity of the disease. CD40 mRNA levels were elevated 1.5-fold in RA patients compared to control subjects; however, no clear tendencies were observed following stratification by genotype. These results suggest that the CD40 SNPs rs1883832 and rs4810485 are not RA susceptibility markers in the western Mexican population. Further studies are needed to clarify their roles in CD40 mRNA expression.

Association of the FTO gene SNP rs17817449 with body fat distribution in Mexican women.

Polymorphisms in the FTO gene are associated with obesity, body mass index, hip circumference, and visceral and subcutaneous fat area. The objective of this study was to analyze the association of the FTO rs17817449 genetic variant (T>G polymorphism) with body fat distribution patterns in women. We included 65 women and 71 healthy subjects in this study. Anthropometric parameters were determined and laboratory studies were performed. The polymorphism was detected by a PCR-RFLP method. The groups were categorized by type of body fat distribution: gynoid (N = 29) and android (N = 36). We found that the FTO gene polymorphism was not associated with body fat distribution according to the type of obesity (P > 0.05). The contribution of G and T alleles among groups indicated no statistically significant differences between the reference and gynoid group [P = 0.93; odds ratio (OR) = 0.97; 95% confidence interval (CI) = 0.46-2.02] and the reference and android group (P = 0.56; OR = 1.20; 95%CI = 0.54-2.82). Thorax circumference and thorax breast circumference were significantly different between the two groups (P = 0.009 and 0.021, respectively) with the genotype TT. We conclude that the FTO rs17817449 TT genotype predisposes individuals to fat deposition in the thoracic and breast region; individuals carrying this genotype had a decrease in thoracic and breast dimensions indirectly causing the gynoid phenotype in Mexican women.

Macrophage migration inhibitory factor: association of -794 CATT5-8 and -173 G>C polymorphisms with TNF-α in systemic lupus erythematosus.

Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory cytokine associated with the pathogenesis of autoimmune inflammatory diseases. There is evidence that MIF functions in a positive feedback loop with TNF-α that could perpetuate the inflammatory process in systemic lupus erythematosus (SLE). In this case-control study we investigated whether commonly occurring functional MIF polymorphisms are associated with SLE as well as with MIF and TNF-α serum levels in a Mexican-Mestizo population. Genotyping of the -794 CATT5-8 (rs5844572) and -173 G>C (rs755622) MIF polymorphisms was performed by PCR and PCR-RFLP, respectively in 186 SLE patients and 200 healthy subjects. MIF and TNF-α serum levels were determined by ELISA. A significant increase of MIF and TNF-α levels was found in SLE patients. According to a genetic model, we found a significant association of genotypes carrying the -794 CATT7 and -173(∗)C risk alleles with susceptibility to SLE and with a significant increase of TNF-α. In conclusion, MIF gene polymorphisms are associated with SLE susceptibility and with an increase of TNF-α serum levels in a Mexican-Mestizo population.

Genetic contribution of CYP2C9, CYP2C19, and APOE variants in acenocoumarol response.

Oral anticoagulants of the coumarin type have an inconveniently narrow therapeutic window, making their use difficult. In Mexico, genetic variables that participate in the heterogeneity of the therapeutic response remain poorly investigated. With the focus on warfarin, extensive pharmacogenomic studies have been performed, including those on the CYP450 family and APOE. The objective of this study was to determine the contribution of CYP2C9, CYP2C19, and APOE polymorphisms to the variations in response to the doses of acenocoumarol, which is the main anticoagulant prescribed to the Mexican population. The polymerase chain reaction-restriction fragment length polymorphism method was applied to identify 2 and 3 of CYP2C9, 2 of CYP2C19, and APOE variants. The genetic distribution of every polymorphism tested showed high variability when compared with other populations worldwide. Our results showed statistical differences only in the CYP2C19 gene between the 1 1 and 1 2 groups, with effective acenocoumarol doses of 2.56 ± 1.34 mg/day vs 1.35 ± 0.84 mg/day (P = 0.005), respectively. Multiple regression analysis, including patient age and both the CYP2C9 and CYP2C19 genes, showed that these variables explained more than 20% of the dose variations. This is the first report in Mexico searching for the relationship between CYP450 and APOE polymorphisms and the dose requirements of acenocoumarol. Our results suggest that, in the Mexican population, CYP2C19 is more involved in acenocoumarol metabolism than CYP2C9 and APOE. Besides considering the age factor, pharmacogenetic testing for CYP2C19 2 before initiating acenocoumarol treatment could lead to a safer anticoagulation therapy in Mexican patients.

Significance of linkage disequilibrium heterogeneous patterns in the 21q22.3 region for mapping 21 trisomy individuals.

Recombination patterns can be indirectly inferred by means of linkage disequilibrium (LD) estimates, since LD is negatively correlated with genetic distance. However, LD does not necessarily have absolute correspondence with genetic distance. We estimated LD at 5 loci located in the 21q22.3 region. These STRs (D21S1440, D21S168, D21S1260, D21S1446, and D21S1411) covered 8.81 Mb of the 21q22.3 region. They were genotyped by conventional PCR. Similar size samples previously validated by sequencing were used as a genotyping control. Three hundred and sixty-nine individuals (62 families) living in Guadalajara, Mexico, were included. As an inclusion criterion, each family had a positive paternity test by autosomal markers for the CODIS core loci. Two hundred and thirty phase known haplotypes were identified by familial segregation. Only those haplotypes whose frequency was higher than 4% were taken into account for LD estimation, expressed as Lewontin's D' coefficient and Bonferroni's correction P values. For all 5 loci, the genetic distributions were in agreement with Hardy-Weinberg expectations. Heterozygosity and haplotype diversity were ≥ 0.69 and 99.58%, respectively. D21S1440-D21S168 (4.51 cM) and D21S1446-D21S1411 (4.58 cM) marker haplotype frequencies were significantly different from those expected by random distribution. The remaining haplotypes, including those with minimal inter-distance (D21S1260-D21S1446, 1.44 Mb), did not show LD. The 5 STRs at the 21q22.3 region in this Mexican population showed a non-homogeneous LD pattern, which demonstrates that recombination or linkage should not be assumed solely on the basis of genetic distance.

Macrophage migration inhibitory factor (MIF): genetic evidence for participation in early onset and early stage rheumatoid arthritis.

Macrophage migration inhibitory factor (MIF) is an upstream pro-inflammatory cytokine that is associated with the pathogenesis of autoimmune inflammatory diseases including rheumatoid arthritis (RA). Two polymorphisms in the upstream region exist in the MIF gene and are associated with RA susceptibility or severity in different populations. In this case-control study, we investigated whether MIF polymorphisms are associated with RA susceptibility or activity in a western Mexican population .The relationship of MIF levels with clinical features of disease also was assessed. Genotyping of the -794 CATT5-8 (rs5844572) and the -173 G>C (rs755622) polymorphisms was performed by PCR and PCR-RFLP respectively on 226 RA patients and 210 healthy subjects. Serum MIF levels were determined by ELISA. We found a significant association between the -794 CATT5-8 6,7 MIF genotype with RA. Moreover, we detected an association between the -794 CATT7 allele with early onset RA. The -794 CATT7 and -173(*)C alleles, which are in linkage disequilibrium, were associated with high disease activity on RA patients. A positive correlation between circulating MIF levels and C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor, anti-citrullinated protein/peptides antibodies and TNFα was detected. MIF levels appear to be associated with disease progression rather than disease activity, which is distinct from the established relationship between disease activity and TNFα levels. In conclusion, the MIF gene and protein are associated with RA in a western Mexican population, with a main contribution onto early onset and early stages of disease.

The -383A>C TNFRI polymorphism is associated with soluble levels and clinical activity in rheumatoid arthritis.

Tumor necrosis factor-alpha (TNF-alpha) plays a central role in inflammation, and it has been directly implicated in the pathogenesis of rheumatoid arthritis (RA). TNF-alpha activity is mediated through TNFRI and TNFRII cell surface receptors, which act as physiological attenuators of TNF-alpha activity. We recruited 190 RA patients and 190 healthy subjects (HS) in order to associate the -383A>C TNFRI polymorphism with sTNFRI levels and DAS28 score in RA. In results, sTNFRI levels were higher in RA patients than HS (P = 0.04). The -383A>C TNFRI polymorphism did not show significant differences in both studied groups. However, in the RA group the sTNFRI levels were significantly elevated (P = 0.004) in A/A genotype carriers. In addition, the A/A genotype carriers had the higher DAS28 score than A/C genotype (P = 0.02). These data suggest that -383A>C TNFRI polymorphism is not a susceptibility marker in RA, whereas the increased levels of sTNFRI could reflect the clinical activity in RA patients.

Circulating TNFRI and TNFRII levels correlated with the disease activity score (DAS28) in rheumatoid arthritis.

OBJECTIVE: To measure levels of soluble tumour necrosis factor alpha (TNFalpha) receptor type I (sTNFRI) and type II (sTNFRII) in order to correlate them with C-reactive protein (CRP), rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and disease activity score (DAS28) in RA patients. METHODS: We recruited 41 RA patients classified according to American College of Rheumatology (ACR) criteria and 38 healthy subjects (HS). sTNFRI and sTNFRII were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Clinical activity in RA patients was evaluated using the Disease Activity Score using 28 joint counts (DAS28). The statistical analysis was realized using SPSS version 10.0. RESULTS: Soluble TNFRI and TNFRII levels were higher in RA patients (p = 0.04 and 0.001, respectively) than HS. Serum levels of sTNFRI correlated with sTNFRII (r = 0.699, p < 0.0001). sTNFRII correlated with DAS28 (r = 0.375, p = 0.017), RF (r = 0.505, p = 0.004), and ESR (r = 0.323, p = 0.042). CONCLUSION: The increased levels of both sTNFRI and sTNFRII suggest a secondary event related to the inflammatory state observed in RA, whereas the correlation of sTNFRII with RF, ESR, and DAS28 reflects the preferential TNFRII shedding induced by TNFalpha. sTNFRII may be useful as an additional inflammatory marker in RA.