Paola Bolli

Genetic analysis of 56 polymorphisms in 17 genes involved in methionine metabolism in patients with abdominal aortic aneurysm

Background: Previous studies suggested an association between abdominal aortic aneurysm (AAA) and hyperhomocysteinaemia, a complex trait determined by genetic and environmental factors. Our hypothesis was that polymorphisms in genes directly or indirectly involved in methionine metabolism may contribute to AAA susceptibility. Method: We studied 56 polymorphisms in MTHFR, MTR, MTRR, CBS, MTHFD1, SLC19A1, NNMT, TCN2, AHCY, BHMT, BHMT2, FOLH1, TYMS, ENOSF1, SHMT1, PON1, PON2 genes according to their demonstrated/putative function, localisation in promoter or regulatory and coding regions and/or heterozygosity values >0.300. Polymorphisms were evaluated by using a primer extension based microarray technology in 423 AAA patients and 423 matched controls. Results: All polymorphisms resulted in Hardy–Weinberg equilibrium in patients and controls. At the multiple logistic regression analysis adjusted for traditional cardiovascular risk factors (sex, age, hypertension, smoking habit, dyslipidaemia, diabetes) and chronic obstructive pulmonary disease (COPD), rs8003379 MTHFD1 (odds ratio (OR) 0.41, 95% confidence interval (CI) 0.26 to 0.65) and rs326118 MTRR (OR 0.47, 95% CI 0.29 to 0.77) polymorphisms resulted in independent susceptibility factor for AAA. Conclusions: After haplotype reconstruction, logistic regression analyses adjusted for traditional risk factors and COPD showed a significant association among AAA and AHCY, FOLH1, MTHFD1, MTR, NNMT, PON1 and TYMS haplotypes. Our findings offer new insights into the pathogenesis of AAA.

Polymorphisms of genes involved in extracellular matrix remodeling and abdominal aortic aneurysm

BACKGROUND Abdominal aortic aneurysm (AAA) has a multifactorial etiology and the relevance of genetic factors is getting increasing interest, in particular those related to the destructive remodeling of extracellular matrix. METHODS We performed a candidate gene association study of polymorphisms in genes coding matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), and elastin (ELN) in AAA. DNA samples from 423 AAA patients and 423 controls were genotyped for 12 polymorphisms in 10 genes: MMP1 (-1607G/GG), MMP2 (-735C/T; -1306C/T; -1575 G/A), MMP3 (5A/6A), MMP9 (-1562C/T), MMP10 (A180G), MMP-12 (-82A/G), MMP-13 (-77A/G), TIMP1 (C434T), TIMP3 (-1296T/C), and ELN (G1355A). RESULTS Genotype distribution was significantly different between patients and controls for the following polymorphisms: -1306C/T MMP2; 5A/6A MMP3; -77A/G MMP-13; G1355A ELN; and C434T TIMP1. In a multivariable logistic regression analysis adjusted for traditional cardiovascular risk factors and chronic obstructive pulmonary disease, -1306C/T MMP2 (odds ratios [OR] = 0.55 [95% confidence interval, CI .34-.85], P < .007) and G1355A ELN (OR = 0.64 ([95% CI .41-.99], P = .046) polymorphisms resulted in independent protective factors for abdominal aortic aneurysm (AAA), whereas 5A/6A MMP3 (OR = 1.82 [95% CI 1.04-3.12], P = .034) and -77 A/G MMP-13 (OR = 2.14 [95% CI 1.18-3.86], P = .012) polymorphisms resulted in independent risk factors for AAA. In a multivariable logistic regression analysis adjusted for traditional cardiovascular factors and chronic obstructive pulmonary disease, the prevalence of the contemporary presence of three or four genetic risk conditions was a strong and independent determinant of AAA disease (OR = 2.96, 95% CI 1.67-5.24, P < .0001). For those polymorphisms independently associated with AAA in this study (-1306C/T MMP2, 5A/6A MMP3, -77A/G MMP-13, and G1355A ELN polymorphisms), we performed a meta-analysis of the available data (this paper and literature data). We found a significant association with an increased risk of AAA for MMP3 (AAA patients n = 1258, controls n = 1406: OR = 1.48 [95% CI = 1.23-1.78], I(2) = 0%) and MMP-13 (AAA patients n = 800, controls n = 843: OR = 1.37 [95% CI = 1.04-1.82], I(2) = 25%) polymorphisms and a trend that did not reach the statistical significance, toward a decreased risk of AAA for MMP2 (AAA patients n = 1090, controls n = 1077: OR = 0.83 [95% CI = .60-1.15], I(2) =7 1%) and ELN (AAA patients n = 904, controls n = 1069: OR = 0.79 [95% CI = .53-1.18], I(2) = 72%) polymorphisms. CONCLUSIONS These findings suggest that polymorphisms in MMP2, MMP3, MMP-13, and ELN genes may independently contribute to the pathogenesis of AAA.

Early-onset ischaemic stroke: Analysis of 58 polymorphisms in 17 genes involved in methionine metabolism

The hypothesis underlying this study is that variations in genes involved in methionine metabolism may contribute to genetic susceptibility for early-onset ischaemic stroke. We investigated 58 polymorphisms in AHCY, BHMT, BHMT2, CBS, ENOSF1, FOLH1, MTHFD1, MTHFR, MTR, MTRR, NNMT, PON1, PON2, SLC19A1, SHMT1, TCN2, TYMS genes on genomic DNA from 501 young patients who survived ischaemic stroke and 1,211 sex and age comparable controls. Genotype distribution was significantly different between patients and controls for the following SNPs: rs10037045 BHMT, rs682985 BHMT2, rs1051319 CBS, rs202680 FOLH1, rs2274976 MTHFR, rs1979277 SHMT1, rs20721958 TCN2. On multiple logistic regression analysis adjusted for traditional risk factors, rs10037045 BHMT, rs682985 BHMT2, rs1051319 CBS, and rs202680 FOLH1 remained independent risk factors for stroke. After haplotype reconstruction, generalised linear model analyses adjusted for traditional risk factors and using the FDR multiple testing correction showed significant associations between ischaemic stroke and BHMT, CBS, FOLH1, MTR, PON2, TCN2 and TYMS haplotypes. This study identifies significant genetic associations between premature ischaemic stroke and haplotypes in BHMT, CBS, FOLH1, MTR, PON2, TCN2 and TYMS genes involved in methionine metabolism.

Atherosclerotic and thrombophilic risk factors in patients with recurrent central retinal vein occlusion

Purpose Atherosclerotic and thrombophilic risk factors may be causes of central retinal vein occlusion (CRVO). The aim of this study was to evaluate the prevalence of the aforesaid risk factors in patients with recurrent CRVOs and patients with a single episode of CRVO. Methods Seventeen patients with recurrent CRVO and 30 with a single episode of CRVO were enrolled. The atherosclerotic risk factors investigated were hypertension, diabetes, smoking, and dyslipidemia. Specific laboratory tests for the following thrombophilic markers were performed: homocystinemia (Hcy), lipoprotein (a), factor VIII, factor II G20210A and factor V G1691A polymorphisms, lupus anticoagulant, anticardiolipin antibodies, plasminogen activator inhibitor-1, and deficit of vitamins B6, B12, and folic acid. A multivariate analysis, adjusted for age, gender, traditional and thrombophilic risk factors, was performed. Statistical significance was set at pp≤0.05. Results Hypercholesterolemia, hypertriglyceridemia, fasting, and postmethionine hyperhomocysteinemia (HHcy) were more prevalent in recurrent CRVO patients (pp<0.001, pp<0.001, p=0.006, and p=0.005, respectively). At multivariate analysis, hypercholesterolemia (OR: 5.04, 95% CI 1.39–18.17; p=0.025), hypertriglyceridemia (OR: 5.60, 95% CI 1.52–20.61; p=0.017), fasting HHcy (OR: 5.77, 95% CI 1.39–23.89; p=0.028), and postmethionine HHcy (OR: 10.88, 95% CI 2.50–47.42; p=0.002) were found to be significantly associated with recurrent CRVO. Conclusions Dyslipidemia and hyperhomocysteinemia are independent risk factors for the occurrence of recurrent CRVO. A complete assessment of atherosclerotic and thrombophilic risk factors is recommended in CRVO patients. In addition, the need for a specific treatment is suggested.

Role of haemorheological factors in patients with retinal vein occlusion

Retinal vein occlusion (RVO) is an important cause of permanent visual loss. Hyperviscosity, due to alterations of blood cells and plasma components, may play a role in the pathogenesis of RVO. Aim of this case-control study was to evaluate the possible association between haemorheology and RVO. In 180 RVO patients and in 180 healthy subjects comparable for age and gender we analysed the whole haemorheological profile: [whole blood viscosity (WBV), erythrocyte deformability index (DI), plasma viscosity (PLV), and fibrinogen]. WBV and PLV were measured using a rotational viscosimeter, whereas DI was measured by a microcomputer-assisted filtrometer. WBV at 0.512 sec(-1) and 94.5 sec(-1) shear rates as well as DI, but not PLV, were found to be significantly different in patients as compared to healthy subjects. At the logistic univariate analysis, a significant association between the highest tertiles of WBV at 94.5 sec(-1) shear rate (OR: 4.91, 95% CI 2.95-8.17; p < 0.0001), WBV at 0.512 sec(-1) shear rate (OR: 2.31, 95% CI 1.42-3.77; p < 0.0001), and the lowest tertile of DI (OR: 0.18, 95% CI 0.10-0.32; p < 0.0001) and RVO was found. After adjustment for potential confounders, the highest tertiles of WBV at 0.512 sec(-1) shear rate (OR: 3.23, 95% CI 1.39-7.48; p = 0.006), WBV at 94.5 sec(-1) shear rate (OR: 6.74, 95% CI 3.06-14.86; p < 0.0001) and the lowest tertile of DI (OR: 0.20,95% CI 0.09-0.44, p < 0.0001) remained significantly associated with the disease. In conclusion, our data indicate that an alteration of haemorheological parameters may modulate the susceptibility to the RVO, by possibly helping to identify patients who may benefit from haemodilution.

High-throughput multiplex single-nucleotide polymorphism (SNP) analysis in genes involved in methionine metabolism.

Hyperhomocysteinemia is a well-known independent marker factor for atherothrombotic diseases and may result from acquired and genetic influences. Several polymorphisms are suspected to be associated with hyperhomocysteinemia, but data are limited and inconsistent. High-throughput genotyping technologies, such as GenomeLab SNPStream, are now available. Moreover, an appropriate selection of SNPs to be analyzed could represent a strong resource to define the role of genetic risk factors. We developed a multiplex PCR-oligonucleotide extension approach by GenomeLab platform. We selected 72 SNPs based on their putative function and frequency in the candidate genes AHCY, BHMT, BHMT2, CBS, ENOSF1, FOLH1, MTHFD1, MTHFR, MTR, MTRR, NNMT, PON1, PON2, SLC19A1, SHMT1, TCN2, and TYMS. We were able to analyze 57 of the SNPs (79%). For MTHFR C677T and A1298C and MTR A2756G SNPs, we compared data obtained with an electronic microchip technology and found 99.2% concordance. We also performed a haplotype analysis. This approach could represent a useful tool to investigate the genotype–phenotype correlation and the association of these genes with hyperhomocysteinemia and correlated diseases.

Platelet aggregability is modulated by eNOS locus in non-type 2 diabetic patients with acute coronary syndrome

BACKGROUND AND AIM Platelet nitric oxide (NO) synthesis is compromised in patients with acute coronary syndrome (ACS), and platelet NO availability may be critically relevant in determining the extent of thrombosis in ACS patients. It has been demonstrated that an impaired responsiveness to the antiaggregatory effects of NO may affect platelet dysfunction in diabetic patients with ACS. Since NO availability may be genetically determined, we have investigated the role of endothelial nitric oxide synthase (eNOS) gene in influencing platelet aggregability in relation to the presence (n=247) or absence (n=883) of type 2 diabetes in ACS patients. METHODS AND RESULTS We have genotyped 1130 consecutive high risk ACS patients on dual antiplatelet therapy, previously investigated in relation to platelet function. eNOS 4a allele frequency was significantly higher in diabetic vs. non-diabetic patients (p=0.02). In non-diabetic patients the eNOS 4a allele significantly modulated platelet aggregability in response to arachidonic acid (AA), but not to collagen and adenosine diphosphate (ADP) stimulus, after Bonferroni correction for multiple testing. After adjustment for age, gender, smoking habit, hypertension and ejection fraction ≤40%, the eNOS 4a allele remained significantly and independently associated with platelet aggregability in response to AA stimulus [β (SE)=0.17 (0.07), p=0.01]. When platelet aggregation values were considered according to the presence or absence of high residual platelet reactivity (RPR) eNOS 4a, but not -786C and 894T, allele was significantly associated with RPR by AA stimulus. The haplotype reconstruction analysis for eNOS gene showed that the -786C/894G/4a and -786C/894G/4b haplotypes significantly influenced platelet aggregation after AA stimulus. CONCLUSIONS Our study indicates that eNOS 4a allele, may be a determinant of higher platelet aggregability and residual platelet reactivity in non-diabetic ACS patients.

eNOS gene influences platelet phenotype in acute coronary syndrome patients on dual antiplatelet treatment.

Nitric Oxide (NO) plays a relevant role in regulating platelet recruitment and eNOS is the major isoform known to be expressed in platelets. Polymorphisms in the eNOS gene with a reduced NO availability might affect platelet phenotype. The aim of our study was to evaluate the role of eNOS–786T > C, 894G > T and 4a/4b polymorphisms in modulating platelet phenotype in 1442 acute coronary syndrome (ACS) patients on dual antiplatelet therapy, previously investigated in relation to platelet function. Platelet aggregation on platelet-rich plasma after collagen (2 µg/mL), ADP (10 µM) and arachidonic acid (AA) (1 mM) stimuli and the genetic analysis of eNOS polymorphisms were assessed. In subjects carrying the eNOS 4a and -786C alleles a significantly higher maximal platelet aggregation value after AA was found (p = 0.02 and p = 0.047, respectively). eNOS 4a but not -786C allele weakly influenced platelet aggregation after collagen stimulus (p = 0.05). eNOS 4a allele significantly and independently influenced AA-induced platelet aggregation (p = 0.01). A significantly higher percentage of patients with AA-induced high residual platelet reactivity (RPR) was found in subjects carrying both eNOS 4a and -786C allele (p = 0.03 and p = 0.04, respectively). At logistic multivariate analysis, the eNOS 4a allele significantly influenced the AA-induced high residual platelet reactivity (p = 0.02). This study evidences a role for eNOS gene in moderately, but significantly, modulating platelet phenotype in a high-risk population on dual antiplatelet treatment.

Improvement in ACE I/D polymorphism detection.

The ACE I/D polymorphism has been reported to influence predisposition to cardiovascular disease. Conflicting results in its detection may be due to mistyping of I/D genotypes as D/D genotypes occurring in the traditional genotyping method. In order to resolve mistyping troubles and to permit a rapid and accurate analysis, we performed a stepdown PCR reaction followed by detection using Nanogen technology, and we compared these results with those obtained from traditional genotyping methods, such as conventional and confirmatory PCR. The Nanogen stepdown method showed a 100% sensitivity and 99.6% specificity, when compared with the confirmatory PCR. Our experiments provide evidence that, by using the Nanogen stepdown method, the DD mistyping was markedly decreased, thus representing a useful tool suitable for performing large-scale screening or research.