Katarzyna Linkowska

Phenotyping vs. genotyping for prediction of clopidogrel efficacy and safety: the PEGASUS-PCI study

Summary.  Background: Prognostic values of genotyping and phenotyping for assessment of clopidogrel responsiveness have been shown in independent studies.Objectives: To compare different assays for prediction of events during long‐term follow‐up.Methods: In this prospective cohort study polymorphisms of CYP2C19*2 and CYP2C19*17 alleles, vasodilator‐stimulated phosphoprotein phosphorylation (VASP) assay, multiple electrode aggregometry (MEA), cone and platelet analyser (CPA) and platelet function analyser (PFA‐100) were performed in 416 patients undergoing percutaneous coronary intervention. The rates of events were recorded during a 12‐month follow‐up.Results: Platelet aggregation by MEA predicted stent thrombosis (2.4%) better (c‐index = 0.90; P < 0.001; sensitivity = 90%; specificity = 83%) than the VASP assay, CPA or PFA‐100 (c‐index < 0.70; P > 0.05; sensitivity < 70%; specificity < 70% for all) or even the CYP2C19*2 polymorphism (c‐index < 0.56; P > 0.05; sensitivity = 30%; specificity = 71%). Survival analysis indicated that patients classified as poor responders by MEA had a substantially higher risk of developing stent thrombosis or MACE than clopidogrel responders (12.5% vs. 0.3%, P < 0.001, and 18.5% vs. 11.3%, P = 0.022, respectively), whereas poor metabolizers (CYP2C19*1/*2 or *2/*2 carriers) were not at increased risks (stent thrombosis, 2.7% vs. 2.5%, P > 0.05; MACE, 13.5% vs. 12.1%, P = 0.556). The incidence of major bleedings (2.6%) was numerically higher in patients with an enhanced vs. poor response to clopidogrel assessed by MEA (4% vs. 0%) or in ultra‐metabolizers vs. regular metabolizers (CYP2C19*17/*17 vs. CYP2C19*1/*1; 9.5% vs. 2%). The classification tree analysis demonstrated that acute coronary syndrome at hospitalization and diabetes mellitus were the best discriminators for clopidogrel responder status.Conclusions: Phenotyping of platelet response to clopidogrel was a better predictor of stent thrombosis than genotyping.

Analysis of LOXL1 single nucleotide polymorphisms in Polish population with pseudoexfoliation syndrome

Acta Ophthalmol. 2011: 89: e64–e66

Interplay between Genetic and Clinical Variables Affecting Platelet Reactivity and Cardiac Adverse Events in Patients Undergoing Percutaneous Coronary Intervention

Several clinical and genetic variables are associated with influencing high on treatment platelet reactivity (HTPR). The aim of the study was to propose a path model explaining a concurrent impact among variables influencing HTPR and ischemic events. In this prospective cohort study polymorphisms of CYP2C19*2, CYP2C19*17, ABCB1, PON1 alleles and platelet function assessed by Multiple Electrode Aggregometry were assessed in 416 patients undergoing percutaneous coronary intervention treated with clopidogrel and aspirin. The rates of major adverse cardiac events (MACE) were recorded during a 12-month follow up. The path model was calculated by a structural equation modelling. Paths from two clinical characteristics (diabetes mellitus and acute coronary syndrome (ACS)) and two genetic variants (CYP2C19*2 and CYP2C19*17) independently predicted HTPR (path coefficients: 0.11 0.10, 0.17, and -0.10, respectively; p<0.05 for all). By use of those four variables a novel score for prediction of HTPR was built: in a factor-weighted model the risk for HTPR was calculated with an OR of 3.8 (95%CI: 3.1–6.8, p<0.001) for a score level of ≥1 compared with a score of <1. While MACE was independently predicted by HTPR and age in the multivariate model (path coefficient: 0.14 and 0.13, respectively; p<0.05), the coexistence of HTPR and age ≥75 years emerged as the strongest predictor of MACE. Our study suggests a pathway, which might explain indirect and direct impact of variables on clinical outcome: ACS, diabetes mellitus, CYP2C19*2 and CYP2C19*17 genetic variants independently predicted HTPR. In turn, age ≥75 years and HTPR were the strongest predictors of MACE.

Mitochondrial DNA Polymerase γ Mutations and Their Implications in mtDNA Alterations in Colorectal Cancer

Mitochondrial DNA was found to be highly mutated in colorectal cancer cells. One of the key molecules involved in the maintenance of the mitochondrial genome is the nuclear‐encoded polymerase gamma. The aim of our study was to determine if there is a link between polymorphisms within the polymerase gamma gene (POLG) and somatic mutations within the mitochondrial genome in cancer cells. We investigated POLG sequence variability in 50 colorectal cancer patients whose complete mitochondrial genome sequences were determined. Relative mtDNA copy number was also determined. We identified 251 sequence variants in the POLG gene. Most of them were germline‐specific (∼92%). Twenty‐one somatic changes in POLG were found in 10 colorectal cancer patients. We have found no association between the occurrence of mtDNA somatic mutations and the somatically occurring variants in POLG. MtDNA content was reduced in patients carrying somatic variants in POLG or germline nucleotide variants located in the region encoding the POLG polymerase domain, but the difference did not reach statistical significance. Our findings suggest that somatic mtDNA mutations occurring in colorectal cancer are not a consequence of somatic mutations in POLG. Nevertheless, POLG nucleotide variants may lead to a decrease in mtDNA content, and consequently result in mitochondrial dysfunction.

Association between Genetic Variants on Chromosome 15q25 Locus and Several Nicotine Dependence Traits in Polish Population: A Case-Control Study

Tobacco smoking continues to be a leading cause of disease and mortality. Recent research has confirmed the important role of nicotinic acetylcholine receptor (nAChR) gene cluster on chromosome 15q 24-25 in nicotine dependence and smoking. In this study we tested the association of smoking initiation, age at onset of daily smoking, and heaviness of smoking with five single nucleotide polymorphisms (SNPs) within the CHRNA5-CHRNA3-CHRNB4 cluster. The group of 389 adult subjects of European ancestry from the north of Poland, including 212 ever (140 current and 72 former) and 177 never smokers with mean age 49.26, was genotyped for rs16969868, rs1051730, rs588765, rs6495308, and rs578776 polymorphisms. Distributions of genotypes for rs16969868 and rs1051730 were identical so they were analyzed together. Further analysis revealed the association between rs16969868-1051730 (OR = 2.66; 95% CI: 1.30–5.42) and number of cigarettes smoked per day (CPD) with heaviness of nicotine addiction measured by the Fagerström Test for Nicotine Dependence (FTND) (OR = 2.60; 95% CI: 1.24–5.43). No association between these polymorphisms and other phenotypes was found. Similarly, the association between rs588765, rs6495308, rs578776, and analyzed phenotypes was not confirmed. This study provides strong evidence for the role of the CHRNA5-CHRNA3-CHRNB4 cluster in heaviness of nicotine addiction.

Analysis of SOD1 polymorphisms in Polish population with pseudoexfoliation syndrome

Editor, T he incidence of pseudoexfoliation (PEX) syndrome varies among ethnic groups – from 0% in Greenland Eskimos to 25% in the Scandinavian countries (Challa et al. 2008; Arnarsson 2009). Three single-nucleotide polymorphisms (SNPs) in the lysyl oxidase-like 1 gene (LOXL1) have been confirmed to be associated with PEX in our previous study (Malukiewicz et al. 2011). In a recent investigation, an association between PEX and two SNPs, rs2107856 and rs2141388 of the contactin-associated protein-like 2 encoded by the CNTNAP2 gene located in intron 11 was found in Germans but not in Italians (Krumbiegel et al. 2011). We investigated the association between the SNPs rs2107856 and rs2141388 and PEX in Polish population. We studied 48 patients (16 men and 32 women), mean age 75 (SD = 7) with PEX and no other ocular diseases, for example, glaucoma. Control group comprised 30 healthy subjects (11 men and 19 women), mean age 76 (SD = 8). Pseudoexfoliation changes were identified as the presence of typical PEX material on the anterior lens surface, iris or corneal endothelium in either eye. Genomic DNA was extracted from blood as described previously (Malukiewicz et al. 2011). Genotypes of the SNPs rs2107856 and rs2141388 were determined using a commercially available TaqMan genotyping assays with the ABI Prism Sequence Detector 7000 (Applied Biosystems, Foster City, CA, USA). Correctness of genotyping was evaluated by direct sequencing for randomly selected samples. The Arlequin software version 3.1. was used to determine the Hardy– Weinberg equilibrium and estimate haplotype frequencies. The Fisher exact test was performed to compare patient and control groups for possible associations between allele frequency and disease state. Odds ratios were also calculated. The significance level for all statistical tests was 0.05. Statistical analysis was performed using Statistica software (version 8). The genotype distribution of rs2107856 and rs214138 was found in Hardy–Weinberg equilibrium in both groups. The allele frequencies in patients with PEX sample were not significantly different from those in the control group, for either rs2107856 (p = 0.38) or rs214138 (p = 0.38) (Table 1). Linkage disequilibrium analysis gave the value of r = 1 which proved that both SNPs are perfectly correlated to each other. There were no significant differences in haplotype frequencies between PEX and control samples. The haplotype (GC), present in 72% of cases and 65% of controls, confers a 1.38-fold of increased disease risk (95% CI, 0.685–2.765; p = 0.367). Krumbiegel et al. (2011) detected a significant association of both SNPs and their haplotype TT with PEX ⁄ PEXG in German but not the Italian cohort. They reported that the TT haplotype confers risk of the disease of 1.42 for German population. In our study, the minor T allele frequency is 0.28 in cases and 0.35 in controls. While the T frequency in our cases is the same as in Krumbiegel et al. (2011), the frequency in controls is higher (0.35 compared to 0.224), and this difference is statistically significant (p = 0.0094). The main cause of these conflicting observations can be either relatively small sizes of Polish samples or genetic heterogeneity of European populations. It was shown that the pronounced population differentiation at the level of haploid markers exists between the two geographically neighbouring countries, Poland and Germany (Kayser et al. 2005). In this respect, it should be emphasized that our study has only 18% power to detect the association, because of the small sample size. These findings strongly support the need for both replication of the CNTNAP2 SNPs association studies with PEX in Polish and other groups of larger sizes and further research into hidden population substructure which may affect the case–control data.

Searching for association of the CAG repeat polymorphism in the mitochondrial DNA polymerase gamma gene (POLG) with colorectal cancer

Mitochondrial DNA polymerase gamma (POLG) is the only DNA polymerase involved in maintaining the mitochondrial genome. Recent studies demonstrated an association of CAG repeat polymorphism in the second exon of POLG gene with the risk of cancer. We investigated the CAG repeat variability in the POLG gene in tumor and non-tumor tissues from colorectal cancer patients and in DNA samples isolated from blood obtained from age-matched healthy persons. Somatically occuring CAG-repeat alterations in cancer tissues have been observed in 10% of patients, but no association has been found between the CAG repeat variants in the POLG gene and colorectal cancer risk.

The Association of Apolipoprotein E Gene Polymorphism With Cognitive Performance in Nondemented Polish Adults Aged 55 to 75

The ε4 allele of the apolipoprotein E (APOE) gene is known as a risk factor for dementia. How APOE ε polymorphism affects cognitive performance in nondemented aging subjects remains less clear. In this study, the relationship between APOE status and cognitive performance across various cognitive domains in adults aged 55 to 75 years (n = 74) without dementia was investigated. E4 carriers (n = 11) performed worse versus noncarriers on forward Digit Span and delayed recall of the Rey-Osterrieth complex figure. General linear model analysis revealed a small but significant main effect of ε4 on Rey-Osterrieth complex figure delayed recall. Comparing ε2 carriers, ε3 homozygotes, and ε4 carriers, ε3/ε3 performed significantly better on Trail Making Test part B and derived score Trail Making Test B-A. The findings support the relation between the APOE ε polymorphism and visual memory, short-term auditory memory, visuospatial attention, and executive functions in an aging sample without dementia.

Evaluation of TGF-Beta 2 and VEGFα Gene Expression Levels in Epiretinal Membranes and Internal Limiting Membranes in the Course of Retinal Detachments, Proliferative Diabetic Retinopathy, Macular Holes, and Idiopathic Epiretinal Membranes

Purpose To evaluate the expression profiles of the VEGFα and TGFβ in the ERMs and ILMs in retinal disorders. Methods In this nonrandomized prospective study, 75 patients (34 females and 41 males) referred to pars plana vitrectomy (PPV) due to different retinal diseases were enrolled to the study. The samples of ERMs and ILMs collected during PPV were immediately put in TRIzol® Reagent (Life Technologies, USA) and stored at −70°C until RNA extraction. Gene expression analysis was done with TaqMan® Gene Expression Assays (Applied Biosystems, USA) following the manufacturers instructions. Results The gene expression levels of VEGFα as well as of TGFβ2 were significantly higher in ERMs than in ILMs in all studied groups. The level of TGFβ2 expression exhibits a significantly lower values in iERMs as compared with the RRD group (p = 0.043). There were differences in TGFβ2 expression in ILM in groups studied: DR versus RRD, p = 0.003; DR versus iERM, p = 0,047; and iERM versus RRD, p = 0.004. Conclusions Our results revealed that factors associated with angiogenesis and wound healing processes in eyes with RRD, PDR, iERM, and MH were more upregulated in ERMs than in ILMs. This may indicate that ILM is not responsible for reproliferation and its peeling should be avoided in routine PPV.

An Analysis of the Association between Epilepsy-Related Genes and Vertigo in the Polish Population

Considering the possibility of a common genetic background of vertigo and epilepsy, we genotyped an affected group of individuals with vertigo and an unaffected group, by studying 26 single-nucleotide polymorphisms (SNPs) in 14 genes which were previously reported to be of particular importance for epilepsy. Significant differences were found between the patients and the control group (χ2 = 38.3, df = 3, p = 1.6 × 10–7) for the frequencies of haplotypes consist ing of 2 SNPs located in chromosome 11 (rs1939012 and rs1783901 within genes MMP8 and SCN3B, respectively). The haplotype rs1939012:C-rs1783901:A, consisting of the minor-frequency alleles was found to be associated with a higher risk of vertigo (OR = 5.0143, 95% CI = 1.6991–14.7980, p = 0.0035). In contrast, the haplotype rs1939012:T-rs1783901:A showed a significant association with a decreased risk of the disease (OR = 0.0597, 95% CI = 0.0136–0.2620, p = 0.0002). Our results suggest that the SNPs rs1939012 and rs1783901 may play a potential role of gene regulation and/or epistasis in a complex etiology of vertigo.