Monika Buraczynska

Candidate gene association study for diabetic retinopathy in persons with type 2 diabetes: The candidate gene association resource (CARe)

PURPOSE To investigate whether variants in cardiovascular candidate genes, some of which have been previously associated with type 2 diabetes (T2D), diabetic retinopathy (DR), and diabetic nephropathy (DN), are associated with DR in the Candidate gene Association Resource (CARe). METHODS Persons with T2D who were enrolled in the study (n = 2691) had fundus photography and genotyping of single nucleotide polymorphisms (SNPs) in 2000 candidate genes. Two case definitions were investigated: Early Treatment Diabetic Retinopathy Study (ETDRS) grades ≥ 14 and ≥ 30. The χ² analyses for each CARe cohort were combined by Cochran-Mantel-Haenszel (CMH) pooling of odds ratios (ORs) and corrected for multiple hypothesis testing. Logistic regression was performed with adjustment for other DR risk factors. Results from replication in independent cohorts were analyzed with CMH meta-analysis methods. RESULTS Among 39 genes previously associated with DR, DN, or T2D, three SNPs in P-selectin (SELP) were associated with DR. The strongest association was to rs6128 (OR = 0.43, P = 0.0001, after Bonferroni correction). These associations remained significant after adjustment for DR risk factors. Among other genes examined, several variants were associated with DR with significant P values, including rs6856425 tagging α-l-iduronidase (IDUA) (P = 2.1 × 10(-5), after Bonferroni correction). However, replication in independent cohorts did not reveal study-wide significant effects. The P values after replication were 0.55 and 0.10 for rs6128 and rs6856425, respectively. CONCLUSIONS Genes associated with DN, T2D, and vascular diseases do not appear to be consistently associated with DR. A few genetic variants associated with DR, particularly those in SELP and near IDUA, should be investigated in additional DR cohorts.

Toll-like receptor 4 gene polymorphism and early onset of diabetic retinopathy in patients with type 2 diabetes

Toll-like receptor 4 (TLR4) is an important mediator of innate immunity. Type 2 diabetes (DM2) might be associated with changed innate immune response. We investigated whether the polymorphisms in the TLR4 gene are associated with diabetic retinopathy (DR). The study group of 864 patients with DM2 and 420 healthy individuals were genotyped. In the patient group 352 subjects were diagnosed with DR. Out of the remaining 512, 140 had DM2 for > or = 10 years but no DR. In the DM2 group 7.4% of patients were heterozygous for the Asp299Gly polymorphism compared with 6.5% controls. For Thr399Ile polymorphism there were 7.2% heterozygotes vs 6.2% controls. In most cases, the linkage disequilibrium between the minor alleles Gly299 and Ile399 was confirmed. Increased frequency of both heterozygous genotypes was observed in patients with retinopathy (11.2% for the Asp299Gly). The frequency of the G allele was significantly higher in patients with early onset retinopathy (n = 80) vs patients without DR (odds ratio = 5.0, and 95% confidence interval = 2.33-10.71). In contrast, in the entire retinopathy group, the odds ratio for the G allele was 1.88 (95% confidence interval = 0.93-3.79). In the multivariate logistic regression analysis, the G allele of Asp299Gly was an independent risk factor of early onset DR (p < 0.001). In conclusion, our results suggest an association between the Asp299Gly polymorphism of the TLR4 gene and early onset of DR in the DM2 patients. Thus the G allele may be a predictor of increased risk of retinopathy.

Polymorphism of the renalase gene in end-stage renal disease patients affected by hypertension

BACKGROUND Renalase is a novel flavin adenine dinucleotide-dependent amine oxidase that is secreted by the kidney. It circulates in the blood and modulates the cardiac function and systemic blood pressure. Insufficiency of renalase in patients with chronic kidney disease may explain the frequent occurrence of hypertension among patients with end-stage renal disease (ESRD) and an increased risk of cardiovascular events in this group. The aim of the study was to assess the relationship of two renalase gene polymorphisms with hypertension in dialysed patients. METHODS Rs2576178 polymorphism was genotyped in 369 patients, rs10887800 polymorphism was genotyped in 421 dialysed patients, using polymerase chain reaction (PCR) and subsequent cleavage with Msp I and Pst I restriction endonucleases. RESULTS Genotype distribution and allele frequencies of rs2576178 polymorphism were compared in the following subgroups of patients: dialysed patients with hypertension: ESRD HY + (n = 200) and dialysed patients without hypertension: ESRD HY - (n = 169). There was a significant difference in the frequency of the G allele carriers. G allele carriers were associated with a 1.55 times higher risk of hypertension [odds ratio (OR) = 1.55; 95% confidence interval (CI): 1.023-2.357, P = 0.039]. Distribution of genotypes and frequencies of alleles of rs10887800 polymorphism were compared in the following subgroups of patients: ESRD HY + (n = 278) and ESRD HY - (n = 143). The G allele carriers were recognized with a significantly higher frequency in ESRD HY + patients (0.46 in ESRD HY + versus 0.37 in ESRD HY - ) [OR = 1.76; 95% CI: (1.159-2.667, P = 0.008)]. CONCLUSIONS Our results are the first to suggest an association between renalase gene polymorphisms analysed and hypertension in dialysed patients. It may be an important step towards gaining a deeper insight into cardiovascular pathophysiology. Furthermore, it might provide an optimal treatment and better prognosis for patients with chronic kidney disease.

Angiotensin II Type 1 Receptor Gene Polymorphism in End-Stage Renal Disease

The genes of the renin-angiotensin system (RAS) are involved in progression of renal failure. We examined the A1166C polymorphism at the angiotensin II type 1 (AT1R) locus in patients with end-stage renal disease (ESRD). The distribution of genotype and allele frequencies was compared in 430 dialysis patients and 260 healthy controls. DNA samples were amplified by the polymerase chain reaction (PCR) and amplification products were digested with BsuRI restriction enzyme. In the presence of cytosine (C) there is a restriction site for this enzyme, giving a fragment of 231 bp (C allele), whereas undigested 255 bp fragment indicates the presence of the A allele. The higher frequency of combined AC and CC genotypes was observed in the patient group than in controls (48.6 vs. 39.5%, p < 0.05). The average time from the onset of renal disease to ESRD in patients with C allele was significantly shorter than in those with AA genotype (6.3 vs. 13 years, p < 0.01). Positive family history of renal disease in patients with AC/CC genotype seems to increase this effect. Our results indicate that the presence of C allele of the AT1R locus polymorphism might be associated with faster deterioration of renal function.

Endothelial Nitric Oxide Synthase Gene Intron 4 Polymorphism in Type 2 Diabetes Mellitus

AbstractIntroduction: Endothelial nitric oxide synthase (ecNOS) is a key regulator of vascular nitric oxide production. Polymorphism in intron 4 of the ecNOS gene is implicated in cardiovascular and renal diseases. We investigated a potential involvement of this polymorphism in the development of type 2 diabetes mellitus and its renal complications. Methods: This preliminary study involved 410 individuals with type 2 diabetes and 330 healthy control subjects. From the diabetes group 178 patients had diabetic nephropathy. All subjects were genotyped for the ecNOS4 polymorphism by the polymerase chain reaction (PCR) followed by gel electrophoresis. Genotype and allele frequencies were compared between diabetes patients with and without nephropathy and the control group. All calculations were performed using the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL, USA) for Windows 5.0. The chi-square test and Fisher’s exact test were used for case-control comparisons. The Kruskal-Wallis test was used for the comparison of subgroups of patients with diabetes. Results: The analysis revealed that patients with diabetes, regardless of their nephropathy status, were significantly different in genotype distribution and 4a allele frequencies compared with controls (p < 0.05). The frequency of aa genotype was 8.2% in diabetic patients without nephropathy, 8.4% with those with nephropathy and 1.2% in controls. The 4a allele showed a significant effect on diabetic nephropathy, with odds ratio of 2.24 (95% confidence interval 1.12–3.40). There were no significant differences in the 4a allele frequency between the normotensive and hypertensive patients with diabetes. Conclusion: Our results suggest that the ecNOS gene polymorphism can serve as a useful genetic marker of increased susceptibility to type 2 diabetes and its renal complications.

Monocyte chemoattractant protein-1 (MCP-1) gene polymorphism as a potential risk factor for cardiovascular disease in hemodialyzed patients

AIM Polymorphism in the monocyte chemoattractant protein-1 (MCP-1) gene (A-2518G) has been associated with functional effects. The aim of the present study was to assess the effect of this polymorphism on end-stage renal disease (ESRD) and cardiovascular disease (CVD) in hemodialyzed patients. METHODS A total of 720 patients with ESRD treated with hemodialysis (450 patients with CVD) and 325 healthy control subjects were genotyped for the MCP-1 -2518 polymorphism by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure. RESULTS There was a significant difference in genotype frequencies between entire group of hemodialyzed patients and controls (p<0.01). The odds ratio for the risk allele was 1.85, 95% CI 1.49-2.32 (p<0.01). Hemodialyzed patients were divided into subgroups with CVD (n=450) and without CVD (n=270). The G allele carriers occurred with significantly higher frequency in patients with CVD (62% vs. 38% in patients without CVD and 36% in controls). The odds ratio for the risk allele for patients with CVD vs. those without CVD was 2.17, 95% CI 1.71-2.79. There was no statistically significant difference in the distribution of MCP-1 genotypes between ESRD patients without CVD and healthy controls. CONCLUSION Our results demonstrate for the first time an association between the polymorphism in the regulatory region of the MCP-1 gene and susceptibility to CVD in hemodialyzed patients.

Association of the Human Bradykinin B2 Receptor Gene with Chronic Renal Failure

AbstractIntroduction: The kallikrein-kinin system plays an important role in blood pressure homeostasis and renal sodium regulation, and some studies have reported that the kinins have a protective effect against hypertension and the development of renal disease. The B2-bradykinin receptor (B2R) mediates the majority of physiological actions of bradykinin. We investigated the effect of the C181→T polymorphism in exon 2 of the B2R gene in patients with end-stage renal disease (ESRD). Methods: This study involved 790 patients with ESRD and 510 healthy controls. All participants were genotyped for the B2R C181→T polymorphism by PCR followed by digestion of a PCR product with TaqI restriction endonuclease. DNA fragments were separated by agarose gel electrophoresis. Genotype and allele frequencies were compared between the groups. All calculations were performed using SPSS® 5.0 for Windows®. Results:B2R genotype distribution in patients and controls was in accordance with Hardy-Weinberg equilibrium. The frequency of the T allele was higher in ESRD patients than in controls. The significant difference was observed in the age at onset of renal disease; for patients with the T allele the mean age at onset was 36.8 years, compared with 52.4 years for those carrying only the C allele (p < 0.001). The frequencies of the T allele and carrier genotypes were not associated with gender, presence of hypertension, or underlying kidney disease. Conclusion: Our results suggest that the B2R polymorphism has a potential role in the earlier development of chronic renal failure in susceptible individuals. We did not confirm the previously published reports that the B2R gene polymorphism has a protective role in the development of ESRD.

X-linked Retinitis Pigmentosa in Two Families with a Missense Mutation in the RPGR Gene and Putative Change of Glycine to Valine at Codon 60

OBJECTIVE This study describes the ophthalmic findings in two unrelated white families with X-linked retinitis pigmentosa (XLRP) caused by a missense mutation in the retinitis pigmentosa GTPase regulator (RPGR) gene. DESIGN Genetic screening and clinical correlation. PARTICIPANTS Thirty-six families with XLRP seen by the authors were screened for a possible mutation in the RPGR gene to identify three affected hemizygotes with retinitis pigmentosa and four heterozygote carriers in one family and one hemizygote and one carrier in a second family. INTERVENTION All nine patients underwent a routine ocular examination, including slit-lamp biomicroscopy and a dilated fundus examination. Goldmann visual field kinetic perimetry, static threshold perimetry, and electroretinography also were obtained. The DNA screening was performed on the three affected male patients and four obligate carriers examined from one family and the two examined patients, plus an additional male and obligate carrier, from the second family to determine the presence of any causative mutation in the RPGR gene. MAIN OUTCOME MEASURES Findings on fundus examination, static threshold and kinetic perimetry, and electroretinography testing were the main outcome measures. RESULTS A G-->T nucleotide change at position 238 in exon 3 of the RPGR gene resulting in a putative substitute of Gly-->Val at codon 60 was shown to segregate with RP in affected males and the carrier state in female heterozygotes in these two families. The ophthalmologic findings in hemizygotes as well as the carriers in this family were within the spectrum of findings characteristically noted in XLRP families. A tapetal-like reflex was not observed in any of the five female carriers. Psychophysical and electrophysiologic testing on the carriers indicated that cone and rod functions were impaired equivalently. When present in the carriers, visual field restriction was most apparent in, or limited to, the superotemporal quadrant, which corresponded to the retinal pigmentary changes that tended to occur in the inferonasal retina. CONCLUSIONS A mutation in exon 3 of the RPGR gene, which would result in a putative glycine to valine substitution at codon 60, is associated with a severe clinical phenotype in male patients and a patchy retinopathy without a tapetal-like reflex in carrier females. In these families, heterozygote carriers showed equivalent impairment of their cone and rod function.

Transcription factor 7-like 2 (TCF7L2) gene polymorphism and complication/comorbidity profile in type 2 diabetes patients

Transcription factor 7-like 2 gene (TCF7L2) has been associated with type 2 diabetes. We investigated the association of the rs7903146 SNP in this gene with clinical profile of type 2 diabetes (T2DM) patients. The study involved 980 patients with diabetic nephropathy (44%), diabetic retinopathy (42%), CVD (65%) and early onset of diabetes (45%) and 924 healthy controls. Subjects were genotyped for rs7903146 by PCR-RFLP. Genotype frequencies significantly differed between T2DM patients and controls (p<0.01, odds ratio (OR) for TT genotype 2.49 (95% confidence interval (CI) 1.84-3.39). An association was observed between rs7903146 and nephropathy (p<0.001), with 22% of TT homozygotes in this subgroup vs. 11% in patients without nephropathy (p=0.006, OR for TT 2.83, 95% CI 1.94-4.13). Association was stronger in patients with early onset of diabetes (34% of TT vs. 12% in the late onset, p<0.001). In DN group 71% of TT homozygotes had an early onset (OR 7.64, 95% CI 4.98-11.73 vs. controls). Our results confirm association of rs7903146 in the TCF7L2 gene with increased risk of type 2 diabetes. The T allele is strongly associated with nephropathy, especially in early onset of diabetes.

TGF-β1 and TSC-22 Gene Polymorphisms and Susceptibility to Microvascular Complications in Type 2 Diabetes

Background/Aim: There is a strong evidence for the involvement of genetic factors in diabetic microvascular complications. The aim of our study was to investigate the role of molecular variants of the TGF-β1 (transforming growth factor beta 1) and the TSC-22 (transforming growth factor beta stimulated clone 22) genes in diabetic nephropathy and diabetic retinopathy in type 2 diabetes. Methods: A case-control study was conducted in 503 patients and 400 healthy subjects. DNA samples were genotyped by polymerase chain reaction and restriction fragment length polymorphism methods. Results: Among the patients, 245 had diabetic nephropathy, 195 had retinopathy, and 168 were free from complications. All subjects were genotyped for T869C and C –509T polymorphisms of the TGF-β1 gene and for –396 polymorphism of the TSC-22 gene. A significantly increased frequency of the CC genotype of the T869C polymorphism was observed in patients with nephropathy and retinopathy (33 and 48%, respectively, vs. 19 and 15%, respectively, in controls and patients free from complications). The frequency of the C allele was also higher (0.58 for nephropathy and 0.64 for retinopathy vs. 0.42 in controls). The G allele of the TSC-22 polymorphism was associated with an increased risk of diabetic nephropathy (frequency 0.15 vs. 0.07 and 0.06, respectively, in patients free from complications and controls). An interaction was observed between the G allele of the TSC-22 polymorphism and the C-allele of the TGF-β polymorphism. Conclusions: Our data suggest the association of TGF-β T869C gene polymorphism with an increased risk of nephropathy and retinopathy in type 2 diabetes patients. It interacts with the TSC-22 gene involved in the TGF-β signaling pathway, promoting the development of diabetic nephropathy.