Association of endothelial nitric oxide synthase Glu298Asp polymorphism with end-stage renal disease

BACKGROUND: Impairment of nitric oxide generation caused by gene polymorphism is considered as a major factor in the deterioration of progressive renal disease, including diabetic nephropathy and hypertension. The aim of the present study was to examine the Glu298Asp polymorphism of endothelial nitric oxide synthase (eNOS) in patients with end-stage renal disease (ESRD). METHODS: The Glu298Asp polymorphism in exon 7 was determined in 100 ESRD patients who were maintained on hemodialysis at Dr. Soetomo Hospital, Surabaya, Indonesia, and in a control group of 100 unrelated healthy individuals. In the patient group, 39 patients had Type 2 diabetes mellitus (DM), 44 hypertension (HT) and 17 miscellaneous conditions. The mean length of time from onset of ESRD to the start of this study was 24.37 +/- 32.37 months (Mean +/- SD). RESULTS: The positivity of Glu298Asp in the ESRD group was significantly higher than that in the control group (p < 0.0001). The odds ratio for this group was 4.57 (95% confidence interval 2.52 - 8.31). The positivity of 298Asp in Type 2 DM ESRD with subgroup was significantly higher than that in healthy controls (p < 0.0001). The positivity of 298Asp in the subgroup of patients with HT-derived ESRD was also significantly higher (males p < 0.036, females p < 0.005) than that in healthy control group. Homozygotes with glutamate to aspartate substitution at nucleotide position 7702 showed a single band at 457 bp. CONCLUSION: It appears that Glu298Asp may be a predisposing factor in DM-derived and HT-derived ESRD.     

Role of the 11beta-hydroxysteroid dehydrogenase type 2 in blood pressure regulation

The renal 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) enzyme inactivates 11-hydroxy steroids in the kidney, thus protecting the nonselective mineralocorticoid receptor (MR) from occupation by glucocorticoids. The gene is highly expressed in all sodium-transporting epithelia, but also in human placenta, pancreas, and thyroid. Mutations in the HSD11B2 gene cause a rare monogenic juvenile hypertensive syndrome called apparent mineralocorticoid excess (AME). In AME, compromised 11betaHSD2 enzyme activity results in overstimulation of the MR by cortisol, causing sodium retention, hypokalemia, and salt-dependent hypertension. Recent evidence suggests a role of the 11betaHSD2 in essential hypertension. We found hypertension with no other characteristic signs of AME in the heterozygous father of a child with AME and in a girl with a homozygous gene mutation resulting in a mild deficiency of 11betaHSD2. Moreover, some studies in patients with essential hypertension showed a prolonged half-life of cortisol and an increased ratio of urinary cortisol to cortisone metabolites, suggesting a deficient 11betaHSD2 activity. These abnormalities may be genetically determined. A genetic association of a microsatellite flanking the HSD11B2 gene and hypertension in black patients with end-stage renal disease has been reported. We recently analyzed a CA-repeat allele polymorphism in unselected patients with essential hypertension, but did not find any correlation between this marker and blood pressure. However, we did find an association between this polymorphic CA microsatellite marker and salt sensitivity. Moreover, the activity of the 11betaHSD2, as shown by elevated mean ratios of urinary cortisol to cortisone metabolites, was decreased in salt-sensitive compared with salt-resistant subjects. These findings indicate that variants of the HSD11B2 gene contribute to the enhanced blood pressure response to salt in humans.     

Impact of IL2 and IL2RB genetic polymorphisms in kidney transplantation

Patient genetic make-up may contribute to a higher risk for acute rejection episodes (AREs). Because interleukin-2 (IL2) and IL2 receptor β (IL2RB) play key roles in immune modulation, we investigated the effect of single-nucleotide polymorphisms (SNPs) in the IL2 gene (rs2069762; T>G, promoter; and rs2069763; G>T, exon 1, Leu38Leu) and IL2RB gene (rs228942: C>A, exon 1, Asp391Glu; and rs228953: C>T, exon 8, Gly250Gly) on renal ARE risk in 61 ARE patients and 276 control renal allograft recipients in Korea.The genotype frequencies of the IL2 and IL2RB SNPs showed Hardy-Weinberg equilibrium in both ARE and control groups. No significant difference in the genotype frequencies of the 2 IL2 SNPs was detected between non-ARE and ARE subjects (P > .05). The occurrence of AREs was significantly associated with genetic variants of the IL2RB gene (rs228942: odds ratio [OR] 2.11, 95% confidence interval [CI] 1.19-3.74; P = .0096, dominant model; rs228953: OR 1.58, 95% CI 1.04-2.38; P = .029, codominant model). In the haplotype-based analysis, the AC haplotype of IL2RB (χ² = 4.738; P = .0295) showed associations with ARE. Our results demonstrate that genetic variants of IL2RB may be associated with the development of AREs and may help predict ARE risk in kidney transplantation patients.     

New amino acid substitutions in the IRS-2 gene in Finnish and Chinese subjects with late-onset type 2 diabetes

We investigated the significance of the variants of the IRS-2 gene in patients with type 2 diabetes. The entire coding part of the IRS-2 gene was screened by single-strand conformation polymorphism analysis in 40 Chinese and 40 Finnish patients with late-onset type 2 diabetes. The association of the variants of the IRS-2 gene with type 2 diabetes was studied in 85 Finnish diabetic patients and 82 Finnish control subjects and in 100 Chinese diabetic patients and 85 Chinese control subjects. The four variants predicting structural changes in the insulin receptor substrate (IRS)-2 protein included an insertion of AAC (Asn) in the Asn repeat sequence centered around codons 29-36 (allele frequencies of 0 vs. 0.6% and 1.5 vs. 0%), the Ala157Thr substitution (0 vs. 0% and 0.5 vs. 0%), the Leu647Val substitution (0.6 vs. 0% and 0 vs. 0%), and the Gly1057Asp polymorphism (31 vs. 31% and 35 vs. 30%) (P = NS for all comparisons). Furthermore, six silent variants were observed (CGC147CGG, CCC155CCG, GCC156GCT, AGT723AGC, TGT816TGC, and CCC829CCT). The Gly1057Asp polymorphism was not associated with insulin resistance or impaired insulin secretion in Finnish subjects with normal glucose tolerance (n = 295) or impaired glucose tolerance (n = 38). These data indicate that structural variants of the IRS-2 gene were uncommon in Finnish and Chinese patients with type 2 diabetes. Thus, the variants in the coding part of the IRS-2 gene are unlikely to have a major role in the development of type 2 diabetes in Finnish or Chinese subjects.     

Low prevalence of nonconservative mutations of serum and glucocorticoid-regulated kinase (SGK1) gene in hypertensive and renal patients.

BACKGROUND: The serum- and glucocorticoid-regulated kinase (SGK1) gene is an important mediator of aldosterone action, regulating the expression of the renal epithelial Na(+) channel. In renal failure, blood pressure (BP) is markedly salt-dependent and increases with decreasing renal function. Mutations of the SGK1 gene affecting phosphorylation could be responsible for salt-mediated increases in BP and hypertension-related progression to end-stage renal disease (ESRD). METHODS: The SGK1 gene was analysed for mutations in the exons 4, 5, 8 and 10-12, because of potential phosphorylation sites, in 591 subjects, including 311 ESRD patients (either dialysis or transplanted). In addition, an intron 6 single-nucleotide polymorphism (SNP) described previously was also investigated in this study. Genotyping was performed either by using a strategy based on single strand conformation polymorphism analysis of polymerase chain reaction (PCR) products and subsequent direct sequencing of identified gel shift variants or by using high throughput 5' nuclease allelic discrimination assay. RESULTS: Two SNPs in coding regions of SGK1 potentially influencing the phosphorylation of Sgk1 were identified. Both SNPs were synonymous. The prevalence of the first variant, a previously reported SNP at codon 240 in exon 8, did not differ between ESRD patients (16.3%) and controls (15.7%). There was no association between the SNP in exon 8 and either BP within the control population or progression of renal disease in the ESRD population. The second SNP at codon 398 in exon 12 was identified in one patient only. Intron 6 and exon 8 SNPs were in strong linkage disequilibrium, but did not show any association with either BP or renal diseases. CONCLUSIONS: Based on statistical analysis homozygosity for nonconservative mutations in the coding region of the SGK1 gene is estimated at < 1/300 000 when a white Caucasian population is considered, arguing against an important role of mutations of this coding region in hypertension and hypertension-associated progression of renal disease.     

Apparent mineralocorticoid excess: report of six new cases and extensive personal experience

In mineralocorticoid target tissues such as the cortical collecting duct in the kidney, the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) is responsible for the peripheral inactivation of cortisol to cortisone, thereby protecting the mineralocorticoid receptor from inappropriate activation by cortisol. Mutations in the HSD11B2 gene cause the syndrome of apparent mineralocorticoid excess, an autosomal recessive form of inherited hypertension in which cortisol acts as a potent mineralocorticoid. Herein are described six new families with mutations in the HSD11B2 gene causing hypokalemic hypertension, with low plasma aldosterone and low renin levels in affected individuals, indicating mineralocorticoid hypertension. Profiling of urinary steroid metabolites showed decreased cortisol inactivation, with urinary tetrahydrocortisol and tetrahydrocortisone ratio (THF + 5alphaTHF)/THE ranging 2.4 to 40 and nearly absent urinary free cortisone in all but one case. Genetic analysis of the HSD11B2 gene from these patients with apparent mineralocorticoid excess revealed distinct homozygous point mutations in four families, a compound heterozygous mutation in one family, and a large 23-bp exonic insert with frameshift and disruption of the amino acid sequence in another family. Expression studies of mutants that were expressed in HEK-293 cells showed marked reduction or abolition of 11betaHSD2 enzymatic activity. These cases are reviewed along with previous ones from the authors' extensive personal experience to highlight the importance of 11betaHSD2 in the understanding of a new biologic principle in hormone action, demonstrating that local metabolism of the glucocorticoid hormones into inactive derivatives by the enzyme 11betaHSD2 is one of the mechanisms that intervene to allow specific aldosterone regulatory effects.     

Identification of PVT1 as a candidate gene for end-stage renal disease in type 2 diabetes using a pooling-based genome-wide single nucleotide polymorphism association study

To identify genetic variants contributing to end-stage renal disease (ESRD) in type 2 diabetes, we performed a genome-wide analysis of 115,352 single nucleotide polymorphisms (SNPs) in pools of 105 unrelated case subjects with ESRD and 102 unrelated control subjects who have had type 2 diabetes for > or =10 years without macroalbuminuria. Using a sliding window statistic of ranked SNPs, we identified a 200-kb region on 8q24 harboring three SNPs showing substantial differences in allelic frequency between case and control pools. These SNPs were genotyped in individuals comprising each pool, and strong evidence for association was found with rs2720709 (P = 0.000021; odds ratio 2.57 [95% CI 1.66-3.96]), which is located in the plasmacytoma variant translocation gene PVT1. We sequenced all exons, exon-intron boundaries, and the promoter of PVT1 and identified 47 variants, 11 of which represented nonredundant markers with minor allele frequency > or =0.05. We subsequently genotyped these 11 variants and an additional 87 SNPs identified through public databases in 319-kb flanking rs2720709 ( approximately 1 SNP/3.5 kb); 23 markers were associated with ESRD at P < 0.01. The strongest evidence for association was found for rs2648875 (P = 0.0000018; 2.97 [1.90-4.65]), which maps to intron 8 of PVT1. Together, these results suggest that PVT1 may contribute to ESRD susceptibility in diabetes.     

Identification of podocin (NPHS2) gene mutations in African Americans with nondiabetic end-stage renal disease

BACKGROUND: Podocin, encoded by NPHS2 and mapped to 1q25.2, is an integral membrane protein exclusively expressed in glomerular podocytes. Mutations in the NPHS2 gene cause autosomal-recessive nephrotic syndrome and have been associated with proteinuria in several populations. Evidence for linkage of end-stage renal disease (ESRD) to chromosome 1q25-31 in the region of NPHS2 has been identified in a genome-wide scan in African American (AA) siblings. METHODS: To investigate the potential role of this gene in ESRD, we sequenced all coding regions and approximately 2 kb of upstream promoter sequence of NPHS2 in 96 unrelated AA nondiabetic ESRD cases and 96 healthy population-based AA controls, and assessed several single nucleotide polymorphisms (SNPs) for association in a larger case-control sample. RESULTS: Fifty-five variants were identified with minor allele frequencies ranging from <1% to 44%. Twenty-three polymorphisms were located in the promoter region, 11 were exonic, 13 were intronic, and 8 were in the 5' and 3'- untranslated regions. Two novel nonsynonymous coding SNPs were identified (A44E and A61V). An insertion polymorphism in intron 3, IVS3+9insA, was detected in 6 ESRD patients and in no controls. This variant, and 4 other common SNPs, were evaluated in a larger sample of 288 AA ESRD cases and 278 AA controls. The overall minor allele frequencies for the insertion allele were 0.018 in cases and 0.002 in controls. Significant evidence of association of IVS3+9insA was observed (P= 0.012), and the haplotype containing the insertion allele in cases was also associated. CONCLUSION: These results suggest that uncommon variants of the NPHS2 gene may play a role in the development of nondiabetic ESRD in AAs.     

T-786C polymorphism of the endothelial nitric oxide synthase gene is associated with albuminuria in the diabetes heart study

Albuminuria demonstrates significant heritability in multiply affected hypertensive and diabetic families. The role of endothelial nitric oxide synthase (eNOS) gene variants as risk factors for albuminuria was investigated in 590 European American siblings from 230 families in the Diabetes Heart Study. Two polymorphisms in the eNOS gene (T-786C in the promoter region and Glu298Asp in exon 7) were genotyped. Albuminuria was defined as an albumin:creatinine ratio (ACR) >/=17 mg/g in men and >/=25 mg/g in women. Tests of association were based on generalized estimating equations, and tests of linkage disequilibrium were based on the quantitative pedigree disequilibrium test. A total of 83% of participants had type 2 diabetes. The median ACR was 10.7 mg/g (interquartile range, 5.1 to 32.8), and 34% (202 of 590) of participants had an elevated ACR. The eNOS -786C allele but not the Glu298Asp was associated with increased ACR (31% increase in absolute level of ACR for each additional copy of the -786C allele; P < 0.0001) and a higher risk for albuminuria (odds ratio, 1.55 for each additional copy of the -786C allele; P = 0.0005). Adjustment for the nongenetic determinants of ACR had no significant effect on the results; neither did stratification by gender, presence of diabetes, and the Glu298Asp genotype. Results were confirmed by quantitative pedigree disequilibrium test analysis and were consistent with haplotype analysis. The -786C eNOS variant was positively correlated with a higher prevalence and a greater degree of albuminuria in European American families in both diabetic and nondiabetic family members.     

Impact of Nitric Oxide Synthase Glu298Asp Polymorphism on the Development of End-Stage Renal Disease in Type 2 Diabetic Egyptian Patients

Abstract

Background: Nitric oxide is an important regulator of renal hemodynamics. This study aimed to investigate the role of endothelial nitric oxide synthase (eNOS) gene polymorphism in type 2 diabetic patients with end-stage renal disease (ESRD) and to elucidate any alteration of nitric oxide synthase (NOS) activity caused by this polymorphism. Methods: The study included 80 patients with type 2 diabetes of >10 years duration (40 with diabetes-derived ESRD, 40 without nephropathy) and 20 healthy controls. Plasma nitrate/nitrite level, and serum NOS activity were measured and eNOS Glu298Asp genotypes were determined. Results: The frequency of Glu/Glu (GG) genotype in diabetics with ESRD was lower than controls. However, the frequency of Asp/Asp (TT) genotype was increased in diabetics with ESRD as compared to those without nephropathy and controls. Diabetics with ESRD had significantly lower nitrate/nitrite level and NOS activity than those without nephropathy. Diabetic patients with TT genotype are at a significant risk for ESRD. Moreover, subjects carrying TT genotype had lower nitrate/nitrite level and NOS activity than those carrying GG genotype. In diabetics with ESRD, creatinine clearance was positively correlated with both nitrate/nitrite level and NOS activity. Conclusions: These results imply that TT genotype of eNOS may be associated with an increased risk of ESRD in Egyptian type 2 diabetics. It could represent a useful genetic marker to identify diabetics at high risk for the development of ESRD. However, larger future prospective studies are required to confirm the role of eNOS gene polymorphism in the progression of diabetic nephropathy to ESRD.     

Assessment of matrix Gla protein, Klotho gene polymorphisms, and oxidative stress in chronic kidney disease

Increased vascular calcification and oxidative stress are considered as extra renal risk factors at the pathogenesis cardiovascular events in chronic kidney disease (CKD). We investigated matrix Gla protein (MGP) (T-138C, Glu60X, Thr83Ala) and Klotho (Cys370Ser) gene polymorphisms, serum MGP levels, and oxidative stress status of 84 CKD patients and 37 healthy controls. The MGP gene Glu60X and Thr83Ala polymorphisms were significantly associated with CKD. The correlation between T-138C genotype of MGP gene, Cys370Ser genotype of Klotho gene, and CKD was not significant (p > 0.05). At the haplotype analysis, the combination of the X allele of Glu60X and the Thr allele of Thr83Ala showed a significantly increased risk of CKD (p < 0.05). X allele, Thr allele, and C allele of T-138C were associated with diabetes mellitus and CKD phenotypes occurring concurrently (p < 0.01). Serum zinc levels were significantly low in end-stage renal disease (ESRD) patients (p = 0.0001). The total comet score frequency of ESRD patients was higher than that of control group (p < 0.05). The urinary 8-hydroxy-2'-deoxyguanosine levels were significantly high in CKD patients (p < 0.05). According to this study, analyzing the distribution of MGP gene and oxidative stress status would be very informative in order to detect their role at CKD.     

Partial characterization of RC-13, a homogeneous fraction isolated from castor bean allergens (CB-1A)

Allergen RC-13's amino acid composition was determined-Lys3, His1, Arg7, Asp6, Thr2, Ser11, Glu29, Pro2, Gly7, Ala4, Cys6, Val5, Met1, Ile3, Leu3, Tyr1; 91 residues. Partial specific volume and minimum molecular weight evaluated from this composition were, respectively, 0.694 cm3/g and 10,194. SDS-polyacrylamide gel electrophoresis performed in the reduced and alkylated form of the allergen revealed a single band, suggesting the existence of only one polypeptide chain in the molecule. Results obtained for carbohydrate analysis by gas-liquid chromatography were: 22.2% galactose and 16.2% mannose--approximately 38% of neutral sugars. Employment of the amino acid methodology for analysis of amino sugars revealed two residues of glucosamine per molecule of the allergen. Hydrolyses with trypsin, alpha-chymotrypsin and type VII protease were tested for sequence studies. Trypsin was considered a suitable enzyme for such purpose.     

Common variants of the endothelial nitric oxide synthase gene and the risk of coronary heart disease among U.S. diabetic men

Endothelial nitric oxide synthase (eNOS) gene represents a promising candidate gene for coronary heart disease (CHD) because of its impact on eNOS activity. We systematically examined the associations of eight variants of the eNOS gene (two potentially functional variants [-786T>C and Glu298Asp] and six tagging single nucleotide polymorphisms) with CHD risk in a large cohort of diabetic patients. Among 861 diabetic men (>97% Caucasian) from the Health Professionals Follow-Up Study, 220 developed CHD, and 641 men without cardiovascular disease were used as control subjects. Genotype distributions of -786T>C and Glu298Asp polymorphisms were not significantly different between case and control subjects. CHD risk was significantly higher among men with the variant allele at the rs1541861 locus (intron 8 A/C) than men without it (adjusted odds ratio 1.5 [95% confidence interval 1.1-2.1]). Moreover, among control subjects, plasma soluble vascular cell adhesion molecule concentrations were significantly higher among carriers of this allele (P 0.019) and carriers of the variant allele of the -786T>C (P 0.010), or the Glu298Asp polymorphism (P 0.002), compared with noncarriers. In conclusion, our data suggested that -786T>C, Glu298Asp, and an intron 8 polymorphism of the eNOS gene are potentially involved in the atherogenic pathway among U.S. diabetic men.     

Assessment of Matrix Gla Protein,Klotho Gene Polymorphisms,and Oxidative Stress in Chronic Kidney Disease

Abstract

Increased vascular calcification and oxidative stress are considered as extra renal risk factors at the pathogenesis cardiovascular events in chronic kidney disease (CKD). We investigated matrix Gla protein (MGP) (T-138C, Glu60X, Thr83Ala) and Klotho (Cys370Ser) gene polymorphisms, serum MGP levels, and oxidative stress status of 84 CKD patients and 37 healthy controls. The MGP gene Glu60X and Thr83Ala polymorphisms were significantly associated with CKD. The correlation between T-138C genotype of MGP gene, Cys370Ser genotype of Klotho gene, and CKD was not significant (p > 0.05). At the haplotype analysis, the combination of the X allele of Glu60X and the Thr allele of Thr83Ala showed a significantly increased risk of CKD (p < 0.05). X allele, Thr allele, and C allele of T-138C were associated with diabetes mellitus and CKD phenotypes occurring concurrently (p < 0.01). Serum zinc levels were significantly low in end-stage renal disease (ESRD) patients (p = 0.0001). The total comet score frequency of ESRD patients was higher than that of control group (p < 0.05). The urinary 8-hydroxy-2′-deoxyguanosine levels were significantly high in CKD patients (p < 0.05). According to this study, analyzing the distribution of MGP gene and oxidative stress status would be very informative in order to detect their role at CKD.     

Diabetes mellitus and late‐onset hypogonadism: the role of Glu298Asp endothelial nitric oxide synthase polymorphism

Nitric oxide has been associated with insulin resistance and type 2 diabetes mellitus (DM). An association has been suggested between a single nucleotide polymorphism (Glu298Asp variant) of the endothelial nitric oxide synthase (eNOS) gene and increased risk of DM. However, the role of this polymorphism in favouring DM has not been investigated in hypogonadism, in which low testosterone and obesity are believed to play the major role. We aimed to evaluate whether eNOS gene single nucleotide polymorphism (Glu298Asp variant) might give a relevant contribution also to the onset of hypogonadism‐associated DM. 110 men affected by late‐onset hypogonadism were retrospectively reviewed. Patients were clinically and biochemically evaluated. Detection of eNOS Glu298Asp polymorphism was performed. After splitting the sample according to the three genetic variants (i.e. eNOS_GG, eNOS_GT, eNOS_TT), no difference was evident in age, body mass index (BMI) and total testosterone. Conversely, DM prevalence, glycaemia and glycated haemoglobin were significantly higher in eNOS_TT than in eNOS_GT and eNOS_GG. Logistic regression analysis showed that, after adjustment for age, BMI and total testosterone, eNOS_TT was positively and significantly associated with DM. Our study suggests that Glu298Asp single nucleotide polymorphism of the eNOS gene may be an independent risk factor for hypogonadism‐associated type 2 DM.     

Mutational analysis of PINX1 in hereditary prostate cancer

BACKGROUND: Telomerase activity is increased in most tumors. PinX1 has recently been identified as a critical component in regulating telomerase activity. The PinX1 gene is located within chromosomal region 8p22-23, a region associated with LOH and potentially linked to increased prostate cancer risk. METHODS: PINX1 was re-sequenced in 159 hereditary prostate cancer (HPC) probands. Four non-synonymous coding variants were genotyped in 159 HPC families. RESULTS: Thirty-nine polymorphisms were identified in the HPC screening panel. Ten coding polymorphisms were identified, seven (Gln50His, Leu91Met, Gln206His, Arg215Ile, Thr220Ala, Ser254Cys, and Glu414Ala) of which were non-synonymous. The most common variants Thr220Ala and Ser254Cys were not significantly over-transmitted from affected parent to affected offspring. CONCLUSIONS: Based on these results, we conclude that PINX1 is not a major factor for HPC risk.     

Lack of Association of the APOL1 G3 Haplotype in African Americans with ESRD

Apolipoprotein L1 gene (APOL1) G1 and G2 variants are strongly associated with progressive nondiabetic nephropathy in populations with recent African ancestry. Selection for these variants occurred as a result of protection from human African trypanosomiasis (HAT). Resequencing of this region in 10 genetically and geographically distinct African populations residing in HAT endemic regions identified eight single nucleotide polymorphisms (SNPs) in strong linkage disequilibrium and comprising a novel G3 haplotype. To determine whether the APOL1 G3 haplotype was associated with nephropathy, G1, G2, and G3 SNPs and 70 ancestry informative markers spanning the genome were genotyped in 937 African Americans with nondiabetic ESRD, 965 African Americans with type 2 diabetes–associated ESRD, and 1029 non-nephropathy controls. In analyses adjusting for age, sex, APOL1 G1/G2 risk (recessive), and global African ancestry, the G3 haplotype was not significantly associated with ESRD (P=0.05 for nondiabetic ESRD, P=0.57 for diabetes-associated ESRD, and P=0.27 for all-cause ESRD). We conclude that variation in APOL1 G3 makes a nominal, if any, contribution to ESRD in African Americans; G1 and G2 variants explain the vast majority of nondiabetic nephropathy susceptibility.     

Liver pyruvate kinase polymorphisms are associated with type 2 diabetes in northern European Caucasians

Pyruvate kinase is a key glycolytic enzyme. Isoforms that are expressed in the red cell, liver, pancreatic beta-cells, small intestine, and proximal renal tubule are encoded by the 12 exons of the PKLR gene, which maps to chromosome 1q23. We hypothesized that common variants of the PKLR gene could account for the linkage of diabetes to this region. We screened the promoter regions, exons and surrounding introns, and the 3' untranslated region for mutations. We identified five single-nucleotide polymorphisms (SNPs), and only one (V506I, exon 11) altered the coding sequence. We tested the five SNPs, a poly-T insertion-deletion polymorphism, and an ATT triplet repeat in 131 unrelated diabetic patients and 118 nondiabetic control subjects. The V506I variant was rare and not associated with type 2 diabetes. The four SNPs and the insertion-deletion polymorphism were associated with diabetes, with a 10% difference between individuals with diabetes and nondiabetic individuals (P = 0.001-0.011, relative risk for minor allele 1.85). The same trend was found for the ATT repeat (P = 0.029). Common variants in the PKLR are associated with increased risk of type 2 diabetes, but because of strong linkage disequilibrium between variants, the actual susceptibility allele may be in a different gene.     

Low fetuin-A levels are associated with cardiovascular death: Impact of variations in the gene encoding fetuin

BACKGROUND: Vascular calcification is common among end-stage renal disease (ESRD) patients and a central characteristic of the atherosclerotic cardiovascular disease observed in dialysis patients. Fetuin-A, a circulating calcium-regulatory glycoprotein that inhibits vascular calcification, is associated with inflammation and outcome in dialysis patients. In the present study, we evaluated the association between fetuin-A, clinical phenotype, and outcome, as well as the impact of fetuin gene (AHSG) polymorphisms on the protein product and outcome. METHODS: In a cohort of 258 (161 males) ESRD patients starting renal replacement therapy [glomerular filtration rate (GFR) 6.8 +/- 0.2 mL/min] aged 52 +/- 1 years the following parameters were studied: presence of malnutrition (subjective global assessment), comorbidity [diabetes mellitus and clinical manifest cardiovascular disease (CVD)], carotid plaques (N= 101), hs-CRP, fetuin-A, S-albumin, interleukin (IL)-6, and single nucleotide polymorphisms (SNPs) in the AHSG gene (N= 215) at amino acid positions Thr248Met (C-->T), Thr256Ser (C-->G), Asp276Asn (G-->A), and Arg317Cys (C-->T). RESULTS: Both all-cause (P < 0.001) and cardiovascular (P < 0.001) mortality were associated with low fetuin-A levels independently of age, smoking, diabetes, S-albumin, CVD, and inflammation (CRP > or =10 mg/L). Inflamed (0.199 vs. 0.247 g/L; P < 0.01) and malnourished (0.207 vs. 0.262 g/L; P < 0.05) patients had significantly lower median fetuin-A than noninflamed and well-nourished ESRD patients, respectively. In a logistic regression model (N= 101), fetuin-A was significantly (P < 0.05) associated with the presence of carotid plaques independently of age, CVD, diabetes, S-albumin, gender, and inflammation. Significant correlations were observed between fetuin-A and both S-albumin (Rho = 0.30; P < 0.0001) and IL-6 (Rho =-0.21; P < 0.01). Patients with the AHSG 256Ser allele had lower serum fetuin-A levels, and higher all-cause and cardiovascular mortality rate if they were inflamed. CONCLUSION: The present study shows that a low fetuin-A level is associated with malnutrition, inflammation, and atherosclerosis (carotid plaques), as well as with increased cardiovascular and all-cause mortality. Because the present study demonstrates an effect of variations in the AHSG gene on both circulating fetuin-A levels and outcome, this indicates that ESRD patients with the AHSG 256Ser allele are at risk of accelerated vascular calcification.     

Incidence of donor and recipient toll-like receptor-4 polymorphisms in kidney transplantation

Toll-like receptors (TLR) comprise an emerging family that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Recently, TLR has been demonstrated to play a role in experimental allograft rejection. However, the TLR-4 gene has a polymorphism that can be associated with a blunted immune response, especially to microbial pathogens. We sought to study the incidence of TLR 4 gene variants among renal transplant donors and recipients from living and deceased organs and then to correlate them with short-term and long-term outcomes. METHODS: Analysis of TLR4 polymorphisms at Asp299Gly and Thr399Ile codons were performed using restriction fragment length polymorphism. Demographic data was obtained from patient records. RESULTS: Among 201 patients, 141 were recipients from related donors (group 1) and 60 recipients from 45 deceased donors (group 2). Patients were followed for 108 +/- 85 months after transplantation. The incidence of polymorphism for TLR-4 Asp299Gly, Thr399Ile or both were 8.9% in recipients and 8.0% in donors. Patients who received a kidney with polymorphism, Asp299Gly, or Thr399Ile, or both, did not show a difference in rate of acute tubular necrosis compared with controls (no polymorphism). Acute rejection occurred in 17.6% of recipients with Asp299Gly/Thr399Ile polymorphisms and in 39.5% of wild-type recipients (P = .400). The incidence of bacterial infection was equal in both groups. CONCLUSION: The incidence of polymorphism in this study was similar in both groups, and donor or recipient polymorphisms were not associated with different renal graft outcomes.