DNA repair gene variants in relation to overall cancer risk: a population-based study.

The hypothesis that germ-line polymorphisms in DNA repair genes influence cancer risk has previously been tested primarily on a cancer site-specific basis. The purpose of this study was to test the hypothesis that DNA repair gene allelic variants contribute to globally elevated cancer risk by measuring associations with risk of all cancers that occurred within a population-based cohort. In the CLUE II cohort study established in 1989 in Washington County, MD, this study was comprised of all 3619 cancer cases ascertained through 2007 compared with a sample of 2296 with no cancer. Associations were measured between 759 DNA repair gene single nucleotide polymorphisms (SNPs) and risk of all cancers. A SNP in O(6)-methylguanine-DNA methyltransferase, MGMT, (rs2296675) was significantly associated with overall cancer risk [per minor allele odds ratio (OR) 1.30, 95% confidence interval (CI) 1.19-1.43 and P-value: 4.1 × 10(-8)]. The association between rs2296675 and cancer risk was stronger among those aged ≤54 years old than those who were ≥55 years at baseline (P-for-(interaction) = 0.021). OR were in the direction of increased risk for all 15 categories of malignancies studied (P < 0.0001), ranging from 1.22 (P = 0.42) for ovarian cancer to 2.01 (P = 0.008) for urinary tract cancers; the smallest P-value was for breast cancer (OR 1.45, P = 0.0002). The results indicate that the minor allele of MGMT SNP rs2296675, a common genetic marker with 37% carriers, was significantly associated with increased risk of cancer across multiple tissues. Replication is needed to more definitively determine the scientific and public health significance of this observed association.

A population-based study of hedgehog pathway gene variants in relation to the dual risk of basal cell carcinoma plus another cancer.

INTRODUCTION: A personal history of basal cell carcinoma (BCC) is associated with increased risk of other malignancies, but the reason is unknown. The hedgehog pathway is critical to the etiology of BCC, and is also believed to contribute to susceptibility to other cancers. This study tested the hypothesis that hedgehog pathway and pathway-related gene variants contribute to the increased risk of subsequent cancers among those with a history of BCC. METHODS: The study was nested within the ongoing CLUE II cohort study, established in 1989 in Washington County, Maryland, USA. The study consisted of a cancer-free control group (n=2296) compared to three different groups of cancer cases ascertained through 2007, those diagnosed with: (1) Other (non-BCC) cancer only (n=2349); (2) BCC only (n=534); and (3) BCC plus other cancer (n=446). The frequencies of variant alleles were compared among these four groups for 20 single nucleotide polymorphisms (SNPs) in 6 hedgehog pathway genes (SHH, IHH, PTCH2, SMO, GLI1, SUFU), and also 22 SNPs in VDR and 8 SNPs in FAS, which have cross-talk with the hedgehog pathway. RESULTS: Comparing those with both BCC and other cancer versus those with no cancer, no significant associations were observed for any of the hedgehog pathway SNPs, or for the FAS SNPs. One VDR SNP was nominally significantly associated with the BCC cancer-prone phenotype, rs11574085 [per minor allele odds ratio (OR) 1.38, 95% confidence interval (CI) 1.05-1.82; p-value=0.02]. CONCLUSION: The hedgehog pathway gene SNPs studied, along with the VDR and FAS SNPs studied, are not strongly associated with the BCC cancer-prone phenotype.

A community-based study of nucleotide excision repair polymorphisms in relation to the risk of non-melanoma skin cancer.

Nucleotide excision repair (NER) is responsible for protecting DNA in skin cells against UVR-induced damage. Using a candidate pathway approach, a matched case-control study nested within a prospective, community-based cohort was carried out to test the hypothesis that single-nucleotide polymorphisms (SNPs) in NER genes are associated with susceptibility to non-melanoma skin cancer (NMSC). Histologically confirmed cases of NMSC (n=900) were matched to controls (n=900) on the basis of age, gender, and skin type. Associations were measured between NMSC and 221 SNPs in 26 NER genes. Using the additive model, two tightly linked functional SNPs in ERCC6 were significantly associated with increased risk of NMSC: rs2228527 (odds ratio (OR) 1.57, 95% confidence interval (CI) 1.20-2.05) and rs2228529 (OR 1.57, 95% CI 1.20-2.05). These associations were confined to basal cell carcinoma (BCC) of the skin (rs2228529, OR 1.78, 95% CI 1.30-2.44; rs2228527, OR 1.78, 95% CI 1.31-2.43). These hypothesis-generating findings suggest that functional variants in ERCC6 may be associated with an increased risk of NMSC that may be specific to BCC.

Association of APOE polymorphism with chronic kidney disease in a nationally representative sample: a Third National Health and Nutrition Examination Survey (NHANES III) Genetic Study.

BACKGROUND: Apolipoprotein E polymorphisms (APOE) have been associated with lowered glomerular filtration rate (GFR) and chronic kidney disease (CKD) with e2 allele conferring risk and e4 providing protection. However, few data are available in non-European ethnic groups or in a population-based cohort. METHODS: The authors analyzed 5,583 individuals from the Third National Health and Nutrition Examination Survey (NHANES III) to determine association with estimated GFR by the Modification of Diet in Renal Disease (MDRD) equation and low-GFR cases. Low-GFR cases were defined as GFR <75 ml/min/1.73 m2; additionally, GFR was analyzed continuously. RESULTS: In univariate analysis, the e4 allele was negatively associated with low-GFR cases in non-Hispanic whites, odds ratio (OR): 0.76, 95% confidence interval (CI): 0.60, 0.97. In whites, there was a significant association between increasing APOE score (indicating greater number of e2 alleles) and higher prevalence of low-GFR cases (OR: 1.21, 95%CI: 1.01, 1.45). Analysis of continuous GFR in whites found the e4 allele was associated with higher levels of continuous GFR (beta-coefficient: 2.57 ml/min/1.73 m2, 95%CI: 0.005, 5.14); in non-Hispanic blacks the e2 allele was associated with lower levels of continuous GFR (beta-coefficient: -3.73 ml/min/1.73 m2, 95%CI: -6.61, -0.84). APOE e2 and e4 alleles were rare and not associated with low-GFR cases or continuous GFR in Mexican Americans. CONCLUSION: In conclusion, the authors observed a weak association between the APOE e4 allele and low-GFR cases and continuous GFR in non-Hispanic whites, and the APOE e2 allele and continuous GFR in non-Hispanic blacks, but found no association with either measure of kidney function in Mexican Americans. Larger studies including multiethnic groups are needed to determine the significance of this association.

Association of single-nucleotide polymorphisms in JAK3, STAT4, and STAT6 with new cardiovascular events in incident dialysis patients.

BACKGROUND: Increasing evidence supports a role for cell-mediated immunity in the pathogenesis of cardiovascular disease. Single-nucleotide polymorphisms (SNPs) in JAK3, STAT4, and STAT6 of the Janus kinase-signal transducer and activator of transcription (Jak-Stat) signal transduction pathway were examined for association with time to new cardiovascular events in incident dialysis patients from the Choices for Healthy Outcomes in Caring for End-Stage Renal Disease Study. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: 764 white (n = 518) and black (n = 246) participants from 79 dialysis centers. PREDICTOR: SNPs in JAK3, STAT4, and STAT6 selected using a pairwise approach to identify a maximally informative set of tag SNPs for populations of European and African descent. OUTCOMES & MEASUREMENTS: Cox proportional hazards models were used to estimate unadjusted and multivariable-adjusted hazard ratios (HRs) for incident cardiovascular disease events after dialysis therapy initiation associated with each race-specific SNP. RESULTS: 2 European tag SNPs (rs3212780 and rs3213409) in JAK3 were associated with new cardiovascular disease events in white patients with unadjusted HRs of 1.92 (P < 0.001) and 1.82 (P = 0.07), respectively. One dual-tag SNP (rs3212752) in JAK3 was associated with new cardiovascular events in white patients with an unadjusted HR of 2.09 (P < 0.001) and in black patients with an HR of 2.07 (P = 0.007). SNP rs3213409 codes for a valine to isoleucine change at amino acid 722, a potentially functional mutation. SNPs in STAT4 and STAT6 were not associated with cardiovascular events after the initiation of dialysis therapy. LIMITATIONS: This study does not provide direct evidence for the mechanism of increased risk. Replication in independent cohorts is necessary. CONCLUSIONS: Genetic polymorphisms in the Jak-Stat signaling pathway are associated with an increased risk of new cardiovascular events in incident dialysis patients.

MYH9 is associated with nondiabetic end-stage renal disease in African Americans.

As end-stage renal disease (ESRD) has a four times higher incidence in African Americans compared to European Americans, we hypothesized that susceptibility alleles for ESRD have a higher frequency in the West African than the European gene pool. We carried out a genome-wide admixture scan in 1,372 ESRD cases and 806 controls and found a highly significant association between excess African ancestry and nondiabetic ESRD (lod score = 5.70) but not diabetic ESRD (lod = 0.47) on chromosome 22q12. Each copy of the European ancestral allele conferred a relative risk of 0.50 (95% CI = 0.39-0.63) compared to African ancestry. Multiple common SNPs (allele frequencies ranging from 0.2 to 0.6) in the gene encoding nonmuscle myosin heavy chain type II isoform A (MYH9) were associated with two to four times greater risk of nondiabetic ESRD and accounted for a large proportion of the excess risk of ESRD observed in African compared to European Americans.

The association of podocin R229Q polymorphism with increased albuminuria or reduced estimated GFR in a large population-based sample of US adults.

BACKGROUND: Rare mutations in nephrosis 2 (NPHS2), encoding podocin, are found in patients with familial and sporadic steroid-resistant nephrotic syndrome and focal segmental glomerular sclerosis. The objective of this study is to assess the contribution of the commonly reported functional podocin polymorphism R229Q to kidney disease in the population at large and replicate a prior study of an association of R229Q and albuminuria in the general population. STUDY DESIGN: Large sample of the Atherosclerosis Risk in Communities (ARIC) Study, a population-based prospective study. SETTING & PARTICIPANTS: 4,424 white and 3,746 black middle-aged adults. PREDICTOR: Genotype at the R229Q polymorphism in podocin. OUTCOMES: Urinary albumin-creatinine ratio (ACR) and decreased estimated glomerular filtration rate (eGFR) as measures of kidney damage/dysfunction. MEASUREMENTS: Crude and multivariable adjusted linear and logistic regression models. RESULTS: R229Q allele frequencies were 3.7% in 4,424 white and 0.6% in 3,746 black individuals. No significant association of R229Q with increased ACR or decreased eGFR was observed (adjusted odds ratio of ACR > or = 30 mg/g in RQ/QQ versus RR carriers, 1.18; 95% confidence interval, 0.76 to 1.84; adjusted odds ratio of eGFR < 60 mL/min/1.73 m(2) in RQ/QQ versus RR carriers, 1.18; 95% confidence interval, 0.76 to 1.83). As expected, the established kidney disease risk factors hypertension and diabetes mellitus were associated strongly with measures of kidney damage/dysfunction, but the R229Q polymorphism was not associated with an additional increase in kidney disease measures. LIMITATIONS: Single measurement of ACR, subsample of all ARIC participants. CONCLUSION: No significant association of the relatively rare R229Q variant and ACR or eGFR was found in either white or black individuals. The phenotypic effect of a variant as R229Q would have to be of great magnitude to meaningfully contribute to the risk of kidney disease on a population level. The importance of such variants in the general population, as well as replication studies, can be evaluated best in large community-based studies that allow for accounting of established disease risk factors.

Reliability of high-throughput genotyping of whole genome amplified DNA in SNP genotyping studies.

Whole genome amplification (wga) of DNA is being widely implemented in many laboratories to extend the life of samples only available in limited quantities for genetic analysis. We determined the reliability of wgaDNA genotypes in three sets of replicates from the same individuals: (i) 23 pairs of genomic DNA (gDNA), (ii) 43 pairs gDNA versus wgaDNA, and (iii) 29 pairs of independently amplified wgaDNA. Amplification was performed using multiple displacement amplification (MDA). Genotyping was successful for both DNA types for 1268 out of 1534 SNPs from 164 cardiovascular candidate genes assayed in a single Illumina panel. Amplified DNA failed for 77 SNPs (6%) that were genotyped successfully with genomic material. Percent of successful SNP calls, and concordance between pairs and kappa statistics (kappa) were determined. A total of 54 110 genotypes from gDNA-wgaDNA pairs were available for concordance analysis. Mean kappa for gDNA-wgaDNA pairs was 0.99. Concordance between gDNA-wgaDNA pairs was higher than amongst wgaDNA pairs (mean kappa for the 29 independently amplified pairs of wgaDNA was 0.95; interquartile range: 0.93-1.00). A statistical analysis of those SNPs which failed to genotype from amplified DNA only revealed that those loci were more likely to be closer to the telomeres and in locally GC-rich sequences. In summary, the MDA method produces wgaDNA samples that can be genotyped using high-throughput technology with a very high reproducibility to the original DNA but with slightly lower call rates. DNA amplification methodologies provide a useful solution for current and future large-scale genetic analyses especially with limited quantities of samples and DNA.

C-Reactive protein haplotype predicts serum C-reactive protein levels but not cardiovascular disease risk in a dialysis cohort.

BACKGROUND: C-Reactive protein (CRP) gene variation has been associated with serum CRP levels in the general population. We examined the associations of CRP gene variation with longitudinal CRP measurements and incident cardiovascular disease (CVD) risk in a cohort of 504 white and 244 African-American incident dialysis patients. METHODS: Seven tagging single-nucleotide polymorphisms in the CRP gene were selected by using the Carlson method (r(2) > 0.65). High-sensitivity CRP was measured every 6 months (mean, 4.6 months). Haplo.glm was used to determine the association of haplotypes with serum CRP levels and CVD risk. Global tests from Haplo.score were conducted to determine statistical significance. RESULTS: Compared with the most common haplotype, 1 haplotype was associated with a 52% lower CRP level at baseline among African Americans (ratio, 0.48; 95% confidence interval [CI], 0.28 to 0.82; global P-value = 0.0005). Furthermore, this haplotype was associated significantly with lower serum CRP levels during 36 months of follow-up. Among whites, this haplotype was associated with an 18% (ratio, 0.82; 95% CI, 0.56 to 1.22; n = 6 carriers) lower CRP level compared with the most common haplotype with a frequency of 1% (global P-value = 0.048). No association was detected between CRP gene variation and CVD risk in either whites or African Americans. CONCLUSION: Compared with the most common haplotype of the CRP gene, 1 haplotype predicts a lower serum CRP level over time, but no association exists between haplotype of CRP gene and incident CVD in this incident dialysis population. Serum CRP level might be a biomarker, rather than a causal factor, in CVD development. CRP variation may lead to susceptibility to inflammation, but not risk for CVD; however, replication in multiple settings is necessary.

Functional variants in the lymphotoxin-alpha gene predict cardiovascular disease in dialysis patients.

TNF-beta that is encoded by lymphotoxin-alpha gene (LTA) regulates adhesion molecules and IL-6. Previously, a genome-wide case-control study showed that LTA gene variants predisposed to cardiovascular disease (CVD). In a prospective study of 775 dialysis patients, LTA and IL-6 gene variants were tested as independent predictors of CVD risk. Four polymorphisms in the LTA gene and one in the IL-6 gene were genotyped. CVD events were ascertained from medical records. During a mean follow-up of 2.6 yr, 294 first-incident CVD events occurred. The LTA 26Asn variant predicted higher adjusted CVD risk (hazard ratio HR 1.33 for each additional copy of Asn allele; 95% confidence interval 1.14 to 1.55; P = 0.0003). Two other nonsynonymous polymorphisms in the LTA, 13Agr and 51Pro, were associated with lower inflammatory activity and CVD risk. LTA haplotypes (based on all four single-nucleotide polymorphisms) were associated with inflammatory markers and predicted CVD risk (P = 0.005) after adjustment. These LTA genotype associations were independent of the IL-6 -174G/C genotype association that was reported recently. LTA and IL-6 gene variants independently predicted risk for CVD among dialysis patients, suggesting that susceptibility in multiple inflammatory pathways contribute to the development of CVD.

Haplotype of signal transducer and activator of transcription 3 gene predicts cardiovascular disease in dialysis patients.

Signal transducer and activator of transcription 3 (STAT3) protein has been linked to cardiovascular disease (CVD) through multiple pathways in experimental and animal studies. STAT3 gene variation was examined as a predictor of incident CVD in a subcohort of 529 incident white dialysis patients. Fifteen single-nucleotide polymorphisms of the STAT3 gene were genotyped. Haplotypes were estimated using software PHASE 2.1, and associations with first CVD event were tested using Cox proportional hazards analysis. Adjusted global tests of haplotype association with incident CVD and inflammation markers were performed using permutated P value in R-package Haplo.score. An a priori specified additive genetic model was assumed for haplotype analysis. Both genotypes (four single nucleotide polymorphisms with P < 0.001) and haplotypes (P = 0.002 overall) were associated with incident CVD. Two major haplotype blocks, blocks A and C, were identified. Compared with common haplotype A-1, A-3 was associated with a hazard ratio (HR) of 0.70 (95% confidence interval [CI] 0.51 to 0.94) for CVD events after adjustment for covariates including C-reactive protein (CRP) and interleukin 6. Compared with common haplotype C-1, C-3 was associated with an adjusted HR of 2.12 (95% CI 1.25 to 3.57) for CVD events. Associations were independent of inflammation markers, but IL-6 levels were 14% lower (geometric mean ratio 0.86; 95% CI 0.77 to 0.96) per copy of haplotype A-3 compared with haplotype A-1 in block A after adjustment for CRP and other risk factors (P = 0.008). Variation in the STAT3 gene is associated with the risk for CVD among white dialysis patients independent of serum IL-6 and CRP levels.

IL-6 haplotypes, inflammation, and risk for cardiovascular disease in a multiethnic dialysis cohort.

It is unknown whether IL-6, a central regulator of inflammation, is a cause of or just a marker of atherosclerosis. Studies of genetic susceptibility to inflammation, however, avoid the potential for reverse causality. Variation in IL6 gene was studied as a predictor of cardiovascular disease (CVD) risk in a cohort of 775 incident dialysis patients, in whom IL-6 levels are elevated. On the basis of published resequencing data on the IL6 gene, a phylogenetic tree with three main branches (clades 1 to 3) was constructed. Two "clade tag" polymorphisms, -174G/C and 1888G/T, and two missense variants, Pro32Ser and Asp162Val, were genotyped. Circulating IL-6 and albumin were measured a median of 5 mo after the start of dialysis. CVD events were ascertained from medical records. During a median follow-up of 2.5 yr, 294 CVD events occurred. The two coding variants, Pro32Ser (present only in black patients, 10% Ser allele) and Asp162Val (present only in white patients, 1% Val), were associated with lower levels of IL-6 and higher levels of albumin. The common variant in the promoter region, -174G/C, was strongly associated with higher CVD risk and weakly with IL-6 levels. Clade 3 (-174C carriers in the absence of 162 Val allele) was associated with higher IL-6 levels (P=0.03) and higher CVD risk (hazard ratio 1.44, P=0.006) after adjustment for covariates. The IL6 gene has functional variants that affect inflammation and risk for CVD among dialysis patients, supporting a causal role for IL6 in CVD.

Beta-fibrinogen haplotypes and the risk for cardiovascular disease in a dialysis cohort.

BACKGROUND: Elevated plasma fibrinogen levels are common in dialysis patients and may be related to an elevated risk for cardiovascular disease (CVD). We tested the hypothesis that genetic variation in the beta-fibrinogen ( FGB ) gene, shown to explain 1% to 5% of fibrinogen level variation in the general population, has an important role in elevated fibrinogen levels and excess CVD risk in dialysis patients. METHODS: Plasma fibrinogen was measured in 735 dialysis patients a median of 3 months from the start of dialysis therapy by using an automated clot-rate assay. Seven polymorphisms of the FGB gene were determined. Haplotype analysis was conducted using the Phase program to estimate haplotypes, with stratification for race. CVD events were ascertained from medical records. RESULTS: During a median follow-up of 2.1 years, 279 CVD events occurred. Genotype frequencies were in Hardy-Weinberg equilibrium. Four common haplotypes identified were not associated with fibrinogen levels or CVD risk in the entire cohort or after stratification by race. The -455A allele, known to increase gene expression in vitro, was marginally associated with fibrinogen levels only in patients without diabetes (regression coefficient [beta], 20 mg/dL [for +1 copy of the A allele; P = 0.06]), adjusted for age, sex, race, smoking, baseline dialysis modality, comorbidity, and history of diabetes and CVD. Post hoc analysis showed that -249C-->T (defining haplotype 3) was associated with greater fibrinogen levels and CVD risk among patients without diabetes and current smokers. CONCLUSION: The FGB gene likely does not have an important role in determining the variation in elevated plasma fibrinogen levels or excess CVD risk in dialysis patients.