Effect of MDM2 SNP309 and p53 codon 72 polymorphisms on lung cancer risk and survival among non-smoking Chinese women in Singapore.

BACKGROUND: Single nucleotide polymorphism (SNP) 309 resulting in a T or G allele in the promoter of MDM2, the negative regulator of p53, has been suggested to affect cancer predisposition and age of onset, primarily in females. However, findings have been inconsistent in various cancers, and ethnicity appears to be a critical factor influencing the effects of the SNP on cancer risk. An increasing trend has been observed in the prevalence of lung cancers in non-smokers, especially females, though the underlying genetic basis is unclear. METHODS: We therefore examined the role of the SNPs in the p53 pathway (p53 codon 72 and MDM2 SNP309) on lung cancer risk and prognosis of a life-time non-smoking female Chinese population, in a hospital-based case-control study of 123 cases and 159 age-matched controls, by PCR analysis. RESULTS: Our findings reveal that the risk of lung cancer among individuals with the MDM2 SNP309 TT genotype was 2.1 (95% CI 1.01-4.36) relative to the GG genotype, contrary to initial expectations that the GG genotype with elevated MDM2 levels will increase cancer risk. Those who had this genotype in combination with the p53 Pro allele had a risk of 2.5 (95% CI 1.2-5.0). There was however no effect of either polymorphism on age at diagnosis of lung cancer or on overall survival. CONCLUSIONS: The results thus demonstrate that the MDM2 SNP309 TT rather than the GG genotype is associated with increased risk of lung cancer in this population, suggesting that other mechanisms independent of increased MDM2 levels can influence cancer susceptibility.

Genetic variation at the SLC12A3 locus is unlikely to explain risk for advanced diabetic nephropathy in Caucasians with type 2 diabetes.

BACKGROUND: Large-scale genotyping efforts performed on Japanese subjects with type 2 diabetes have implicated polymorphisms in solute carrier family 12 (sodium/chloride transporters) member 3 (SLC12A3) as being associated with advanced diabetic nephropathy. However, it is not known whether these polymorphisms confer a risk for this complication in type 2 diabetic Caucasians. METHODS: A case-control study was conducted that consisted of 295 cases with advanced diabetic nephropathy and 174 controls who have remained normoalbuminuric despite >or=7 years of diabetes. A total of 11 single nucleotide polymorphisms (SNPs) spanning the SLC12A3 locus was analysed including +34372G>A (Arg913Gln) that was the marker previously showing the strongest evidence for disease association in type 2 diabetic Japanese. Power calculations indicated that with an alpha of 0.05, our study has >90% power to detect disease associations of the magnitude previously reported for +34372G>A (Arg913Gln). RESULTS: Allele and genotype distributions for all 11 SNPs were found to be comparable between cases and controls, consistent with the absence of disease association. This negative result was reiterated in subgroup analysis after taking into account potentially important covariates including gender, diabetes duration, blood pressure and glycaemic control. No significant disease associations were likewise found for SLC12A3 haplotypes. Allele, genotype and haplotype distributions were similar in cases regardless of whether they were proteinuric or had developed chronic renal failure/end-stage renal disease. CONCLUSIONS: Genetic variation at the SLC12A3 locus is unlikely to explain the risk for advanced diabetic nephropathy among type 2 diabetic Caucasians.

A disease haplotype for advanced nephropathy in type 2 diabetes at the ACE locus.

Previous investigations of the ACE gene as a susceptibility factor for diabetic nephropathy have primarily focused on its insertion/deletion (Ins/Del) polymorphism. In a departure from these earlier studies, we used three tagging markers (A-5466C, T-3892C, and Ins/Del) at the ACE locus to test for disease haplotype associations. A case-control study design was used where case subjects were type 2 diabetic patients with advanced diabetic nephropathy, as indicated by the presence of proteinuria or chronic renal failure/end-stage renal disease, while control subjects were normoalbuminuric, despite >6 years of diabetes. None of the individual markers showed significant disease association when considered on their own. However, haplotype analyses revealed a near doubling in the prevalence of the A.T.D risk haplotype in case subjects (0.136) compared with control subjects (0.075) (P = 0.009), thus providing first evidence for a disease haplotype for advanced diabetic nephropathy at the ACE locus.

Saliva as a viable alternative source of human genomic DNA in genetic epidemiology.

BACKGROUND: Saliva is a potentially useful but untapped source of genomic DNA for genetic epidemiological studies. However, current commercial methods are mainly concerned with DNA extraction and do not address important issues concerning saliva preservation and storage. As such, we evaluated how various saliva storage conditions affected DNA yield and quality obtained using a new commercially available method that proposes to integrate these aspects in a single kit. METHODS: The conditions involved the extraction of the DNA immediately after saliva collection (condition 1) or when stored at air-conditioned room temperature (20 degrees C) for 1 month (condition 2) and 6 months (condition 3) as well as at -80 degrees C for 6 months (condition 4). The effect of incorporating an additional incubation of saliva samples at 30 degrees C for 2 weeks was also examined. RESULTS: Overall average DNA yield from 2 ml of saliva was 35.5 microg (8.5-85.2 microg). DNA yield was unaffected by incubation of saliva at 30 degrees C but DNA yield under condition 3 was significantly higher compared to conditions 1 and 2. OD260/280 values were acceptable and comparable across all conditions. Differences in storage conditions did not impact DNA quality in real time PCR experiments and genotyping fidelity remained undiminished. CONCLUSION: Saliva is a viable alternative source of human genomic DNA for genetic epidemiological studies and that this new commercial method and possibly other related techniques can be effective means towards this end.

Joint effect of asthma/atopy and an IL-6 gene polymorphism on lung cancer risk among lifetime non-smoking Chinese women.

Recent evidence suggests that inflammatory pathways are important mediators of carcinogenesis. Asthma, allergic rhinitis and atopic dermatitis are clinical manifestations of a systemic atopic disorder, which is associated with airway hyper-responsiveness and inflammation. We examined the effect of a history of asthma/atopy among 132 lung cancer cases (of which 72% were adenocarcinomas) and 163 controls, all of whom were non-smoking Chinese women, in combination with a single nucleotide polymorphism (-634C/G) in the interleukin-6 (IL-6) gene which regulates secretion of a pro-inflammatory cytokine found to be predominant in lung tumour tissue. We observed a slight increase in risk of lung cancer [odds ratio, OR = 1.5, 95% confidence interval (95% CI) = 0.8-2.6] and of adenocarcinoma (OR = 1.6, 95% CI = 0.9-3.1) with asthma/atopy alone. There was no effect of the IL-6 CG/GG genotype on lung cancer risk on its own. Among individuals with both asthma/atopy and the IL-6 -634 G allele, however, risk was increased at least 3-fold (OR = 3.1, 95% CI = 1.2-8.3 for all cancers and OR = 4.2, 95% CI = 1.5-11.6 for adenocarcinomas) relative to individuals with no asthma/atopy and the CC genotype. On stratified analysis, a significant increase in risk with asthma/atopy was restricted to those with the at-risk genotype (Pint < 0.05). Our findings are consistent with the role of chronic inflammation as an aetiologic factor among non-smoking Asian women, and suggest that asthma/atopy is a risk marker for susceptibility to the development of lung cancer.

The fatty acid-binding protein-2 A54T polymorphism is associated with renal disease in patients with type 2 diabetes.

The intestinal fatty-acid binding protein-2 (FABP2) gene codes a protein responsible for the absorption of long-chain fatty acids. To test whether FABP2 is a candidate gene for renal disease in patients with type 2 diabetes, a functional A54T polymorphism was genotyped in 1,042 Brazilians with type 2 diabetes. Patients were classified as having normoalbuminuria (urinary albumin excretion [UAE] <20 microg/min; n = 529), microalbuminuria (UAE 20-199 microg/min; n = 217), or proteinuria (UAE >199 microg/min; n = 160). Patients with end-stage renal disease (ESRD) (n = 136) were also included. The prevalence of the TT genotype was higher in patients with renal involvement compared with those with normoalbuminuria (odds ratio [95% CI] 2.4 [1.1-5.4]) following adjustment for type 2 diabetes duration, BMI, hypertension, A1C, and cholesterol levels. The risk was similar considering different stages of renal involvement. In a second independent patient sample (483 type 2 diabetic Caucasians residing in Massachusetts), a significant association was also observed between the TT genotype and proteinuria or ESRD (2.7 [1.0-7.3]; P = 0.048). This study thus provides evidence that FABP2 confers susceptibility to renal disease in type 2 diabetic patients.

Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva.

BACKGROUND: Saliva is a potentially useful source of genomic DNA for genetic studies since it can be collected in a painless and non-invasive manner. We sought to determine whether different storage conditions of saliva samples impact our ability to extract genomic DNA that is of sufficient quality for use in the polymerase chain reaction (PCR). METHODS: Saliva was collected from healthy volunteers and 2-ml aliquots subjected to different storage conditions: S1--washing of saliva using phosphate-buffered saline (PBS) and extraction of DNA on the same day of collection; S2--washing and centrifugation to yield a pellet, which was stored at-70 degrees C for 1 week prior to DNA extraction; S3--storage of whole saliva at 4 degrees C for 7 days, followed by washing and extraction of DNA; S4--storage at 4 degrees C for 7 days, followed by washing and pellet formation. The pellet was stored at -70 degrees C for 1 month before extraction of the DNA; S5--storage at-70 degrees C for 1 month, followed by washing and extraction of DNA. DNA yield and purity was determined by spectrophotometry at 260 and 280 nm. Twenty nanograms of genomic DNA was used for the polymerase chain reaction, and the resulting PCR band was captured by digital photography and quantified. RESULTS: The amounts of DNA extracted from 2 ml of saliva varied widely under the different storage conditions, while purity of the DNA extraction, based on OD(260/280) ratios, was good and comparable. PCR resulted in the presence of a single specific product of the correct size from all samples regardless of saliva storage conditions. Quantification of PCR bands showed significant differences between the various storage conditions (P<0.05). Compared to S1 samples, PCR bands from conditions S2 and S3 were not as strong, while those amplified from S4 and S5 samples were the weakest. Post-hoc analyses showed that the means for conditions S4 and S5 were significantly different from S1-S3. Qualitatively similar results were obtained when the PCR experiment was repeated. CONCLUSIONS: Saliva can act as a useful source of genomic DNA, even when stored under less than optimal conditions.

Scrutiny of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) locus reveals conserved haplotype block structure not associated with diabetic nephropathy.

Glutamine-fructose-6-phosphate transaminase 1 (GFAT) is the rate-limiting enzyme of the hexosamine pathway that has been implicated in the pathogenesis of diabetic nephropathy. As such, we hypothesized that GFPT1, which encodes for GFAT, may confer genetic susceptibility to this complication among Caucasians. Screening of all known functional regions of GFPT1 revealed six single nucleotide polymorphisms (SNPs) that were located in the promoter, introns, and 3' untranslated region. The approximately 60 kb GFPT1 locus was encompassed in a single conserved haplotype block, and two tagging SNPs were sufficient to capture >90% of the haplotype diversity. Analysis of these SNPs in a case-control study made up of type 1 diabetic subjects (324 case subjects with diabetic nephropathy and 289 control subjects with normoalbuminuria despite >15 years of diabetes) revealed no significant association even after stratification by sex, diabetes duration, glucose control, and blood pressure. Similar results were obtained among type 2 diabetic subjects (202 case and 114 control subjects). Genetic variation in GFPT1 is thus unlikely to have a major impact on susceptibility to diabetic nephropathy.