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

Daniel P. K. Ng 1 2 3 Siti Nurbaya 1 2 Serena Choo 1 2 David Koh 1 2 3 Kee-seng Chia 1 2 3 Andrzej S. Krolewski 0 2 4 0 Section on Genetics and Epidemiology, Joslin Diabetes Center 1 Centre for Molecular Epidemiology, National University of Singapore , Singapore 2 Community, Occupational and Family Medicine, National University of Singapore , Singapore 3 Department of Community, Occupational and Family Medicine 4 Department of Medicine, Harvard Medical School , Boston, MA, USA C The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: - 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 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. Introduction Preliminary large-scale genotyping efforts have been carried out among Japanese subjects with type 2 diabetes to identify genes for advanced diabetic nephropathy. In an initial report based on an analysis of 56 648 single nucleotide polymorphisms (SNPs), polymorphisms in the solute carrier family 12 (sodium/chloride transporters) member 3 (SLC12A3) were associated with advanced diabetic nephropathy that was defined as the presence of proteinuria or chronic renal failure secondary to diabetes [1]. The association was strongest for the +34372G>A SNP in exon 23 that causes a non-conservative Arg/Gln amino acid change. Although the functional impact of this SNP on the SLC12A3 protein function has not been established, the implication of this gene may potentially yield novel insights into the aetiology of diabetic nephropathy. There are, however, several unresolved issues. First, the evidence implicating SLC12A3 was derived from the genotyping of a vast number of SNPs. This leads to an obvious issue of multiple hypothesis testing that will inadvertently cause false positive findings (statistical type 1 error). Secondly, the importance of SLC12A3 has only been studied in Asian populations [2,3]. Thus, the importance of this gene among other human populations such as Caucasians remains unknown. In this present study, therefore, we sought to determine if genetic variation at the SLC12A3 locus does confer susceptibility to advanced diabetic nephropathy [proteinuria or chronic renal failure/end-stage renal disease (CRF/ESRD)] among Caucasians with type 2 diabetes. Subjects, materials and methods Study groups Since 1998, individuals with type 2 diabetes have been recruited for studies of the genetics of nephropathy from among patients attending the Joslin Clinic in Boston, MA. Diabetes has been classified as type 2 if it was diagnosed Controls Clinical characteristics Number (n) Gender (male/female) Age at diabetes diagnosis (years) At the enrolment into the study Age BMI (kg/m2) Known duration of diabetes (years) Haemoglobin A1c (%) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) ACR (mg/g)a Glomerular filtration rate (ml/min)b Percentage of cases with CRF/ESRD Data are presented as mean (SD). N/A, not applicable. aMedian (25th75th percentiles). bEstimated using the MDRD formula [5]. between ages 35 and 64 years and was treated for at least 2 years with diet or oral hypoglycaemic agents. Only patients younger than 75 years of age at enrolment are included in the study. Diagnosis of diabetic nephropathy Diabetic nephropathy was determined on the basis of the medical records of the Joslin Clinic (supplemented with records of other physicians if necessary) and results of routine analyses, including measurements of the albumin to creatinine ratio (ACR) in spot urine samples and serum concentration of creatinine [4]. The diagnosis of type 2 diabetes is generally established many years after the onset of hyperglycaemia. Patients were classified as controls if they had type 2 diabetes with the duration of at least 7 years and the ACR (in mg/g) was <17 (men) or <25 (women) in at least 2 out of the last 3 urine specimens spanning at least a 2-year interval. Patients with microalbuminuria or intermittent proteinuria were not included in this study. Patients were considered cases if they had persistent proteinuria or if they had ESRD due to diabetic nephropathy. Persistent proteinuria was defined as 2 out of 3 successive urinalyses positive by either reagent strip (>2+ on Multistix, Bayer Corporation, Diagnostics Division, Elkhart, IN, USA) or an ACR (in mg/g) >250 (men) or >355 (women). Patients with persistent proteinuria and serum creatinine >2.0 mg/dl were considered as cases with CRF. Using serum creatinine values, we estimated the glomerular filtration rate (GFR) according to the Modification of Diet in Renal Disease (MDRD) formula [5]. At the time of this study, genomic DNA was available for 295 cases and 174 controls. Examination of study participants All patients selected for the genetic studies were examined at the clinic or at their homes. After consenting to participate in the study, each subject had a standardized physical examination and provided a diabetes history regarding its diagnosis, treatment and complications. Each individual provided a blood sample for DNA extraction. Patient medical records were thoroughly reviewed to minimize the possibility of the presence of non-diabetic kidney disease and patients were also directly questioned whether they were ever diagnosed for non-diabetic kidney disease by MDs (...truncated)