Skip Navigation


NDT Advance Access originally published online on February 10, 2008
Nephrology Dialysis Transplantation 2008 23(7):2260-2264; doi:10.1093/ndt/gfm946
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/7/2260    most recent
gfm946v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Ng, D. P. K.
Right arrow Articles by Krolewski, A. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ng, D. P. K.
Right arrow Articles by Krolewski, A. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please e-mail: journals.permissions@oxfordjournals.org


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

Daniel P. K. Ng1,2, Siti Nurbaya2, Serena Choo2, David Koh1,2, Kee-seng Chia1,2 and Andrzej S. Krolewski3,4

1 Department of Community, Occupational and Family Medicine 2 Centre for Molecular Epidemiology, National University of Singapore, Singapore 3 Section on Genetics and Epidemiology, Joslin Diabetes Center 4 Department of Medicine, Harvard Medical School, Boston, MA, USA

Correspondence and offprint requests to: Daniel P. K. Ng, Department of Community, Occupational and Family Medicine, National University of Singapore, Singapore. E-mail: cofnpkd{at}nus.edu.sg



   Abstract
Top
 Abstract
Introduction
 Subjects, materials and methods
 Results
 Discussion
 Supplementary data
 References
 
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.

Keywords: end-stage renal disease; genetic susceptibility; haplotype block; multiple hypothesis testing; proteinuria



   Introduction
Top
 Abstract
 Introduction
Subjects, materials and methods
 Results
 Discussion
 Supplementary data
 References
 
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
Top
 Abstract
 Introduction
 Subjects, materials and methods
Results
 Discussion
 Supplementary data
 References
 
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 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. The Committee on Human Subjects of the Joslin Diabetes Center approved the protocols and informed consent procedures for our studies.

Genotyping
We analysed 11 SNPs spanning the 48 kb SLC12A3 gene. Among these SNPs included all that were previously associated with diabetic nephropathy among Asians including the +34372G>A (Arg913Gln) SNP that had showed the strongest disease association [1–3]. Genotyping was performed either by Taqman-based assays or by PCR-RFLP as appropriate (Supplementary Table 1). The median success rate in genotyping was 97.4% (range: 93.6–99.6%).

Power calculations and statistical analysis
Data on the study groups were compared using {chi}2 and Student's t-tests for categorical and continuous variables, respectively (The SAS® system for windows version 9.1, SAS Institute Inc., Cary, NC, USA). A comparison of means across multiple groups was performed using ANOVA. Haplotype blocks were defined according to the method of Gabriel et al. [6] with |D'| ≥ 0.8 being considered as evidence of strong linkage disequilibrium (LD). Estimation of haplotype frequencies was performed using the expectation–maximization algorithm [7]. Statistical significance of haplotype association with disease was evaluated using a score test based on unphased genotypic data [8]. A P-value of <0.05 was considered statistically significant after applying the Bonferroni correction for the number of allele, genotype and haplotype comparisons. Power calculations were performed to determine our ability to detect the magnitude of disease association reported by Tanaka et al. [1]. With an alpha of 0.05, our study has >90% power to detect disease association with the SLC12A3 +34372G>A (Arg913Gln) ‘G’ allele, given the reported odds ratio (OR) of 2.53 [1]. Additional power calculations revealed that our study had power exceeding 90%, 80% and 70% to detect ORs of 2, 1.9 and 1.7 respectively.



   Results
Top
 Abstract
 Introduction
 Subjects, materials and methods
 Results
Discussion
 Supplementary data
 References
 
Clinical characteristics
A total of 174 controls and 295 cases were analysed in this study. Both groups were similar in terms of gender composition, age at diabetes diagnosis and HbA1c values (P = NS) (Table 1). Besides a longer known diabetes duration (P < 0.0001), cases had higher systolic blood pressure (P < 0.0001) but slightly lower diastolic blood pressure compared to controls (P < 0.001) (Table 1). Fifty three point two per cent of cases had already developed CRF/ESRD by the time of enrolment into this study.


View this table:
[in this window]
[in a new window]

  		Table 1 Patient characteristics

 
Linkage disequilibrium across SLC12A3
All SNPs were in Hardy Weinberg equilibrium among cases and controls after taking into account the number of markers studied. Measures of LD for SLC12A3 SNPs are shown in Table 2. The SNPs could be partitioned into 3 haplotype blocks on the basis of |D'| values according to the method of Gabriel et al. [6] (Table 3). Block 1 consisted of –1991C>A, –950A>G, +1784C>T and +1918A>G, while block 2 comprised +18806C>T, +21822C>T and +34372G>A (Arg913Gln). SNPs +39375C>T and +41727C>T constituted block 3 while the remaining +39143G>A and +39240C>T were not assigned to any blocks.


View this table:
[in this window]
[in a new window]

  		Table 2 Linkage disequilibrium across SLC12A3 in Caucasians

 

View this table:
[in this window]
[in a new window]

  		Table 3 Genotype and allele distributions of SLC12A3 SNPs

 
SNP-disease association
None of the 11 SNPs showed any significant association with advanced diabetic nephropathy both in terms of allelic or genotypic distributions (Table 3). Consideration of potentially important covariates including gender, blood pressure, glycaemic control and diabetes duration did not reveal any association between any of the SNPs and advanced diabetic nephropathy (data not shown). Haplotypes were constructed from unphased genotypic data separately for each of the haplotype blocks. Estimated haplotype frequencies were comparable between cases and controls (Table 4). Consistent with these estimates, results of haplotype score tests yielded no significant evidence to support any association with diabetic nephropathy (Table 4). Allele, genotype and haplotype distributions were similar among cases regardless of whether they were proteinuric or had developed CRF/ESRD (data not shown).


View this table:
[in this window]
[in a new window]

  		Table 4 SLC12A3 haplotype distribution

 


   Discussion
Top
 Abstract
 Introduction
 Subjects, materials and methods
 Results
 Discussion
Supplementary data
 References
 
In this present study, we found that SLC12A3 SNPs, which were previously associated with advanced diabetic nephropathy in Japanese diabetic patients, do not appear to contribute to such a disease risk in Caucasians with type 2 diabetes. Although this is not an unfamiliar situation in studies on diabetic nephropathy [9] and other genetically complex diseases [10], various possibilities may explain these discrepant findings.

One of the possibilities may be that the first evidence implicating SLC12A3 may potentially be spurious. With >55 000 SNPs having been studied in that genome-wide investigation, this is a distinct possibility. Moreover, while the reported association with +34372G>A (Arg913Gln) had a small P-value of 0.00002, it is not clear if this would have remained significant after correction for multiple hypothesis testing.

It may be postulated that the Japanese findings reflect the effect of a ‘winner's curse’, a phenomenon in which the first reported positive study tends to demonstrate an association whose effect size is biased upwards of its true magnitude [11]. However, there is no direct biological evidence in the current literature that we are aware of that would have spurred the investigation of SLC12A3 as a candidate gene. A winner's curse is therefore unlikely to explain the positive findings of Tanaka et al. [1], although publication bias may potentially influence subsequent reports on this issue.

Another possibility is that the effect of SLC12A3 could be population specific, a notion that is certainly worthwhile to investigate. Supporting this notion, a cohort study on type 2 diabetic Japanese subjects suggested that +34372G>A (Arg913Gln) ‘GG’ genotype could confer a risk for the progression of diabetic nephropathy [2]. This positive finding is, unfortunately, not without caveats. First, the study is based on a small cohort of 264 subjects with only 71 progressors after a 10-year follow-up. This small study size is also reflected by the fact that of the 264 subjects, only 20 individuals were not homozygous for the SLC12A3 ‘G’ risk allele. Secondly, it is not explicit whether the SLC12A3 SNP was associated with advanced diabetic nephropathy per se as a mixed case definition was used to define disease progression [2]. Specifically, progressors included those who developed overt proteinuria from microalbuminuria (n = 22) or normoalbuminuria (n = 11), as well as those who were normoalbuminuric at baseline but later became microalbuminuric (n = 38) [2]. More studies among type 2 diabetic Japanese patients would be useful to more firmly establish the importance of SLC12A3 as a susceptibility gene in this ethnic group.

There is yet another study of late demonstrating an association of SLC12A3 with ESRD due to diabetic nephropathy among type 2 diabetic Koreans. In this case, +34372G>A (Arg913Gln) also turned out to be the most significant SNP. Intriguingly, however, this study found that it was the ‘A’ rather than the ‘G’ allele of +34372G>A that conferred a risk for ESRD. In further analyses, the Korean researchers also reported that a specific haplotype (which was termed ‘BL2-ht4’) was associated with ESRD [3]. In attempting to replicate their results, however, we did not find any statistically significant evidence to support such an association in Caucasians with type 2 diabetes (data not shown).

Our findings are not without caveats. Since the primary aim of our study was to replicate the findings reported in earlier studies, we focused on the 11 SNPs that were studied previously (including Arg913Gln and all others that were found to have some evidence for association) [1–3] as this allowed us to unambiguously compare our results with the previous reports. On the other hand, these SNPs were not deliberately chosen as tagging SNPs and thus may not completely capture all possible haplotypes at this locus. Still, a substantial degree of the common haplotype diversity would inadvertently have been covered since the average SNP density was 1 SNP per 4.4 kb. At the same time, the 11 SNPs were predominantly independent of each other with low pairwise r2 values (Table 2) and therefore when combined together would be informative in haplotype analysis. Indeed, in addition to the SNPs that were directly studied, 10 common haplotypes were also tested for association with diabetic nephropathy (Table 4).

In conclusion, our present findings suggested that risk for advanced diabetic nephropathy among type 2 diabetic Caucasians is unlikely to be explained by genetic variation at the SLC12A3 locus. The observation that SLC12A3 on chromosome 16q13 is not located within all prospective major susceptibility loci reported to date based on linkage scans in this ethic group would seem to be consistent with this conclusion [12].



   Supplementary data
Top
 Abstract
 Introduction
 Subjects, materials and methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary data is available online at http://ndt.oxfordjournals.org



   Acknowledgments
 
We thank Adam Smiles (Joslin Diabetes Center) for maintaining the clinical and genotypic databases. A.S.K. received funding from National Institutes of Health (DK058549 [GenBank] ) and D.P.K.N. from National Medical Research Council (NMRC/1018/2005).

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects, materials and methods
 Results
 Discussion
 Supplementary data
 References
 

  1. Tanaka N, Babazono T, Saito S, et al. Association of solute carrier family 12 (sodium/chloride) member 3 with diabetic nephropathy, identified by genome-wide analyses of single nucleotide polymorphisms. Diabetes (2003) 52:2848–2853.[Abstract/Free Full Text]
  2. Nishiyama K, Tanaka Y, Nakajima K, et al. Polymorphism of the solute carrier family 12 (sodium/chloride transporters) member 3, SLC12A3, gene at exon 23 (+78G/A: Arg913Gln) is associated with elevation of urinary albumin excretion in Japanese patients with type 2 diabetes: a 10-year longitudinal study. Diabetologia (2005) 48:1335–1338.[CrossRef][Web of Science][Medline]
  3. Kim JH, Shin HD, Park BL, et al. SLC12A3 (solute carrier family 12 member sodium/chloride] 3) polymorphisms are associated with end-stage renal disease in diabetic nephropathy. Diabetes (2006) 55:843–848.[Abstract/Free Full Text]
  4. Warram JH, Gearin G, Laffel L, et al. Effect of duration of type I diabetes on the prevalence of stages of diabetic nephropathy defined by urinary albumin/creatinine ratio. J Am Soc Nephrol (1996) 7:930–937.[Abstract]
  5. K/DOQI. clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis (2002) 39(Suppl_1):S1–S266.[CrossRef][Web of Science][Medline]
  6. Gabriel SB, Schaffner SF, Nguyen H, et al. The structure of haplotype blocks in the human genome. Science (2002) 296:2225–2229.[Abstract/Free Full Text]
  7. Excoffier L, Slatkin M. Maximum-likelihood estimation of molecular haplotype frequencies in a diploid population. Mol Biol Evol (1995) 12:921–927.[Abstract]
  8. Schaid DJ, Rowland CM, Tines DE, et al. Score tests for association between traits and haplotypes when linkage phase is ambiguous. Am J Hum Genet (2002) 70:425–434.[CrossRef][Web of Science][Medline]
  9. Ng DPK, Tai BC, Koh D, et al. Angiotensin-I converting enzyme insertion/deletion polymorphism and diabetic nephropathy: a meta-analysis of studies reported during 1994–2004 and comprising 14,727 subjects. Diabetologia (2005) 48:1008–1016.[CrossRef][Web of Science][Medline]
  10. McCarthy MI, Groop PH, Hansen T. Making the right associations. Diabetologia (2005) 48:1241–1243.[CrossRef][Web of Science][Medline]
  11. Lohmueller KE, Pearce CL, Pike M, et al. Meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease. Nat Genet (2003) 33:177–182.[CrossRef][Web of Science][Medline]
  12. Ng DPK, Krolewski AS. Molecular genetic approaches for studying the etiology of diabetic nephropathy. Curr Mol Med (2005) 5:509–525.[CrossRef][Web of Science][Medline]
Received for publication: 12.10.07
Accepted in revised form: 19.12.07


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Ther Adv Cardiovasc DisHome page
S. Maeda
Review: Genetics of diabetic nephropathy
Therapeutic Advances in Cardiovascular Disease, October 1, 2008; 2(5): 363 - 371.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
23/7/2260    most recent
gfm946v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Ng, D. P. K.
Right arrow Articles by Krolewski, A. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ng, D. P. K.
Right arrow Articles by Krolewski, A. S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?