Role of {alpha}-adducin DNA polymorphisms in the genetic predisposition to diabetic nephropathy

Bryan R. Conway1, Rosalind Martin1, Amy-Jayne McKnight1, David A. Savage1, Hugh R. Brady2 and Alexander P. Maxwell1

1 Nephrology Research Group, Queen's University of Belfast, Northern Ireland and 2 Conway Institute of Biomolecular and Biomedical Research, University College Dublin and the Dublin Molecular Medicine Centre, Irish Republic

Correspondence and offprint requests to: Dr Bryan Conway, Regional Nephrology Unit, Belfast City Hospital, Lisburn Road, Belfast BT9 7AB, Northern Ireland. Email: BryanConway{at}ntlworld.com



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. There is substantial evidence for genetic susceptibility to diabetic nephropathy. In particular, genes that predispose to hypertension in the general population may confer susceptibility to nephropathy in patients with diabetes. A Gly460Trp variant in the {alpha}-adducin gene has been associated with essential hypertension. Our aim was to screen the {alpha}-adducin gene for polymorphisms and to determine if any variants predisposed patients with diabetes to nephropathy. A secondary objective was to assess for association between the Gly460Trp variant and hypertension.

Methods. The exons of the {alpha}-adducin gene were resequenced in 30 individuals. Selected variants were then genotyped in 155 patients with type 1 diabetes and nephropathy (cases) and 216 persons with type 1 diabetes but no evidence of nephropathy (controls) from Northern Ireland and in 95 cases and 118 controls from the Irish Republic.

Results. Eleven polymorphisms were detected, of which six were novel and three caused amino-acid substitutions. The Gly460Trp and a novel Ser617Cys polymorphism were in strong linkage disequilibrium (D' = 0.98). Neither the genotype nor allele frequencies for the Gly460Trp polymorphism (P = 0.89 and 0.93 respectively) or the Ser617Cys polymorphism (P = 0.46 and 0.76) were significantly different between cases and controls when the Northern Ireland and Irish Republic sample groups were combined. Carriage of the 460Trp allele was not significantly associated with systolic or diastolic blood pressure in either the cases (P = 0.48 and 0.06, respectively) or in the controls (P = 0.50 and 0.94, respectively).

Conclusions. Variation in the {alpha}-adducin gene does not play a major role in the development of nephropathy in persons with type 1 diabetes in the Irish population. Furthermore, the Gly460Trp variant was not associated with hypertension in this population.

Keywords: {alpha}-adducin; diabetic nephropathy; genetic susceptibility; hypertension; polymorphism



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The incidence of diabetic nephropathy has more than doubled in the past 10 years, accounting for 44% of all new cases of end-stage renal failure in the US in 1999 [1]. This has significant implications, both for individuals with diabetes and for national healthcare budgets. It is imperative that new treatments are developed, based on a better understanding of the pathogenesis of the disease, in order to prevent nephropathy or reduce the rate of progression in diabetic patients. In addition, new methods of screening for susceptibility to nephropathy are required, so that interventions can be targeted towards those type 1 diabetic patients who are at greatest risk of developing nephropathy.

Unlike diabetic retinopathy, which occurs in the majority of patients with longstanding type 1 diabetes, diabetic nephropathy only ever occurs in approximately one-third of diabetic patients [2,3]. Furthermore, the incidence of nephropathy is greatest in the second decade following diagnosis of type 1 diabetes and beyond 20 years it declines to ~1% per annum, despite the accumulating glycaemic burden. Hence, it appears that only a subset of patients with type 1 diabetes are susceptible to nephropathy and this subset will tend to develop nephropathy within 20 years of the diagnosis of diabetes. Further support for an inherited predisposition to diabetic nephropathy comes from studies showing that nephropathy tends to cluster within families [4,5].

There is increasing evidence that genes that predispose to hypertension in the general population may, in part, mediate the susceptibility to nephropathy in patients with type 1 diabetes. Several studies have shown that non-diabetic parents of patients with type 1 nephropathy have higher blood pressure than the parents of matched type 1 diabetic patients who do not have nephropathy [6–8]. The mean blood pressures of the proteinuric patients correlated closely with that of their parents, implying that the hypertension susceptibility genes may mediate predisposition to nephropathy via their effect on blood pressure [6].

Adducin is a cytoskeletal protein that regulates the number and activity of transmembrane ion pumps. It is a heterodimeric protein composed of two of three different subunits (either {alpha}/ß- or {alpha}/{gamma}-heterodimers), each of which is encoded by separate, but homologous, genes. Mutations in the {alpha}-subunit of the adducin gene have been associated with increased renal sodium reabsorption and salt-sensitive hypertension in Milan hypertensive rats [9]. In humans, the {alpha}-adducin gene is located on chromosome 4 and a G -> T polymorphism at position 217 in exon 10 of the gene results in a glycine to tryptophan substitution at amino-acid position 460 (Gly460Trp) in the protein. This polymorphism has been linked to hypertension in some [10–12] but not all studies [13,14].

The {alpha}-adducin gene previously has been screened for the presence of DNA polymorphisms in African and European-American populations [15]. We wished to screen critical regions of the gene for variation in a sample of the Northern Irish population. The principal aim of the investigation was to employ a case-control study to test for association between selected polymorphisms and diabetic nephropathy. We wished to attempt to replicate the findings in an independent case-control study from the Irish Republic. A secondary objective was to test for association between the Gly460Trp polymorphism and blood pressure in patients with type 1 diabetes, with and without nephropathy.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
Ethical approval was obtained from the appropriate University Research Ethics Committees and written, informed consent was obtained from all subjects prior to conducting this study. We recruited diabetic patients with nephropathy (cases) and without nephropathy (controls) from Northern Ireland. The clinical characteristics of these patients have been described previously [16]. All cases (n = 155) and controls (n = 216) were Caucasians and were diagnosed with type 1 diabetes before 35 years of age. Patients with nephropathy had diabetes for ≥10 years prior to the onset of proteinuria (>0.5 g/24 h), had diabetic retinopathy and exhibited no clinical, serological or radiological evidence of non-diabetic renal disease. The control patients had diabetes for >15 years, had no evidence of microalbuminuria on repeated testing and were not receiving antihypertensive medication. In an attempt to replicate the findings of the Northern Ireland case-control study, 95 patients with type 1 diabetes and nephropathy and 118 patients with type 1 diabetes but without nephropathy were also recruited from the Irish Republic, employing identical diagnostic criteria. The clinical characteristics of the combined sample group are shown in Table 1. We also recruited 400 sex-matched, healthy, non-diabetic controls from the Irish population.


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Table 1. Clinical characteristics of the combined Northern Ireland and Irish Republic sample groups. Values are expressed as the mean (±SD) if normally distributed or as the mean (range) where the distribution is skewed. P-values for the differences between the cases and controls were calculated using t-test and Mann–Whitney statistics for normal and skewed distributions, respectively

 
Resequencing the {alpha}-adducin gene
All 16 exons of the {alpha}-adducin gene, including ~100 bp of intronic sequence on either side of each exon, the 5'- and 3'-untranslated regions and 276 bp upstream of the transcription start site were screened for DNA polymorphisms. This was achieved by resequencing 15 cases and 15 controls from the Northern Irish sample on an ABI 3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA). This sample size provided >95% power to detect polymorphisms with a minor allele frequency of >5%.

Blood pressure measurements
The systolic and diastolic blood pressures for patients with nephropathy and diabetic controls were measured prior to blood sampling. The blood pressure readings from the previous two clinic attendances were obtained and the mean of the three systolic and diastolic blood pressure readings was determined. The number of antihypertensive medications that were prescribed to patients with nephropathy was also recorded.

Genotyping
Based on the minor allele frequency and potential functional effects of the DNA polymorphisms identified, two polymorphisms were selected for genotyping: the previously known Gly460Trp polymorphism and a novel Ser617Cys polymorphism.

Genomic DNA samples from the Northern Irish cases and controls were genotyped for the Gly460Trp polymorphism using an amplification refractory mutation system method, essentially as described previously [13]. Patient samples from the Irish Republic case-control study and the 400 healthy non-diabetic controls were genotyped by pyrosequencing. The region containing the polymorphism was amplified using the following primers: forward: 5'–biotin–CCTTTGCTAGTGACGGTGA-3'; reverse: 5'-CGTGTGTAATGTTCGTCCAC-3'. The polymerase chain reaction (PCR) was performed in a 15 µl reaction containing 10 ng genomic DNA, 1x PCR buffer, 2 mM MgCl2, 0.3 units Hotstar Taq (Qiagen, Crawley, UK), 0.15 mM dNTPs and 0.2 µM of each primer. Thermocycling was performed on a MJ Tetrad Thermal cycler (MJ Research Inc., Waltham, MA, USA) under the following conditions: 95°C for 15 min, then 45 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s, with a final extension at 72°C for 7 min. Pyrosequencing was performed under standard conditions on a PSQTM HS 96A System (Pyrosequencing AB, Uppsala, Sweden) employing the sequencing primer 5'-TGCTTCCATTCTGCC-3'.

The Ser617Cys polymorphism was genotyped in both the Northern Ireland and Irish Republic sample groups using a Taqman assay (Applied Biosystems, Foster City, CA, USA). The region surrounding this polymorphism was PCR-amplified using the following primers: forward: 5'- GCAGGGAGGTGGAGAGGAA-3'; reverse: 5'-CGTGCCCAGGACCACAA-3'. The PCR reaction also contained two fluorescently labelled, allele-specific probes: 617Ser: 5'–VIC–ACCTTCAGAGCCCTT-3'; 617Cys: 5'–FAM–CCTTCACAGCCCTT-3'. The C -> G + 152 polymorphism causing the Ser617Cys amino-acid substitution is marked in bold italics. The PCR was performed in a 15 µl reaction containing 25 ng genomic DNA, 1x universal mastermix (Applied Biosystems), 100 µM of each primer and 5 µM of each probe. Thermocycling conditions were 95°C for 10 min followed by 40 cycles of 92°C for 15 s and 60°C for 1 min. Fluorescent signals were detected using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems).

Statistical analysis
Genotype and allele frequencies in the cases and controls were compared using the {chi}2-test, with the level of significance set at P<0.05. The Mann–Whitney test was used to compare the mean systolic and diastolic blood pressures in carriers vs non-carriers of the 460Trp allele in both the case and control groups individually. It was also used to compare the mean number of antihypertensive agents prescribed to carriers vs non-carriers of the 460Trp allele in patients with nephropathy. The estimated haplotype (EH) frequencies program was used to calculate Lewontin's D', a measure of the degree of linkage disequilibrium, between the Gly460Trp and Ser617Cys polymorphisms.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In total, almost 8 kb of the {alpha}-adducin gene were resequenced in 30 individuals. Eleven polymorphisms were detected, six of which occurred on more than one occasion (Table 2). Three polymorphisms were located in the coding sequence, all of which resulted in an amino-acid substitution. Five of the eleven (45%) polymorphisms identified were also present in the National Center for Biotechnology Information SNP database (dbSNP, Build 116) and each of these occurred in more than one individual in our screen. All four polymorphisms with an estimated minor allele frequency of >5% in our population were present in dbSNP. Only 5 of 19 polymorphisms from the dbSNP were detected in our population, hence the positive predictive value of dbSNP in predicting the presence of polymorphisms in the Northern Irish population was 26%. The C11152-38T->G, C1378G -> T, C1757C -> G and C1861+574C->T polymorphisms had similar minor allele frequencies. In each of these, the minor allele tended to occur in the same individuals, suggesting a high degree of linkage disequilibrium between them.


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Table 2. Polymorphisms detected in the adducin gene. Numbering is based on reference sequence NM001119 (Genbank database), with the A of the translation initiation codon designated as +1

 
On genotyping both the Northern Ireland and Irish Republic sample groups, the Gly460Trp and Ser617Cys polymorphisms were confirmed to be in strong linkage disequilibrium (D' = 0.98). For both polymorphisms and in both sample groups, the distributions of genotypes in cases and controls were in Hardy–Weinberg equilibrium. There was no significant difference in the allele or genotype frequencies between cases and controls for either polymorphism in both sample groups (Table 3). Similarly, when the data from the two sample groups were combined, the allele and genotype frequencies in the cases and controls did not differ significantly for either the Gly460Trp (P = 0.89 and 0.93, respectively) or the Ser617Cys polymorphism (P = 0.46 and 0.76, respectively). When a subgroup of cases who had reached end-stage renal failure (n = 79) was compared with a subgroup of controls who had more than a 20 year duration of diabetes (n = 242), there remained no statistically significant difference in the genotype or allele distributions for the Gly460Trp polymorphism (P = 0.11 and 0.12, respectively).


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Table 3. Distribution of the {alpha}-adducin genotypes and alleles in cases and controls in both the Northern Ireland and Irish Republic sample groups

 
Furthermore, for the Gly460Trp polymorphism, there was no significant difference in the genotype or allele frequencies between the type 1 diabetic patients (combining both cases and controls in both sample groups) and the 400 healthy non-diabetic controls (P = 0.13 and 0.07, respectively).

As expected, there was a highly significant difference in the mean systolic (148.3 vs 125.4 mmHg; P<0.0001) and diastolic blood pressures (85.4 vs 76.2 mmHg; P<0.0001) between cases and controls when the two sample groups were combined. In the combined sample group, there was no association between carriage of the 460Trp allele and systolic or diastolic blood pressure in either the patients with nephropathy (P = 0.48 and 0.06) or in the non-nephropathic controls (P = 0.50 and 0.94, respectively; Table 4). Furthermore, the number of antihypertensive medications prescribed to patients with nephropathy was not significantly greater for carriers of the 460Trp allele than for non-carriers (1.72 vs 1.95, respectively; P = 0.10). Finally, for the Gly460Trp polymorphism, there was no significant difference in the genotype or allele frequencies between hypertensive diabetic patients and the healthy controls (P = 0.21 and 0.12, respectively).


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Table 4. Mean systolic and diastolic blood pressure in patients with and without nephropathy according to 460Trp carriage status in the combined sample

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
While there has been much interest in the association between variants in the {alpha}-adducin gene and essential hypertension, no large studies have addressed its role in susceptibility to diabetic nephropathy. In a case-control study comprising over 150 cases and over 200 controls from Northern Ireland, no association was detected between the Gly460Trp and Ser617Cys polymorphisms in the {alpha}-adducin gene and the development of nephropathy. These findings were replicated in a further case-control study from the Irish Republic. Considered together, the two studies provide ~80% power to detect association with nephropathy with odds ratio of 1.5 and a minor allele frequency of 20%, at the 5% level of significance. Therefore, it appears unlikely that common variation in the {alpha}-adducin gene plays a major role in susceptibility to nephropathy in the Irish population. These results are consistent with a smaller UK case-control study that showed no association between the Gly460Trp polymorphism and nephropathy in patients with type 1 diabetes [17].

Many previous studies investigating the association between genetic variants and diabetic nephropathy have examined a single polymorphism within each gene. As a consequence, good candidate genes may be discarded prematurely without considering all polymorphisms that may potentially affect gene function. In the current study, we comprehensively screened the key regions of the {alpha}-adducin gene for polymorphisms in 30 individuals. This provided >95% power to detect variants with a minor allele frequency of >5%. If the current dbSNP had been used as a tool to predict the presence of polymorphisms in the {alpha}-adducin gene in the Northern Irish population, it would have had a sensitivity of 45% and a positive predictive value of only 26%. Similar results have been found in other studies [18]. Therefore, until gene-centric, population-specific SNP discovery programs are complete, resequencing a sample of the population under investigation is essential, prior to selecting candidate polymorphisms for genotyping.

A further advantage of resequencing is that it permits identification of the patterns of linkage disequilibrium between polymorphisms. From the patterns observed in the initial resequencing, all four polymorphisms with an allele frequency of >5% appeared to be in very strong linkage disequilibrium, lying along a single haplotype. This was confirmed for the Gly460Trp and Ser617Cys polymorphisms when larger-scale genotyping was performed. Therefore, there are only two common haplotypes in this region in the Northern Irish population. By genotyping any one of the four common polymorphisms, the alleles present at the others can be inferred. This method, known as ‘haplotype tag SNPs’ [18], is likely to be the most efficient method of comprehensively screening the common variation within genes for association with disease. While the 460Trp variant has been associated with salt-sensitive hypertension, it is in strong linkage disequilibrium with at least three other polymorphisms, including one that causes an amino-acid substitution. Therefore, the potential role of these variants on the function of the {alpha}-adducin gene warrants further study.

One possible confounding factor in the study is that the 460Trp allele of the {alpha}-adducin gene has been associated with increased susceptibility to cardiovascular disease, particularly in hypertensive patients [19]. Therefore, the presence of the 460Gly may confer a survival bias, particularly in patients with nephropathy who are at high risk of cardiovascular disease. In the current study, more than one-third of the patients with nephropathy had already progressed to end-stage renal failure by the time of recruitment. Carriers of the 460Trp allele may have died prematurely; therefore, the 460Trp allele may be under-represented in this group, masking any true association between the 460Trp allele and nephropathy. A prospective cohort study would be required to conclusively discount this possibility.

As expected, there was a highly significant difference in the systolic and diastolic blood pressures between the patients with nephropathy and the non-nephropathic controls. Within each group, however, carriage of the 460Trp allele was not associated with higher blood pressure. The hypertensive effect of the 460Trp allele may have been masked by the use of anti-hypertensive medications in the patients with nephropathy; however, there was no significant difference between the number of antihypertensive agents prescribed and carriage of the 460Trp allele. While a multitude of additional factors, such as fluid overload, degree of renal dysfunction and degree of proteinuria, might influence blood pressure in the nephropathy group, these were not present in the control group. Hence, it appears that the 460Trp allele exerts minimal effect, if any, on blood pressure in this group of patients. This is in keeping with a recent meta-analysis of 18 studies that examined the association between the Gly460Trp polymorphism and essential hypertension and failed to find evidence for replication of the original report [20].

In conclusion, in this study, the largest to date to investigate the role of the {alpha}-adducin gene in genetic susceptibility to diabetic nephropathy, we have found no evidence of association between variation in the {alpha}-adducin gene and the development of nephropathy in the Irish population. While we cannot exclude the possibility of a minor gene effect, it is unlikely that common variation within the {alpha}-adducin gene plays a major role in the genetic predisposition to diabetic nephropathy in this population.



   Acknowledgments
 
We thank Dr Chris Patterson, Department of Epidemiology, Queen's University, Belfast, for his help with the statistical analysis. This study was supported by funding from the Northern Ireland Kidney Research Fund and a North–South Co-operative Grant from the Research and Development Office (Northern Ireland)/Health Research Board (Irish Republic).

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. US Renal Data System. USRDS 2002 Annual Data Report: Atlas of End-Stage Renal Disease in the United States. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2002
  2. Krolewski AS, Warram JH, Christlieb AR, Busick EJ, Kahn CR. The changing natural history of nephropathy in type I diabetes. Am J Med 1985; 78: 785–794[ISI][Medline]
  3. Krolewski AS, Warram JH, Rand LI et al. Risk of proliferative diabetic retinopathy in juvenile-onset type I diabetes: a 40-yr follow-up study. Diabetes Care 1986; 9: 443–452[Abstract]
  4. Seaquist ER, Goetz FC, Rich S, Barbosa J. Familial clustering of diabetic kidney disease. Evidence for genetic susceptibility to diabetic nephropathy. N Engl J Med 1989; 320: 1161–1165[Abstract]
  5. Quinn M, Angelico MC, Warram JH, Krolewski AS. Familial factors determine the development of diabetic nephropathy in patients with IDDM. Diabetologia 1996; 39: 940–945[CrossRef][ISI][Medline]
  6. Viberti GC, Keen H, Wiseman MJ. Raised arterial pressure in parents of proteinuric insulin dependent diabetics. Br Med J 1987; 295: 515–517[ISI][Medline]
  7. Barzilay J, Warram JH, Bak M et al. Predisposition to hypertension: risk factor for nephropathy and hypertension in IDDM. Kidney Int 1992; 41: 723–730[ISI][Medline]
  8. Fagerudd JA, Tarnow L, Jacobsen P et al. Predisposition to essential hypertension and development of diabetic nephropathy in IDDM patients. Diabetes 1998; 47: 439–444[Abstract]
  9. Bianchi G, Tripodi G, Casari G et al. Two point mutations within the adducin genes are involved in blood pressure variation. Proc Natl Acad Sci USA 1994; 91: 3999–4003[Abstract]
  10. Cusi D, Barlassina C, Azzani T et al. Polymorphisms of alpha-adducin and salt sensitivity in patients with essential hypertension. Lancet 1997; 349: 1353–1357[CrossRef][ISI][Medline]
  11. Province MA, Arnett DK, Hunt SC et al. Association between the alpha-adducin gene and hypertension in the HyperGEN Study. Am J Hypertens 2000; 13: 710–718[CrossRef][ISI][Medline]
  12. Grant FD, Romero JR, Jeunemaitre X et al. Low-renin hypertension, altered sodium homeostasis, and an alpha-adducin polymorphism. Hypertension 2002; 39: 191–196[Abstract/Free Full Text]
  13. Kato N, Sugiyama T, Nabika T et al. Lack of association between the alpha-adducin locus and essential hypertension in the Japanese population. Hypertension 1998; 31: 730–733[Abstract/Free Full Text]
  14. Bray MS, Li L, Turner ST, Kardia SL, Boerwinkle E. Association and linkage analysis of the alpha-adducin gene and blood pressure. Am J Hypertens 2000; 13: 699–703[CrossRef][ISI][Medline]
  15. Halushka MK, Fan JB, Bentley K et al. Patterns of single-nucleotide polymorphisms in candidate genes for blood-pressure homeostasis. Nat Genet 1999; 22: 239–247[CrossRef][ISI][Medline]
  16. Fogarty DG, Harron JC, Hughes AE et al. A molecular variant of angiotensinogen is associated with diabetic nephropathy in IDDM. Diabetes 1996; 45: 1204–1208[Abstract]
  17. Harvey JN, Mead P, Leitch H et al. The Gly460Trp polymorphism of the alpha-adducin gene in diabetic nephropathy. Diabetic Med 2002; 19 [Suppl 2]: 101
  18. Johnson GC, Esposito L, Barratt BJ et al. Haplotype tagging for the identification of common disease genes. Nat Genet 2001; 29: 233–237[CrossRef][ISI][Medline]
  19. Morrison AC, Bray MS, Folsom AR, Boerwinkle E. ADD1 460W allele associated with cardiovascular disease in hypertensive individuals. Hypertension 2002; 39: 1053–1057[Abstract/Free Full Text]
  20. Lohmueller KE, Pearce CL, Pike M, Lander ES, Hirschhorn JN. Meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease. Nat Genet 2003; 33: 177–182[CrossRef][ISI][Medline]
Received for publication: 16. 9.03
Accepted in revised form: 6. 5.04





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