G-protein ß3-subunit C825T genotype and nephropathy in diabetes mellitus

Joachim Beige1, Jens Ringel1, Armin Distler1 and Arya M. Sharma2,

1 Department of Internal Medicine, Division of Endocrinology and Nephrology, Universitätsklinikum Benjamin Franklin, Free University of Berlin, Berlin, Germany and 2 Department of Nephrology, Hypertension and Genetics, Franz-Volhard-Klinik, Humboldt-University, Berlin, Germany



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Genotyping
 Statistical analysis
 Results
 Discussion
 References
 
Background. Recent studies have identified a novel polymorphism (C825T) of the gene encoding the ß3 subunit of heterotrimeric G proteins (Gß3) which is associated with enhanced activation of G-proteins and appears to be more common in hypertensive patients and possibly contributes to decreased kidney allograft survival.

Methods. In the present study we examined the relationship between this genetic variant, type 1 and type 2 diabetes and renal complications of diabetes in 1008 Caucasian patients recruited from an outpatient diabetes clinic and four dialysis centres. We also studied 1940 healthy controls.

Results. After multivariate adjustment and in univariate statistics, the Gß3 825TT genotype was not associated with a significantly enhanced risk of diabetes or renal complications.

Conclusions. These findings indicate that the Gß3 825T allele apparently does not contribute to the development of diabetes or associated renal complications in patients with type 1 or type 2 diabetes mellitus.

Keywords: diabetic nephropathy; Gß3; G-proteins; genetics; HbA1C; polymorphism



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Genotyping
 Statistical analysis
 Results
 Discussion
 References
 
Diabetes mellitus is a complex multicausal disorder characterized by decreased insulin liberation in individuals suffering from type 1 diabetes or impaired insulin-mediated glucose utilization in patients with type 2 diabetes. The pathophysiological mechanisms involved, include immunological impairment of islet cell function in type 1 diabetes [1], as well as a more complex genetic susceptibility to type 2 diabetes [2], clearly exacerbated by environmental factors. Particularly abdominal obesity is known as a major risk factor influencing both the development of type 2 diabetes and hypertension [3].

Recently, a novel single-nucleotide polymorphism (C825T) in exon 10 of the gene encoding the ß3 subunit of heterotrimeric G proteins (Gß3) has been identified, and was found to be significantly associated with enhanced G-protein activation and essential hypertension in patients recruited from Essen, Berlin, and Heidelberg [4]. Moreover, in a recent study we reported that the TT genotype in kidney donor organs was associated with a 30% decreased kidney allograft survival in transplant recipients [5].

The 825T variant is associated with the occurrence of a splice variant Gß3-s, in which the nucleotides 498–620 of exon 9 are deleted, resulting in the loss of 41 amino acids and one WD repeat domain of the Gß subunit. This splice variant was found to be predominantly expressed in fibroblasts from individuals carrying the T allele, and appears to result in significantly enhanced activation of G proteins. It was postulated that expression of Gß3-s may contribute to the development of hypertension by increasing proliferation of smooth-muscle cells, thus resulting in the development of vascular hypertrophy. It is thus readily conceivable that this new marker of genetic vascular risk may accentuate the mechanisms associated with the development of renal impairment in diabetes mellitus.

In the present study we therefore examined the relationship between the Gß3 genotype and the presence of diabetic nephropathy in a large group of Caucasian patients with type 1 and type 2 diabetes mellitus.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Genotyping
 Statistical analysis
 Results
 Discussion
 References
 
Patient selection and clinical investigation
Diabetic patients (n=1008) of Caucasian ethnicity were enrolled in the study from one large diabetes clinic and, in order to enrich the population with patients with advanced renal impairment, from four dialysis centres in Berlin. On selected days, determined by laboratory capacity, all patients with diabetes presenting in the diabetes clinic were approached, of whom more than 70% agreed to participate in the study. Clinical data were recorded by review of clinical records. Classification of diabetes was based on American Diabetes Association criteria [6]. The diagnosis of nephropathy was based on the documentation of more than 30 mg/24 h or 20 µg/min albumin excretion on at least two occasions or the presence of terminal renal failure necessitating renal replacement therapy (dialysis or transplantation). Three subgroups of patients were analysed, and defined either as diabetic patients without albuminuria, with albuminuria, or as patients with end-stage renal failure. Other causes of increased albumin excretion were excluded by appropriate clinical investigation. As a control group, we selected 1940 healthy blood donors and members of the hospital staff (age 38.5±12.6, body mass index 23.6±2.9 kg/m2, resting blood pressure 126.9±12.5/79.0±8.4 mmHg, 55% men). HbA1c was determined by standard laboratory techniques. Measurement of blood pressure was done by sphygmomanometer and in a sitting position. Hypertension was defined as a systolic blood pressure >140 mmHg and a diastolic blood pressure >95 mmHg noted in the medical records on at least two separate occasions or on the prescription of antihypertensive medication, excluding diuretics.



   Genotyping
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 Abstract
 Introduction
 Subjects and methods
 Genotyping
 Statistical analysis
 Results
 Discussion
 References
 
Genomic DNA was prepared from peripheral white blood cells using a standard column-extraction technique (Qiagen, Hilden, Germany). Subsequently, the Gß3 C825T genotype was determined by polymerase chain reaction (PCR) amplification of the relevant region followed by restriction with BseD1 (Fermentas) as described previously [4]. Complete restriction of the PCR product (268 bp=TT genotype) generates bands of 116 bp and 152 bp (CC genotype).



   Statistical analysis
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 Abstract
 Introduction
 Subjects and methods
 Genotyping
 Statistical analysis
 Results
 Discussion
 References
 
All data are presented as means±standard deviation or proportions. Continuous variables were compared by two-sided Student's t-test. Categorical data were assessed by {chi}2 statistics. For multivariate analysis and adjusted correlations, the Statistical Package for Social Sciences® (SPSS) was used.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Genotyping
 Statistical analysis
 Results
 Discussion
 References
 
Genotype distribution in diabetic patients with end-stage nephropathy (CC/TC/TT: 22/22/4) and in patients with albuminuria (188/159/49) did not differ significantly from non-albuminuric diabetics (282/230/53) or from healthy controls (950/831/159). Clinical and anthropometrical characteristics of both patients and controls are presented in Figure 1Go. In a multivariate analysis using nephropathy as independent variable, duration and type of diabetes, age, and systolic blood pressure, but not Gß3 genotype were significantly associated with nephropathy and/or end-stage renal disease in diabetic patients (Table 1Go).



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Fig. 1. Boxplots presenting median and quartiles of systolic BP, age, BMI and duration of diabetes in case subgroups and control individuals by Gß3 genotype.

 

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Table 1. Multivariate logistic regression analysis of factors influencing the risk of nephropathy in patients with type 1 or 2 diabetes by forwarded conditional mode

 
Neither the Gß3 TT genotype nor the presence of T allele were significantly associated with either the presence of type 1 (qT=0.32) or type 2 (qT=0.29) diabetes (controls: qT=0.30; 950/831/159). This was also true after controlling for age and sex. HbA1C values were comparable between the genotype groups (Table 2Go) even after adjustment for body mass index (BMI) and duration of diabetes. In controls as well as in patients, genotype distribution and T-allele frequency were similar between women and men and did not differ between BMI quartiles (data not shown). All groups and subgroups were in Hardy–Weinberg equilibrium. Hypertension was more common in patients with type 2 than in those with type 1 diabetes and was associated with age (P<0.0001) and duration of diabetes (P=0.0002). Nephropathy was significantly associated with hypertension in both groups (P<0.0001; P=0.015). However, neither the Gß3 genotype nor the 825T allele were associated with hypertension or blood pressure values in either group.


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Table 2. Patient characteristics by Gß3 genotype in type 1 and type 2 diabetes

 



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Genotyping
 Statistical analysis
 Results
 Discussion
 References
 
In this large and ethnically homogeneous group of patients and controls we found no relationship between the Gß3 genotype distribution or presence of the T allele and the prevalence of type 1 or type 2 diabetes or the presence of renal complications. Together, these data do not support the hypothesis that the Gß3 C825T polymorphism is a clinically important marker of an increased risk for the development of diabetes or renal complications in diabetes.

The rationale for our study was based on the assumption that the 825T variant leads to increased activation of G-proteins, a finding that has been reported in immortalized B-cell lines from IDDM patients with diabetic nephropathy [7]. Moreover, increased expression of a functional splice variant of the Gß3 subunit (Gß3-s) associated with this mutation, which apparently changes intracellular signal transduction, may accentuate morphological alterations, resulting in atherosclerotic vascular–renal complications. In our study, however, diabetes and related vascular–renal complication were not associated with the Gß3 genotype. This negative finding is consistent with a recent report by Fogarty et al. [8], who failed to find an association between the Gß3 genotype and diabetic nephropathy in a small population with type 1 diabetes.

Negative studies usually raise the question of statistical power to discard the null hypothesis. However, assuming that the Gß3T-allele at least doubles the risk for either diabetes or renal diabetic nephropathy, our study had a power of about 0.85 to exclude such an effect of the genotype (P=0.05).

Nevertheless, some limitations of our study have to be addressed. The average age of our healthy control group was lower compared to the diabetic case group. However, since the principal aim of our study was to investigate the association between diabetic nephropathy and Gß3 genotype, we compared the genotype distribution between subgroups of diabetics with and without nephropathy after exclusion of those with end-stage nephropathy. No significant association was found despite adjustment for age and duration of diabetes.

It is probably also important to note that this genetic variant, which leads to the increased expression of a splice variant of the Gß3 subunit, was identified in cell lines derived from hypertensive individuals selected on the basis of an increased activity of the Na/H exchanger in immortalized lymphoblasts and cultured fibroblasts [4]. Increased activity of the Na/H antiporter has been found in approximately 50% of patients with essential hypertension and has also been reported in immortalized lymphocytes [9] and fibroblasts [10] from diabetic patient with nephropathy, but whether or not these in vitro abnormalities of ion transport are directly related to the pathogenesis of vascular complications or clinically relevant manifestations of the disease remains unclear.

In summary, our study does not support the hypothesis that individuals bearing the Gß3 825T allele have a markedly increased risk of developing either type 1 or 2 diabetes or related nephropathy.



   Acknowledgments
 
We are indebted to Dr Austenat of the Berlin Diabetes Center and Dr W. Pommer for their support in recruiting the patients, and to Klaus Schlotter and Brigitte Egbers for their expert technical help in genotyping. Genotyping was in part supported by the Deutsche Forschungsgemeinschaft (DFG Sh35/2-3).



   Notes
 
Correspondence and offprint requests to: Professor Dr Arya M. Sharma, Medizinische Klinik IV, Klinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Genotyping
 Statistical analysis
 Results
 Discussion
 References
 

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Received for publication: 23. 8.99
Revision received 21. 4.00.