© 1997 American Heart Association, Inc.
Articles |
The Angiotensin-Converting Enzyme Gene and the Angiotensin II Type I Receptor Gene as Candidate Genes for Microalbuminuria
A Study in Nondiabetic and Non–Insulin-Dependent Diabetic Subjects
From the Departments of Medicine (J.S.Y., C.A., V.M.-A., A.P.) and Primary Health Care (M.G., A.P.H.), Whittington Hospital; and the Division of Cardiovascular Genetics, Department of Medicine, Rayne Institute (S.H., P.T.), University College London Medical School, UK.
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Abstract |
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Abstract Familial clustering of microalbuminuria with cardiovascular disease suggests a possible common genetic antecedent. We have tested the hypothesis that the angiotensin-converting enzyme (ACE) DD genotype and the angiotensin II type I receptor (AT1R) gene C allele represent the common link between microalbuminuria and coronary heart disease. The frequency of polymorphisms of the ACE and AT1R genes were investigated in 509 nondiabetic white subjects and in 86 non–insulin-dependent diabetic white patients. There was no significant difference in albumin excretion rate between the genotypes in nondiabetic subjects on either a daytime or an overnight sample or in diabetic subjects expressed as a normalized albumin concentration on an untimed morning urine collection. We have found no evidence for an association between polymorphism of the ACE or AT1R genes and microalbuminuria in two groups of subjects without insulin-dependent diabetes.
Key Words: cardiovascular disease • microalbuminuria • non–insulin-dependent diabetes • angiotensin-converting enzyme gene • angiotensin II type I receptor gene
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Introduction |
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Microalbuminuria is increasingly recognized as an important predictor of ischemic heart disease in subjects with non–insulin-dependent diabetes mellitus (NIDDM) and insulin-dependent diabetes mellitus (IDDM).1 2 The Islington Diabetes Survey of nondiabetic subjects found an odds ratio for ischemic heart disease of 5.7 in microalbuminuric subjects, and a 24-fold increased mortality over a period of 3.5 years.3 The predisposition to renal disease, and perhaps its relationship with ischemic heart disease, may involve genetic mechanisms.4 The levels of serum angiotensin-converting enzyme (ACE) are highly variable between individuals with around half of this interindividual variability determined by a major gene effect associated with a deletion polymorphism in intron 16 of the ACE gene.5 There are conflicting data concerning the associations between the deletion polymorphism of the ACE gene and myocardial infarction in nondiabetic and diabetic populations.6 7 8 A polymorphism has been identified in the 3' untranslated region of the angiotensin II type I receptor (AT1R) gene corresponding to an A
C substitution
at nucleotide position 1166 of the mRNA
sequence.9 Tiret et al9 have recently
described a synergism between the DD genotype of the ACE gene
and the C allele of the AT1R gene in their association
with myocardial infarction in a large European case-control study. We have postulated that variation in the ACE gene or of the AT1R gene may represent an explanation for the association of cardiovascular disease with microalbuminuria in nondiabetic and diabetic subjects. Part of this material has been previously published.10
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Methods |
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Subjects and Clinical Methods
Studies were performed on two groups of subjects, one nondiabetic and one with NIDDM. The nondiabetic subjects were a subsample of subjects investigated during the Goodinge Study.11 12 In brief, 1046 individuals aged 40 to 75 years were recruited from the age-sex register of a north London general practice, with 62.3% responding. The subjects were weighed, and height was recorded on a Seca balance for calculation of body mass index (wtxheight-2). A 2-hour timed urine collection was made at the time of screening, and a timed overnight urine sample was requested from all participants. Albumin excretion rate was calculated from these samples. A 75-g oral glucose tolerance test was performed. Blood for genotyping was collected during the second half of the study and was available on 509 of 959 subjects (53%), who were similar in age, gender distribution, and albumin excretion rate to those subjects who were not genotyped.
Urinary albumin concentration was assayed by an in-house modified competitive enzymoimmunoassay,13 which was validated against a commercial radioimmunoassay (r=.96; Pharmacia-LKB). Microalbuminuria was defined as an albumin excretion rate of 20 to 200 µg/min, without urinary infection, on a 2-hour or overnight sample during the screening phase of the study.
The diabetic subjects comprised 86 white patients aged 35 to 73 currently followed up in the department as part of the UK Prospective Diabetes Study.14 All subjects had been diagnosed on the basis of a fasting plasma glucose concentration >6.0 mmol/L on two occasions and had been randomly allocated to therapy with diet, oral hypoglycemic agents, or insulin. These subjects had weight and height recorded. For calculation of albumin excretion rate, an untimed early morning urine sample was collected and urinary albumin and creatinine measured using an immunoturbidimetric assay and by the Jaffé method, respectively. The urinary albumin excretion rate was expressed as a normalized urinary albumin concentration,15 which adjusts urinary albumin concentration for creatinine concentration. Using this method of expressing urinary albumin excretion, microalbuminuria is defined as a normalized urinary albumin concentration of 50 to 300 mg/L, while levels of >300 mg/L are considered as proteinuria.14 For most of the analyses, these two categories have been combined.
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Genetic Studies |
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Extraction of DNA was performed by the salting-out method.16 The ACE gene was amplified by a three-primer method by the polymerase chain reaction, as described by Evans at al,17 thus overcoming mistyping of the heterozygotes, using two oligonucleotide primers (ACE1 and ACE3) outside the insertion sequence in intron 16 and a primer (ACE2) inside the sequence.18 The two alleles were resolved on a 7.5% polyacrylamide microtitre array diagonal gel electrophoresis (MADGE).19 The AT1R A1166
C was detected by polymerase chain reaction and
[
32P]ATP-radiolabeled allele-specific
oligonucleotides according to Tiret et
al.9 Positive controls of DNA from individuals of known
genotype (the two homozygotes and a heterozygote in the case of
both ACE and AT1R genes) were included in each gel. The
AT1R genotypes were not available on 17 nondiabetic
and 2 diabetic subjects because of failed polymerase chain
reaction.
Statistical Methods
The data were entered into a database computer software package
and analyzed using SPSS for Windows. Logarithmic transformation
was used to reduce positive skewness. Associations were tested using
2, Student's t test, or
analysis of variance by genotype, with ethnicity as an
additional independent variable in the diabetic subjects. Data are
expressed as mean±SD for normally distributed, or as median
(interquartile range) for skewed data. Significance is taken as
P<.05, although more rigorous criteria might be appropriate
for investigations of candidate genes involving multiple comparisons or
groups.
The numbers of nondiabetic subjects provided an 80% power to detect a 24% difference in geometric mean albumin excretion rate between subjects with and without the ACE DD genotype and a 37% difference between those with and without the AT1R CC genotype, at the 5% level, and the same power to detect a 1.14-fold (ACE DD) and 4.0-fold (AT1R CC) difference in geometric mean normalized albumin concentration in diabetic subjects at the same level.
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Results |
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The characteristics of the subjects are shown in Table 1
. The diabetic subjects were older and
had a higher prevalence of microalbuminuria than the
nondiabetic subjects. In both groups, the ACE and AT1R
genotypes were in Hardy-Weinberg equilibrium and the
frequencies were similar to those in published studies.6 7 8
Albumin excretion rate as a continuous variable was
compared between the three ACE gene genotypes, and the results
are shown in Table 2. There was no
difference in albumin excretion rate between either set of
genotypes in either group, whether or not the nondiabetic
albumin excretion rate was calculated on the basis of daytime,
overnight, or geometric mean. These analyses were unaffected by
controlling for age, gender, smoking, and systolic blood
pressure; by grouping subjects with and without the ACE DD
genotype or the AT1R AA genotype; or by
excluding subjects treated with ACE inhibitors (data not
shown). The analyses were repeated using a categorical
variable of microalbuminuria, and there were also no
significant associations with either ACE or AT1R
genotype in either group, the odds ratio for
microalbuminuria in ACE-DD subjects being 1.75 (95% CI
0.65-4.60) and 1.32 (95% CI 0.71-2.45) in diabetic and nondiabetic
subjects, respectively, while these odds ratios in subjects with the
AT1R CC genotype were 0.25 (95% CI 0.03-1.88 ) and
0.77 (95% CI 0.27-2.19), respectively.
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As previously reported, there was no association of genotypes of the ACE gene with either coronary heart disease or levels of blood pressure in either the diabetic or nondiabetic subjects.20 There was also no association between ATR1 genotype and either coronary heart disease or blood pressure.
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Discussion |
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Genetic studies have suggested a possible role of the polymorphisms of the ACE gene, which influences plasma ACE levels,5 on cardiovascular risk in both nondiabetic6 and diabetic7 subjects, although more recent studies have cast doubt on these associations.8 Two studies have found associations of polymorphisms of the ACE gene with nephropathy in IDDM subjects,21 22 although other studies in IDDM and NIDDM groups have found less straightforward results.23 24 25 26 27 28 Two recent studies suggest that the DD genotype may affect prognosis and response to treatment in diabetic nephropathy.29 30 To date, no studies have been reported of relationships between microalbuminuria and ACE gene polymorphisms in nondiabetic subjects.
The recent observation that polymorphisms of the angiotensin II type 1 receptor interact with those of the ACE gene in influencing risk of myocardial infarction in nondiabetic subjects9 has not been supported by other studies.31 32 Two studies have investigated the role of the AT1R gene in nephropathy in patients with insulin-dependent diabetes, one finding an effect only in poorly controlled subjects33 and the other no relationship.34
We have investigated associations between ACE gene and AT1R gene polymorphisms and microalbuminuria in nondiabetic and NIDDM subjects. We found no association between either polymorphism and either albumin excretion rate or microalbuminuria in either diabetic or nondiabetic subjects. Because of variability of albumin excretion rate,35 we have repeated the analyses using the geometric mean albumin excretion rate of two timed collections and have still found no association with genotype. Our observations could suggest possible differences in the pathogenesis of microalbuminuria between subjects with and without IDDM or that the previous suggestions of associations were chance findings.
In conclusion, we have found no evidence of any powerful association between polymorphisms of the ACE gene or the AT1R gene and albumin excretion rate in nondiabetic or diabetic subjects.
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Acknowledgments |
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This study was supported by grants from the Wellcome Trust, the Medical Research Council, and the British Diabetic Association. The Cardiovascular Genetics Group is supported by the British Heart Foundation.
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Footnotes |
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Reprint requests to John Yudkin, Departments of Medicine and Primary Health Care, University College London Medical School, Whittington Hospital, Archway Rd, London N19 3UA, United Kingdom.
Presented in part at the American Diabetes Association meeting, 1995.
Received March 4, 1996; accepted December 12, 1996.
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References |
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