From the Research Division, Joslin Diabetes Center, and Department of
Medicine, Harvard Medical School, Boston, Mass.
A molecular variant of the AGT gene (with a threonine substituted for
methionine at amino acid residue 235) has been associated with elevated
levels of serum angiotensinogen and essential hypertension
in nondiabetics.11 Recently, this
polymorphism has been found to be in complete linkage
disequilibrium with a sequence difference at position 6 in the promoter
region. The latter affects the expression of the AGT
gene,12 with the highest tissue levels of
angiotensinogen being found in homozygotes
T235/T235.12
Two case-control studies in IDDM patients have shown that the
homozygotes 235T/235T had an elevated risk of developing diabetic
nephropathy.13 14 Recently, this
association has been confirmed in our family study using
TDT.15 The 235T effect was found among men but
not among women. Other case-control studies have not found any
association with M235T polymorphism.16 17 A
gender-specific effect of the AGT polymorphism, however, was not
examined in those studies.
Because these studies were carried out mainly in IDDM patients, we
conducted a case-control study to evaluate whether the M235T
polymorphism in AGT gene plays a role in predisposition to diabetic
nephropathy in NIDDM. We specifically tested the effect of
this polymorphism according to gender.
Determination of the Presence or Absence of Nephropathy
Examination of Case and Control Subjects
Examinations were performed by trained family recruiters, and the
examinations took place at the Joslin Diabetes Center or in the home of
the study participant. In addition to a standardized interview about
the diagnosis of diabetes and history of its treatment, measurements of
height, weight, and blood pressure were obtained. From all examinees,
urine samples were obtained and blood was drawn for biochemical
measurements and extraction of DNA. The protocol for this study was
approved by the Human Subjects Committee of the Joslin Diabetes
Center.
Determination of Clinical Characteristics
Measurements of height and weight were obtained during the examination
and used to calculate percent ideal body
weight.21 Blood pressure was measured twice, 5
minutes apart, according to a standard protocol while the patient was
resting in a sitting position. Hypertension was diagnosed if a patient
was being treated with antihypertensive drugs at the time of
examination or the average blood pressure obtained during the
examination was
Level of creatinine in serum and in urine was measured
using alkaline picric colorimetry (modified Jaffe
reaction) on an Astra7 (Beckman Instruments). Albumin
concentration in urine was measured by immunonephelometry on a BN-100
with the N Albumin Kit (Behring) as described
previously.18
DNA Analysis
Statistical Analysis
The genotypes and allele frequencies of the AGT M235T
polymorphism are shown in Table 2
In contrast, a distinctive pattern of differences in the distributions
of both genotypes and alleles emerged when the study groups
were analyzed separately for men and women (Table 3
To examine whether the effect of the 235T/235T homozygosity on
the development of diabetic nephropathy is mediated through
elevated systemic blood pressure, the analysis was carried out
separately among individuals without and with hypertension. 235T/235T
homozygotes (Table 4
Finally, the effect of the M235T polymorphism was estimated by
logistic regression models that included gender and type of diabetes
and interactions between gender and the M235T polymorphism and type
of diabetes. There was little difference in comparison with the results
of univariate analyses. The relative OR for men
homozygous for the 235T allele at the AGT locus increased to 6.9
compared with 4.8 in univariate analysis (Table 3
We found that a molecular variant of the AGT gene, arising from the
substitution of C for T at nucleotide 714 and changing
methionine to threonine (M235T), is associated with increased risk of
the development of diabetic nephropathy in NIDDM.
Nondiabetic individuals homozygous for this variant have elevated
levels of angiotensinogen.11 It is
postulated that the level of intrarenal angiotensin II is
influenced mainly by the level of substrate,
angiotensinogen, and is minimally affected by levels of
angiotensin-converting enzyme. A recent study demonstrated
that homozygotes for the T235 allele have a blunted renal vascular
response to exogenous angiotensin II, a finding that could
be explained by higher prevailing levels of angiotensin II
in the kidney of these patients.24 This effect
was evident specifically in men but not in women. It is interesting,
therefore, that we found the risk of nephropathy in our
study was associated with the T235 allele in men only.
In two recent studies of patients with IDDM, homozygotes for the T235
allele at the AGT locus had an elevated risk of the development of
diabetic nephropathy,13 14 while in
other studies this finding was not
confirmed.16 17 None of these studies, however,
analyzed the results in men and women separately.
Interestingly, in our recent family-based study, we found evidence that
the T235 allele was preferentially transmitted from heterozygous
parents to individuals with nephropathy, but only among
men.15
The mechanisms underlying the association between T235 homozygosity and
diabetic nephropathy are unknown. The association is
restricted to hypertensive NIDDM patients, so the TT genotype
by itself does not seem to be sufficient to increase susceptibility to
diabetic nephropathy. However, in the presence of
hypertension (only a minor portion of which is related to the M235T
polymorphism), T235 homozygosity and its associated high levels of
angiotensinogen in kidney may interact with the high blood
pressure, possibly through a further increase of
intraglomerular pressure.24
Finally, some limitations of this study should be mentioned. In this
case-control study, we enrolled patients who were being seen at a
clinic. Most likely, this population was depleted of patients with
coronary artery disease and case patients who progressed
rapidly to end-stage renal disease and died. This might have reduced
the differences in the distribution of the M235T alleles
between case and control groups. Although the present study
identified positive associations between the development of diabetic
nephropathy and T235 homozygosity at the
angiotensinogen locus, it is clear that the proportion of
cases of nephropathy accounted for by this association is
small, with the larger proportion being due, presumably, to as yet
unidentified genes.
Received July 8, 1997;
first decision August 4, 1997;
accepted December 8, 1997.
2.
Seaquist ER, Goetz FC, Rich SS, Barbosa J. Familial
clustering of diabetic kidney disease: evidence for genetic
susceptibility to diabetic nephropathy. N Engl
J Med. 1989;320:1161–1165.[Abstract]
3.
Borch-Johnsen K, Norgaard K, Hommel E, Mathiesen ER,
Jensen JS, Deckert T, Parving HH. Is diabetic nephropathy
an inherited complication? Kidney Int. 1992;41:719–722.[Medline]
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4.
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.[Medline]
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5.
Viberti GC, Keen H, Wiseman MJ. Raised
arterial pressure in parents of proteinuric insulin
dependent diabetics. BMJ. 1987;295:515–517.
6.
Krolewski AS, Canessa M, Warram JH, Laffel L,
Christlieb AR, Knowler WC, Rand LI. Predisposition to hypertension and
susceptibility to renal disease in insulin-dependent diabetes mellitus.
N Engl J Med. 1988;318:140–145.[Abstract]
7.
Stattin EL, Rudberg S, Dahlquist G. Hereditary risk
determinants of micro- and macroalbuminuria in young IDDM
patients. Diabetologia. 1996;39:299. Abstract.
8.
Fagerudd J, Tarnow L, Jacobsen P, Stenman S, Nielsen
FS, Pettersson-Fernholm K, Gronhagen-Riska C, Parving HH, Groop PH.
Predisposition to hypertension and development of diabetic
nephropathy in IDDM patients. J Am Soc
Nephrol. 1997;8:110. Abstract.
9.
Schmidt S, Ritz E. Genetics of the
renin-angiotensin system and renal disease: a progress
report. Curr Opin Nephrol Hypertens. 1997;6:146–151.[Medline]
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10.
Cusi D. Genetic renal mechanisms of hypertension.
Curr Opin Nephrol Hypertens. 1997;6:192–198.[Medline]
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11.
Jeunemaitre X, Soubrier F, Kotelevtsev YV, Lifton RP,
Williams CS, Charru A, Hunt SC, Hopkins PN, Williams RR, Lalouel JM,
Corvol P. Molecular basis of human hypertension: role of
angiotensinogen. Cell. 1992;71:169–180.[Medline]
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12.
Inoue I, Nakajima T, Williams CS, Quackenbush J,
Puryear R, Powers M, Cheng T, Ludwig EH, Sharma AM, Hata A, Jeunemaitre
X, Lalouel JM. A nucleotide substitution in the promoter of
human angiotensinogen is associated with essential
hypertension and affects basal transcription in vitro. J
Clin Invest. 1997;99:1786–1797.[Medline]
[Order article via Infotrieve]
13.
Fogarty DG, Harron JC, Hughes AE, Nevin NC, Doherty CC,
Maxwell AP. A molecular variant of angiotensinogen is
associated with diabetic nephropathy in IDDM.
Diabetes. 1996;45:1204–1208.[Abstract]
14.
Doria A, Onuma T, Gearin G, Freire MBS, Warram JH,
Krolewski AS. Angiotensinogen polymorphism M235T,
hypertension, and nephropathy in insulin-dependent
diabetes. Hypertension. 1996;27:1134–1139.
15.
Rogus JJ, Moczulski D, Freire MBS, Yang Y, Warram JH,
Krolewski AS. Diabetic nephropathy is associated with AGT
polymorphism T235: results of a family-based study.
Hypertension. 1998. In press.
16.
Tarnow L, Cambien F, Rossing P, Nielsen FS, Hansen BV,
Richard S, Poirier O, Parving HH. Angiotensinogen gene
polymorphisms in IDDM patients with diabetic
nephropathy. Diabetes. 1996;45:367–369.[Abstract]
17.
Schmidt S, Giebel R, Bergis KH, Strojek K, Grzeszczak
W, Ganten D, Ritz E, Diabetic Nephropathy Study Group.
Angiotensinogen gene M235T polymorphism is not
associated with diabetic nephropathy. Nephrol Dial
Transplant. 1996;11:1755–1761.
18.
Warram JH, Gearin G, Laffel L, Krolewski AS. Effect of
duration of type 1 diabetes on the prevalence of stages of diabetic
nephropathy defined by urinary
albumin/creatinine ratio. J Am Soc
Nephrol. 1996;7:930–937.[Abstract]
19.
Grubin CE, Daniels T, Toivola B, Landin-Olsson M,
Hagopian WA, Li L, Karlsen AE, Boel E, Michelsen B, Lernmark A. A novel
radioligand binding assay to determine
diagnostic accuracy of isoform-specific glutamic acid
decarboxylase antibodies in childhood IDDM. Diabetologia. 1994;37:344–350.[Medline]
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20.
Christie MR, Vohra G, Champagne D, Delovitch TL.
Distinct antibody specificities to a 64-kD islet cell antigen in type I
diabetes as revealed by trypsin treatment. J Exp Med. 1990;172:789–794.
21.
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diagnosis of diabetes mellitus and other categories of glucose
intolerance. Diabetes. 1979;28:1039–1057.[Medline]
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22.
Miller SA, Dykes DD, Polesky HF. A simple salting
out procedure for extracting DNA from human nucleated cells.
Nucleic Acids Res. 1988;16:1215.
23.
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24.
Hopkins PN, Lifton RP, Hollenberg NK, Jeunemaitre X,
Hallouin MC, Skuppin J, Williams CS, Dluhy RG, Lalouel JM, Williams RR,
Williams GH. Blunted renal vascular response to angiotensin
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© 1998 American Heart Association, Inc.
Scientific Contributions
Gender-Specific Association of M235T Polymorphism in Angiotensinogen Gene and Diabetic Nephropathy in NIDDM
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—This study examined the
association between the development of nephropathy in
non–insulin-dependent diabetes mellitus (NIDDM) patients and M235T
polymorphism in the angiotensinogen gene. White NIDDM
patients with diabetic nephropathy (case subjects, n=117)
and patients without any evidence of nephropathy and 
10
years of NIDDM (control subjects, n=125) were selected from among
patients of the Joslin Diabetes Center and examined. In addition to a
standardized examination, blood was drawn for DNA and determination of
M235T genotypes at the angiotensinogen locus. For
the angiotensinogen gene, the frequency of the
genotype 235T/235T, known to be associated with essential
hypertension, was higher among case subjects with
nephropathy than in control subjects without this
complication. This difference, expressed as the odds ratio for
nephropathy among 235T/235T homozygotes in comparison with
all other genotypes, was 2.2 (95% confidence interval, 1.1 to
4.4). The difference, however, was confined to men (odds ratio, 4.8;
95% confidence interval, 1.5 to 14.9), with the distribution of
genotypes in case and control subjects being equal among women
(odds ratio, 1.1). DNA polymorphism M235T in the
angiotensinogen gene, which is associated with higher
expression of this gene, contributes to the risk of diabetic
nephropathy in NIDDM men but not in women.
Key Words: nephropathy • angiotensinogen • polymorphism • diabetes
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Recent studies have
provided evidence that the development of renal complications in
diabetes may be determined by genetic factors.1
The evidence includes clustering of diabetic nephropathy in
families,2 3 4 as well as an association between
predisposition to essential hypertension and the development of
diabetic nephropathy.5 6 7 8 It is
hypothesized that DNA sequence differences in regulatory or structural
parts of genes encoding for proteins of the RAS may contribute to
essential hypertension in nondiabetic persons and to hypertension and
renal damage in the presence of diabetes.9 10
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Study Population
Among the patients of the Joslin Diabetes Center in 1991, there
were 6600 Massachusetts residents whose diabetes was diagnosed at
between ages 30 and 59 years. In 1993, a questionnaire regarding the
family history of diabetes and presence or absence of diabetic
complications was sent to a 33% random sample of this subset of the
Joslin population. We obtained responses from 1429 patients (65%
response rate). For all these patients, we retrieved results of
urinalyses (albumin reagent strips as well as determinations of
the albumin/creatinine ratio) obtained during
routine clinic visits during the 3-year interval of January 1991
through December 1993.
During routine clinic visits to the Joslin Diabetes Center, a
random specimen of a patient's urine is examined with a reagent strip
(Multistix, Ames Division, Miles Laboratories). Since January 1, 1991,
urine samples with a Multistix reading <2+ have generally been
examined for the albumin/creatinine ratio as well.
For the present study, the Multistix and
albumin/creatinine ratio results obtained during
the 3-year interval were used to classify patients with regard to the
presence or absence of diabetic nephropathy as described
previously.18 If Multistix readings were 2+ in
two of three samples, patients were considered to have overt
proteinuria; otherwise, measurements of the
albumin/creatinine ratio (micrograms per milligram)
were used to classify patients. Two of three ratios 250 for men or
355 for women were considered indicative of overt proteinuria.
Microalbuminuria was defined as a ratio of albumin
to creatinine in the intermediate range, 17 to 249 for men
and 25 to 354 for women. Patients with an
albumin/creatinine ratio <17 in men and <25 in
women were considered to be normoalbuminuric. In total, 617
individuals had normoalbuminuria, 232 had
microalbuminuria, 236 had proteinuria, and 345 had
insufficient measurements (Multistix or
albumin/creatinine ratio) to determine diabetic
nephropathy status.
All patients selected for this study were white. For the control
group, we selected a 33% systematic sample of patients (n=150) from
among patients with normoalbuminuria and 10-year duration
of diabetes (as of January 1994). For the case group, we selected a
66% systematic sample of patients with persistent proteinuria (n=156).
Between 1994 and 1997, we examined 125 of the controls (83%) and 117
of the cases (75%).
To evaluate what proportion of these patients might have IDDM,
we determined the interval between the diagnosis of diabetes and the
beginning of insulin treatment, as well as the level of IDDM-associated
antibodies in the serum at the time of examination. Antibodies to
GAD65Ab were measured by the GAD65Ab assay as previously
described.19 IAAs were measured by the IA2-icAb
assay as previously described.20 For both
antibodies, the criterion for a positive assay was an index >0.1,
which is >2 SD above the mean for normal control subjects. In the
Combined Autoantibody Workshop 1995 (Denver, Colo), the GAD65Ab assay
was 100% specific and 61% sensitive, and the IA2-icAb was 88%
specific and 68% sensitive. The GAD65Ab was 91% specific and 89%
sensitive in the Second International Glutamic Acid Decarboxylase
Antibody Workshop. 95 mm Hg diastolic or 160
mm Hg systolic.
DNA was extracted from leukocytes according to standard
protocols.22 For 7 control and 2 case subjects,
insufficient blood was obtained for DNA extraction. Polymorphism
M235T in the AGT gene was determined by a DGGE protocol that was
developed in our laboratory and described
previously.14 Briefly, the 3' portion of exon 2
of the AGT gene was amplified by polymerase chain reaction with the
same primers described by Jeunemaitre et al.11 To
facilitate the analysis by a DGGE protocol, a 6-bp
"GC-clamp" at the 5' end was attached to the antisense primer.
Polymerase chain reaction products (354 bp) were electrophoresed in
10% polyacrylamide gels with a linearly increasing
concentration of denaturant from 35% to 70% (100% denaturant: 40%
formamide, 7 mol/L urea, 1x TAE). The gels were electrophoresed for
600 volt-hours (150 Vx4 hours) in 1x TAE buffer at a constant
temperature of 60°C. After staining with ethidium bromide, gels were
exposed to UV light and photographed. The allele coding for a
threonine (235T, codon ACG) stopped lower in the gel (higher
concentration of denaturant, 48.5%) than the allele coding for a
methionine (235M, codon ATG; concentration of denaturant, 48%).
Homozygotes were detected as a single band at 48% or 48.5% of
denaturant concentration, whereas heterozygotes were identified as two
bands.
The distribution of genotypes were compared between case
and control subjects using 
2 tests. The
association between genotypes and outcomes was evaluated by
computing ORs and 95% CIs. The homogeneity of ORs was tested using the
Breslaw-Day test.23 Multiple logistic regression
was used to estimate the effect of the AGT genotypes after
adjustment for other variables.23
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Clinical characteristics of the study groups are summarized in
Table 1
. We enrolled 117 diabetic
patients with diabetic nephropathy (cases) and 125
individuals with long-duration diabetes and
normoalbuminuria (controls). Both groups had similar
average duration of diabetes and age at the time of examination. The
proportion of each group with IDDM was similar regardless of whether
IDDM was defined on the basis of clinical presentation
(insulin required within 2 years of diagnosis and percent ideal body
weight <130) or the presence of immunological markers of IDDM. Case
subjects were significantly heavier, and the case group had a higher
frequency of hypertension and a larger proportion of individuals
treated for hypertension. Patients with nephropathy had
higher serum creatinine than controls.
View this table:
[in a new window]
Table 1. Characteristics of Patients With NIDDM According to
Nephropathy Status
according to study group. The genotype distributions differed
significantly (2=6.04, 2 df,
P<.05), with the genotype 235T/235T being more
frequent among cases with nephropathy than among controls.
For these 235T homozygotes, the risk of nephropathy
expressed as the OR in comparison with all other genotypes was
2.2 (95% CI, 1.1 to 4.37). The difference in genotype
frequencies was not strong enough, however, to be reflected as a
significant difference in the distribution of AGT alleles.
View this table:
[in a new window]
Table 2. Distribution of M235T AGT Genotypes and
Alleles in Study Groups ). All of the difference between cases
and controls was confined to men (OR, 4.8; 95% CI, 1.5 to 14.9), while
the distribution of genotypes among women was quite similar for
cases and controls (OR, 1.1; 95% CI, 0.4 to 3.0). The difference
between the ORs in men and women was statistically significant
(P=.05), indicating that the increased risk of
nephropathy associated with 235T/235T homozygosity was
specific to men. The pattern of OR was not changed when the
analysis was carried out after excluding patients who could
have adult-onset IDDM, ie, those with positive GAD65Ab or IAA
antibodies or nonobese patients (ideal body weight <130%) treated
with insulin within 2 years of the diagnosis of diabetes (data not
shown).
View this table:
[in a new window]
Table 3. Distribution of AGT Genotypes in Case and
Control Subjects According to Gender ) did not have an
increased risk of diabetic nephropathy if they were
normotensive (OR, 1.2; 95% CI, 0.4 to 3.6), but they were at
particularly high risk for diabetic nephropathy if they
were hypertensive (OR, 3.4; 95% CI, 1.1 to 10.8). The difference
between the two ORs, however, was not statistically significant
(P=.21).
View this table:
[in a new window]
Table 4. Distribution of AGT Genotypes in Case and
Control Subjects According to Hypertension Status 
),
and as before, the interaction term for the difference between the
relative OR in men and women had a value of P=.05.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Recent studies have shown that the development of diabetic
nephropathy may be associated with susceptibility to
essential hypertension.1 Because the RAS plays a
critical role in blood pressure homeostasis, DNA sequence differences
in the regulatory or structural parts of genes encoding for the RAS
proteins have been proposed as candidate genes for the development of
essential hypertension in nondiabetics and of hypertension and renal
damage in the presence of diabetes.1 9 10 The
present study examined this hypothesis in white patients with
NIDDM.
Selected Abbreviations and Acronyms
AGT
=
angiotensinogen gene
CI
=
confidence interval
DGGE
=
denaturing gradient gel electrophoresis
GAD65Ab
=
glutamic acid decarboxylase 65
IAA
=
insulin autoantibody
IA2-icAb
=
IA2 intracellular period antibody
IDDM
=
insulin-dependent diabetes mellitus
NIDDM
=
non–insulin-dependent diabetes mellitus
OR
=
odds ratio
RAS
=
renin-angiotensin system
TAE
=
tris acetate EDTA
TDT
=
transmission disequilibrium testing
Acknowledgments
This research was supported by grants from the Baxter Healthcare
Co and the Joslin Diabetes Center. Dr Freire was supported by a
postdoctoral fellowship from the Brazilian government (CNPq Conselho
Nacional de Desenvolvimento Cientifico e Tecnologico).
Footnotes
Reprint requests to Andrzej S. Krolewski, MD, PhD, Section on Epidemiology and Genetics, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Fogarty DG, Krolewski AS. Genetic
susceptibility and the role of hypertension in diabetic
nephropathy. Curr Opin Nephrol Hypertens. 1997;6:184–191.[Medline]
[Order article via Infotrieve]
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