From the Joslin Diabetes Center, Section on Epidemiology and Genetics,
Boston, Mass (J.J.R., D.M., M.B.S.F., Y.Y., J.H.W., A.S.K.); Harvard School of
Public Health, Program for Population Genetics, Boston (J.J.R.); and Harvard
Medical School, Department of Medicine, Boston (D.M., M.B.S.F., Y.Y., J.H.W.,
A.S.K.).
Correspondence to John Rogus, ScD, Program for Population Genetics, Harvard School of Public Health, FXB-101, 665 Huntington Ave, Boston, MA 02115-6096. E-mail jrogus{at}ppg.harvard.edu
About a decade ago, a clue about the genetic basis of DN surfaced: The
genetics of DN and essential hypertension may overlap.4 5
Consequently, genes involved in blood pressure regulation are logical
candidates for DN susceptibility. Although the genetic basis of
hypertension is still unfolding, many studies have suggested putative
hypertension susceptibility genes. One widely studied example is the
angiotensinogen gene (AGT) on chromosome 1. The initial
report implicating AGT in hypertension was made by Jeunemaitre et
al6 and confirmed by Caulfield et al.7 This
finding was replicated by many but not all subsequent
studies.8
Several recent studies have tested AGT polymorphisms, most notably
M235T (characterized by a threonine [T] to methionine [M]
substitution at position 235) for a role in DN. For example, using
study subjects from Northern Ireland, Fogarty et al9 found
an excess of TT genotypes among 95 IDDM patients with DN
compared with 100 nephropathy-free individuals with IDDM.
Moreover, Marre et al10 have reported an interactive
effect of AGT with the ACE gene. Other case-control
studies11 12 13 14 have found no AGT effect.
Successful application of this type of case-control study design,
however, is dependent on the ability to identify a group of cases and
control subjects from the same gene pool. If allele frequencies of
cases and control subjects differ for any reason not relevant to the
disease process, such as population stratification, then false-positive
or false-negative results may occur. While steps can be taken to
minimize this problem (eg, drawing cases and control subjects from the
same relatively homogeneous population as done in Fogarty
et al9 , the only foolproof way of eliminating potential
bias is by application of family-based analytic methods.
We report in this article the results of a family-based study that
investigates AGT for a possible role in DN. Specifically, we test the
M235T T allele for evidence of association with DN by using the
TDT.15 In addition to aggregate analysis, we also
stratify by the presence or absence of ESRD, a measure of disease
severity. Finally, in light of a recent report16
concluding that the AGT TT genotype causes blunted renal
vascular response to angiotensin II in males but not in
females, we also stratify by gender.
Examination of IDDM Patients and Parents
Definition of IDDM
Diagnosis of DN
Criteria for Family Inclusion
Genetic Markers and Genotyping
Statistical Analysis
The cornerstone of our analytic strategy is the TDT.15
Typically, the TDT statistic is found by identifying all parents
heterozygous for a particular variant and determining the number of
times that variant is and is not transmitted to an affected child. The
difference divided by the square root of the sum (ie, the square root
of McNemar's statistic) follows a standard normal distribution under
the null hypothesis, and large values are indicative of allelic
association. To apply this procedure to DN, we must account for the
fact that a susceptibility gene will be transmitted in excess to
DN+ children, whereas there will be deficient transmission
to DN- children. Thus we divide our overall sample by DN
status and carry out separate analyses conditional on renal
status.
In addition to separate assessment of allele transmission in
DN+ and DN- samples, we also compared the
DN+ transmission rate with the DN-
transmission rate by using a Pearson
Allele transmission for both tests was determined by use of
the publicly available computer program ANALYZE
(ftp://linkage.cpmc.columbia.edu/software/analyze). To avoid
asymptotic approximations, all corresponding P values were
calculated with exact methods. For standard TDT, where transmission
from each heterozygous parent may be viewed as a Bernoulli trial, exact
P values were found by determination of the upper tail
binomial probabilities with a spreadsheet. For the Pearson
Table 3
This gender-dependent trend was even more pronounced when the
transmission ratios in DN+ and DN- trios were
compared directly by
Stratification of the DN+ sample by ESRD also revealed
potential evidence for association of the T allele with DN.
Specifically, while no transmission difference was observed in those
without ESRD (P=.65), 38 heterozygous parents transmitted T
25 times to those with ESRD (P=.04).
Gender-specific analysis of the ESRD+ sample did
not reveal any additional insight: The T allele was transmitted to
15 ESRD+ males from 22 heterozygous parents (68%) and to
10 ESRD+ females from 16 heterozygous parents (63%).
To explore further the role of AGT, we identified family trios
and tested for nonrandom allele transmission from parents to
children. In our analysis, we considered two types of trios,
those with a DN+ child and those with a DN-
child. The addition of DN- trios increases specificity
because positive results using only DN+ trios may be due to
IDDM as well as DN genes or even another mechanism leading to
segregation distortion that is independent of nephropathy
status.
In aggregate, the M235T T allele displayed no significant evidence
of association with DN. However, we did uncover an apparent association
within certain subgroups. For example, we observed preferential
transmission of the T allele to those with ESRD (P=.04).
We can thus speculate that the T allele is important primarily in
the most severe cases of DN. Alternatively, those without ESRD may
simply tend to be more frequently misclassified with respect to DN.
We have also considered analysis stratified by gender.
Recently, Hopkins et al16 found gender to be an important
covariate in the relationship of AGT with blunted renal vascular
response, a potential promoter of hypertension. Specifically, an effect
due to a TT genotype was strongly evident for males but
nonexistent for females. Thus our finding of preferential transmission
of T to DN+ males compared with DN- males
(P=.05) may be of particular interest. Furthermore, this
finding is consistent with our investigation of patients with
non–insulin-dependent diabetes mellitus, which revealed a strong
relationship between the TT genotype and DN only in men and not
in women.19a
Assuming that the M235T T allele is indeed associated with a subset
of DN, it is still unclear whether the relationship is causative. If DN
susceptibility is in fact related to blood pressure–driven alterations
in hemodynamics, then causality would imply that the
presence of T alleles could increase blood pressure directly.
Although such a functional role has neither been definitively confirmed
nor refuted, Jeunemaitre et al20 have recently conducted
an extensive investigation of AGT diallelic polymorphisms in two
large populations (French Caucasian and Japanese) and concluded that
(1) with the exception of G-6A, no other known diallelic
polymorphism is consistently associated with hypertension
and (2) the G-6A G and A alleles are virtually synonymous with the
M235T M and T alleles, respectively. Notably, Inoue et
al21 have shown that the defining nucleotide
substitution of G-6A affects the basal transcription rate of AGT, which
could potentially account for essential hypertension susceptibility.
The only multiallelic marker reported to show strong
association with hypertension is the AGT-GT
polymorphism.7 For completeness, we also
genotyped this marker but found no alleles to be either
independently associated with DN or helpful in gaining further insight
into transmission from M235T homozygous parents (data not shown).
Because we lacked systematic blood pressure measurements on
parents and also on subjects at the time of diabetes, we cannot rule
out the possibility that the M235T T allele (or the G-6A A
allele) modifies renal hemodynamics directly (ie,
independent of blood pressure) in the presence of IDDM. Further studies
will therefore be necessary, not only to confirm our association
results but also to investigate more fully the biology underlying renal
complication of IDDM.
Received July 8, 1997;
first decision August 4, 1997;
accepted September 19, 1997.
© 1998 American Heart Association, Inc.
Scientific Contributions
Diabetic Nephropathy Is Associated With AGT Polymorphism T235
Results of a Family-Based Study
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—Diabetic nephropathy is a serious and
frequent complication of insulin-dependent diabetes mellitus (IDDM)
that has a strong genetic component. Several case-control studies have
reported conflicting results with regard to the role of
angiotensinogen gene polymorphisms, specifically the
M235T T allele, in the development of diabetic
nephropathy. The primary limitation of the case-control
approach is that bias may be introduced by unrecognized differences in
the populations selected for cases and control subjects. In contrast,
family-based approaches, such as the transmission/disequilibrium test,
assess whether a particular variant, or allele, is transmitted
preferentially from a parent having a single copy of that allele.
Thus each family provides its own control, thereby eliminating spurious
results caused by mismatched population samples. To take advantage of
this study design for further investigation of M235T, we collected from
the Joslin Diabetes Center in Boston 148 IDDM patients with diabetic
nephropathy, 62 nephropathy-free patients with
long-duration IDDM, and, very importantly, parents of all these
individuals. We found that among males (but not females) the T
allele of the M235T polymorphism was transmitted preferentially
to those with nephropathy compared with IDDM patients
without nephropathy (P=.05). Moreover, the T
allele was transmitted preferentially to patients with the most
severe manifestation of nephropathy, end-stage renal
disease (P=.04). In conclusion, results obtained in our
family-based study support a role of the angiotensinogen
gene M235T polymorphism, and specifically the T allele, in the
development of diabetic nephropathy in IDDM.
Key Words: angiotensinogen • nephropathy, diabetic • diabetes, insulin-dependent
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Diabetic
nephropathy is a serious complication of diabetes, which
frequently results in hemodialysis, renal transplant, or death.
Although common among all individuals with IDDM; (35% lifetime risk),
DN is extremely common among IDDM patients with DN siblings (71%
lifetime risk).1 Similar familial aggregation has been
found in other studies.2 3 Because familial clustering of
risk factors such as poor glycemic control is insufficient to explain
such a high risk in the sibling of an affected patient, genetic factors
are strongly suspected to play a large role in susceptibility to DN.
However, little progress has been made in identifying genes relevant to
this disease process.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Study Population
Families for this study were selected from two rosters assembled
as part of our ongoing research at the Joslin Diabetes Center. The
first roster focuses on families with multiple cases of IDDM and
includes 137 families (examined 1991 to 1996) having two or more
siblings with 15+ years of IDDM. The second roster is based on a
large-scale DN screening effort at the Joslin Diabetes
Center.17 During 1991 to 1993, roughly half of Joslin's
IDDM patients who were 15 to 44 years old were screened for DN,
resulting in 201 diagnoses of persistent proteinuria or more advanced
stages of DN.
All IDDM patients and parents (if available) on these rosters
had previously undergone physical examination including height, weight,
and blood pressure measurement. They also provided demographic
information and medical history (eg, IDDM diagnosis/treatment and late
diabetic complications) through a standardized questionnaire. Each
individual provided a random urine sample for urinalysis and for
albumin/creatinine ratio determination and a blood
sample for biochemical analysis and DNA isolation. All
examinations were performed according to institutional guidelines by
specially trained recruiters in either the subjects' homes or the
Joslin Diabetes Center. The protocol for the study was approved by the
Human Subject Committee at the Joslin Diabetes Center. All subjects
gave informed consent.
Only white patients with IDDM were considered for the study.
Diabetes was considered IDDM if it was diagnosed before age 30 years
and treatment with insulin began within 1 year and continued
thereafter.
IDDM patients were classified with regard to various stages of
DN on the basis of questionnaires, medical records (from Joslin or
other institutions), and measurements of
albumin/creatinine ratio. Methods for
albumin/creatinine ratio determination and
classification have been described previously.17 IDDM
patients without any history of DN having <17 (males) or <25
(females) µg albumin/mg creatinine were
considered normoalbuminuric (DN-). IDDM patients
with clear evidence of renal disease (renal transplant or dialysis),
persistent proteinuria (Albustix 1+ or
albumin/creatinine ratio 250+ [males] or 355+
[females] on two of three determinations), or persistent high
microalbuminuria (albumin/creatinine
ratio 65+ [males] or 92+ [females] on two of three determinations)
were considered to have DN (DN+) if a review of all
available medical information revealed no evidence of nondiabetic renal
disease. IDDM patients who did not fit one of these categories were
considered unclassifiable and were not included in this study.
For this study, we included only families with at least one
DN-classifiable IDDM patient whose parents had both been examined. From
these families, we identified our final study population (summarized in
Table 1
) of 148 DN+ trios
(DN+ child plus parents) and 62 DN- trios
(DN- child with 15+ years of IDDM plus both parents).
View this table:
[in a new window]
Table 1. Summary of Families Used for Family-Based
Association Testing
The AGT exon 2 polymorphism M235T6 was
genotyped according to the previously described denaturing
gradient gel electrophoresis protocol.18
Because of the potential for bias in case-control studies, we
chose to focus exclusively on family-based association testing.
Consequently, instead of comparing distributions of alleles in
cases and control subjects, our procedure tests for preferential
transmission of a genetic variant, or allele, from a parent who has
only one copy of that variant. If a variant is relevant to the disease
process (or is a marker for a disease allele), it will be
preferentially transmitted to affected children and not transmitted to
unaffected children. Because parental DNA is crucial to avoid biased
results,19 the relevant unit of analysis for this
type of analysis is a family trio (ie, a child and both
parents). 
2 statistic. This
alternative statistic was proposed by Spielman et al15 as
a safeguard against false-positives caused by segregation distortion, a
phenomenon characterized by preferential transmission of an allele
to all children (in this context, both DN+ and
DN-). However, aside from eliminating potential bias
caused by segregation distortion, this statistic also allows us to
consider all trios simultaneously, adding not only to the
sample size but also to the test specificity, because searching for
opposite transmission patterns in DN+ and DN-
patients prevents IDDM loci from being detected (as they might be in a
DN+ sample alone). 2 statistic, P values reflecting the exact
probability of obtaining the observed statistic or larger under the
null hypothesis were calculated in STATXACT (CYTEL Software Corp).
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Table 2 shows the clinical
characteristics of the DN+ (n=148) and DN-
(n=62) IDDM patients who, together with their parents, make up the
family trios for this study. Both groups had similar age at
examination, had diabetes diagnosed at a young age, and had long
duration of IDDM. Twenty-five percent of patients with
nephropathy had ESRD (8% on dialysis or 17% with kidney
transplant).
View this table:
[in a new window]
Table 2. Clinical Characteristics of IDDM Patients Used for
TDT According to Their Nephropathy Status summarizes the results of the
standard TDT analysis that we performed separately on
DN+ and DN- samples. For each
analysis, this table shows the number of informative parents
(ie, parents having genotype MT as opposed to MM or TT), the
number of times these heterozygous parents transmitted T versus M, and
the exact P value corresponding to the probability of
observing equal or greater transmission of T under the null hypothesis
of random transmission. In aggregate, neither the 148 DN+
trios nor the 62 DN- trios demonstrated significant
evidence for association of the M235T T allele. In particular, T
was transmitted to DN+ children 76 times from 143
heterozygous parents (P=.25) and to DN-
children 32 times from 70 heterozygous parents (P=.28). (The
remaining parents, numbering 153 [DN+] and 54
[DN-], were MM or TT homozygotes and thus
noninformative.) However, when stratified by gender, a different
picture emerged. The T allele was transmitted 41 of 73 times to
DN+ males (P=.17) and only 12 of 33 times to
DN- males (P=.08). Thus among males, there was
some suggestion that the T allele is associated with DN. In
contrast, T was transmitted 35 of 70 times to DN+ females
(P=.55) and 20 of 37 times to DN- females
(P=.74), suggesting random transmission.
View this table:
[in a new window]
Table 3. Allele Transmission From M235T Heterozygous
Parents to IDDM Offspring According to DN Status 2 analysis (Table 4). Now, instead of testing each sample
individually for deviation from random transmission, the T/M
transmission ratio in the DN+ sample is compared with that
in the DN- sample. Among females, the DN+
35/35 ratio is not significantly different from the DN-
20/17 ratio (P=.73). Among males, however, with both
transmission ratios consistent with association (41/32 for
DN+ and 12/21 for DN-), direct comparison of
them is now statistically significant (P=.05).
View this table:
[in a new window]
Table 4. Allele Transmission (T/M) From M235T
Heterozygous Parents to IDDM Offspring According to DN Status and
P Values Based on Comparison of DN+ and
DN- Samples
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Previous investigations of AGT as a candidate gene for DN
have been mixed. While several groups have published negative
results,11 12 13 14 Fogarty et al9 found the M235T
TT genotype to be associated with DN, and Marre et
al10 observed an interactive effect of M235T and the ACE
I/D polymorphism. Adding to this ambiguity, a previous study in our
laboratory found that the T allele occurs more frequently among
patients with either overt proteinuria or microalbuminuria
compared with those with normoalbuminuria, but the level of
evidence did not reach statistical significance.18
Selected Abbreviations and Acronyms
ACE
=
angiotensin I-converting enzyme
AGT
=
angiotensinogen gene
DN
=
diabetic nephropathy
ESRD
=
end-stage renal disease
IDDM
=
insulin-dependent diabetes mellitus
M235T
=
methionine/threonine polymorphism (at position 235) of the AGT
gene
TDT
=
transmission/disequilibrium test
Acknowledgments
This research was supported by National Institutes of Health
grants DK-41256 and P50-HL55000 and Juvenile Diabetes Foundation
grant 194193.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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