(Investigative Ophthalmology and Visual Science. 2000;41:4064-4068.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
Polymorphisms of the Aldose Reductase Gene and Susceptibility to Retinopathy in Type 1 Diabetes Mellitus
Andrew Demaine,
Deborah Cross and
Ann Millward
From the Department of Molecular Medicine, Plymouth Postgraduate Medical School, Tamar Science Park, Plymouth, United Kingdom.
 |
Abstract
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PURPOSE. Aldose reductase (ALR2) is the first and rate-limiting enzyme of the
polyol pathway and is involved in the pathogenesis of diabetic
retinopathy. Polymorphisms of the ALR2 gene are
associated with susceptibility to diabetic retinopathy in Chinese and
Japanese patients with type 2 diabetes. There are no reports
investigating these polymorphisms in white patients with type 1
diabetes from either Western Europe or North America. A CA dinucleotide
repeat polymorphism (5'ALR2; located at -2100 bp) as well as a novel
C(106)T polymorphism was investigated in 229 white patients with type 1
diabetes, with or without retinopathy.
METHODS. The DNA was typed for these polymorphisms using conventional polymerase
chain reaction techniques.
RESULTS. There was a highly significant increase in the frequency of the
Z-2 5'ALR2 allele and Z-2/X (where X is not Z+2) genotype in
patients with diabetic retinopathy (n = 159) compared
with those without who had diabetes of 20 years’ duration
(uncomplicated, n = 70; 
2 = 17.0,
P < 0.0001). There was a similar decrease in the
Z+2/Y genotype (where Y is not Z-2; 2 = 30.1,
P < 0.000,001) in the patients with retinopathy
compared with the uncomplicated diabetes group. The C/Z-2 C(-106)T/5'
ALR2 haplotype was found in 33.3% of the patients with retinopathy and
8.7% of the patients with uncomplicated diabetes.
CONCLUSIONS. These results confirm previous studies in other populations and in type
2 diabetes showing that polymorphisms in the promoter region of the
ALR2 gene are associated with susceptibility to diabetic
retinopathy.
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Introduction
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Retinopathy is a leading cause of visual impairment in patients
with type 1 diabetes mellitus.1
It is well known that
long-term exposure to hyperglycemia is a major cause of diabetic
retinopathy, and strict glycemic control may help to prevent or delay
the onset of this complication of diabetes.2
3
4
It is
becoming clear that genetic factors may also play a role in the
pathogenesis of diabetic retinopathy. At present, little is known about
these genetics factors, although recent studies have implicated the
gene encoding aldose reductase (ALR2).5
6
7
8
9
10
11
12
13
Aldose reductase is the first and rate-limiting enzyme of the polyol
pathway and converts glucose to sorbitol in an reduced nicotinamide
adenine dinucleotide phosphate (NADPH)–dependent reaction. Sorbitol is
subsequently converted to fructose by the enzyme sorbitol dehydrogenase
(SORD) with the cofactor nicotinamide adenine dinucleotide
(NAD)+.14
15
16
Under hyperglycemic
conditions there is increased flux through the polyol pathway, and this
in turn leads to a number of metabolic and vascular abnormalities that
may ultimately cause tissue ischemia.
It has recently been shown that a polymorphism (5'ALR2)
consisting of a (CA)n repeat located 2.1 kb upstream of the initiation
site of ALR2 is associated with diabetic nephropathy and
neuropathy.17
18
19
Chinese and Japanese patients with type
2 diabetes and retinopathy have been found to have an increased
frequency of the Z-2 5'ALR2 allele consisting of 23 CA
repeats.8
12
Similar findings have been reported for young
white adolescents from Australia with type 1 diabetes and an early
onset of retinopathy.9
However, to date there are no
studies investigating a population of patients with type 1 diabetes
from either Western Europe or North America. Therefore, the purpose of
this study was to investigate the 5'ALR2 locus in a large population of
patients with type 1 diabetes with established retinopathy.
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Methods
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Patients and Normal Control Subjects
DNA samples from 229 white British patients with type 1 diabetes
were randomly taken from the freezer for analysis. A total of 114 DNA
samples from normal healthy white British subjects were randomly taken
from the freezer to obtain control frequencies. The normal control
subjects consisted of DNA from cord blood samples collected
sequentially after normal obstetric delivery from the Obstetric
Department, Derriford Hospital, Plymouth (UK). Local ethics committee
approval was obtained. The study protocol adhered to the tenets of the
Declaration of Helsinki. The patients were classified according
to their microvascular complications, as previously
described.17
18
These are summarized as follows.
Patients with Uncomplicated Diabetes
These patients (n = 70) had had type 1 diabetes for at
least 20 years but remained free of retinopathy (fewer than five dots
or blots per fundus) and proteinuria (negative results in urine
Albustix testing [Bayer, West Haven, CT] on three consecutive
occasions over 12 months).
Patients with Retinopathy
These patients (n = 159) had retinopathy defined as
more than five dots or blots per eye; hard or soft exudates, new
vessels, or fluorescein angiographic evidence of maculopathy or
previous laser treatment for preproliferative or proliferative
retinopathy; and maculopathy or vitreous hemorrhage. Ninety of these
patients also had proteinuria. Fundoscopy was performed by both a
diabetologist and ophthalmologist. The clinical features of the
patients are shown in Table 1
.
Preparation of DNA and Analysis of ALR2 Promoter
Region Polymorphisms
For the 5'ALR2 locus, DNA samples were taken at random
from the freezer, and an aliquot (50–100 ng) was used for
amplification of the DNA. Briefly, a pair of amplimers was constructed
that flanks a 138-bp region that contains the dinucleotide (CA)n
repeat.8
The antisense amplimer was labeled with
32P-deoxyadenosine triphosphate (dATP) by T4
polynucleotide kinase. A 50-µl reaction mix was prepared using the
end labeled 5'ALR2 antisense and the sense amplimer, dNTPs and
Taq polymerase. The samples were subjected to 30 cycles of
amplification, which consisted of denaturing for 1 minute at 95°C,
annealing for 1 minute at 61°C, and extension for 1 minute at 72°C.
An aliquot of the amplified genomic DNA was electrophoresed through a
5% formamide-urea gel at 90 W for 3 to 4 hours and dried, and
autoradiography was performed using radiographic film (X-Omat; Eastman
Kodak, Rochester, NY) with intensifying screens at -85°C overnight.
A C(-106)T polymorphism upstream of the ALR2 gene was typed
by taking 50 to 100 ng of genomic DNA together with amplimers that span
the polymorphic site to amplify the region using previously
published sequences.9
The following conditions were used:
The samples were denatured for 2 minutes at 96°C and then amplified
for 35 cycles at 94°C for 30 seconds and 70°C annealing-extension
for 2 minutes. The amplification products were then purified using a
commercial system (Wizard PCR Prep DNA purification system; Promega,
Madison, WI) and a laboratory vacuum manifold (Vac-Man;
Promega).
The C(-106)T substitution creates a new BfaI restriction
endonuclease site. To detect the C(-106)T polymorphic site, 20 µl of
purified product was digested to completion using 10 U of
BfaI and incubated in the appropriate buffer at 37°C for 2
hours. Digested products were run out on a 3.5% agarose gel containing
ethidium bromide at 150 V against a 50-bp DNA molecular weight marker
and visualized under a UV illuminator.
Statistical Analysis
The number of 5'ALR2 and C(-106)T alleles was obtained using gene
counting. The frequency of 5'ALR2 and C(-106)T alleles and genotypes in
the patient and normal control groups were compared using the
2 test and contingency tables. The
probability was corrected for the number of comparisons made
(Pc) using the Bonferroni inequality method.20
C(-106)T/5'ALR2 haplotype frequencies were obtained by the
gene-counting method using the DNA of those subjects who were
homozygous for either one or both loci.
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Results
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The frequency of the 5'ALR genotypes in the patients and normal
control subjects are shown in Table 2
. The frequencies of the 5'ALR2 and the C(-106)T genotypes in the
patient subgroups as well as the normal control subjects were found to
be in Hardy–Weinberg equilibrium. Nine alleles were detected; Z+8,
Z+6, Z+4, Z+2, Z, Z-2, Z-4, Z-6, and Z-8 where Z is the most common
allele and consists of 24 CA repeats. Of the 159 patients with
retinopathy, 49.1% had the Z-2/X 5'ALR2 genotype (where X is not Z+2)
compared with only 20.0% in the uncomplicated diabetes group
(2 = 17.0, P < 0.0001). In
contrast, the Z+2/Y genotype (where Y is not Z-2) was found in only
16.4% of the patients with retinopathy but in 51.4% of the patients
with uncomplicated diabetes (2 = 30.1,
P < 0.00,001). The normal control subjects had
intermediate frequencies of these genotypes. There were no other
differences in the frequency of the 5'ALR2 genotypes between the
patient subgroups.
The frequency of the 5'ALR2 alleles is shown in Table 3
. The patients with retinopathy had an increased frequency of the Z-2
allele compared with the patients with uncomplicated diabetes (33.6%
and 14.3%, respectively; 2 = 18.1,
P < 0.0001). Conversely, the Z+2 allele was decreased
in the patients with retinopathy compared with the patients with
uncomplicated diabetes (13.2% and 33.6%, respectively;
2 = 39.8, P < 0.00,001).
There were no other significant differences, although it is interesting
that there was a small increase in the frequency of the Z-4 allele in
the patients with retinopathy compared with the patients with
uncomplicated diabetes (6.6% and 2.9%, respectively). The normal
control subjects had intermediate Z-2 and Z+2 allelic frequencies
compared with those for the uncomplicated diabetes and retinopathy
groups.
Table 4
shows the frequency of the C(-106)T ALR2 alleles and genotypes in the
patients with type 1 diabetes and normal control subjects. The
retinopathy group had an increased frequency of the C/C(-106) ALR2
genotype compared with the uncomplicated diabetes group (46.7% and
25.0%, respectively; 2 = 5.18,
P < 0.025, Pc = 0.05). Consequently,
the frequency of the C(-106) allele was increased in the retinopathy
group compared with the uncomplicated diabetes group (71.9% versus
55.6%, respectively; 2 = 6.7,
P < 0.01).
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Table 4. Frequency of C(-106)T ALR2 Alleles and Genotypes in Patients
with Type 1 Diabetes Mellitus and Diabetic Retinopathy
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It is possible to deduce the 5'ALR2/C(-106)T haplotypes in those
subjects who were homozygous for at least one of these loci. In the
retinopathy group, it was possible to determine 155 of the 318
haplotypes; similarly, 46 of the 140 haplotypes in the uncomplicated
diabetes group were identified. The frequency of the 5'ALR2/C(-106)T
haplotypes is shown in Table 5
. The most common haplotype in the retinopathy group was the Z-2/C,
which was present in 31.0% compared with only 8.7% of the
uncomplicated diabetes group (2 = 9.2,
P < 0.001, Pc = 0.005) and with a
frequency of 14.3% in the normal control subjects. In contrast, the
Z+2/C as well as the Z+2/T haplotypes occurred with a reduced frequency
in the retinopathy group compared with the uncomplicated group (9.0%
and 41.3%, respectively, 2 = 26.6,
P < 0.0001, Pc = 0.0005). The
frequency in the normal control subjects was 18.5%.
View this table:
[in this window]
[in a new window]
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Table 5. Frequency of 5' ALR2/C(-106)T Haplotypes in Patients with Type 1
Diabetes Mellitus and Diabetic Retinopathy
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Discussion
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We have recently shown that the Z-2 5'ALR2 allele is strongly
associated with the susceptibility to diabetic nephropathy and
neuropathy.17
18
However, in our previous report, although
the frequency of the Z-2 allele was increased in patients with
retinopathy, the change did not attain significance. In this study we
have presented data on a greatly expanded patient population and find a
highly significant increase in the frequency of the Z-2 allele in
those patients with retinopathy compared with patients with long-term
uncomplicated diabetes. To our knowledge, this is the first report of a
study investigating these genetic markers in a white patient population
with retinopathy from Western Europe or North America. The results
presented here confirm the recent reports studying young adolescents
with type 1 diabetes and retinopathy from Australia as well as studies
in Chinese and Japanese patients with type 2 diabetes. However, in
contrast to these studies we also found a protective effect of the Z+2
allele in our population. Indeed, the decreased frequency of this
allele in our patients with retinopathy are more significant than the
increase in the frequency of the Z-2 allele. The discrepancy between
findings in the studies may reflect the allelic frequencies between the
different ethnic groups. For instance, the Z+2 allele is relatively
uncommon in Japanese populations. An alternative explanation is
duration of diabetes in the uncomplicated diabetes groups used in these
studies. The duration of disease for the uncomplicated diabetes group
in our study was at least 20 years. Although this does not exclude the
possibility that retinopathy will develop in some of these patients in
the future, the risk is clearly much lower than in those individuals
who have had diabetes for only 5 to 10 years, which is the cutoff point
that was used in many other studies.
In the diabetic patients with retinopathy the C(-106) allele was
associated with the Z-2 5'ALR2 allele. Conversely, in the
uncomplicated group, both the C and the T-allele was associated with
the Z+2 5' ALR2 allele. This suggests that the 5' ALR2, together with
the C(-106)T loci is close to the region that confers susceptibility to
retinopathy. These results are consistent with those of Kao et
al.9
who studied a young adolescent population of patients
with type 1 diabetes and retinopathy in Australia. However, Moczulski
et al.25
recently showed that in an North American
population of patients with type 1 diabetes and nephropathy the T(-106)
allele was associated the Z-2 allele, and this haplotype was found
more often in this group of patients than in those without proteinuria
(but not necessarily retinopathy). None of these studies included any
normal control frequencies. In our normal control population the C/Z-2
haplotype was relatively common, suggesting that these two markers may
display linkage. However, family studies are needed to confirm this.
It has recently been shown that the Z-2 5' ALR2 allele is associated
with the increased mRNA expression in patients with nephropathy
compared with patients without the allele.19
Therefore,
the C/Z-2 haplotype may identify individuals who have enhanced levels
of ALR2 mRNA, and the presence of this haplotype may lead to increased
flux through the polyol pathway. In contrast, the C/Z+2 and T/Z+2
haplotypes may be associated with reduced gene expression. Ultimately,
increased gene expression would result in excessive production of
sorbitol and fructose, metabolic and vascular abnormalities, and
oxidative stress in the cell.
The polymorphisms in the current study are in a region of the
ALR2 gene that is known to contain a number of osmotic
response elements (OREs).21
22
23
We have recently described
the sequencing of those patients with or without nephropathy as well as
sequencing of the region using a cosmid clone.24
We did
not find any sequence differences between those patients with
microvascular disease and those without. These observations together
with those of Shah et al.19
and Ikegishi et
al.10
suggest that the 5'ALR2 may be directly involved in
modulating the expression of the ALR2 gene. It is possible
that the length of the CA repeat may influence the accessibility of the
binding sites for the transcription factors. The length of the
microsatellites has been shown to modify the expression of other genes
including interferon-
.26
Further studies are now
required to explore the functional relevance of these polymorphisms in
the susceptibility to diabetic retinopathy. Further, it is still
possible that the association with this region is due to linkage with
an adjacent susceptibility gene. In this respect, it is interesting
that the endothelial nitric oxide synthase (eNOS) gene has also been
localized to chromosome 7q35, the same region as
ALR2.27
Recent studies have shown that
polymorphisms of eNOS are associated with diabetic microvascular
disease.28
In conclusion, we have investigated two polymorphic regions in the
promoter region of the ALR2 gene and confirm the role of the
gene in the susceptibility to and possibly the protection from diabetic
retinopathy. Individuals with retinopathy have a significantly
increased frequency of the Z-2/C haplotype compared with those with no
retinopathy after diabetes of 20 years’ duration.
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Acknowledgements
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The authors thank Angela Heesom for technical support.
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Footnotes
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Supported by grants from the British Council for the Prevention of Blindness, British Diabetic Association, and Northcott Devon Medical Foundation.
Submitted for publication May 11, 2000; revised July 31, 2000; accepted August 17, 2000.
Commercial relationships policy: N.
Corresponding author: Andrew G. Demaine, Department of Molecular Medicine, Plymouth Postgraduate Medical School, ITTC Building, Tamar Science Park, Plymouth PL6 8BX, Devon, UK. ademaine{at}plymouth.ac.uk
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Top
Abstract
Introduction
