The document analyzes the association between polymorphisms in the AKR1B1 gene and diabetes type 2 leading to complications. It finds that the five AKR1B1 gene variants studied are not implicated in disease progression. However, two variants, rs2259458 G/T and rs2670230 C/A, show significant association with risk of diabetes leading to complications. The rs2259458 G/T variant is associated under an additive genetic model, while rs2670230 C/A is associated under a co-dominant genetic model. Analysis of haplotypes also finds three haplotypes to be significantly associated with risk of diabetes complications.

1. Int J Med Health Sci. Oct 2015,Vol-4;Issue-4 430
International Journal of Medical and Health Sciences
Journal Home Page: http://www.ijmhs.net ISSN:2277-4505
Αssociation between Polymorphisms and Haplotypes in AKR1B1 and Diabetes Type 2
leading to Complications
Sophia V Tachmitzi1*
, Evangelia E Tsironi1
, Maria G Kotoula1
, Efthimios Dardiotis2
, Theodoros Eleftheriadis3
,
Dimitrios Z Chatzoulis1
, Paraskevi Xanthopoulou4
, Maria Tziastoudi4
, Aristotle G Koutsiaris1,5
, Anatoli
Fotiadou1
, Georgios M Hadjigeorgiou2
, Ioannis Stefanidis3
, Elias Zintzaras6
Departments of 1
Ophthalmology, 2
Neurology, 3
Nephrology and 4
Biomathematics, University of Thessaly School of
Medicine, Larissa, Greece; 5
Bio-medical Informatics and Engineering Lab., School of Health Sciences, T.E.I. of
Thessaly, Larissa, Greece; 6
The Institute for Clinical Research and Health Policy Studies, Tufts Medical Center, Tufts
University School of Medicine, Boston, MA, USA.
ABSTRACT
Background: A candidate-gene association study in a south eastern Mediterranean population was conducted to investigate the
association of five AKR1B1 gene variants (rs2259458 G/T, rs2734653 G/A, rs2670230 C/A, rs1790998 C/A, rs17188118 A/C)
with i) diabetes progression and ii) risk of diabetes leading to microvascular complications. Materials and Methods: The cohort
consisted of 169 diabetic cases with complications, 107 diseased controls and 315 healthy controls. The disease progression was
tested using the generalized odds ratio (ORG). The risk of diabetes leading to complications was tested using the ORs of the
additive and co-dominant models. The mode of inheritance was assessed using the degree of dominance index. Results: The
analysis showed that the five AKR1B1 gene variants are not implicated in disease progression. However, the same AKR1B1
variants are associated with the risk of diabetes leading to complications. Significant results were derived for the additive model of
the variant rs2259458 G/T [OR= 1.87 (1.01-3.50)] and the co-dominant model of the variant rs2670230 C/A [OR=1.45 (1.01-
2.04)]. The modes of inheritance for the variants rs2259458 G/T and rs2670230 C/A were “non-dominance” and “dominance of
allele A”, respectively. The frequencies of three haplotypes (T-G-A-C-A, G-G-C-C-A and G-A-C-C-A) were significantly different
(P≤0.05) between cases and healthy controls. Conclusion: Genetic variation in AKR1B1 gene may alter susceptibility to diabetes
leading to complications.
KEYWORDS: Aldose reductase gene variants, Diabetic Nephropathy, Diabetic Retinopathy
INTRODUCTION
Diabetic nephropathy (DN) and diabetic retinopathy (DR)
are considered as two major microvascular complications of
diabetes (type 1 and 2) [1]. DN is the primary cause of end-
stage renal failure and is characterized by a progressive
clinical course, ultimately leading to death [2,3]. Diabetic
retinopathy (DR) represents one of the leading causes of
adult acquired blindness [4]. The main risk factor for
developing microvascular complications in diabetes is the
poor glycemic control; though, patients with relatively good
glycemic control develop also complications [1]. A familial
clustering of DN and DR indicated that a genetic
predisposition is implicated in the pathogenesis of
microvascular complications [5-7]. Thus, there is evidence
that genetic factors may contribute to the development of
complications in diabetes. However, the genes conferring
susceptibility have not been identified yet [2,8-10].
Aldo-keto reductase family 1, member B1 (aldose
reductase-AR) is an enzyme that in humans is encoded by
the AKR1B1 gene which is located at 7q35 [11]. This gene
encodes a member of the aldo/keto reductase superfamily
Original article

2. Int J Med Health Sci. Oct 2015,Vol-4;Issue-4 431
which catalyzes the reduction of a number of aldehydes,
including glucose. Therefore, AKR1B1 might be implicated
in the development of microvascular complications in
diabetes (such as DN and DR) by catalyzing the reduction of
glucose to sorbitol [11].
The present candidate-gene study examined the hypothesis
of association of five common AKR1B1 gene variants-SNPs
(rs2259458 G/T, rs2734653 G/A, rs2670230 C/A, rs1790998
C/A, rs17188118 A/C) and the progression of diabetes type 2
(i.e. from healthy status to diabetes without complications or
to diabetes leading to complications). Thereafter, the
association of the AKR1B1 variants with the risk of diabetes
leading to complications was tested [2]. The former
hypothesis was tested using the generalized linear odds ratio
(ORG) [12, 13]. The later hypothesis was tested using the co-
dominant and additive models [14-16] and the magnitude of
associations was expressed in terms of odds ratios (ORs) as
a genetic model-free approach. In addition, the mode of
inheritance was estimated based on the degree of dominance
index (h-index) [15,16]. Lastly, an analysis of haplotypes
was conducted.
MATERIALS AND METHODS
Subjects
The cohort consisted of 169 diabetic cases (type 2) with
microvascular complications (23% with DR, 18% with DN
and 59% with both DR and DN), 107 diseased controls
(diabetics type 2 without microvascular complications) and
315 healthy controls.
The population consisted of males and females (in cases,
diseased and healthy controls the males were: 52%, 51%
and 52%, respectively). The distribution of age was: above
60 years old 89%, 85% and 79% of subjects in cases,
diseased and healthy controls, respectively. In 42% of cases
and 21% of diseased controls, the diabetes duration was
more than 10 years and all subjects were whites of Greek
origin. The study was approved by the University of
Thessaly Ethics Committee. A verbal informed consent was
received from all patients. Τhe Ethics committee does not
require a written consent since in the report forms the full
name of the participants is not recorded.
Genotyping
Genomic DNA was extracted from peripheral blood
leukocytes using a salting out method. Based on Hapmap
data for CEU population (Release 27, Phase II+III, Feb09,
on NCBI B36 assembly, dbSNP b126), tag SNPs across the
AKR1B1gene (16.78Kbp spanning from 133777647 to
133794428 in chromosome 7) were selected using the tagger
algorithm (http://www.broadinstitute.org/mpg/tagger/) with
a pair wise approach, an r2
cut-off of ≥ 0.8 and a minor
allele frequency > 0.05.
A total of 5 tag SNPs in four distinct gene regions were
retrieved: in the intronic region between exons 1 and 2
(intron 1-2) (rs17188118, rs1790998), in the intron 3-4
(rs2670230), in the intron 5-6 (rs2734653) and the intron 8-
9 (rs2259458). Tag SNPs genotyping was performed with
TaqMan allele specific discrimination assays on an ABI
PRISM® 7900 Sequence Detection System and analysed
with SDS software (Applied Biosystems, Foster City, USA).
At least 10% of the samples were selected randomly for
repeated genotyping, as an internal control. Genotyping was
performed by laboratory personnel blinded to clinical status.
Data Analysis
The association between genotype distribution and clinical
status (i.e. disease progression) was tested using the chi-
square test. The association between disease progression and
genotype distribution was also examined using the
generalized odds ratio ORG [12,13]. The ORG expresses the
probability of a subject being more diseased relative to the
probability of being less diseased, given that diseased
subjects have higher mutational load.
In investigating the susceptibility to diabetes leading to
complications, the co-dominant and additive models of
cases were compared to healthy controls using a logistic
model. These two models were selected since they are
orthogonal [14-16]. The magnitude of associations was
expressed in terms of odds ratios (ORs) unadjusted and
adjusted for age and gender with the corresponding 95%
confidence interval (CI).
In healthy controls, deviation of the genotype distribution
from the Hardy-Weinberg equilibrium (HWE) and existence
of linkage disequilibrium (LD) between polymorphisms
were evaluated using exact tests according to Weir [17,18].
The mode of inheritance was estimated using the degree of
dominance index (h-index) [15,16]. A result was considered
statistically significant when p < 0.05.
The unadjusted and adjusted ORs were calculated using
SPSS (SPSS Inc. Version 11.5, Chicago). HWE and LD
were tested using the Genetic Data Analysis (GDA)
software [19]. The haplotype frequencies were estimated
and compared by SHEsis [20]. ORG was calculated using
ORGGASMA (http://biomath.med.uth.gr) [12].
RESULTS
Disease progression
The genotype distributions of the five variants in cases,
diseased controls and healthy controls, and the respective
ORGs, are shown in Table 1. The healthy controls were
conformed to HWE for all variants (P ≥ 0.05). None of the
five variants showed significant association between disease
progression and genotype distribution (P ≥ 0.05); a marginal
association at P = 0.08 was only shown for rs2670230 C/A.
The ORG produced non-significant results for all variants,
indicating that the risk of disease progression is not related
to the mutational load of the variants (Table 1).

3. Int J Med Health Sci. Oct 2015,Vol-4;Issue-4 432
Table: 1 Results from testing the association of the AKR1B1 variants with diabetes progression
Variant Genotype Cases
N (%)
Diseased
Controls
N (%)
Healthy
controls
N (%)
P-value ORG (95% CI)
rs2259458 G/T
GG 56(33.9) 43(40.6) 115 (37.6)
0.65 1.11 (0.80-1.53)GT 80(48.5) 50(47.2) 138(45.1)
TT 29(17.6) 13 (12.3) 53(17.3)
rs2734653 G/A
GG 92(57.1) 63(59.4) 177(57.8)
0.93 1.00 (0.70-1.44)GA 60(37.3) 38(35.8) 108(35.3)
AA 9(5.6) 5(4.7) 21(6.9)
rs2670230 C/A
CC 50(30.5) 25(23.8) 108(35.2)
0.08 1.02 (0.75-1.40)CA 87(53.0) 60(57.1) 134(43.6)
AA 27(16.5) 20(19.0) 65(21.2)
rs1790998 C/A
CC 75(45.5) 30(29.1) 123(39.4)
0.13 0.82 (0.59-1.13)CA 64(38.8) 52(50.5) 133(42.6)
AA 26(15.8) 21(20.4) 56(17.9)
rs17188118 A/C
AA 148(90.2) 93(92.1) 267(88.4)
0.65 0.83 (0.45-1.55)AC 14(8.5) 8(7.9) 33(10.9)
CC 2(1.2) 0(0.0) 2(0.7)
Genotype distribution of AKR1B1 gene variants among cases, diseased controls and healthy controls. The P-values and the generalized odds
ratio (ORG) for testing the association between genotype distribution of each variant and disease progression (healthy controls, diabetics without
microvascular complications-diseased controls, diabetics with complications-cases) are shown.
Diabetes leading to complications
Single-locus analysis: Table 2 shows the association results
for diabetes leading to complications. The model-free
approach (OR) produced a marginally significant
association (P = 0.05) for the additive model of the variant
rs2259458 G/T [ORadjusted = 1.87 (1.01-3.50)] and the co-
dominant model of the variant rs2670230 C/A [OR=1.45
(1.01-2.04)].
For the variant rs2259458 G/T the mode of inheritance is
“non-dominance” (h=0.21, Table 3), indicating that the
heterozygote GT has a risk of being diseased that lies in the
middle of the risk-protected GG and risk-exposed TT
homozygous genotypes. For the variant rs2670230 C/A, the
mode of inheritance is “dominance of allele A” (h=3.53),
indicating that the homozygous AA has a greater risk of
being diabetic with complications than the homozygous CC,
and that the heterozygote CA has a risk of diabetes leading
to complications closer to the AA homozygote than to the
midpoint between the two homozygotes (Table 3).
Linkage disequlibrium analysis: Table 4 shows the P-values,
and the respective D primes, for testing LD between pairs of
two variants for patients with diabetes leading to
complications and healthy controls. All variants were in LD
(P < 0.05), except for variant rs2670230 C/A for both
populations and the variant rs2734653 G/A for the patients
with diabetes leading to complications.
Analysis of haplotypes: The distribution of the estimated
haplotype frequencies of the five variants, both for cases and
healthy controls, is presented in Table 5. The overall
difference between cases and healthy controls was
marginally significant (Global P = 0.06). T-G-A-C-A, G-G-
C-C-A and G-A-C-C-A haplotypes derived significant results
(P ≤ 0.05).

4. Int J Med Health Sci. Oct 2015,Vol-4;Issue-4 433
Table :2 Results from testing the association of the AKR1B1 variants with diabetes leading to complications
SNP Genetic model OR (95% CIs) p-value ORadjusted (95% CIs) P-value
rs2259458 G/T Additive 1.12 (0.65-1.96) 0.68 1.87 (1.01-3.50) 0.05
Co-dominant 1.15 (0.78-1.67) 0.48 1.14 (0.75-1.72) 0.55
rs2734653 G/A Additive 0.83 (0.36-1.87) 0.65 0.94 (0.38-2.32) 0.89
Co-dominant 1.09 (0.73-1.61) 0.67 0.98 (0.64-1.52) 0.95
rs2670230 C/A Additive 0.90 (0.51-1.57) 0.70 0.77 (0.42-1.43) 0.41
Co-dominant 1.45 (1.01-2.04) 0.05 1.39 (0.91-2.13) 0.12
rs1790998 C/A Additive 0.87 (0.66-1.15) 0.33 0.80 (0.59-1.08) 0.14
Co-dominant 0.85 (0.58-1.25) 0.42 0.74 (0.49-1.12) 0.16
rs17188118
A/C
Additive
1.34 (0.50-3.60) 0.56 1.04 (0.37-2.91) 0.94
Co-dominant 0.76 (0.40-1.47) 0.41 0.63 (0.31-1.30) 0.21
Odds Ratio (OR) and the corresponding 95% Confidence Intervals (CIs) for testing the association of the AKR1B1 gene variants with diabetes
leading to complications for the additive and co-dominant models. The ORs adjusted for age and sex are also shown.
Table:3 The h-index and the respective mode of inheritance for the significant variants.
SNP h-index Mode of inheritance
rs2259458 G/T 0.21* None-dominance (additiveness)
rs2670230 C/A 3.53† Dominance of allele A
*
based on adjusted values, †based on unadjusted values
Table:4 P-value/D for testing linkage disequilibrium between pairs of variants in cases and healthy controls (in brackets).
rs2734653 rs2670230 rs1790998 rs17188118
rs2259458 <0.01/1
(<0.01/1)
<0.01/0.75
(<0.01/0.80)
<0.01/0.72
(<0.01/0.72)
0.02/1.00
(0.01/1.00)
rs2734653 <0.01/0.94
(<0.01/1.00)
<0.01/0.70
(<0.01/0.60)
0.07/0.66
(0.02/0.79)
rs2670230 0.45/0.09
(0.05/0.04)
0.04/0.76
(<0.01/0.82)
rs1790998 <0.01/1.00
(<0.01/1.00)

5. Int J Med Health Sci. Oct 2015,Vol-4;Issue-4 434
Table: 5 Estimated haplotype frequencies for the five AKR1B1 gene variants (SNP1- SNP2 - SNP3 - SNP4 - SNP5)
Haplotype
SNP1-SNP2-SNP3-
SNP4-SNP5
Estimated frequencies
Cases Healthy
controls
P-value P-value
global
G-A-A-A-A 0.02 0.05 0.07
0.06
G-A-A-A-C 0.01 0.00 0.45
G-A-A-C-A 0.21 0.19 0.63
G-A-C-C-A 0.01 0.00 0.05
G-G-A-A-A 0.12 0.10 0.34
G-G-A-A-C 0.00 0.00 0.86
G-G-A-C-A 0.02 0.05 0.07
G-G-C-A-A 0.12 0.14 0.41
G-G-C-A-C 0.05 0.06 0.58
G-G-C-C-A 0.03 0.01 0.05
T-G-A-A-A 0.02 0.03 0.74
T-G-A-C-A 0.02 0.01 0.04
T-G-C-A-A 0.01 0.02 0.64
T-G-C-A-C 0.00 0.00 0.21
T-G-C-C-A 0.36 0.35 0.81
Estimated haplotype frequencies for the five AKR1B1 gene variants (SNP1: rs2259458 G/T, SNP2: rs2734653 G/A, SNP3: rs2670230 C/A, SNP4: rs1790998 C/A,
SNP5: rs17188118 A/C). The P-values for comparing each haplotype between cases (diabetics with complications) and healthy controls, and the global P-value for
comparing the overall difference in haplotypes are shown.
DISCUSSION
The present study investigated whether certain AKR1B1
gene variants are associated with the diabetes disease
progression and with the development of diabetes leading to
complications.
In examining the association of AKR1B1 gene variants with
diabetes progression, the results showed that none of the
variants is implicated in disease progression.
In examining the association of AKR1B1 gene variants with
diabetes leading to complications, two variants (rs2259458
G/T and rs2670230 C/A) were found to be associated with
diabetes leading to complications. The degree of dominance
index (h-index) indicated that the mode of inheritance is
“none-dominance” for the variant rs2259458 G/T and
“dominance of allele A” for the variant rs2670230 C/A
[15,16]. The genotype distributions of the examined variants
conform to HWE in the healthy control group indicating
lack of population stratification and genotyping mistakes
[21].
Haplotype analysis revealed that three haplotypes are
implicated in the development of complications in diabetes
(Table 5). The haplotype G-G-C-C-A may confer protection
for diabetes leading to complications, whereas allele T of
rs2259458 G/T and allele A of rs2670230 C/A may
contribute to the risk of diabetes leading to complications
when haplotypes are considered. The same applies for allele
A of rs2734653 G/A and allele A of rs1790998 C/A.
DN and DR are complex diseases with multifactorial
etiology and involve epistatic and gene-environment
interactions. Therefore, in addition to hypothesis-driven
studies (i.e. the gene-candidate association studies),
hypothesis-free studies such as GWAS [14,22,23],
microarrays gene expression analyses [24,25] and whole
genome linkage scans [26,27] may assist in providing more
conclusive evidence regarding the significance of AKR1B1
as a marker in diabetes leading to complication. This can be
achieved by examining the genomic convergence of these
different types of studies [23].

6. Int J Med Health Sci. Oct 2015,Vol-4;Issue-4 435
Although GWAS represent a superior strategy for
unraveling genetic complexity [22], the findings of gene-
candidate association studies may be supportive in
replicating existed evidence and in revealing genuine
genetic effects that could merit prioritization in future
studies. GWAS themselves lack replication and therefore,
replication of their findings from different investigators and
different methodologies (such as gene-candidate association
studies) are essential to interpret the mass of associations
likely to result from GWAS [14,26,28]. In addition, this
work fulfills the minimum requirements for the association
study to be informative [23].
In conclusion, the present study showed that genetic
variation in AKR1B1 gene may alter susceptibility to
diabetes leading to complications; though, it is not
implicated in disease progression. The results suggest that
AKR1B1 variants and haplotypes are involved in the
pathogenesis of diabetes leading to complications. However,
additional studies (including gene-gene and gene-interaction
studies [14,29] and a genetic convergence analysis of
different data sources are needed in order to produce more
conclusive claims of the association between AKR1B1 and
disease progression or diabetes leading to complications.
Acknowledgements
We thank Almpanidou Pavlina for technical assistance. This
work was supported in part by the grant (code:2989) of the
University of Thessaly Research Committee. EET was the
principal investigator of the study and responsible for the
study conduct. ET, DZC, GMH, IS conceived and designed
the study. SVT, EET, MGK, TE, DZC, AF, IS screened the
patients and collected the blood samples and assembled the
GAS patient database. SVT, ED, AGK, GMH performed the
genotyping. VX and MT assisted in the statistical analysis of
the data. GMH, IS and AGK assisted in drafting the
manuscript. EZ performed the statistical analysis of the data
and he drafted the manuscript.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing if the paper.
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____________________________________________
*Corresponding author: Sophia V Tachmitzi
E-Mail: sophitach@yahoo.gr