The Relationship of Haptoglobin Polymorphism and Cardiovascular Diseases in Some of Iranian Diabetic Patients
In this study, we found that allelic polymorphism in
Hp gene acts as a major determinant of susceptibility for the development
of diabetic cardiovascular complications. We studied this gene in 122
Iranian diabetic cardiovascular patients. The results showed that distribution
of the Hp phenotypes were found to be in Hardy-Weinberg equilibrium. By
using the chi square test we determined the association between Hp
allele and diabetic cardiovascular diseases (χ2 = 52.98
p<0.001) and this finding was independent of gender (χ2
= 0.39 p>0.05). The chance of having allele two in diabetic patients
with cardiovascular disease was 22.31 (95% CI, 6.18 to 80.5) times more
than those with allele one (p<0.0001).
Haptoglobin (Hp) is a hemoglobinbinding polymorphic plasma glycoprotein
present in all vertebrates. There are two classes of alleles for haptoglobin,
denoted class one and class two and three potential genotypes denoted
Hp 1-1, Hp 2-1 and Hp 2-2 (Quaye, 2008). Hp consists
of two different polypeptide chains, α-chain and β-chain. β-chain
is heavier than α-chain and is identical in all Hp phenotypes (Langlois
and Delanghe, 1996).
Oxidation of low-density lipoprotein is catalyzed by heme and leads to
vascular endothelial cell damage and atherosclerosis (Vercellotti et
al., 1994). Haptoglobin by binding to hemoglobin and removing it from
the circulation prevents iron stimulated formation of oxygen radicals
and has an important role as an antioxidant (Quaye, 2008). The antioxidant
capacity of Hp 2-2 in circulation is lower than that of Hp 1-1 because
Hp 2-2 binds to hemoglobin with lower binding affinity than Hp1-1
In some races individuals who were homozygous for Hp allele two
(Hp 2-2) were found to be at significantly greater risk of developing
myocardial infraction, stroke and cardiovascular death (UK Prospective
Diabetes Study Group, 1998; Carter and Worwood, 2007 ). In this study,
we found the relationship between Hp phenotypes and the risk of cardiovascular
diseases (CVD) in some Iranian diabetic patients.
MATERIALS AND METHODS
In 2007, 122 consecutive patients (61 men, 61 women) who presented with
diabetic cardiovascular disease were enrolled in this study. These patients
were recruited from Alghadir Hospital (Tehran-Iran) for participation
in this trial. In control group (50 cardiovascular patients) Hp
genotyping was determined.
Blood samples were collected in the presence of EDTA and the plasma was
stored at -70 °C. Genomic DNA was extracted from whole blood using
DNGTM plus solution (Fermentas). Two sets of polymerase chain
reaction (PCR) were performed (Koch et al., 2002):
||With primers A (5`- GAGGGGAGCTTGCC TTTCCATTG- 3`) (forward)
and B (5-` GAGATTTTTG AGCCCTGGCTGGT-3`) (reverse) for the amplification
of a 1757-bp Hp allele one -specific sequence and a 3481-bp
Hp allele two -specific sequence (Annealing temperature 63
°C, cycle 30)
|| Primers C (5`-CCTGCCTCGTATTAACTGCACCAT-3`) (forward) and D (5`
CCGAGTGCTCCA CATAGCCATGT3`) (reverse) amplified a 349-bp fragment
that is generated only in the presence of Hp allele two template
(Annealing temperature 61 °C, cycle 30)
Statistical analysis: Chi-square test was used to determine association
between Hp allele and diabetic CVD and CVD diseases and gender
effect on distribution Hp genotypes in those patients. Odds ratio were
calculated together with their 95% confidence intervals. The proportion
test was used to compare percentage of two genotypes in diabetic cardiovascular
disease. For all statistical analyses, p<0.05 was considered significant
(the calculations were manual).
After electrophoresis of the reaction products in 1% agarose gel, Hp
genotyping-specific banding patterns were obtained.With primers A and
B, Hp 1-1 and Hp 2-2 genotypes were characterized by single
bands representing the 1757 and 3481 bp products, respectively and Hp
2-1 genotype was characterized by the presence of both the 1757 and
3481 bp products (Fig. 1). Primers C and D were used
for detecting the 349 bp Hp allele two-specific product (Fig.
The distribution of three major Hp genotypes in diabetic cardiovascular
patients was 1.6% (1-1), 52% (2-1), 45% (2-2) in the men and 3.27% (1-1),
54.09% (2-1), 42.62% (2-2) in the women. These prevalence were shown to
be in Hardy-Weinberg equilibrium. In the no diabetic Iranian cardiovascular
patients these prevalence were respectively 36% (1-1), 62% (2-1), 2% (2-2)
(data not shown).
The Chi-square test determined the association between Hp allele
and diabetic cardiovascular diseases (χ2 = 52.98 p<0.001)
and this finding was independent of gender (χ2 = 0.39
p>0.05). Odds ratio shows that the chance of having Hp 2-2 phenotype
in diabetic patients (men) with CVD were estimated about 504 (95% CI,
29.6 to 8577.7) times more than those with Hp 1-1 phenotype ( p<0.0001)
and 27.12 (95% CI, 3.47 to 211.73) times more than those with Hp 2-1 phenotype
(p = 0.0008). The chance of having Hp 2-1 phenotype in the same patients
were calculated about 18.58 (95% CI, 2.33 to 147.74) times more than those
with Hp 1-1 phenotype (p = 0.02). The chance of having Hp 2-2 phenotype
in diabetic patients (women) with CVD were 234 (95% CI, 19.7 to 2779)
times more than those with Hp 1-1 phenotype (p<0.0001) and 24.42 (95%
CI, 3.12 to 190) times more than those with Hp 2-1 phenotype (p = 0.001).
The chance of having Hp 2-1 phenotype in the same patients were calculated
about 9.58 (95% CI, 2.05 to 44.7) times more than those with Hp 1-1 phenotype
(p = 0.0019). The alterations of odds ratio in both sex were homogenous
||Haptoglobin genotyping with primer A and B. (Lane 1)
DNA size marker (Lane 2) genotype Hp 1-1 (Lane 3) genotype
Hp 2-2 (Lane 4) genotype Hp 2-1
||Haptoglobin genotyping with primer C and D. (Lane 1)
DNA size marker, (Lanes 2 and 3) Hp 2-specific amplification
||The relationship between diabetic cardiovascular disorder
and Hp genotypes
It is concluded that there is no difference between percentage of Hp
2-2 and Hp 2-1 phenotype in diabetic patients with cardiovascular diseases
(Z = 1, p>0.05).
The oxidative modification hypothesis of atherosclerosis states that
oxidative modification of low-density lipoprotein (LDL) or other oxidative
events within the blood vessel wall promotes the development and progression
of atherosclerotic lesion (Brown and Goldstein, 1983; Steinberg and Witzum,
2002). Hemoglobin (Hb) is an oxidant of LDL by the Fenton reaction (Kristiansen
et al., 2001; Mowri et al., 2000). Hb is released from red
blood cells with intravascular hemolysis in endothelial cells and injury
may enter it into the sub endothelial space (Fernandez et al.,
The amount of hemoglobin penetrating into the blood vessel wall is increased
in diabetes because of an increased turnover of red blood cells (Venerando
et al., 2002) and an increase in endothelial cell dysfunction and
injury (Laight et al., 2000).
So the generation of reactive oxygen species has an important role in
the development of diabetic vascular complication (Melamed-Frank et
al., 2001; Suzuki et al., 1999). Haptoglobin is an antioxidant
that is able to prevent hemoglobin-driven oxidation (Langlois and Delanghe,
1996; Melamed-Frank et al., 2001). The mechanism by which haptoglobin
serves as an antioxidant is believed to be via stabilization of the heme
moiety within the hemoglobin protein (Quaye, 2008). Ability of haptoglobin
to protect against hemoglobin-driven oxidative stress is lost when hemoglobin
becomes heavily glycosylated. Glycosylated Hb is markedly increased in
the diabetic state (Asleh et al., 2003). In diabetic white, South
Asian and Afro-Caribbean patients have been shown that Hp 2-2 increases
the risk of developing CVD (UK Prospective Diabetes Study Group, 1998;
Carter and Worwood, 2007).
This study for the first time in Iran suggests that Hp genotype
may contribute to developing CVD in diabetic patients and we showed that
the presence of Hp allele two in diabetic individuals increases
the risk of developing CVD.
The authors wish to thank Alghadir Hospital and Alzahra University for
their grateful support of this work.
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