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ABSTRACT

Diabetes
mellitus (DM) is the chronic and heterogenous group of disorder in which
insulin is formed in minimum quantity or resistance in insulin action. Both
types of diabetes; Type 1 (insulin-dependent) and Type 2
(non-insulin-dependent) are associated with long term complications. Diabetic
nephropathy is one of the microvascular complications of diabetes in which
kidney damages occur due to excess glucose in blood. Initial sign of
nephropathy is microalbuminuria that is the excretion of urine albumin at the
rate of 30-300 mg/24 hour. Approximately 75% of type 2 diabetic patients have
microalbuminuria. Superoxide dismutase is one of the enzymatic antioxidant
which catalyzes the conversion of oxygen radical into either hydrogen peroxide
or oxygen molecule. It plays a very important role in preventing cellular and
histological damages caused by reactive oxygen species. The present study will
find the role of MnSOD (Val16Ala) polymorphism and its association with
microalbuminuria in type 2 diabetic patients of local population of Pakistan.
Blood and urine samples will be collected of from 200 patients and 200 from
healthy controls. Estimation of albuminuria will be done by immunoturbidometric
assay by the commercially available kit. Genomic DNA  from blood will be extracted for MnSOD
(Val16Ala)  polymorphism and PCR-RFLP
will be done for detection of (Val16Ala) alleles. If association is found
between MnSOD (Val16Ala) polymorphism with microalbuminuria in type 2 diabetes
it will be benefit in diagnosis and treatment of diabetes and its
complications.

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INTRODUCTION

Diabetes
mellitus (DM) is the chronic and heterogenous group of disorder in which
insulin is formed in minimum quantity or resistance in insulin action.
Metabolic deficiency of fat, protein, carbohydrates and prolonged
hyperglycemia proved it a metabolic syndrome 1 which disturbing about 6 %
people around the world 2. Many of the organs like kidney, eyes, nerve, and
blood vessels are compromised by the higher concentration of glucose in blood
or chronic hyperglycemia 3. Diabetes mellitus is one of the ancient disease, first
reported in Egyptian documents around three thousand years ago. The ratio of patients
with diabetes mellitus in 2011 was 366 million and expected to increase this
ratio to 552 million by 2030. More than 80% of the people are affected with
type 2 diabetes in developed and under developed countries 4. From another
report the ratio of diabetes was 135 million in 1995 and would be increased to 300
million by 2025 and this proportion will be increased in Asian countries 5.
In a survey held at national level. It was reported that there is higher chance
for men as compared to women to suffer with diabetes millitus 6. The
prevalence of diabetes mellitus vary from race to race, age to age, and from
one geographical area to another one 1. There are two main types of diabetes
mellitus, type 1 or insulin dependent diabetes mellitus and type 2 diabetes mellitus
which is also known as non-insulin dependent diabetes mellitus. The main cause
of type 1 is the damage of beta cells of pancreas and this type is also known
as juvenile as it arises adolescently and about 5-10% people are affected with
type 1 diabetes. The other form of diabetes is type 2 diabetes or adult onset
diabetes. It is most common form of diabetes mellitus characterized by insulin
resistance and relative insulin deficiency. Type 2 diabetes is primarily depends
upon the life style and hereditary factors 7. Obesity and sedentary life
style also cause insulin resistance. It occurs at the age of 40 and about
90-95% of the diabetic patients have this type of diabetes 8. There are many
complication of diabetes but majorly classified into 2 groups: micro and macro
vascular complications. Diabetic retinopathy, neuropathy and nephropathy are
included in micro-vascular complications while atherosclerosis in
macro-vascular complications. These complications are related to type1 and type
2 diabetes 9. Persistent diabetes mellitus (DM) results in diabetic nephropathy
which leads to the end stage renal disease which ia a major cause of death
10. Excretion of albumin in the urine is the characteristics diabetic
nephropathy 11.

Approximately
30 % of the diabetic patients are affected with diabetic nephropathy and this
is most common cause of end stage renal disease 12. The common symptoms of
diabetic nephropathy are decrease rate of glomerular filtration, albuminuria,
hypertension and hyperlipidemia 14. Albuminuria is further classified into
two groups microalbuminuria and macro-albuminuria. If the value of albumin in
24 hours collected urine is in between 30-300 mg then this condition is known
as microalbuminuria. If the value of albumin in urine sample is >300mg/24h
then this condition is known as overt albuminuria or macro-albuminuria15. The
other diagnostic criteria for microalbuminuria in the patients of diabetic
nephropathy is measuring the albumin to creatinine ratio (ACR) in a spot urine
sample. In such patients 75% have raised microalbuminuria and 70-80% have
hypertension 13.The severity of diabetic nephropathy is increased when
microalbuminuria and hypertension occurs simultaneously 16. 

The
condition in which increased production or less removal of reactive oxygen
species (ROS) occur is known as oxidative stress 17. Oxidative stress is
induced by the elevation in the levels of free fatty acids and glucose that has
the key role in pathogenesis of both types of diabetes. As the excessive
production of ROS is a lethal factor which leads towards the damage to DNA,
lipid and proteins. Increased production of ROS and disturbance in antioxidants
defense in DM leads to the oxidative damage to cells in several tissues
involving eyes, nervous system and kidney 18. Highly complex antioxidant
system (enzymatic or non-enzymatic) is present in humans. Both types of
antioxidants might be supplied from exogenously as a part of diet or produced endogenously.
Common antioxidants includes the glutathione , Vitamins A, E , C and the
enzymes catalase, superoxide dismutase, glutathione peroxidase and glutathione
reductase 17. Antioxidants counter the action of free radicals by several
mechanisms. These mechanism includes the protein like transferrin that can
binds the metal which stilmuate the formation of free radicals, the enzymes
which degrade free radicals and the antioxidants like vitamins A and E which acts
as the free radical scavengers 18. The antioxidants defense systems work in
collaboration with each other to eliminate the excessive reactive oxygen
species and maintains the balance between oxidation and antioxidation in normal
condition in order to protect cells or organ from damages 19.

Superoxide
dismutase is one of the enzymatic antioxidant which catalyzes the conversion of
oxygen radical into either hydrogen peroxide or oxygen molecule. It plays a very
important role in preventing cellular and histological damages caused by
reactive oxygen species. Superoxide dismutase has high turnover rate.
Superoxide dismutase is further classified into three types, copper zinc SOD
(SOD 1) present in cytosol, manganese superoxide dismutase (SOD 2) in
mitochondria and extracellular SOD (SOD 3) 20. Superoxide dismutase 2 or
MnSOD gene is located on q arm of chromosome 6 at 25 position. The structure or
functional SNP of MnSOD gene is of high importance for maintenance of cellular
reactive oxygen species level. Around 190 single nucleotide polymorphisms have
been identified for SOD 2 gene 21.  But
the Ala16Val (rs4880) is the most studied and functional polymorphism present
on exon 2 of SOD2 gene 20. This substitution of T to C (GTT to GCT) that is valine
to alanine results in structural alterations in the mitochondrial targeting
domain from ?-sheet to ?-helix which induces a 30-40% increase in MnSOD
activity in mitochondria 22. This Ala16Val polymorphism is result into the
etiology of the T2D and might be risk factor for diabetes in Japanese Americans
23.

 

 

 

 

 

 

 

 

 

 

JUSTIFICATION AND LIKELY BENEFITS

The
number of people with diabetes is growing in Pakistan day by day as the result
of obesity and sedentary life. In Pakistan, diabetes mellitus is the major
health problem and its prevelance vary from 5-15 % among urban and rural
population of Pakistan 24. Pakistan is considered as one of the country with
largest population with diabetes and ranked seventh globally by the IDF
(international diabetes foundation) 1. The prevalence of type 2 diabetes in
Pakistan is accountable to cause threats to the quality of life as well of the
economy of Pakistan due to the poor glycemic controls and its complication 2.
It is very difficult to identify those genes which are involved in the
pathogenesis of type 2 diabetes. It is very important to screen the candidate
genes for the nucleotide variants that are associated with diabetes mellitus
and its related complication. The literacy rate of Pakistan is low and most of
the people are unaware of the complications which are associated with
uncontrolled diabetes 25. Diabetic nephropathy is the leading cause of the
chronic renal failure and approximately 7-53% of all the diabetic patients
develop diabetic nephropathy. The prevalence of diabetic nephropathy varies from
population to population and race to race, 42% in south Africa, 13.6% in
Pakistan, 9% in UK population and 36.7% in India 24,26. Microalbuminuria is
considered as the first maker of diabetic nephropathy 15. There are many
factors involved in diabetes mellitus and oxidative stress in one of them. Oxidative
stress is induced by the elevation in the levels of free fatty acids and
glucose that has the key role in pathogenesis of both types of diabetes 18. Superoxide
dismutase is one of the enzymatic antioxidant which catalyzes the conversion of
oxygen radical into either hydrogen peroxide or oxygen molecule. It plays a
very important role in preventing cellular and histological damages caused by
reactive oxygen species 20. Many studies has been done internationally on the
association of Val16Ala polymorphism of MnSOD gene with microalbuminuria in
type 2 diabetes 21. The aim of my research is to investigate the distribution
of this polymorphism in Pakistani diabetic patients and evaluate the association
of this polymorphism to the etiology of T2D and with diabetic nephropathy.  If this gene is positively correlated with
diabetic nephropathy in type 2 diabetes then it may give the better way to
treat the diabetes and to deal with vascular complications.

OBEJECTIVES

 

The
objectives of the study will be:

1.      To
determine the levels of ACR ratios in type 2 diabetic patients and healthy
individuals.

2.      To
Determine the Val16Ala polymorphism of MnSOD gene in type 2 diabetes and
controls.

3.      To
determine the association of microalbuminuria with Val16Ala polymorphism gene
in type 2 diabetic patients.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LITERATURE
REVIEW

 

Zheng
et al, 27 determined the
relationship of Ala16Val polymorphism in SOD2 gene with type 2 diabetes and
diabetic nephropathy in Chinese population. This case control study included
total 429 subjects from which 172 were normal subjects and 257 had type 2
diabetes. Among type 2 diabetes 154 had diabetic nephropathy and 103 did not.
The patients of diabetic nephropathy were further divided into 2 groups with
microalbuminuria included 92 individuals and 62 patients with overt
albuminuria. The PCR-RFLP was used to detect the genotypes of the Ala16Val
polymorphism for all subjects. The AA+VA allele frequencies of patients with
diabetic nephropathy were significantly lower than that of the non-diabetic
nephropathy patients (11.6% and 24.3%). It was suggested that Val allele was a
risk factor and Ala allele is the protective factor for diabetic nephropathy in
type 2 diabetes in Chinese population. The result of the study suggested that A16V
polymorphism in the SOD 2 gene is associated with decreased risk of diabetic
nephropathy in type 2 diabetes in Chinese population.

Many
of the SNP of the genes that linked to the oxidative stress had been evaluated
as the intracellular ROS are positively associated with the development of
diabetes mellitus and its microvascular complications. Lee et al 28 performed a case control study to investigate whether
this V16A  polymorphism in MnSOD gene is
associated with the stages of albuminuria and whether this polymorphism is
related to the pathogenesis of diabetes in Korean population. Total 549
individual were included in a study from which 178 were healthy subjects and
371 had type II diabetes. The type 2 diabetic patients were further divided
into 3 groups, normoalbuminuria included 244 subjects, 86 in microalbuminuria
and 41 subjects were included in macroalbuminuria. The PCR-RFLP was used to
determine the V16A genotypes. The patients with diabetic nephropathy had
significantly lower  A allele frequency,
longer duration of diabetes, greater ACR and higher prevalence of hypertension
as compared to the patients without diabetic nephropathy. In the patients of
type 2 diabetes A allele carriers had lower prevalence of hypertension as
compared to the A allele non-carriers. This study showed that the V16A
polymorphism is not linked to pathogenesis of diabetes but it is positively
associated with stages of albuminuria in T2D in Korean Population

Manganese
superoxide dismutase protects the cells from oxidative damage as oxidative
stress leads to the development of diabetic nephropathy. The levels of
antioxidants increased by smoking which may disturb the balance between
radicals and antioxidants. Stefan et al
29 determined whether the Val /Ala polymorphism in SOD2 gene alone or in
combination with smoking could contribute to the production of diabetic
nephropathy in type 1 diabetic in Finnish and Swedish population. Total 1510
subjects were included in a study from which 555 were healthy subjects and 955
had diabetic nephropathy. The patients of diabetic nephropathy were further
divided into 2 groups overt diabetic nephropathy included 619 patients having
the albumin excretion rate >200 g/min and incipient diabetic nephropathy
having AER 20-200 g/min. The duration of diabetes, age at onset, sex, A1C,
smoking and V/V genotype was associated with high risk of DN. The result of the
study showed that smoking and Val/Val allele had positive association in the
progression of DN which further supports the hypothesis that oxidative stress
contributed to the development of diabetic nephropathy in type I diabetes.

The
relationship of an Ala16Val polymorphism MnSOD gene with diabetes and diabetic
nephropathy in type 2 diabetes in Japanese population by Nomiyama
et al, 30. In this case control study, 478 individuals had type 2 diabetes
and 261 were normal healthy subjects. The Val16Ala genotyping of SOD 2 gene was
done by PCR-RFLP using Bsaw I restriction enzyme. The genotyping and allele
frequency of diabetic patients were not much different from those of the
healthy subjects. The VV type showed a significantly higher frequency in
diabetic patients with diabetic nephropathy as to the AA and VA type. The
result of the study showed that Val16Ala polymorphism in SOD 2 gene is not
related to the etiology of type 2 diabetes but it is positively associated with
DN in type 2 diabetes in Japanese population.

Mona
et al 31 studied the role of SOD2
gene in a progression of diabetic nephropathy and neuropathy in Egyptian
population. Total 210 children included in a study from which 40 were normal
healthy control, 65 individuals had diabetes, 40 subjects were suffering in
diabetic nephropathy and 45 had diabetic neuropathy. PCR-RFLP was used for the
detection of polymorphism. Amplified product was digested with AgeI enzyme to
detect Ala16Val polymorphic sites. The results of their study showed that the
frequency of Ala and Ala/Ala genotype (OR=0.43 and 0.26) were lower in diabetic
neuropathy. In contrast, the frequency of Val or Val/Val allele (OR=2.2 and
6.6) were higher in diabetic patients with neuropathy than diabetes without
neuropathy. Though the Val allele (OR=3.2) was higher in diabetic nephropathy
patients than without nepropathy but their difference was non-significant
statistically. Therefore, the substitution (Ala-9Val) in SOD 2 was associated
with diabetic neuropathy in Egyptian diabetic children but not the significant
factor in the patients with nephropathy.

Cruz
et a,l 32 assessed the association
of this polymorphism in MnSOD gene and macro-albuminuria in the sample of 994
un-related Mexican T2D patients. This study was included 119 individuals with
urinary albumin >300 mg/dL and 875 individuals with urinary albumin ? 30
mg/dL. Real Time PCR was used for the detection of polymorphism. The result of
their study showed that TT genotype (6.7%) was higher in participants with
macro-albuminuria (16.8%) as to normo-albuminuria (10.1%). They observed that
the CC genotype had significantly lower risk of macro-albuminuria than TT
genotype. The conclusion of this study showed that there is positive association
of this polymorphism of MnSOD gene with macro-albuminuria in Mexican T2D patients.

 

 

 

 

 

 

 

 

PROPOSED
METHODOLOGY

 

Study
design                     

It will be a case control analytical
study.

Study
Location         

Mayo Hospital Lahore, Diabetes
Management Centre Services Hospital Lahore, Kinnaird College for Women Lahore.

Study
population      

Patients
with type-2 diabetes mellitus for more than 10 years including both sexes
between the ages of 40-60 years. The patients with diabetes mellitus will be
divided into two groups (i) diabetes without nephropathy (normal albumin
excretion) (ii) diabetes with nephropathy (microalbuminuria/macroalbuminuria).

Comparison
group   

Healthy
subjects with no history of diabetes mellitus, hypertension, renal failure,
ischemic heart disease and any other chronic disease were selected from general
population. The age and sex should be matched with the Patients.

Inclusion criteria      

Type-2
diabetes mellitus patients of 40-60 years of age having disease for more than
10 years ages between 40-60 years.

Exclusion criteria     

Patients
with other disease like chronic liver disease, hematuria, pyuria and end-stage
renal disease will be excluded from the study.

Ethical Review                   

 The  
methodology   of   the  
study   will be approved by the
Kinnaird college of Women Lahore and Institutional Review Board of King Edward
Medical University (KEMU).

 

Determination of Body mass index

 

Weight and height will be measured
to calculate BMI. The following formula will be used to calculate BMI:

 

Formula:      Weight (kg)

                      

                    
Height (m2)

 

Data
collection          

Personal information of the patients
will be recorded on the prescribed Performa after obtaining the informed
consent (Annexure I) from all the subjects.

Sample
collection & storage

Total 3 ml of blood will be drawn in EDTA
vacutainer tubes from diabetic patients and healthy controls. Samples will be
stored at -4oC for further DNA extraction. For the examination of
urine, it will be collected in urine container.

Estimation
of Albumin and Creatinine Ratio

For
the examination of urine, it will be collected in urine container. After
centrifugation, supernatant will be separated and stored at -80oC
until analysis. Evaluation of microalbuminuria will be done on spot urine
sample using albumin: creatinine ratio (ACR) by an immunoturbidometric assay by
the commertially available kit.

DNA
extraction

The genomic DNA will be extracted
using conventional phenol-chloroform method 30 from peripheral blood cells
and the quantity and quality of DNA will be analyzed on 1% agarose gel.

Primer
designing

Sequence of genomic DNA of manganese
superoxide dismutase gene (MnSOD) will be retrived from NCBI and primer will be
selected from the previous literature 27,28,30. The location of primer
binding on DNA, Tm, GC content, primer dimer formation and self-complementarity
of the primers will be determined. The fragment size produced after restriction
analysis will be calculated.

PCR-PFLP

For the determination of genotyping
of Val16Ala of MnSOD, PCR-RFLP will be used. Genomic DNA fragment that contains
V16A polymorphic site will be amplified. After optimizing the conditions like
concentration of template, duration, concentration of MgCl2 and melting
temperature. Amplified PCR product will be digested with BsawI restriction
enzyme. The restriction fragments will be analyzed on agarose gel to determine
the polymorphism.

Data
Analysis

Data will be analyzed using
statistical system SPSS 18 and MiniTab by applying the statistical tests.
Quantitative variables e.g. age, BMI, microalbuminuria will be illustrated by
means +/- SD and qualitative variables e.g. sex and polymorphism will be
depicted by frequency and percentages. Comparisons of the laboratory and
clinical parameters between the type 2 diabetic patients and healthy subjects
will be analyzed by applying student T-test or chi square test. The subgroups
of type 2 diabetes mellitus will be compared with a one way analysis of
variance (ANOVA). The p-value less than 5% will be considered as significant. 

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