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CONTENTS

Sl. No. Title Page no.

1 Introduction 3

2 Review of Literature 5

3 Aim and Objective 9

4 Materials and methods 9

5 Results and discussion 14

7 Conclusion 15

8 References 16

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 INTRODUCTION
A chronic condition is a human health condition or disease that is persistent or otherwise
long-lasting in its effects or a disease that comes with time. The term chronic is often applied
when the course of the disease lasts for more than three months. Chronic diseases are also
the major causes of premature adult deaths in many parts of the World. According to the
World Health Organization (WHO), almost half of all chronic disease deaths occur in people
under the age of 70.

TYPES OF CHRONIC DISEASE:

Common chronic diseases include

 Bipolar mood disease

 Cardiac failure
 Chronic obstructive pulmonary disease
 Chronic kidney disease
 Coronary artery disease
 Crohn's disease
 Diabetes mellitus (type 1 and type 2)

CHRONIC KIDNEY DISEASE:

Chronic kidney disease is a general term for heterogeneous disorders affecting kidney
structure and function. Disease and management are classified according to stages of disease
severity, which are assessed from glomerular filtration rate (GFR), and albuminuria, and
clinical diagnosis (cause and pathology). Chronic kidney disease can be detected with
routine laboratory tests, and some treatments can prevent development and slow disease
progression, reduce complications of decreased GFR and risk of cardiovascular disease, and
improve survival and quality of life. Even if one kidney stops functioning, the other can
carry out normal functions. It is not usually until the disease is fairly well advanced and the
condition has become severe that signs and symptoms are noticeable; by which time most of
the damage is irreversible.

It is not unusual for people to realize they have chronic kidney failure only when their
kidney function is down to 25 percent of normal.As kidney failure advances and the organ's
function is severely impaired, dangerous levels of waste and fluid can rapidly build up in the
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 body. Treatment is aimed at stopping or slowing down the progression of the disease - this is
usually done by controlling its underlying cause

Figure 1.1 normal kidney vs. diseased kidney

GST GENE:

Glutathione S-transferases (GSTs) are a very important family of enzymes that catalyze the
detoxification of a wide variety of active metabolites of tobacco carcinogens such as
benzopyrene and other polycyclic aromatic hydrocarbons, monohalometahnes, etc.
Subsequently, epoxide intermediates are formed, for example benzopyrenediol epoxide,
which release free radicals and may bind and modify DNA. Therefore, variations in the
expression of GSTs due to heritable genetic polymorphisms probably modulate the process
of carcinogenesis by altering the exposure levels of tobacco-derived carcinogenesis. Five
different groups of GSTs have been described within the GST super family; (A), (M), (T),
(P), and (Zeta). Genetic polymorphisms relevant to altered GST expression have been
demonstrated in GST M1, GSTM3, GST T1 and GSTP1 genes. GSTs conjugate glutathione
to DNA-damaging electrophiles which render them hydrophilic and nontoxic.

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 Figure 1.2 Structure of GST gene

Both GSTM1 and GSTT1have deletion variants which occur at relatively high frequencies
in human populations from diverse ethnic backgrounds. A common homozygote deletion of
the GSTM1 gene nullifies its activity completely leading to a non-functional phenotype.
Similarly, a deletion polymorphism in GSTT1 leads to lack of enzyme activity.

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 REVIEW OF LITERATURE

10% of the population worldwide is affected by chronic kidney disease (CKD), and millions
die each year because they don’t have access to affordable treatment. According to the 2010
Global Burden of Disease study, chronic kidney disease was ranked 27th in the list of causes
of total number deaths worldwide in 1990, but decrease to 18th in 2010.

It is estimated that number of cases of kidney failure will increase disproportionately in

developing countries like China and India, where the elderly people are increasing. In
people aged 65 to 74 worldwide it is estimated that one in five men and in four women have
CKD. Non communicable diseases (such as heart disease, diabetes, kidney disease) have
replaced communicable disease (such as influenza, malaria, or AIDS) as the most common
causes of premature death worldwide. An estimated 80% of this burden occurs in low- or
middle- income countries, and 25% is in people younger than 60 years. Chronic Kidney
disease is a worldwide health crisis. For example in the year 2005, there were approximately
58 million deaths worldwide, with 35 million attributes to chronic disease, according to the
World Health Organization. Chronic kidney disease can be treated with early diagnosis and
treatment, it is possible to slow or stop the progression of kidney disease.

GST gene polymorphism may exert an effect on the functioning of enzymes encoded by these
genes through the change in both the level of gene expression and activity of the protein
itself. In this way it has an influence on the possibility of detoxification of carcinogens, and
consequently, the level of DNA damage; thus it may have an indirect effect on the risk of
development of cancer and other chronic diseases .Polymorphism of GSTM1 and GSTT1
genes observed in the human population consists in hereditary homozygous null deletion of a
gene fragment, resulting in lack of the protein product, which is related to the total loss of
enzymatic activity. The GSTM1 gene is located on chromosome 1p13.3. Deletion of a
fragment of this chromosome of the length of 20 kb developed in the course of evolution as a
result of recombination between two highly homologous fragments flanking the gene locus.
In this way, there developed the deletion GSTM1 null genotype. Deficiency of GSTM1
transferase activity resulting from the above-mentioned genotype is observed in 30–50% of
humans, according to population studies. GSTM1 null genotype seems to be related to low
capacity for the detoxification of selected xenobionts and reduced capability for controlling

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oxidative stress, which is equivalent to the damage to the cell caused by the activity of free
radicals.

Adverse effects of xenobiotics, which are foreign substances, are excreted via covalent
interactions between intermediate metabolites and genetic materials or proteins and their
related metabolites. Enzymatic reactions of xenobiotic metabolism are needed to avoid
accumulation of lipophilic xenobiotics in cells and tissues. These reactions can be divided
into 2 phases during the activation-deactivation sequence, namely phase-I and phase-II
enzymatic reactions. During these reactions, toxic metabolites are generated which might be
processed by phase-II enzymes. Phase-II enzymes complete detoxification by neutralizing
reactive electrophiles or by acting as indirect antioxidants. NQO1 and GSTs are both phase-II
detoxifying enzymes which play important roles in preventing carcinogen-induced disorders.
Glutathione S transferase M1 (GSTM1) and glutathione S transferase T1 (GSTT1) are cancer
susceptibility genes because of their ability to regulate the conjugation of carcinogenic
compounds to excretable hydrophilic metabolites. Deletion variants lacking in enzyme
activity exist for both genes. Individuals with homozygous deletions in the GSTM1 or
GSTT1 genes are supposed to have less ability to metabolize carcinogens and may therefore
be more susceptible to many chronic diseases.

SYMPTOMS OF CHRONIC KIDNEY DISEASE:

The most common signs and symptoms of chronic kidney disease include:

1. Anaemia
2. Blood in urine
3. Dark urine
4. Decreased mental alertness
5. Decreased urine output
6. Edema - swollen feet, hands, and ankles (face if edema is severe)
7. Fatigue (tiredness), etc.....

STAGES OF CHRONIC KIDNEY DISEASE: Changes in the GFR rate can assess how
advanced the kidney disease is.

Stage 1 - GFR rate is normal. However, evidence of kidney disease has been detected.

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Stage 2 - GFR rate is lower than 90 millilitres, and evidence of kidney disease has been
detected.
Stage 3 - GFR rate is lower than 60 millilitres, regardless of whether evidence of kidney
disease has been detected.
Stage 4 - GRF rate is lower than 30 millilitres, regardless of whether evidence of kidney
disease has been detected.
Stage 5 - GFR rate is lower than 15 millilitres. Renal failure has occurred.

DIAGNOSIS OF CHRONIC KIDNEY DISEASE:

 Blood tests: Kidney function tests look for the level of waste products, such as
creatinine and urea, in your blood.

 Urine tests: Analyzing a sample of your urine may reveal abnormalities that point to
chronic kidney failure and help identify the cause of chronic kidney disease.

 Imaging tests: Your doctor may use ultrasound to assess your kidneys' structure and
size. Other imaging tests may be used in some cases.

 Removing a sample of kidney tissue for testing: Your doctor may recommend a kidney
biopsy to remove a sample of kidney tissue.

TREATMENT OF CHRONIC KIDNEY DISEASE:

Depending on the underlying cause, some types of kidney disease can be treated.
Often, though, chronic kidney disease has no cure.
 VITAMIN D:
Patients with kidney disease typically have low levels of vitamin D. It is essential for
healthy bones.
 SKIN ITCHING:
Antihistamines such as chlorophenamine may help alleviative symptoms of itching.
 ANTI-SICKNESS MEDICATION
If toxins build up in the body because the kidney does not work properly patients may
feel sick, medications such as cyclizine or metaclopramide help relieve sickness.
 DIALYSIS:
Dialysis artificially removes waste products and extra fluid from your blood when
your kidneys can no longer do this.

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 AIM AND OBJECTIVE

AIM:

Identification of GST gene polymorphism in chronic kidney diseases.

OBJECTIVE:

Null deletion of the GST gene polymorphism by using polymerase chain reaction (PCR).

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 MATERIALS AND METHODOLOGY

SAMPLE COLLECTON:

We have collected 50 blood samples which consists of 24 control subjects and 25 patients
with chronic kidney diseases. After genomic DNA extraction, multiplex PCR assay was
performed using specific primers and the result suggest that the frequency of null
polymorphism in GSTM1 were higher in patients when compared to controls.

DNA ISOLATION:

The isolation of DNA is a fundamental first step in an array of molecular techniques involved
in genetic identity analysis. The isolation of DNA from a sample source can be tedious
involving steps that yield the highest quality DNA at the expense of speed and convenience
.Many techniques currently used for DNA isolation leads to significant dilutions of the
sample material or require the precipitation of material especially difficult. This consideration
is especially important in the field of forensic analysis and genetic identity where DNA is
extracted from extremely small amounts of starting material often recovered from suboptimal
storage conditions.

BLOOD:

The technique presented in this report can be used to isolate DNA from a variety of sample
media, including fresh or dried blood and as little as 10ml of blood. In addition, the sample
and throughput of the technique enable large number of samples to be processed quickly.
This report outlines the basic protocol used to isolate DNA a variety of human sources. The
quality of the isolated DNA is demonstrated by spectrophotometric analysis and performance
in the gene print power plex system.

MATIERALS:

Pipettes, eppendorf tubes, tips, micro centrifuge, vortex, gloves and spirit. TKM1 solution,
TKM2 solution, protein precipitation solution, DNA hydration solution, ethanol and distilled
water, triton-X.

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 PREPARATION OF REAGENTS:

All the stock reagents required for DNA isolation were presented below:

1) TKM1 SOLUTION:
Tris Hcl, PH 7.6,Potassium chloride(KCl),magnesium chloride(MgCl2),,ethylene
Diamine Tetra Acetic Acid(EDTA)make the final volume of solution to 100ml with
distilled water.
2) TKM2 SOLUTION:
Weigh tris Hcl, MgCl2, EDTA, NaCl sodium Dodecyl sulphate (SDS) make the final
volume of the solution to 100ml with distilled water.

PROCEDURE FOR DNA ISOLATION FROM BLOOD:

To 400µl of heparinised blood samples, and 400µl of TKM1 solution, vortex and mix by
inversion.

Centrifuge at 10000 RPM for 5 minutes discard the supernatant. To the pellet add 300µl of
TKM1 solution and triton-X 40µl of vortex until the pellets dissolve completely.

Again centrifuge at 10000 RPM for 5 minutes discard the supernatant.

Repeat step 1 for two times

Add 150µl of TKM2 solution and add 50µl of SDS.

Add 150µl of TKM2 and 40µl of NaCl and incubate for 15 min to precipitate protein.

Centrifuge at 10000 RPM for 5 min and discard the supernatant.

Transfer the supernatant into the eppendorf tube containing 300µl of ethanol; invert the
eppendorf tube several times slowly until the DNA precipitates.

Centrifuge at 10000 RPM for 5 minutes and discard the supernatant and keep it for air-
drying.

After air-drying, add 25µl of TE buffer and put paraffin paper and store it in freezer. Observe
the DNA fragments in agarose gel electrophoresis.

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 POLYMERASE CHAIN REACTION (PCR):

Figure 1.3: PCR

VOLUME(µl)
PCR water 15
Master mix 20
Forward primer(B,G) 3,3
Reverse primer(B,G) 3,3
DNA 3
Total 50

Table 1.4: components added for PCR

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 PCR CONDITIONS:

The entire mixture was added and was incubated at the following thermal conditions.

TEMPERATURE(ºC) TIME
Initial denaturation 96 4 minutes
Denaturation 95 45 seconds
Annealing temperature 62 45 seconds
Amplification temperature 72 55 seconds
Extension temperature 72 8 minutes

Table 1.5: Incubation temperature for PCR

The entire mixture was carried out for 35 PCR cycles.

GEL ELECTROPHORESIS DETERMINATION:

The concentration of DNA was checked by using agarose gel (2%: 0.6 gm of agarose
dissolved in 35ml of 1x TBE buffer) and allowed to boil. After complete dissolution of
agarose, Etbr (20µl) was added and casted the gel in pre-set gel caster. Combs were kept in
the gel caster to form wells and the gels were allowed to solidify. Electrophoresis was
performed at 50 volts for 45 mins and the fluorescence was emitted (due to the chelating of
bases of DNA by Etbr).

AGAROSE GEL ELECTROPHORESIS:

Reagent preparation:

Tris-borate-EDTA (TBE) buffer (1x):

For Preparation of 1000ml of 10x TBA, mixed 108gm of tris ,55gm of boric acid and
7.44gm of EDTA or 40ml of 0.5M EDTA .

6x Sample Loading Dye:

It was prepared by adding 0.25gm of bromophenol blue, 0.25gm of xylene cyanol, and 4ml
glycerol into 100ml distilled water.

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 Figure 1.6: Agarose gel

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 RESULTS AND DISCUSSION

CASE CONTROL
Sample size(n) 25 24
Female 10 9
Male 15 15
Age (Female) 40-60 yrs 27-45 yrs
Age (Male) 26-65 yrs 27-67 yrs
Creatinine range(F) 1.0mg/dl-8.8mg/dl 0.7mg/dl-0.9mg/dl
Creatinine range(M) 2.0mg/dl-14.5mg/dl 0.6mg/dl-0.9mg/dl

Table 1.6: characterization of study

Case Control Odd ratio X2 P- Value

(n=25) (n=24)
Normal 9 18 1 1 1
Deletion 16 6 5.33 (18.30- 7.5282 0.0060
1.55)

Table 1.7: The association of GSTM1 gene polymorphisms

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 DISCUSSION

GST polymorphism and its association with CKD are not yet to be well established. We have
collected 49 blood samples which consists of 24 control subjects and 25 patients with chronic
kidney diseases. After genomic DNA extraction, multiplex PCR was performed using
GSTM1 and positive control beta globin primers. Among the control, 6 had null deletion and
18 were normal, and in patients 16 had null deletion and 9 had CKD without null deletion.
The result suggests that the frequency of null polymorphism in GSTM1 were higher in
patients when compared to controls.

Most previous studies concentrated on association of these polymorphisms with Glutathione

S-transferase M1 deletion is a risk factor for developing end-stage renal disease in diabetic
patients (Datta SK, et al, 2010). GSTM1 deletion is associated with increased oxidative stress
in CKD patients. The blood samples collected from these patients were analyzed to study the
genetic polymorphism. GSTM1 null genotype, was significantly associated with CKD
irrespective of the presence of diabetes. The null alleles of GSTM1 are strong predisposing
risk factors for CKD. After observation, we suggested GSTM1 null allele as risk factor for
CKD.

Among the genotypes, the GSTM1− group had the least GST levels pointing toward the
additive effect of these deletions. Because of small sample size, further studies are needed to
evaluate the influence of their polymorphism on the risk of CKD.

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 CONCLUSION

 Our study demonstrated that GSTM1 polymorphism showed higher risk in CKD
patients.
 The probability of the presence of GSTM1 gene is 0.0060 which is less than 0.05 so it
is significant.
 Controls have less number of people with deletions of this gene compared to patients .
Hence we conclude that the null deletion is responsible for changes in DNA and this
leads to Chronic Diseases.

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 REFERENCES

1. Association of glutathione S-transferase M1 and T1 gene polymorphism with oxidative

stress in diabetic and nondiabetic chronic kidney disease ( Sudip K. Datta,Vivek
Kumar,Rahul Pathak,Ashok K. Tripathi,Rafat S. Ahmed,Om P. Kalra )
2. Mannervik B, Awasthi YC, Board PG, Nomenclature for human glutathione
transferases. Biochem J

3. Prohaska JR. The glutathione peroxidase activity of glutathione S-transferases. Biochem

Biophys Acta.
4. Harrison DJ, Kharbanda R, Cunningham DS, McLellan LI, Hayes JD. Distribution of
glutathione S-transferase isoenzymes in human kidney: Basis for possible markers of
renal injury. J Clin Pathol
5. Yang Y, Kao MT, Chang CC, Glutathione S-transferase M1 deletion is a risk factor for
developing end-stage renal disease in diabetic patients. Intern J Mol Med.
6. Kim JH, Moon MK, Kim SW, Glutathione S-transferase M1 gene polymorphism is
associated with type 2 diabetic nephropathy. J Korean Diabetes Assoc.
7. Agarose Gel Electrophoresis B. G. Johansson
8. Chronic Kidney Disease and the Risks of Death, Cardiovascular Events, and
HospitalizationAlan S. Go, M.D.,Glenn M. Chertow, M.D., M.P.H.,Dongjie Fan,
M.S.P.H.,Charles E. McCulloch, Ph.D.,and Chi-yuan Hsu, M.D.
9. A multiplex PCR procedure for polymorphic analysis of GSTM1 and GSTT1 genes in
population studies Sherif Z. Abdel-Rahman Randa A. El-Zein Wagida A. Anwar

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