This slide show that how GSTM1(Glutathione S-transeferase M1) initiate sporadic breast cancer.Also show what is breast cancer,their types and causes.This is a short case control study on breast cancer cases and healthy population.

1. GSTM1 gene Polymorphism in Sporadic
Breast Cancer Patients
Sateesh Chandra Gupta

2. Overview
 Cancer
 Breast cancer
 sporadic ,familial and hereditary breast cancer
 Types of Breast Cancer
 Stages of Breast cancer
 causes of breast cancer
 Genetic, Environmental, Life style and hormonal factor
 Polymorphism
 Classes of polymorphism
 Role of polymorphism in breast cancer
 glutathione S-transferase (GST)
 Glutathione S-transferase Mu1 ( GST M1)
 Work Flow
 Result
 Reference

3. BACKGROUND

4. Cancer
 Cancer is a group of more than 100 disease that developed across
time and involved the uncontrolled cell division of body.
 Cancer are arise from the loss of normal growth control.
 In normal tissue, the rate of new cell growth and old cell death are
balance with the process known as cell division and apoptosis
respectively.
 In cancerous cell this balance is disrupted.

5. Breast cancer
 Breast cancer is the most common type of cancer (29%) among
women in the world
 An estimated 40,730 breast cancer deaths (40,290 women, 440 men)
are expected in 2015. Breast cancer ranks second as a cause of cancer
death in women (after lung cancer).
 The risk of breast cancer increases with age and most breast cancers
occur after the age of 50.
(America Cancer Society, 2015 )
 Breast Cancer is a malignant tumor that start in the cells of breast.
 A malignant tumor is a group of cancer cells that can grow(invade)
into surrounding tissue or spread (metastasize) nearby area of the
body.

6. Breast Cancer
The disease occur almost entirely in the women but men also get it.
 Most commonly from inner lining of milk duct or lobules that supply
ducts with milk.
 Cancer originating from duct are known as ductal carcinoma, while
those originating from lobules are known as lobular carcinoma.

7. Hereditary
Breast cancer
Sporadic Familial
 Approximate 10%
 Clustering within
family.
 Not hereditary
 Early onset than
sporadic cancer.
 Risk factor - same life
style of a family.
 Approximate 85 %
 Late onset
 Risk factor –age
exposure to
environmental
carcinogens,
Hormone and
Lifestyle .
 Approximate 5-7 %.
 Same type of cancer
in the family.
 Early onset.
 Bilateral.
 Having strong
family history.
 results from same
genetic and
environment or
lifestyle factors.

8. Breast carcinoma
Invasive Breast CarcinomaNon-invasive Breast Carcinoma
Ductal Carcinoma.
Lobular Carcinoma. Invasive Ductal Carcinoma
Invasive Lobular Carcinoma
Inflammatory Carcinoma
Secretary Carcinoma
Tubular Carcinoma

9. Receptor specific Breast cancer
Receptor
Hormone Receptor
(HR)
Tyrosine Kinase
Receptor
Estrogen Receptor(ER) Progesterone Receptor(PR) HER2 Receptor
ER Positive ER Negative PR Positive PR Negative
HER2 Positive HER2 Negative
Triple Negative Breast Cancer are Estrogen Receptor(ER),
Progesterone Receptor(PR) and Human Epidermal Growth Receptor
2(HER2) negative ,Hence called as triple negative breast cancer

10. Causes of Breast
Cancer

11. GENETIC
 The genome is susceptible to damage by both intrinsic( like Cell
division errors) and extrinsic factors(carcinogens and radiations) .
 Certain genes called Tumor Suppressor genes (BRCA1, BRCA2,
TP53, ATM and PTEN) are involved in maintenance of genomic
stability.
 Mutations that impair their function predisposes an individual to
develop cancer.
 Accumulation of mutations causing DNA damage transforms a normal
cell to a cancerous cell.
( Emel Ergul Ali Sajki et al. 2000 )

12. EnvironmentalFactor
 Natural or synthetic substances in environment that can cause
cancer are called environmental carcinogens.
 Divided into
 Chemical agents - Lead, cobalt, asbestos, nitrosamine and many type
of pesticide.
 Physical agents – X- rays, Gamma rays and Ultraviolet rays
 Light at Night (LAN Hypothesis)
May decrease the synthesis of melatonin- change in melatonin may
affect the level of estrogen and initiate the breast cancer.
(Richard G Stevens 2009)

13. LIFE STYLE
SMOKING and ALCOHOL DRINKING
 Each cigarette contains a mixture of carcinogens including
polycyclic aromatic hydrocarbons (PAHs), N-nitrosamines, NO2 and
free radicals .
 Cytochrome P-450 enzymes (P-450s) convert the carcinogens to
more reactive forms which bind to DNA and form DNA adduct.
 Alcohol in the body converts into the aldehyde. Acetaldehyde
converts into the Reactive Oxygen Species (ROS),which react with
DNA to form DNA adducts, which may initiate cancer.
 Alcoholic beverage consumption in women also causes an increase in
levels of endogenous estrogens hormone.

14. Smoke & alcohol carcinogens in cancer..
Metabolic
Activation
Metabolic
Detoxification Repair
Excretion Normal DNA
Uptake of
Smoke &
Alcohol
Carcinogen
DNA
Adduct
Mutation
in DNA
repair
genes and
Tumor -
suppresso
r genes
Loss of
normal
growth
control
mechanis
m
Cancer

15. HORMONAL
 Estrogens and progesterone are essential hormone for normal breast
development. The main estrogens are estradiol and estrone, as well as
16-hydroxyestradiol (estriol).
Estrone and estradiol
hydroxylated at positions
C2, C4 and C16
(2 hydroxyestrone, 4 hydroxyestrone
2-hydroxyestradiol, and 4-
hydroxyestradiol), and 16a-
hydroxyestrone.
carcinogenic potential by
damaging DNA

16. Estrogen Catabolism...
COMT GST
CYP450 CYP450 DNA
CYP450 1A2
CYP450 1B1
CYP450 CYP450 DNA
COMT GST
2-Methoxy Estrogens
2-Hydroxy estrogen
Estrogen
E,2,3 semi Q E,2,3 Q
Glutathione Conjugate
4-Hydroxy estrogen E,4,3 semi Q E,4,3 Q DNA Adduct
Glutathione Conjugate4-Methoxy estrogen
CANCER
DNA Adduct

17. Premenopausal Postmenopausal
Estrogen
Ovaries adipose tissue, muscle
liver
ER
ER
Breast Cancer
Ovarian removal Aromatase Inhibitors
Tamoxifen Tamoxifen
Receptor Mediated Carcinogenesis of Estrogen

18. xP0LYMORPHISm
 polymorphism is a DNA sequence variation that seen in more than 1%
in normal population. In this case no single allele is regarded as the
standard sequence. There are two or more acceptable alternatives.
Classes of polymorphism
 Single Nucleotide Polymorphisms (SNP): is a genetic variation
when a single nucleotide (i.e., A, T, C, or G) is altered .
 Variable Number of Tandem Repeats or VNTR: is a location on
genome where a short nucleotide sequence organized as tandem repeat.
 Microsatellites or STR or SSR: short tandem repeat (2 – 6 bp long)
 Minisatellites : simple sequence repeats (10 – 40 bp long)

19. Cont…
Example of SNP
Example of STR
ATCGACTGCGATCATGCATGCATGCATGCATGC
ATGCATGCATGCATGCATGCCTACGTGACTGAC
Here sequence CATG are repeated several times.
Ex. of Minisatellit
CCATCACATATATTCCATATATTCACATATATTACCA
GACTCACATATATTCACATATATTCACATATATTCTA
Here sequence CACATATATT are repeated several times.
C T T C A T C G A T C G G
C T T C A T G G A T C G G

20. Role of polymorphism in Development of breast cancer
 Polymorphisms consist of minor changes in DNA sequence -modify
the structure, expression or activity of the proteins.
 Polymorphisms in xenobiotic or carcinogenic metabolic genes give
rise to variable enzyme activity - differing abilities in the metabolism
of xenobiotic or carcinogen.
 When the activity of xenobiotic metabolizing enzyme is impaired by
polymorphism it will not metabolize the Xenobiotic and carcinogenic
compound.
 xenobiotic or carcinogenic compounds react with DNA and form the
DNA adduct - causes mutation and initiation of breast cancer.

21. GlutathioneS-transferase(GST)
 Glutathione S-Transferase (GSTs), is a phase ll xenobiotic detoxifying
enzyme.
 GSTs catalyses the conjugation of glutathione (GSH) to a wide
variety of endogenous and exogenous electrophilic compounds .
Alpha class
Mu Class
Pai Class
Theta Class
Zeta Class
Omega Class
Sigma Class
GSTA1, GSTA2, GSTA3, GSTA4, GSTA5
GSTM1,GSTM2,GSTM3,GSTM4,GSTM5
GST P1
GSTT1, GST T2
GST Z1
GST O1, GST O2
GST S1
Cytosolic
Mitochondrial
Microsomal (MAPEG)
GST
Kappa Class
MGST
GST K1
MGST1, MGST2, MGST3

22. Glutathione S –Transferase Mu 1 (GST M1)
 Located on chromosome 1p13.3.
Having 8 exons.
Gene size is 5490 bp.
 GSTM1 is polymorphically expressed, and three alleles have been
identified:GSTM1*0,GSTM1a and GSTM1b.
 The GSTM1a and GSTM1b are functionally identical, but differ
by single nucleotide substitution C >G at base position 519 or
position aa173 Lys convert in the asparagine.
 Homologous deletion results in the loss of this gene thus loss of
function of the enzyme.

23. Blood sample collection
DNA Extraction By Phenol
-Chloroform Method
Genotyping By Multiplex
PCR
Sample loading on 1%
agarose gel
Work Flow
Lymphocytes Separation
Blood sample collection
Lymphocytes Separation

24. Multiplex PCR- Amplification of many desired DNA fragments.
 We used two sets of primers: IFN gene primer pairs and GSTM1 primer
pairs. IFN primers were used to detect that whether PCR has worked or not.
Condition for PCR
Initial Denaturation Temperature
95 0c For 5 Minutes
Denaturation Temperature
950c for 45 second
Annealing Temperature
600c for 45 second
Extension temperature
720c for 45 second
Final extension temperature
720c 5 minutes
34 cycles

25. Master Mix for PCR
Component 1X(for One Reaction) nX (for n reaction)
MilliQ water 12.4µl 12.4n µl
10X PCR Buffer (Axygen) 2.5µl 2.5n µl
25mM dNTP 1.0µl 1.0n µl
10pmol IFN (forward Primer)
5′ GGCACAACAGGTAGTAGGCG 3′
1.0µl 1.0n µl
10pmol IFN (Reverse Primer)
5′ GCCACAGGACGTACTGACAC 3
1.0µl 1.0n µl
10pmol GSTM1 (Forward Primer)
5′CTGGATAGTAGCAGATCATGC 3′
1.0µl 1.0n µl
10pmol GSTM1 (Reverse Primer)
5′ CTGCCCTACTTGATTGATGGG 3′
1.0µl 1.0n µl
Sample DNA (20ng/µl) 5.0µl 5.0n µl
Taq. Polymerase (10U/µl) 0.1µl 0.1n µl

26. 100 bp Ladder- 1
Result
Rrrrr
Wild Type Wells- 3, 5
1 2 3 4 5
Null Type Wells-2, 4
GST M1
(273 bp)IFN
(173 bp)
500 bp
1
Result
100 bp Ladder Well -1

27. Frequency of GST M1 Homozygous Null Genotypes in worldwide Populations
Frequency of GST M1 Homozygous Null Genotypes in ACTREC Cohort
Resultcontd....
GST M1 Joanne E. Curran et al.
Australia (2000)
Christine B. Ambrosone
et al. New York ( 1995)
Samson et al.(2007)
South India
Genotype Null Type Wild Type Null Type Wild Type Null Type Wild Type
SC 56 (43% ) 73 (57% ) 84(48%) 93 (52%) 65(28.3%) 185(71.7%)
SN 57 (44% ) 72 (56% ) 116(50%) 117(50%) 110(22%) 390(78%)
Genotype Case (SC)
180
Control (SN)
188
Odds Ratio 95% CI P Value
Null 68(37.7%) 60(37.3%) 1.0235 0.6710 –
1.5611 P =0.9142Wild 112(62.3%) 118(62.7%)
All the Analytical analysis are calculated by using online software www.medcalc.net

28.  Correlation of different variable in development of breast cancer
in case and control.
Association between Breast Cancer Risk and Food Preference among Case and
Control population:
Vegetarian OR P Value Non Vegetarian OR P Value
Case Control 1.117
95% CI
0.506 –
2.457
P=0.784
Case Control
0.936
95% CI
0.556 –
1.576
0.803
Null 17 26 45 42
Wild 24 41 87 76
There are no Statistical significance have been found ( OR =1.117, P
=0.784 and OR =0.936, P = 0.803) between Breast cancer case and
healthy control population in ,correlation with food preference
Vegetarian and non Vegetarian Respectively.

29.  Association between Breast Cancer Risk and Tobacco
consumption Among Case and Control
Tobacco User OR P Value Non User OR P Value
Case Control
0.7955
95% CI
0.2362 -
2.6788
P=0.711
1
case Control
0.9951
95% CI
0.5976–
1.6570
P=0.985
0
Null 7 11 51 41
Wild 12 15 95 76
There are no Statistical significance have been found ( OR =0.7955,
P =0.7111and OR =09951, P = 0.9850) between Breast cancer case
and healthy control population in ,correlation with Tobacco
consumption, tobacco user and non user Respectively.

30.  Association between Breast Cancer Risk and Menopause status
among Case and Control
Premenopause OR P Value Postmenopause OR P Value
Case Control
0.997
95% CI
0.556 –
1.789
P=0.993
Case Control
0.927
95% CI
0.488 –
1.759
P=0.817
Null 33 39 29 29
Wild 56 66 55 51
There are no Statistical significance have been found ( OR =0.997, P
=0.993 and OR =0.927, P = 0.817) between Breast cancer case and
healthy control population in ,correlation with Menopause
status,premenopause and Postmenopause Respectively.

31. conclusion
 The frequency of null polymorphism in breast cancer patients and
healthy individual is almost similar (37%) null and wild genotype
frequency (63%).
 Our finding are in coherence with the previously studies
worldwide.
 The present study suggest that ,the null polymorphism in GSTM1
may not be involved in sporadic breast cancer susceptibility, But
have the modifier effect in initiation of breast cancer in
combination with other detoxifying enzyme, like GSTT1,
GSTP1,CYP450,NAT and COMT.

32. Conclusion cont..
 We also correlate breast cancer case and healthy control with
many variable ,like food preference, Tobacco consumption and
menopause status, but there are no statistical significant value
have been found.
 Further, we need to extend our study in a larger cohort to stabilise the
precise role of null polymorphism in GSTM1 in sporadic breast
cancer susceptibility.

33. Reference
 Christine B. Ambrosone, Cytochrome P4501A1 and Glutathione S-Transferase
(M1) Genetic Polymorphisms and Postmenopausal Breast Cancer Risk.
 A Khedhaier, Glutathione S-transferases (GSTT1 and GSTM1) gene deletions
inTunisians: susceptibility and prognostic implications in breast carcinoma.
 Joanne E. Curran, Polymorphisms of glutathione S-transferase
genes(GSTM1, GSTP1 and GSTT1) and breast cancer susceptibility
 Hamed Samavat, Estrogen metabolism and breast cancer.
 Stephen S. Hecht, Tobacco smoke carcinogens and breast cancer.
 Ramona G. Dumitrescu, The etiology of alcohol-induced breast cancer
 Alison M. Dunning, A Systematic Review Of Genetic Polymorphisms and Breast
Cancer Risk
 S.Zhong, Relationship between the GSTM1 genetic polymorphism and
susceptibility to bladder, breast and colon cancer

34. THANK YOU