Nateriab and Nethods

The test of all knowledge zu experzme7it Rzchard P. Feynnzan

4.1. Materials

4.1.1. Reagents

4.1.1.1. DNA extraction

Ammonium bicarbonate (ICN Biomedicas, I ~ c . , OH, USA), ammonium chloride (Merck

Iimlted, Mumbai), chloroform (Qualigens fine chemicals, Mumbai), EDTA disodium

hydrate (Amersco, OH, USA), ethanol absolute, 99.9% (S.d. fine-chem Itd., Murnbai),

hydrochloric acid (Qualigens fine chemicals, Mumba~), 2-mercaptoethanol (Merck,

Darmstadt, Germany), 1-octanol (Sigma-Aidrich Co. MO, USA), phenol (SRL),

proteinase K (Genei, Bangalore), sodium chloride ultrapure (USE Corporation, OH,

USA), tri-sodium citrate (Qualigens flne chemicals, Mumbai), sodium lauiyisuphate

(SISCO research laboratories pvt. itd, Mumbai), sodium hydroxide pellets (Ranbaxy

laboratorles, S.A.S. Nagar), tris buffer (Himedia laboratorles itd., hlumbai)

4.1.1.2. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

PCR

Primers (Alpha DNA, Montreal, Canada), Taq DNA polymerase (Genecraft. Germany),

dNTPs (Fermentas Life Sciences), magnesum chlor~de (Sigma, Missourie, USA), DMSO

(Sigma, Missourie, USA)

Restriction digestion (Enzymes)

Msp I (MBI Fermentas), Nco I (New England Biolabs, Ispswich, MA, USA), Dra I (hlBI

Fermentas ), Taq I (MBI Fermentas), Rsa I (MBI Fermentas). Pst i (MBI Fermentas),

BsmA I (MBI Fermentas), Dpn iI (New England B~olabs, Ispswich, MA, USA).

Horizontal &Vertical gel electrophoresis

Acrylamide: bisacrylamide 29:1, 30% (Genei, Bangalore), agarose (Sigma, Missourle,

USA), ammonium persulphate (SISCO research laboratories pvt Itd., Mumbai), Boric

acid (Ranbaxy fine chemicals Itd., S.A.S. Nagar), disodium EDTA (Amersco, OH, USA),

eth~dium bromide (Medox biotech India pvt. Itd., Chennai), glacial acetic acid (Merck

iimited. Mumba~). TEMED (AppliChem GmBH, Darmstadt, Germany), tris base (Himedia

laboratories Ltd., Mumbai), bromophenol blue (S.d.fine chem Itd., Mumbai), Ficoll

(Amersham biosciences AB, Uppsala, Sweden), xylene cyanoi (Loba Chemie, Mumbai),

lambda DNA (New England biolabs, ipswich, MA, USA), molecular weight marker Vlll

(Roche diagnostics GmBH, Mannheim, Germany), low molecular we~ght DNA ladder

(New England biolabs, Ipswlch, MA, USA), 100bp DNA ladder (New England

biclabs,lpswich,MA, USA)

4.1.2. Instruments

Elix Ri03 water purification system (Miiipore, USA ), MIIII-Q Biocel water purification

sytem (Millipore, USA ), thermal cycler (Eppendorf Mastercycler Gradent), horizontal

electrophoresis system (Appeiex, Paris, France), vertical eiectrophoresis system

(Shelton scient~fic, Iowa USA and Biorad, Protean I1 Xi), gel documentation system

(Vilber Lourmat, France), gel rocker (Genei, Bangalore), microwave oven (Kenstar ),

deep freezer (-80°C) (Sanyo ultralow, Gumma, Japan), microcentrifuge (Hettich,

Fohrenstr, Tuttlingen), microppettes and accessories (Eppendorf, Hamilton, Finpipette,

Nichipette), spectrophotometer (Hitatchi technologies America lnc., California. USA),

orbital shaker (Scigenics, Chennai), centrifuge (Remi instruments itd., Mumba~, Hettich,

Fohrenstr, Tuttlingen), dry bath (Genei, Bangalore), electronic balance (Precisa,

Dietkon, Switzerland), pH meter (Digisun Electronics, Chennai), vortex mixer (Remi

cyclemixer, Mumbai), U.V. steril~zation chamber (Genei, Bangaiore).

4.2. Methods

4.2.1. Subjects

The retrospectlve case-control study was conducted between December 2003 and

November 2006 on genetically unrelated subjects at JiPMER hospital, Puducherry,

india. The subjects selected for the study were residents of south Indian state of

Tam~lnadu or Puducherry (representative of Tamilnadu) for upto two previous

generations based on their family history. They should be between 20 to 70 years of

age of either sex.

The case group consisted of histopathologicaily confirmed 408 patients (269 males and

139 females) with squamous ceil carcinoma of UADT. The patients recruited were

subjects who were diagnosed for UADT cancers viith~n eight months of the disease

occurrence. This was ma~nly to avoid possible disparity associated with the tumor

outcome measures among the subjects. They were diagnosed at the Departments of

ENT and Radiotherapy of the hospital.

A total of 220 controls (148 males and 72 females) were recruited for the study. Control

subjects were considered eligible if they were age and sex matched with the cases, who

came to JIPMER hospital during December 2003 to November 2006, for treatment of

various diseases other than malignancy. It was also ensured that they did not have

history of previous or present malignant disease. The controls were matched for the age

based on the groups of 20-30 yrs, 31-40 yrs, 41-50 yrs, 51-60 yrs and 61-70 yrs. The

male: female ratio in the case and control groups was 2:l.

Through an in-person interview, each of the subjects were asked to provide detailed

demographic information about the duration of disease, severity of disease, frequency

and duration of smoking, tobacco chewing and alcohol consumption, socio-economic

status, dietary habits, life style (physical activity) and family history of cancer. Subjects

73

were interviewed for the number of cigarettes smoked per day, the age they began

smoking and duration of smoking in years. The lifetime smoking consumption was

expressed in pack-years. One pack-year corresponds to smoking one pack of cigarettes

(10 cigarettes) per day for one year. Data on lifetime frequency of alcohol consumption

and tobacco chewing were also estimated. The severity of the cancer was estimated

based on the tumour characteristics viz., initial tumor extension, presence of nodes,

distant metastasis (TNM classification) and histoiogical grades were also recorded. In

addition to the above data, cancer remssion and recurrence were aiso recorded

The study was approved by the Institute Research Council and Institute Ethics

Committee (human), JIPFV1ER. Afler explaining the purpose and protocol of the study,

the written informed consent was obtained from all the subjects.

4.2.2. Study protocol

4.2.2.1. Collection of blood samples

About 5 milliliters of blood was collected from each volunteer tnto polypropylene tubes

containing 100 11 of 10% ethylene diamine tetra acetic acid (EDTA) as anticoagulant.

4.2.2.2. Genomic DNA extraction

The DNA was extracted from the peripheral leucocytes by standard phenol: chloroform

extraction method2M. The blood samples coiiected in poiypropylene tubes were

subjected to centrifugation at 2500 rpm for 10 minutes at 4°C. The plasma was

separated, and the celiuiar fraction was used for DNA extraction. The DNA extraction

according to the above method is a two day procedure. On day one, afler adding 10 ml

of RBC lysis solution into the tubes, it was mixed for 3 minutes and kept in a freezer at - 20°C for 10 minutes. Then it was centrifuged at 2500 rpm for 10 minutes at 4°C. The

upper layer was discarded. Again RBC lysis solution was added, mixed, centrifuged and

upper layer was discarded. This was repeated tiil the RBCs were lysed and removed

completely (when the solution appeared colourless). To the remainng cellular portion,

2.25 mi of WBC lysis solution was added and mixed gently. This was foilowed by the

addition of 125 p1 of 10% SDS, 50 pl of proteinase K, and 75 pl of Milli-Q water. It was

mixed well again and incubated at 37"C, for overnight.

On the second day, the soiution was checked for complete lysis (On complete lysis, the

solution would be homogenous and freely moved when mixed). If iysis was incomplete,

the tube was gently inverted 10 times and then incubated at 37°C for 3-4 hours. After

ensuring compiete iyss, 1 ml of saturated sodium chloride was added and mixed gently.

This was foilowed by addtion of 3 5 mi of chioroform and mixed till a homogenous

solution was formed. it was centrifuged at 4000 rpm at 4°C for 20 minutes. The upper

aqueous layer was gentiy aspirated with a Pasteur pipette without d~sturbing the lower

layer and the aqueous layer was carefully transferred to a fresh tube. into the aqueous

layer, 2.5 ml of equilibrated phenol was added, mixed gentiy for 5 minutes and

centrifuged at 4000 rpm for 20 minutes at 4°C. Similar to the previous step, the

supernatant was transferred to a fresh tube. Then 2.5 ml of chioroform: octanol (24:l)

was added to the tube and mixed gently for 5 minutes. It was then centrifuged at 4000

rpm for 20 minutes. The supernatant was transferred to another fresh tube nto which 2

volumes of ice-cold absoiute ethanol was added and mixed by inverting the tubes many

times. High molecular weight DNA was precipitated which was removed using a clean

pipette tip and washed with 70% ethanol by tapping the tube five to six times. The DNA

was removed from the ethanol tube and air dried and it was transferred to a micro

centrifuge tube containing 200 PI of 1XTE buffer (pH 7.4). After proper mixing, the micro

centrifuge tube containing the extracted DNA was incubated at 37'C overnight.

[Appendices 1 and 21.

The optical dens~ty (OD) of the extracted DNA was measured at 260, 270 and 280 nm

usng spectrophotometer. OD at 260 nm corresponds to DNA, 270 nm corresponds to

phenol and 280 nm corres?onds to proteins QuantiRcetion of DNA (nglpl) = OD at 260

nm x d x 50, where d is diut~on factor; 50 corresponds to 10D of DNA= 50 ngipl of

double stranded DNA.

The various geres invest~gated in the study and their chromosomal location, single

nuceotide polymorphism and ihe influence of the polymorphism on the enzyme activity

are glven ~n Table 11.

Table 11. Description o f the genes

Genes Chromosome Nucleotide change Enzyme activity

CYPIA1 CYPfA1*2A 3801T>C increased 15q22-q24

CYPfA1*2C 2455A>G Increased

CYPZEI CYP2EfYB t896C>G Increased

GST GSTM? lp13.3 Gene delet~on Absent

GSTTI 22q11.2 Gene deieyion Absent

GSTPI l l q 1 3 313A>G Decreased

ABCBI ABCB? 7q21.12 3435C>T Decreased 3435C>T

4.2.2.3. Bioinformatics

The retrieval of gene sequences flanking the polymorphic sites of interest was done by

aligning the upstream and downstream primers with the reference gene sequences from

ENSEMBL Genome Browser. The selection of su~table restriction endonucieases was

done by NEB cutler software.

4.2.2.4. Genotyping

The genotyping of CYPIAI'ZA, CYPlAI?C, CYPPEI':B, CYPZEI'SB, CYPZE1'6,

GSTMI, GSTTI, GSTPl and ABCBl were done by polymerase chain

reaction-restriction fragment iength polymorphism (PCR-RFLP) and multiplex PCR

methods, using the Eppendorf Mastercycle& gradient. The PCR products were

checked for amplification on 1% agarose by horizontal gel electrophoresis [Appendix 31

Ampiified samples were subjected to restnct~on digestion by appropriate restriction

enzymes which recognized the specific polymorphisms. The digested PCR products

were separated by vertical gei electrophoresis or polyacrylamide gel electrophoresis

(PAGE) [Appendix 41 using 8% polyacrylamide gel and stained with ethidium bromide.

The genotypes were identified based on the size of the DNA fragments.

Genotyping of CYPlAl

The CYPIAITA and CYPIAl*ZC genotypes were determined according to the

methods described by Matthias et and Chacko et respectively with a few

modificat~ons [Tables 12 and 131. The polymorphism of CYPIA1*2A gives rise to an

Msp I restriction site In the 3'-noncoding region of exon 7 at nucieot~de position 3801

resulting in a T-C transition. The primers used were 2AF and 2AR that bind to the

specific sequences in 3'-noncoding region, flanking a 343 bp segment [Figure 231. The

genotyping of CYPIAI'ZA was carried out in a 2 5 ~ 1 reaction mixture containing

30-50ng of genomic DNA, 5p1 of 10X buffer (500mM KC1 1 100mM tris-HCI pH 8.31 15

mM MgCIZ), 0.2 mM dNTPs, 12.5 prnol of each primer (2AF and 2AR) and 2 U of Taq

polymerase. Amplified samples were subjected to restriction digestion by Msp i

restriction endonuciease at 37°C overnight. The Msp I digestion produced either 210

and 133 bp bands or 343,133 and 210 bp bands for the homozygous and heterozygous

variants respectively and an undigested 343 bp band for the wild type genotype

[Figure 241.

CYPlA1'2C polymorphism consists of an amino acid substitution at codon, l ~ e ~ ~ ~ ~ a l that

result in A-G transftlon in the heme-binding region of exon 7 of the gene. For the

genotyping of CYPlAl?C, 2CF and a 2CR primers were used. A mismatch reverse

primer was used to introduce a restriction site for recognition of the enzyme Nco I. The

fonvard primer binds to intron 6, and the reverse primer to exon 7, thereby spanning a

region 333 bp long [Figure 251. The genotyping was carried out in a 2 5 ~ 1 PCR mixture

containing 30-50ng of genomic DNA, 5111 of 10X buffer (500mM KC1 IlOOmM tris-HCI

pH 8.31 15 rnM MgCIP), 0.3 mM dNTPs, 13 pmol of each primer (2CF and 2CR) and 2.5

U of Taq polymerase The amplified PCR products were digested wlth NGO I restriction

enzyme at 37°C. The RFLP resulted in the digestion of a 333bp PCR product

irrespective of the genotype status due to an additional Nco I site. The digestion yieided

a 232 bp product forthe wild type and 264 bp for the polymorphic variant [Figure 261.

Genotyping of CYPZEI

The CYPZEl*lB, also known as Taq I polymorphism in intron 7 has a base change at

9896C>G was determined by PCR-RFLP method as described earlier by Hu et aizS6

[Tables 12 and 131. The primers used were 1BF and I B R which bind to introns 6 and 8

of CYPPEI respectively, flanking a 969 bp segment [Figure 27). For the identification of

CYP2El*lB genotypes, PCR was carried out in a 25111 reaction mixture containing

30-50ng of genomic DNA, 511 of 10X buffer (500mM KC1 1100mM tris-HCi pH 8 31 15

mM MgCI2). 0.2 mM dNTPs, 12.5 pmol of each primer ( IBF and IBR) and 2 U of Taq

polymerase. The amplified PCR products were digested with Taq I restriction enzyme at

65°C for 2 hours. The presence of the restriction site resulted in two fragments, 639

and 330 bp indicating the wild type genotype. The fragments of 969, 639 and 330 bp

represented heterozygous and an undigested 969 bp indicated the rare genotype.

[Figure 281.

CYP2E1'5B and CYPZEl'o' genotypes were delermlned by the methods described by

Matthias et a/"' with minor modifications [Tables 12 and 131. CYP2E1'58 constitutes

Rsa I (-1053C>T) and Pst I (-1293G>C) polymorphisms that are present n the

5' promoter region of the gene. 5BF and 5BR primers fiank both the polymorphic sites

resulting in a 413 bp long DNA sequence of interest [Figure 291 PCR for CYP2E1*58

was carried out in a 50p1 reaction mixture containing 5G100ng of genomic DNA, 10 pI

of 10X buffer (500mM KC1 I 100mM tris-HCI pH 8.31 15 mM MgCi2). 0.45 mM dNTPs,

24.4 pmoi of each primer (5BF and 5BR) and 4 U of Taq poiymerase. The amplif~ed

PCR products were separately digested with Rsa I and Psf I restriction enzymes at 37°C

overnight [Figures 30 a & b]. The presence of restriction sites yieided fragments of 352

and 61 bp for the Rsa I and 118 and 295 bp for the Pst I.

The CYP2E1'6 polymorphism is associated with a base change at 7632 T2A in intron 6

of the gene. The primers, 6F and 6R aligned to the intron 6 sequences bordering a

376 bp long DNA segment [Figure 311. PCR for CYP2E1'6 was carr~ed out in a 25 p1

reaction mixture containing 30-50ng of genomic DNA, 5 p of 10X buffer (500mM KC1

1100mM tris-HCI pH 8.31 15 mM MgCIZ), 0.25 mM dNTPs, 12.5 pmol of each primer (6F

and 6R) and 2.5 U of Taq polymerase. The amplified PCR products were digested w~th

restriction endonuclease, Dra I at 37°C overnight. The presence of an undigested

376 bp fragment and the presence of digested fragments of sizes, 251 and 125 bp due

to restriction site were indicative of the CC and DD genotypes, respectively. The

presence of 376, 251 and 125 bp DNA fragments identified the CD genotype

[Figure 321.

Genotyping of GST

To detect the deletions of the GSTMI and GSTTI gene loci, the multiplex PCR method

as described by Chen eta?'' was used. Albumin gene was used as an internal control

[Tables 12 and 131. The primers, M I F and M l R bind to the exons, 6 and 7 flanking a

215 bp segment. shows the presence of GSTMl gene [Figure 331; while for GSTTI,

TIF and T I R aligned to the 5!%nd 6"xons that covers the DNA segment of 480 bp

long [Figure 341. The PCR mixture (25111) was prepared containing 30-50ng of DNA,

5p1 of ?OX buffer (500rnM KC1 I lO0mM tris-HCI pH 8.3115 mM MgCP). 0.35 mM

dNTPs, 14 pmol each of forward primers and reverse primers of GSTMl (MI F & MI R),

GSTTI (TIF & T?R) and 12.5 prnol each of primers of albumin (AlbF & AlbR) and 2.5 U

of Taq polymerase. Agarose gel electrophoresis (1%) resolved amplified DNA fragments

of480 bp, 380 bp and 215 bp for GSTTI, aihumin end GSTMl respectively. Absence of

DNA fragments of 215 bp and 480 bp indicates GSTMl null and GSTTI null genotypes

respectiveiy [Figure 351.

GSTPl was identified according to the method described by Kote-Jarai e t ai2"

[Tables 12 and 131. The polymorphism at codon 105 where an adenine to guanine

(313 A>G) transition causes an isoleucine to valine substitution (1105V) at exon 5

creates a BsmA i restriction enzyme cleavage site. The forward primer, P I F blnds

partially to intron 4 and exon 5 while the reverse primer, P IR binds to intron 5 of GSTPI

gene, flanking a 176 bp segment [Figure 361. The PCR mixture consists of 25pl

containing 30-50ng of DNA. 5pl of 10X buffer (500mM KCi i 100mM tris-HCI pH 8.3 I 15

mM MgCIZ), 2.5 rnM dNTPs, 12 pmol each of the forward primer, P?F and reverse

primer, P IR and 2 U of Taq polymerase. Digestion of the 176 bp amplicon using

BsmA I restriction enzyme at 37'C overnght resuited in either retention of the 176 bp

product or complete digestion to 93 bp and 83 bp fragments corresponding to

individuals homozygous for the ilellle or ValNai genotypes respectively [Figure 371.

Genotyping of ABCBI

ABCBI 3435C>T polymorphism at exon 26 was determined using the PCR-RFLP assay

described earlier by Calado ef a?'' [Tables 12 and 131. The forward prim* -r , B1 F binds

to intron 25 and reverse primer, B1R aligned partially to each of exon 26 and intron 26

of the ABCBI gene thereby spanning a region of 244 bp long [Figure 381. For the

identification of the genotypes, PCR mixture (25111) was prepared containng 30-50ng of

DNA, 5p1 of 10X buffer (500mM KC1 I 100mM tris-HCI pH 8.31 15 mM MgCIZ), 0.2 mM

dNTPs. 12.5 pmol each of primers, B IF and B1R and 2U of Taq polymerase. The

amplified PCR products upon digestion using Dpn II restriction enzyme at 37°C

overnight resulted in two fragments of 172 and 72 bp that constitutes the wild type, the

fragments of 244, 172 and 72 bp and an undigested 244 bp indicated heterozygous and

homozygous genotypes, respectively [Figure 391.

Table 12: Details of primers and restriction enzymes

- Genotypes Primers Restriction

enzymes

8'-CAG TGA AGA GGT GTA GCC GCT-3' Msp l

5'-TAG GAG TCT TGT CTC ATG CCT-3'

5'-GAA AGG CTG GOT CCA CCC TCT-3 Nco l

5'-CCA GGA AGA G M AGA CCT CCC AGC GGG

CCA-3

5'-GGA TGA TGG GTG GAT GCC-3' Taq l

5'-CAC ATG TGG AGG GGA GAT -3'

5'-CCA GTC GAG TCT ACA TTG TCA -3' Rsa I, Ps! I

5'- TTC ATT CTG TCT TCT AAC TGG -3'

5'-AGT CGA CAT GTG ATG GAT CCA-3 Dra l

5'-GAC AGG GTT TCA TCA TGT TGG -3'

GSTMI AlbF 5'-GCC CTC TGC TAA CAA GTC CTA C-3' hlultiplex & PCR GSTTI AlbR 5'-GCC CTA AAA AGA AAATCG CCA ATC-3 abumn

as internal 5'-GAA CTC CCT GAA AAG CTA AAG C-3 con:rol

8'-CTT GGG CTC AAA TAT ACG GTG G-3

5'-TTC CTT ACT GGT CCT CAC ATC TC-3'

5'-TCA CCG GAT CAT GGC CAG CA-3'

5'-ACC CCA GGG CTC TAT GGG AA-3 BsmA I

5'-TGA GGG CAC A4G AAG CCC CT-3

%GAT CTG TGA ACT CTT GTT TTC A-3 Dpn II

5'-GAA GAG AGA CTT ACA TTA GGC-3

Table 13. D e t a i l s o f P C R c o n d i t i o n s and size of t h e P C R p r o d u c t s

Genotypes PCR Cond i t i ons Size o f PCR product (bp)

CYPIA1'ZA 94% 5 min * l W 1 A -343

94°C- 1 min, 61'C-1 m n , '1Al'ZA-343 + 210 + 133

72'C- 30 sec x 30 cycles '2W2A -210 + 133

CYP1AIi2C 94°C- 5 min lleiile-232 t 69 + 32

94'C-1 min, 66.5"C-1 min. lleRfal-264 + 232 + 69 + 32

72°C- 30 sec x 30 cycles ValNal-264 t 69

CYPZEI"1B 94'C- 2 min A2A2-639 + 330

94°C- 30 sec, 58'C- 1 mln, A2A1-969 + 639 + 330

72°C- 1 min x 30 cycles AlA1-969

CYPZEI'SB 94°C- 2 mln Rsa I. c lc l -352 + 61

94°C- 20 sec, 55°C- 30 sec, clc2-413 + 352 + 61

72°C- 30 sec x 30 cycles c2c2-413

P s t l : c l c l -413

c lc2-413+118+295

c2c2-118tZQ5

CYP2EIe6 94% 2 m ~ n DD-2511125

94°C- 20 sec, 64'C- 30 sac, DC-376125 1+125

72°C -30 sec x 30 cycles CC-376

GSTMI 94'C- 5 min GSTTI-460

8 84°C- 2 mln Aibumln-380

GSTTI 72°C- 30 sec GSThll-215

94°C- 20 sec

64°C- 20 sec x 30 cycles

GSTPI 94°C- 2 min iiellie-176

94% 20 sec. 59% 30 sec, IleNa-176 + 83 t 93

72°C- 30 sec x 30 cycles ValNal-83t93

ABCBI 94'C- 2 min CC-172+72

94°C- 20 sec, 5YC. 30 sec, CT-244 t 172 + 72

72°C- 30 sec x 30 cycles TT-244

Figure 23. Alignment o f primers restriction enzyme f o r CYPIAI'ZA [3801TzC]

ATTGGTCTCCCTTCTCTACACTCTTGTAATAAAATGTCTATTTTTAATGTTTGTACACM

C C T G A G C T A A A T A A A G A T A T T G T T C A G A A A T C C T A T A G G T ATGATTCATCACTCGTCTAAATACTCACCCTGAACCCCATTcTGTGTT~TTTTAcTGT AGGGAGULAGAAGAGGAGGTAK~G~~~~?XGGT "YZhGpCCk2TGCACTTAAGCAGTCTGT

2 3 r TTGAGGGACAAGACTCTATTTTTTGAGACAGGGTCCCCAGGTCATCCAGGCTGGAGTGCA

CTW;TACCATTTTGTTTCACTGTAACCTCCACCTCC'<IGCGCTCACACGATTCTCCCAC lntron 7 3% '7.C

4313L?MTCACTGTGCTGACTTACGCCCTCTCTAACTTATCACTGATGGTA TTCTCCGAATTAGGCAATAAGGGTATCMGTGAATATAGTACAGTCCCTGCTTTCAAGAA

Length of PCR product' 343 bp

Msp I sequence: 5' ... CXCGG ... 3'

Figure 24. Representative gel picture o f CYPIAI'ZA

[Lane 1 is io,v molecular weight DNA ladder, lane 2 is undigested PCR product, lane 3 is

'2AI*2A, lane 4 IS " IA I * lA and lane 5 IS "IAI"2AJ

Figure 25. Alignment o f primers 8 restriction enzyme for CYPIAI'ZC [2455 A>G]

AAGGCCCCATGGAGCCACTGCTGTCTGTTACTGATCTTACTCCWC~CATACCTGATT AGGGTTAGTGGGA~CACGGCATGGGAGACAGGGAGATTTGCCTGTTGCCCTGAGCCT G A C T G A G C T T C C T T T C T C C C T A G C A C A A C A A G A G A C A C A A G T T T G ~ ~ T T T A ~ T Exon 6 CCCAAGGGGCGTTGTGTCTTTGTAAACCAGTGGCAGATCAACCATGACCAGTAAGTTCAG AGATGCAGAGGAAAQGCTGGGTCCP.CC:~?C'1'TAARAUCTCTTATATATGATTAATACAATCA

2CF lntron 6 TTGCATTGATCCTCCTGTCCATGGGCTGCTTGCCTGTCCTCTATCCTTTGGGXTGAGC T C C A C T C A C T T G A C A C T T C T G A G C C C T G A A C T G C C A C T T C AGGAAGCTATGGGTCPACCCATCTGAGTTCCTACCTGAACGTTTCTCACCCCTGATGGT GCTATCGACAAGGTGTTAAGTGAGEAGGTGATTATCTTTGXATGGGCAAGCGGAAGTGT

ATCGGTGAGAC~<C~TGGCC~JGC~~GG~".".~~;TTJT'~~CCC~~~C(:T~~!XTATCCTGCTGCAAC - Exon i4SSAtG SCR

GCCCTGAGGCCTAGACTCTGTCTACCTGGTCTGGTTW;GCAGCCAGACCAGCAW;CT~

Length of PCR product: 333 bp

Nco I sequence. 5' ... CXCATGG.. .3'

Figure 26. Representative gel picture of CYPIA1*2C

[Lane 1 is low molecular we~ght DNA ladder, lane 2 is undigested PCR product lanes 3 5 8 6

are 'llellle, lane 4 is VaiNal and lane 7 is IleNal]

Figure 27. Alignment of primers 8 restriction enzyme for CYP?Ef*fB [9896 C>Gl

AATAGATGGGT1CL4'.iP~TCCGT;1C~u2 ;CCCAACTGGCCAGGAACCAATCCCTGAAATTTG 15-

TCCCATTCATATCTTGGCAGAGAAGCTCCATGAAGAAATTGACAGGGTGATTGGGCCAAG c C G A A T C C C T G C C A T C A A G G A T A G G C A A G A G A T G C C C T A C A T T G T T A T Exon7 GATTCAGCGGTTCATCACCCTCGTGCCCTCCAACCTGCCCCATGAAGCAACCCGAGACAC CATTTTCAGAGGATACCTCATCCCCAAGGTTAAGCAATGAGCCTGCAGCACACAGCATGA ACACCATCCTATCAGCTAATCGCCTTCCTGCCAGGGAGCAGGATGGGCCCCAAGACCC TTCCCTTTGGCAGGGGTCACTGAGGGGAAGGGCTGGCCCCACTCCCACCCTGTGGGATAC TGCATCTCCAGGAGTGCTCACATTGGCCTGGTGACCAGAGAGGTGGAGGAAATCTGGA.U AGAGCCTCAGCAGATAGTGCCTGGGACTGTAGTGAATTCTAATGCCAGGAACRPACTATC ACAACCAGCCCTGGGGTTAATCCTGTGAGAAGATTAGGGCTTTCATCTTCATTTAGACCT G A C C C C T G A C T G C T T T C T A T C T A A T C C T T C I L C T ~ G C ~ C T C C T T C ~ C T : < ~ ~ ~ T A T

5846C>0 ACTATCCTATATAGCATAATATTCAAAACTACATTCTTCACT~TTTCCAGATGAAA G C C C A C A T T T T G T T A A C A T G A C T C A C T G A G A C A G T C T T T G A G T C G Exon TAGTGCCMCTCTGGACTCTGTTTTGTATGACAACCAAGAATTTCCTGATCCA~GT TTAAGCCAGAACACTTCCTGEATGAAAATGGAAAGTTTAAGTACAGTGACTATTTCAAGC CATTTTCCACAGGTGAGAAAGATCAGAGGCAGTACCTTCCCTTGAGGAL%AGCCCACP.CT CCTCATCTCCCCTCCACATGCGCTCTGCCCTCGTCCCAGGCACCCACTGACACCCCAAAC

Length of PCR product. 969 bp

Taq I sequence 5 ' . . .TXCGA ... 3'

Figure 28. Representative gel picture of CYP2El*IB

[Lane i is molecular weight rnarke- Vlll lanes 2 & 4 are A2A2 lane 3 is A2A1 and lare 5 is

A I A i ]

Figure 29. Al ignment of pr imers restriction enzymes for CYP2EIb5B [-1293G>C/-1053C>T]

TGGCTAATAAATTGTCPAGAGAAWACTGGGTTAGAATGCAATATATAGTATGTAGTCTC promoter ATTTTTGTATAAATACAAGTATAGAATGGCATAACTCPAAATCCACAAGTGATTTGGCTG ,,,ion

Length of PCR product 413 bp

Pst I sequence. 5' ... C T G C A X G ... 3'

Rsa I sequence 5 ' ... G T X A C ... 3'

Figure 30. Representative gel p icture of CYPZEVSB [a) Rsa I & b) Pst I]

[Rsa I lane : s molecular we~glt marker Vl l l lane 2 &4 - c l c i i n d ane 3 1s c l c l

F j t I lane 1 IS molecular weght marker Vlll, lare 2 - c 1 ~ 2 and lanes 3 2 L represent c l c l j

a) Rsa I b) Psf I

Figure 31. Alignment of primers 8 restriction enzyme for CYP2E1'6 [7632T>A]

G T G G T C T T A A G G C T C G T C A G T T C C T W L A A G C A G G T A T T A T T T T C C C CCAAWGTC:G>~CP.TG?OAZX-P.'FCCAGGGTCAGACCCTGGCCTTTTCTTGTTCTTTCC

6F

TGCCCAGGCTGGAGTGCGGTGATGCAATCATGGCTCATTGTAGCTTCTACCTATTG~G~T CAAGCGATCCTCCCACCTCAGCCTCCCAAGTAACTGGGCCACAGGTGCACACCACCACAC lntron 6 CCAGCTGATT~TTTKAA.~AAATTATTTTGGCTCTGT

1632- iP AATCCTGGCACTTTGGGAGGCTGAGGCAGGCGATCACGAGGTCAGGAGTTC~~CCTTC CTGGC~C~?CE~T*CC"ITSTCTCTCCTAAAATACAAAAAAGTAC~GTA~CWTGTGGTG

6R GCACGCGCCTATAGTCACAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTCAACCTCA GAGGCACAGGGTGCAGTGATCCGAGATTGCACCCCACTGCACTCTAGCCTGACAACAGAG

Length of PCR product 376 bp

Dra I seqilence 5' ... TTTXAAA ... 3'

Figure 32. Representative gel picture of CYP2E7'6

[Lane 1 1s 100 bp DNA ladder, a l e 2 IS undigested PCR product, lane 3 IS DC, lane 4 is DD and

lane 5 s CC genotype]

Figure 33. Aiignment of primers for GSTMI

RefSeq ENSGO0000134184

ATCCAATTGAAGCCTGGGCACTGCCCCAGTTCCAGCTTGGGGAAGATGGCTGCTTGCCCA TGGCCAGCCTGGGCCGTCCACAGCCCCGGGGAGGCCACGTCTGTGCAGGGAGCTTTTGTC CGAGGGTGGTGACAGCTGTTTTCTGCCTCAGGAGAAACTGAAGCCAAAGTACTTGGAGGA Exon6 , \ 3 :CSCi5 .9R~AE~P- i iPGCTCTACTCAGAGTTTCT~KGGCCATGTTTGCAGG AAACAAGGTAAAGGAGGAGTGATATGGGGA7iTGAGATCTGTTTTGCTTCACGTGTTATGG intron 6 AGGTTCCAGCCCACATATTCTTGGCC1TCTGCAGATCACTTTTGTAGATTTTCTCGTCTA TGATGTCCTTGACCTCCACCG~KTAT~IC1F3C::CA4GTGCTTGGACGCCTTCCCAAATCT ~~~~7 GAAGGACTTCATCTCCCGCTTTGAGGTGATGCCCCCATCCTCCTTTCTCTTTGATGCCCC TTGTTCCGTTACCTCCTTTCAGATGTTTTCCCAGTCCTGGCTC

Length of PCR product. 215 bp

Figure 34. Alignment of primers for GSTTI

RefSeq. ENSGO0000184674

TGGATGTGACCCTGCAGTTGCTCGAGGACAAGTTCCTCCAGAACAAGGCCTTCCTTACTO SICC2CAJATC?CCTTAGCTGACCTCGTAGCCATCACGGAGCTGATGCATGTaGTGCTG TGGGCAGGTGAACCCACTAGGCAGGGGGcCCTGGCTAGTTGCTGA&GTCCTGCTTATGCT GCCACACCGGGCTATGGCACTGTGCTTAAGTGTGTGTGCATCTGT Intron 4 GGTCCCCAAATCAGATGCTGCCCATCCCTGCCCTCACAACCATCCATCCCCAGTCTGTAC CCTTTTCCCCACAGCCCGTGMjTGCTGGCTGCCA9GTCTTCGAAGGCCGACCCAAGCTGG CCACATGGCGGCAGCGCGTGGAGGCAGCAGTCGGGAGGACCTCTTCCAGGAGGCCCATG

AGGTCATTCTGAAGGCCAACGACTTCCCACCTGCAGACCCCACCATAAAGCAGAAGCTGA TGCCCTGGGCGCTGGCCATGBTCCC*JC*J~~GCTGGGAPACCTCACCCTTGCACcGTCcTcA Exon GCAGTCCACAAAGCATTTTCATTTCTAATGGCCCATGGGAGCCAGGCCCAGAAAGCAGGA ATGGCTTGCCTAAGACTTGCCC~GTCCCAGAGCACCTCACCTCCCWIAGCCAccATccc

Length of PCR product: 480 bp

Figure 35. Representative gel picture of GSTMI and GSTTI

[Lane 1 is 100 bp DNA ladder, lane 2 is absence of botb GSTMI and GSTT1, lane 3 is presence

of both GSTTI and GSTMI, lane4 IS presence of GSTT1 and iane 5 IS presence of GSTMI]

Figure 36. Alignment of primers & restriction enzyme for GSTPl [313AzG]

TTGCCCTGTGCCAGGCTGCCTCCCAGGTGTCAGGTGAGCTCTGAGCACCTGCTGTGTGGC AGTCTCTCATCCTTCCACGCACATCCTCTTCCCCTCCTCCCAGGCT~TCACAGACA lntron 4 GCCCCCTGGTTGGCCCATCCCCAGTGACTGTGTGTTGATCAGGCGCCCAGTCACGCGGCC TGCTCCCCTCCACCCRC.CCC~4IrGW:TCTAP?I'j:GGW.GGACCAGCAGGAGGCAGCCCTGGT

P I F Exon 5 GG~~CATGGTGAATGACCGCGTGGAGGACCTCCGCTGCAAATACATCTCC*~<CTCATCWC

3i3PS>G ACCAP.CTATGTGAGCATCTGCACCAGGGTTGGGCACTGGGGGCTGAACAAAGAABGAAB~X1Ki-, ., , ,? . ..nrnirlppr-ii-a , r I.,&;. .,.L~.CCCCCCTTACCCCTCAGGTGGCTTGM;CTGACCCCTTCTTGGGTCA

P I R GGGTGCAGGGGCTGGGICAGCTCTGGGCCAmCCAmCTGGGACAAGACACAAc CTGCACCCTTATTGCCTGGGACATCAACCAGCCAAGTAACGW3TCATGGGGGCGAGTGCA AGGACAGAGACCTCCAGULACTGGTGGTTTCTGATCTCCTGGGGTGGCGAGGGCTTCCTG lntron 5 G A G T A G C C A G A G G T G G A G G A G G A T T T G T C G C C A G T T T C T G T T T

Length of PCR product 176 bp

BsmA I sequence 5' .,. GTCTC(N),X ... 3'

Figure 37. Representative gel picture of GSTPI

[Lane 1 IS molecular bveght marker V I lane 2 IS IleNiia, lane 3 IS VallVal and lane 4 IS llellle

genotyce]

Figure 38. Alignment of primers & restriction enzyme forABCB1 [3435C>r3

RefSeq ENSGO0000085563

AGGATGATCTGTTTTCTTGCTTGTGGCCCCTTCCTCCATTTTCAAAACCAGCAGTCGCTG GTTGAGTCTTTCTCACACTGCATTTTGCTGATACTCTTCTCCCTCCTTCTTCTTCAGTTAA G A G C C C T T A T G A T T A C A T T A G T T T C A C C A A G A T A A T T C A G G T C A G C T G A T T A G C A A C C T T A C A T C T A C T A C T T T A G T T T C T T T T T C A C A G G A T C C A G G G A T T A G G A C A C A G A T G T C T T G T G G G C TACCACATGCATACATCAGAAACCATGGTTGAAACACAGGAAACATGACAGTTCCTCAAG GCATACAATTATGACCTTGTTGGGTTRACCTTCACTATCCAAATTTTAATCACACAAACT T T T C C T T A A T C T C A C A G T A A C T T G G C A G T A A T G T G CTACATTCAAAGTGTGCTGGTCCTGAAGTTG~nlFZ~=SPJi~1T~lCfTT~CAGCTGCTTG

B1 F

GCCGCGTGGTGTCACAGGAAGAXGAT~GTGAGGG~GCEAAGGAGGCCRACP~TACATGC 34356>V

CTTCATCGAGTCAC~GCC~AATGTmGCr.TCCP~~PPC~TAAACAGCCTGGGAGCATTGTG 51 R

GCAGCCTCTCTGGCCTATAGTTTGATTTATAA-TGGTCTCCCAGAAGTGAAGAGAA A T T A G C A A C C A A A T C A C A C C C T T A C C T G T A T A C A A G C

Length of PCR product 244 bp

Dpn ll sequence 5' ... XGATC ... 3'

Figure 39. Representative gel picture of ABCBI

[Lane 1 is low molecular welght DNA ladder. lane 2 is undigested PC9 product, lane 3 is CT

iane 4 IS CC and lane 5 IS TT genotype]

4.2.2.5. DNA sequencing

Genotyping procedures were validated by sequencing of representative samples of

each genotype. Prior to sequencing, the PCR products were purified using the PCR

product purification kit (Genei. Bangalore, india). Sequencing was done by the Dideoxy

chain termination method of Sanger et alz7%sing the ABI Prism@ 377 sequencer

(Appl~ed Biosystems, Foster City, CA, USA) and ABI Prism BigDyee Terminator v3.0

cycle sequencing kit. Sequencing was done at the INSERM U-458, Paris, France and

at Macrogen, Korea.

4.2.2.6. TNM staging

The severlty of the disease was est~mated based on the tumour characteristics viz.,

initial tumor extension, presence of nodes and distant metastasis. The lymph node

status and metastasis are same for all the UADT sites while primary tumor size varies

[Tables 14.221'~'.

4.2.2.7. Histopathological differentiation

The UADT tumor samples obtained by fine needle aspirat~on cHology were

histologically examined for SCC and graded as follows:

G I - Well differentiated, G2- Moderately differentiated, G3- Poorly dfferentiated

4.2.2.8. Cancer remission and recurrence

Cancer remisson in the study was defined as the cases free of UADT cancers for a

period of one year. Tumor recurrence was defined as reappearance of signs or

symptoms of the disease at the same site or in another iocation of UADT following a

previous negative follow-up of the disease for one year. For each patient, we calculated

recurrence-free survival time as the time from remission to recurrence or death. The end

point for the disease-free survival analysis was cancer recurrence/metastasis or death

related to UADT cancer.

Table 14. Lymph node (N)

NX Regional nodes cannot be assessed

NO No regionai lymph node metastasis

N1 Metastasis in a single ipsilaterai lymph node, 3 cm or iess in greatest dimension

N2 Metastasis in a single ipsiiatera lymph node, more than 3 cm but not more than

6 cm in greatest dimension, or in muitipie ipsiiateral iymph nodes, none more

than 6cm in greatest dimension; or in bilaterai or conlraiaterai iymph nodes, none

more than 6 cm in greatest dimension

N2a Metastasis in a single ipsilateral lymph node, more than 3 cm but not more than

6 cm in greatest dimension

N2b Metastasis in multiple lpsiiateral iymph nodes, none more than 6 cm in greatest

dimension

N2c Metastaals !n bilateral or contralaterai iymph nodes, none more than 6 cm in

greatest dimension

N3 Metastasis in a lymph node, more than 6 cm in greatest dimension

Table 15. Metastasis (M)

MX Distant metastasis cannot be evaluated

MO No distant metastasis (cancer has not spread to other parts of the body)

M I Distant metastasis (cancer has spread to distant parts of the body)

Table 16. Primary Tumor (T) -. TX Primary tumor cannot be assessed

TO No evidence of primary tumor Common for all the UADT sites

Tis Carcinoma insltu

Table 17. Lip a n d oral cavity

T I Tumor 2 cm or less in greatest dimension

T2 Tumor more than 2 cm but not more than 4 cm in greatest dimension

73 Tumor more than 4 cm in greatest dimension

T4a Lip: Tumor invades through coriicai bone. inferior aiveoiar nelve, floor of

mouth, or skin (ch~n or nose)

T4a Oral cavity: Tumor invades adjacent structures (through cortical bone, into

deeplextrinsic muscle af the tongue (genioglossus, hyoglossus, palatoglossus

and stylogiossus). max~iiary sinus, skin of face)

T4 b Lip and oral cavity: Tumor invades masticator space, pterygoid plates. or skuli

base or encases internal carotid artery

Pharynx

Table 18. Oropharynx

T I Tumor 2 cm or less in greatest dimension

T2 Tumor more than 2 cm but not more than 4 cm in greatest dimension

T3 Tumor more than 4 crn in greatest dimension

T4a Tumor invades any of the following: larynx, deeplextrinsic muscle of tongue,

medial pterygoid, hard palate and mandible

T4 b Tumor invades any of the following: lateral pterygoid muscle, pterygod plates

lateral nasopharynx skuli base, prevertebral fascia or encases the carotid artery

Table 19. Hypopharynx

T1 Tumor limited to one subsite of hypopharynx and 2 cm or less in greatest

dimension

T2 Tumor invades more than one subsite of hypopharynx or an adjacent slte, or

measures more than 2 cm but not more than 4 cm in greatest diameter without

fixation of hemiiarynx

T3 Tumor more than 4 cm in greatest dimension, or with fixation of hemiiarynx

T4a Tumor invades any of the foilowing- thyroidicricod cartilage, hyold bone. thyroid

gland, oesophagus, cenlrai compartment of soft tissue

T4b Tumor invades prevertebra fascia, encases carotid artery, or invades mediastinal

structures

Larynx

Table 20. Supraglottis

T i Tumor iimited to one subsite of supraglottis with normal vocal cord mobiiity

T2 Tumor invades mucosa of more than one adjacent subsite of supragiottlc or

glottis or region outside the supraglotis (e.g mucosa of base of tongue.

valiecula. medial waii of plriform sinus) without fixation of the larynx

T3 Tumor iimited to larynx with vocal cord flxation andlor invades any of the

foilowing: postcricoid area, pre-epiglottic tissues, paraglottic space, andlor with

minor thyroid cartiiage erosion (e.g., inner cartex)

T4a Tumor invades through the thyroid cartilage, andlor wades tissues beyond the

larynx e.g., trachea, soft tissues of the neck including deep extrillsic muscle of

tongue (geniogiossus, hyoglossus, paiatogiossus and styloglossus), strap

muscles. thyroid, oesophagus

T4b Tumor wades prevertebral space, mediastinal structures, or encases carotid

artery

Table 21. Glottis

T1 Tumor iimited to vocal cord(s) (may involve anterior or posterior comm~ssure)

with normal mobility

Tla Tumor iimited to one vocal cord

T l b Tumor involves both vocal cords

72 Tumor extends to supraglottis andior subglottis, andior wllh impaired vocal cord

mobility

T3 Tumor limited to larynx with vocal cord fixation andior invades paragiottic space,

andlor with minor thyroid cartt!age erosion(e g , inner cortex)

T4a Tumor invades through the thyroid cartilage. andior invades tissues beyond the

larynx. e.g , trachea, soft tissues of the neck inciuding deep extrinsic muscle of

tongue (geniogiossus, hyoglossus, paiatogiossus and styloglossus), strap

muscles, thyroid, oesophagus

T4b Tumor ~nvades prevertebral space, mediastinal structures, or encases carotid

artery

Table 22. Subglottis --

T I Tumor limited to subglottis

T2 Tumor extends to vocal cord(s) with normal or impaired mobility

T3 Tumor limited to larynx with vocal cord fixation

T4a Tumor invades through cricoid or thyroid cartilage, andlor invades into tissues

beyond the larynx, e.g., trachea, soft tissues of the neck including deep extrlnslc

muscle of tongue (genioglossus, hyoglossus, palatoglossus and stylaglossus),

strap muscles, thyroid, oesophagus

T4 b Tumor invades prevertebral space, mediastinal structures, or encases carotid

artery

4.2.3. Statistical analysis

Data were analysed using the Statistical Package for Social Sciences stalistical

software (SPSS Windows verslon release 13). The association behveen the CYPIAI,

CYP2E1, GST and ABCBI genotypes and UADT cancer risk was analyzed by

calculating the crude odds ratio (OR) and 95% confidence interval (95% CI) using the

~2-test. The adjusted OR was calculated using unconditional logistic regression analysis

w~th the low risk genotype designated as the referent category. Observed genotype

frequencies among the cases and controls were analyzed separately and compared

with the predicted frequencies according to the Hardy-Weinberg law using the chi-

square test.

For analyzing the gene-gene and gene-environment interactions (genotype x genotype

and genotype x environmental factor respectively), stratified variables were generated

and Included in the iogistlc model simuitaneously with the appropriate ind~cator

variables. Gene-gene interaction anaiysls was conducted using the homozygous

wild-type individuals for both the genes as the reference group. For gene-environment

interaction analysis, the reference group cons~sts of those homozygous wild-type

individuals who had not been exposed to environmental factors. Stratified analysis was

used to assess departure from the multiplicative effects among major potential risk

factors, including tobacco chewing, tobacco smoking and alcohol drinking as well as

mutant genotypes. A multiplicative interaction was suggested when: OR.,+> OR,;. X

ORs, where OR,;, = OR when both factors are present, OR,,. = OR when only factor 1

is present, OR.,, = OR when only factor 2 is present'7'~272. When the observed OR

(OR,,,) was more than the expected OR(OR,. X OR,,), then an interaction is said to be

present.

A logistic regression model was used to examine the data for associations between

individual genotypes and each tumor outcome parameter. Ail the outcome parameters

were transformed to binary data (TlTr2 versus T3iT4, nodes present versus nodes

absent and GllG2 versus G3). The role of genotypes in cancer remission and

recurrence was also determined by unconditional logistic regression method. For each

patient, we calculated recurrence-free survlval time as the time from remission to

recurrence or death. The end pont for the disease-free survivai analysis was cancer

recurrencelmetastasis or death related to UADT cancer. The recurrence-free survival in

relation to various genotypes was compared by Kaplan-Me~er survival function and log-

rank tests. Multivariate anaiysis using the Cox proporiional hazards model was used to

assess the effect of individual SNPs on the risk for end point events. The reference

hazard ratio of 1 was set for the w~ld type genotype. All the P values were two-sided and

P values < 0.05 were considered statlsticaily significant.