SUSCEPTIBILITY TO ORAL SQUAMOUS CELL CARCINOMA: CORRELATION WITH VARIANTS OF CYP1A1-MspI, GSTT1, GSTM1, ALDH2, EC-SOD AND LIFESTYLE FACTORS
Dong T-T, Wang L-J, Liu L-Z, Ma S-N
*Corresponding Author: Mrs. Ting-Ting Dong, General Hospital of Daqing Oil Field, No. 9 Zhongkang Street, Saertu District, Daqing 163001, Heilongjiang Province, People’s Republic of China. Tel: +86-459-599-4114. Fax: +86-459-580-5247. Email: tingtingdong_139@163.com
page: 61

PARTICIPANTS AND METHODS

Participants. This prospective study included 750 patients who were admitted to our hospital from June 2011 to May 2015. Another 750 participants who received physical examinations during the same time period were selected as healthy controls; the physical examination showed no cancer or hereditary diseases. There was no statistically significant difference in age, gender, place of origin or nationality, and the subjects were unrelated. Participant demographic data, smoking history, alcohol drinking history, occupational history and family tumor history were collected. The smoking status was evaluated using the smoking index (SI) that was the product of the daily number of cigarettes multiplied by number of years of smoking. Based on SI values, the participants were divided into the following categories: non smokers, individuals with SI ≤400, and individuals with SI 400. Alcohol consumption was evaluated with the drinking index (DI) that was the product of the daily amount of drinking (in grams) multiplied by the number of years of drinking. Based on DI values, the participants were classified as non drinkers, those with DI ≤3000, and those with DI 3000. The study was approved by the General Hospital of Daqing Oil Field, Daqing, Heilongjiang Province, People’s Republic of China (PRC). Informed written consent was obtained from all participants. Genotyping. From each participant, 3 mL blood was collected in vacutainers with EDTA as anticoagulant. The QIAmpDNA extraction kit (Qiagen GmbH, Hilden, Germany) was used to extract DNA from white blood cells. Extracted DNA was stored at –30 °C. Polymerase chain reaction (PCR) was used to amplify the DNA to the levels required for restriction fragment length polymorphism (RFLP) analysis. For all reactions, a total volume of 25 μL comprised 2.5 μL 10 × buffer, 2.5 μL dNTP, 20 pmol upstream primers, 20 pmol downstream primers (see below), 0.75 μL Taq DNA polymerase (all PCR reagents from Promega, Madison, WI, USA), and 100 ng template DNA. Reactions were performed on PE480 thermocycler (Perkin Elmer?, Norwalk, CT, USA), as follows: initial denaturation at 94 °C for 4 min., and 35 cycles of denaturation at 94 °C for 40 seconds, annealing at 55 °C for 40 seconds, and extension at 72 °C for 50 seconds, and final extension at 72 °C for 5 min. The PCR products were digested with restriction endonucleases as appropriate (described below). A total reaction volume of 20 μL comprised 1 ng PCR product, 2 μL 10 × NEB (New England Biolabs, Ipswich, MA, USA) reaction buffer, and 10 U endonuclease; reactions were performed at 37 °C for 3 hours. Digestion products were separated by 100V electrophoresis on a 3.0% agarose gel, for 1 hour. After 30 min. in ethidium bromide, bands were detected by ultraviolet light. The CYP1A1-MspI polymorphism was detected using the following primer sequences: upstream primer (5’-CAG TGA AGA GGT GTA GCC GCT-3’), downstream primer (5’-TAG GAG TCT TGT CTC ATG CCT-3’ (synthesized by TaKaRa, Dalian, China). Restriction digestion with MspI produced three genotypes: wild-type (m1/m1) with a band at 340 bp, heterozygous (m1/m2) with bands at 340, 200 and 140 bp, and homozygous mutant (m2/m2) with bands at 200 and 140 bp. The EC-SOD polymorphism was detected using primer sequences as reported in a previous study [7]: upstream primer (5’-GCA ACC AGG CCA GCG TGG AGA ACG GGA A-3’), and downstream primer 5’-CCA GAG GAG AAG CTC AAA GGC AGA-3’). The PCR product was digested with restriction endonuclease PauI. Two genotypes were produced: homozygous C/C with bands at 111 and 109 bp; and heterozygous C/G with bands at 220, 111 and 109 bp. The GSTT1 polymorphism was detected using primer sequences according to Wilson et al. [8]: upstream primer (5’-TCT CCT TAC TGG TCC TCA CAT CTC-3’), and downstream primer (5’-TCA CCG GAT CAT GGC CAG CA-3’). GSTT1 positive [+] was indicated by the presence of a 480 bp fragment after PCR amplification; GSTT1 negative [–] meant lack of PCR product. The control β-globin gene was detected with upstream primer (5’-CAA GAG CCA ACC ACA GGT AC-3’) and downstream primer (5’-GAA GAG CCA AGG ACA GGT AC-3’). The GSTM1 polymorphism was detected using primer sequences selected according to Nguyen et al. [9]: upstream primer (5’-GAA CTC CCT GAA AAG CTA AAG C-3’), and downstream primer (5’-GTT GGG CTC AAA TAT ACG GTG G-3’). GSTM1 positive [+] indicated the presence of bands at 230 and 219 bp; GSTM1 negative [–] indicated the presence of only one 219 bp band. The control β-globin gene was detected with upstream primer (5’- CAA GAG CCA ACC ACA GGT AC-3’), and downstream primer (5’-GAA GAG CCA AGG ACA GGT AC-3’). The ALDH2 polymorphism was detected using primer sequences selected according to Ishibashi et al. [10]: upstream primer (5’-CCC TTT GGT GGC TAG AAG ATG-3’), and downstream primer (5’-CCA CAC TCA CAG TTT TCT CTT-3’). The PCR products were digested with the restriction endonuclease MboI. The amplified fragment was 91 bp and three genotypes could be seen after digestion: homozygous G/G with a band at 55 bp, heterozygous G/L with bands at 65 and 55 bp, and homozygous L/L with a band at 65 bp. The ALDH2 (G/L) and ALDH2 (L/L) were combined and marked as ALDH2 (non G/G) for co-analysis. Statistical Methods. The difference in genotype distributions between the patient and control groups was determined using the χ2 test. A non conditional logistic regression model was used to analyze the adjusted odds ratio (OR) for risk of oral squamous cell carcinoma with different genotypes, as well as the combined effects between smoking, drinking, and genotype. The 95% confidence interval (CI) is also reported. The Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) version 11.0 was used for statistical analysis. Statistical significance was accepted at a p value of <0.05.



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