Analysis On The Variation Of Smoking Behavior, Body Mass Index And Methylation On Monozygotic Twins From High Incidence Area For Esophageal Cancer

1Background and PurposeEsophageal cancer (EC) is one of the six most common malignant tumors worldwide. Taihang Mountain areas at the junction Henan, Hebei and Shanxi provinces in China have been well documented as the high incidence area for EC. Smoking and low body mass index have been demonstrated as risk factors for esophageal squamous cell carcinoma (ESCC) in these regions. Recent studies have showed that smoking and low body mass index (BMI) could increase the risk for EC in Chinese population. Genetic factor has been indicated to play an important role in smoking behavior and BMI. Many studies find that smoking can lead to BMI decrease, but there are also reports that smoking can cause the BMI increased. Different genetic and environment background may explain these controversy findings. Two members from monozygous twin have the same genetic materials, which may be better to address these issues. On the other hand, DNA methylation has been considered as one of the indicators for interation between environmental and genetic factors for diseases, including cancers. DNA methylation and histone modifications abnormal changes can affect the regulation of cell growth, differentiation, transformation and apoptosis gene transcription. Promoter region aberrant methylation of tumor suppressor gene is an early frequent event in tumorigenesis, often leading to inactivation of gene expression. Tumors involving the relevant organs secrete body fluids (peripheral blood, saliva, sputum, etc.) have been detected the tumor tissue gene promoter aberrant methylation. This discovery provides desirable clues for early cancer detection.Abnormal methylation of tumor-related genes has significant genes-tumor types. The traditional methylation detection methods can not meet the research needs. High-throughput screening cancer markers are imperative. Epigenetic sequencing of whole genome has become possible, but requires a huge support of labour, material and financial. High-throughput microarray technology has become the preferred screening of tumor markers at this stage, for high throughput detection, loci clear, short test cycle and low-cost. The subject of traditional study has differences genetic background, cause great interference for study at screening of tumor markers.In this study, smoking behavior and BMI and peripheral venous blood DNA methylation were investigated on monozygous twins, EC patients and healthy people from high incidence areas for EC, to further determine the effect of genetic and environmental factors on BMI and smoking behavior, and DNA methylation. To provide the basic data for the pathogenesis research of EC. 2Materials and Methods2.1TwinsIn this study,1492pairs of twins were enrolled. In addition,7696healthy subjects were also enrolled for futher validation and comparsion. In one pedigree with one pair of monozygous twin sufferred from EC, another three members were detected with EC. All these subjects were from Anyang, Xinxiang and Hebi in Henan province, the high incidence areas for EC. Physical examination and questionnaire survey were applied to eliminate chronic diseases or malignant tumors.2.2Questionnaires and Physical examinationWe took questionnaire and home visits to collected information about twins and healthy people from above-mentioned areas, including personal circumstances, disease history, smoking history, living habits and general physical examination. For the twin survey, member similarity, twin family history, fingerprint examination were recoded. Height and weight were measured for each subject and patient in fasting and barefoot.5ml fasting peripheral venous blood (EDTA-K2anticoagulant) was collected from each twin member. Endoscopic and biopsy examinations were performed on family members within esophageal cancer pedigree. 2.3Zygosity diagnosis of twinsSimilar diagnostic method and short tandem repeat sequences were taken to identify the egg-types.2.4DNA extraction and methylation microarrayDNA extraction was performed using Qiagen blood kit. Infinium Human Methylation450BeadChip was applied to detect methylation. After quality controls for extracted DNA, the genomic DNA was further treated, including bisulfite-convert, amplification, digestion, precipitation and suspension, chip hybridization and washing, single-base extension, dyeing and protection, scanning, etc.2.5StatisticsT-test, Chi-Square test and Wilcoxon rank sum test (SPSS18.0software) were applied to the high incidence twins and healthy people data, the level of the test (a) was0.05.Used GenomestudioV2011software to preliminary analysis of microarray data, then used SAS software to analysis the data which was filtered out points of the bead number<3and Detection Pval>0.05. Screening criteria:the absolute value of the average methylation level difference between patients with esophageal cancer and normal controls≥0.1and P<0.0001. 3Results3.1Smoking behavior and BMI in twinsThe heritability of BMI in twins was0.781. The heritability of smoking in male twins was0.142. There were no differences in average and different grading BMI between the twins with and without smoking (P>0.1). There were differences in average and different grading BMI between the healthy subject with and without smoking (P<0.001), the smoking subject of low weight rate greater than non-smokers subject, and the overweight and obesity rate were lower than non-smokers subject. The rate for the smoking in the healthy male and female was56.7%and1.0%, respectively. The rates for low-weight, overweight and obesity in males and females were5.0%,29.1%,6.5%and8.0%,29.4%,8.7%, respectively.3.2Methylation detection for members in esophageal cancer pedigreeThe58differentially methylated CpG sites were found between the Patients with esophageal cancer and normal members. Of these,33CpG sites were corresponded to19genes, i.e., MYOM2ARHGEF10, VCX3B, GDPD1MBP, BNIP3, MIR886, RPH3A, ADAMTS12, STK32C, NTM, DNM1LDDX12, PQLC1, CNDP1CNDP2, KLK3, MGC87042and TBCD. 4Conclusions4.1Smoking behavior and BMI in twinsGenetic factors have obvious impact on BMI, but not on smoking behavior; long-term heavy smoking may be related with low BMI. The overweight, obesity rate and male smoking rate of healthy population are higher than the national level.4.2The methylation on members in esophageal cancer pedigree with twinSix kinds of frequent methylated genes have been found in twin esophageal pedigree methylation analysis, i.e., MBP, BNIP3, DNMlL, ADAMTS12, KLK3and MIR886. This discovery provides important clues on establishment of candidate molecular biomarkers for high-risk subject screening and early detection of EC.