Risk stratification of synchronous gastric cancers including alcohol-related genetic polymorphisms.

BACKGROUND AND AIM: We previously identified that ever-smoking and severe gastric atrophy in pepsinogen are risk factors for synchronous gastric cancers (SGCs). This study aimed to determine the association of alcohol drinking status or alcohol-related genetic polymorphism with SGCs and also stratify their risk. METHODS: This multi-center prospective cohort study included patients who underwent endoscopic submucosal dissection for the initial early gastric cancers at 22 institutions in Japan. We evaluated the association of alcohol drinking status or alcohol dehydrogenase 1B (ADH1B) and acetaldehyde dehydrogenase 2 (ALDH2) genotypes with SGCs. We then stratified the risk of SGCs by combining prespecified two factors and risk factors identified in this study. RESULTS: Among 802 patients, 130 had SGCs. Both the ADH1B Arg and ALDH2 Lys alleles demonstrated a significant association with SGCs on multivariate analysis (odds ratio, 1.77), although alcohol drinking status showed no association. The rates of SGCs in 0-3 risk factors in the combined evaluation of three risk factors (ever-smoking, severe gastric atrophy in pepsinogen, and both the ADH1B Arg and ALDH2 Lys alleles) were 7.6%, 15.0%, 22.0%, and 32.1%, respectively. The risk significantly increased from 0 to 3 risk factors on multivariate analysis (P for trend <0.001). CONCLUSIONS: Both the ADH1B Arg and ALDH2 Lys alleles were at high risk for SGCs. The risk stratification by these three factors may be a less invasive and promising tool for predicting their risk.

Unique Genotyping Protocol of CYP2D6 Allele Frequency Using Real Time Quantitative PCR from Japanese Healthy Women.

CYP2D6 is an important drug-metabolizing enzyme involved in the metabolism of 20-25% of commonly prescribed drugs. Genetic polymorphism of CYP has clinically significant modifications in patients' drug-metabolizing capacities. Since gene copy number variation (CNV) and single nucleotide polymorphism (SNP) frequently occur in the CYP2D6 gene, which the activity of CYP2D6 particularly depend on the genetic factors. This study aimed to investigate the frequencies of CYP2D6 genotypes in a Japanese female subject of 216 healthy volunteers. The volunteers were genotyped for CNV Exon 9 and four CYP2D6 genetic variants (*2, *5, *10, *14, *41) performed by TaqMan® genotyping assays. The CNV allele frequencies were 82.9% for two copies, 11.6% for one copy, 4.6% for three copies and 0.9% for zero copy, respectively. The frequencies of CYP2D6*1, *2, *5, *10, *14, and *41 were 38.7, 16.7, 6.3, 34.7, 0.2, and 1.2%, respectively. CYP2D6*5 and *14 were the major defective alleles. However, this genotyping is labor intensive, time consuming, and costly. We report an optimized novel protocol for the determination of CNV and SNP in CYP2D6 gene by real-time quantitative PCR. This can lower the cost and accurately determine CNV and SNP in the CYP2D6 gene with a higher output and enabling reliable estimates of disease prediction in large epidemiological samples.

Development of an Inexpensive and Rapid Operation Device for High-Throughput Real-Time Quantitative PCR-Based CYP2D6 CNV Genotyping.

The CYP2D6 gene is the most well characterized gene involved in drug metabolism and is known to have both gene duplication and deletion variants. We report an optimized method for the determination of copy number variation (CNV) in the CYP2D6 gene by a novel purification process for a real-time quantitative PCR. This high-throughput low-cost method accurately determines CNV in the CYP2D6 gene enabling reliable estimates of disease prediction in large epidemiological samples.

[Development of Novel Genotyping Protocol and Its Application for Genotyping of Alcohol Metabolism-related Genes].

A new single nucleotide polymorphisms (SNP) genotyping method has been developed and validated using biological specimens directly as templates for TaqMan PCR without general DNA extraction and purification procedure from dried saliva samples attached on water-soluble papers. This new method can set up at ease and complete PCR analysis including data interpretation in under two hours with additional advantages of application for large-scale clinical research, diagnostics, and epidemiological studies at low cost. Specifically, SNP genotyping of alcohol metabolism-related genes ADH1B (rs1229984) and ALDH2 (rs671) were demonstrated by TaqMan PCR assay using dried saliva samples in the present investigation. In this protocol, by simplifying experimental operations and improving efficiency, omitting and simplifying the time and laborious DNA purification process, it is possible to shorten the experiment time and reduce the risk of human error such as contamination. Furthermore it became possible with great cost reduction. We succeeded in dramatically improving the judgment rate and accuracy of SNP genotyping by the master mix reagent for commercial available real-time TaqMan PCR. Moreover, it becomes possible to stably introduce template DNA into the reaction system, and it will be possible to apply it to copy number variation (CNV) by TaqMan probe method. The SNP analysis process using this optimized water-soluble paper will be applied to gene polymorphism analysis of drug metabolizing enzyme gene CYP, etc., to help efforts to realize personalized medicine.

Rapid and Cost-Effective Genotyping Protocol for Angiotensin-Converting Enzyme Insertion/Deletion (Ins/Del) Polymorphism from Saliva.

DNA extraction and purification have been generally considered to be required for PCR assay. We demonstrated a new protocol using biological specimens directly as templates for real-time PCR with melting curve analysis. We confirmed the melting curve analysis was particularly suitable for the identification of the insertion/deletion (Ins/Del) polymorphism of the angiotensin-converting enzyme (ACE) gene. The new protocol we developed can be set up using simple and complete PCR analysis including data interpretation in under four hours with additional advantages of application for large-scale clinical research, diagnostics, and epidemiological studies at low cost.