Standardization of DNA amount for bisulfite conversion for analyzing the methylation status of LINE-1 in lung cancer.

Highly methylated Long Interspersed Nucleotide Elements 1 (LINE-1) constitute approximately 20% of the human genome, thus serving as a surrogate marker of global genomic DNA methylation. To date, there is still lacking a consensus about the precise location in LINE-1 promoter and its methylation threshold value, making challenging the use of LINE-1 methylation as a diagnostic, prognostic markers in cancer. This study reports on a technical standardization of bisulfite-based DNA methylation analysis, which ensures the complete bisulfite conversion of repeated LINE-1 sequences, thus allowing accurate LINE-1 methylation value. In addition, the study also indicated the precise location in LINE-1 promoter of which significant variance in methylation level makes LINE-1 methylation as a potential diagnostic biomarker for lung cancer. A serial concentration of 5-50-500 ng of DNA from 275 formalin-fixed paraffin-embedded lung tissues were converted by bisulfite; methylation level of two local regions (at nucleotide position 300-368 as LINE-1.1 and 368-460 as LINE-1.2) in LINE-1 promoter was measured by real time PCR. The use of 5 ng of genomic DNA but no more allowed to detect LINE-1 hypomethylation in lung cancer tissue (14.34% versus 16.69% in non-cancerous lung diseases for LINE-1.1, p < 0.0001, and 30.28% versus 32.35% for LINE-1.2, p < 0.05). Our study thus highlighted the optimal and primordial concentration less than 5 ng of genomic DNA guarantees the complete LINE-1 bisulfite conversion, and significant variance in methylation level of the LINE-1 sequence position from 300 to 368 allowed to discriminate lung cancer from non-cancer samples.

Methylation Profiles of BRCA1, RASSF1A and GSTP1 in Vietnamese Women with Breast Cancer

Objective: This study investigated the DNA promoter methylation profiles of BRCA1, RASSF1A and GSTP1 genes, both individually and in an integrative manner in order to clarify their correlation with clinicopathological parameters of breast cancer from Vietnamese patients, and establish new potential integrative methylation biomarkers for breast cancer detection. Material and methods: The methylation frequencies of BRCA1, RASSF1A and GSTP1 were analyzed by methylation specific polymerase chain reaction (MSP) in 70 specimens of breast carcinomas and 79 pairs of tumor and matched adjacent normal tissues from breast cancer patients. Results: All the three analyzed genes showed a concordance concerning their promoter methylation in tumor and adjacent normal tissue. The methylation of BRCA1, RASSF1A and GSTP1 was found in 58.23 %, 74.68 % and 59.49 % of tumor tissues and 51.90 %, 63.29 % and 35.44 % of corresponding adjacent tissues, respectively. When each gene was assessed individually, only the methylation of GSTP1 was significantly associated with tumor tissues (p=0.003). However, the methylation frequency of at least one of the three genes and the methylation frequency of all the three genes both showed significant association with tumor (p=0.008 and p=0.04, respectively). The methylation of BRCA1 was found to be significantly associated with tumor grade (p=0.01). Conclusion: This study emphasized that the panel of the three genes BRCA1, RASSF1A and GSTP1 can be further developed as potential biomarkers in diagnosis and classification of breast cancer in Vietnamese women.

Analysis of Common ß-Thalassemia Mutations in North Vietnam.

Available and flexible choice of methods for screening and detecting ß-thalassemia (ß-thal) can promote control of thalassemia in developing countries. In this study, two methods, the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and reverse dot-blot hybridization assays were developed to detect common ß-thal mutations in 244 thalassemia patients and 152 healthy people in North Vietnam. The most common mutation was codon 26 (G>A), also known as Hb E (HBB: c.79G>A), accounting for 26.4% of the total studied chromosomes, followed by codons 41/42 (-TCTT) (HBB: c.126_129delCTTT) and codon 17 (A>T) (HBB: c.c.52A>T), accounting for 19.4 and 16.4%, respectively. In addition, codon 95 (+A) (HBB: c.c.287_288insA) that is known as the Vietnamese mutation, accounted for 0.6%. Moreover, the heterozygous state of the four mutations was also found in healthy people, of which Hb E was again the most common mutation with a frequency 3.0%. The results of this study provide available methods and indicative data for preventive and control strategies concerning the genetic diagnosis of thalassemia.

Methylation profile of BRCA1, RASSF1A and ER in Vietnamese women with ovarian cancer.

DNA methylation is considered a promising biomarkers for diagnosis of cancer in general and of ovarian cancer in particular. In our study, we validated the accuracy of methylation specific polymerase chain reaction (MSP) to analyze the methylation pattern of BRCA1, RASSF1A and ER in 59 and 10 Vietnamese patients with epithelial ovarian cancer (EOC) and benign ovarian tumors, respectively. We found methylation of BRCA1, RASSF1A and ER in 11/59 (18.6%), 40/59 (67.8%) and 15/59 (25.4%) of EOC cases, while methylation of BRCA1 was only detected in 2/10 (20%) benign ovarian patients. Forty five out of the 59 EOCs (78%) demonstrated methylation at one or more genes. The methylation frequency of RASSF1A was significantly associated with EOC (p<0.0005). No significant association was observed between methylation status of these genes and the clinical and pathological parameters of tumors collected from Vietnamese women suffering from ovarian cancer.