Complement receptor type 1 and 2 (CR1 and CR2) gene polymorphisms and plasma protein levels are associated with the Dengue disease severity.

The pathological outcome of dengue disease results from complex interactions between dengue virus (DENV) and host genetics and immune response. Complement receptor types 1 and 2 (CR1 and CR2) mediate complement activation through the alternative pathway. This study investigated the possible association of genetic polymorphisms and plasma levels of CR1 and CR2 with dengue disease. A total of 267 dengue patients and 133 healthy controls were recruited for this study. CR1 and CR2 gene polymorphisms were analyzed by Sanger sequencing, while plasma CR1 and CR2 levels were measured by ELISA. The frequency of the CR1 minor allele rs6691117G was lower in dengue patients and those with severe dengue compared to healthy controls. Plasma CR1 and CR2 levels were decreased in dengue patients compared to healthy controls (P < 0.0001) and were associated with platelet counts. CR1 levels were lower in dengue patients with warning signs (DWS) compared to those without DWS, while CR2 levels were decreased according to the severity of the disease and after 5 days (T1) and 8 days (T2) of follow-up. CR2 levels were decreased in dengue patients positive for anti-DENV IgG and IgM and patients with bleeding and could discriminate DWS and SD from dengue fever patients (AUC = 0.66). In conclusion, this study revealed a reduction in CR2 levels in dengue patients and that the CR1 SNP rs6691117A/G is associated with the dengue severity. The correlation of CR2 levels with platelet counts suggests that CR2 could be an additional biomarker for the prognosis of severe dengue disease.

An Internal Control for Evaluating Bisulfite Conversion in the Analysis of Short Stature Homeobox 2 Methylation in Lung Cancer.

Objective: The methylation status is considered as powerful diagnostic, prognostic, and predictive biomarkers. However, the limited DNA amount and conversion efficiency after bisulfite treatment are considerable hindrances in quantitative methylation analysis. In this study, we designed an artificial internal control (IC) system that contained the cytosine-free fragment (CFF) following CpG sequences of the SHOX2 promoter whose methylation status has been described as a valuable biomarker of lung cancer. Its performance in quantifying DNA recovery and bisulfite conversion efficiency as well as in detecting false-positive SHOX2 methylation was determined on samples from lung cancer patients. Material and Methods: The IC system is composed of two pConIC and pUnIC plasmids that both contain a cytosine-free (CF) sequence derived from the CFF and the CpG containing SHOX2 sequences. They are identical in sequence, except that in the ConIC insert, all cytosines have been converted into thymines. Thus, the ConIC can be used as calibrator of 100% bisulfite conversion efficiency, while the UnIC is the indicator in order to evaluate the DNA recovery, bisulfite conversion efficiency of the SHOX2 promoter sequence by quantitative real time PCR. Results: The copy number of the target sequences impacted on both DNA recovery rates and bisulfite conversion efficiency. An amount of 0.005 ng pUnIC (106 copies) showed recovery rate of 18%, similar to that of pConIC, and a bisulfite conversion efficiency of the SHOX2 reaching 98.7%. On the contrary, higher copy number of pUnIC showed incomplete conversion (<85%) and over recovery (~42%). Using this calibrator/indicator couple, we were able to detect false-positive SHOX2 methylation (3.77% instead of 0.03%) due to incomplete bisulfite conversion.Conclusion: Our results proposed a customizable internal control using the ConIC/UnIC as calibrator/indicator to quantify simultaneously and accurately the DNA recovery and bisulfite conversion efficiencies of individual sequence as well as whole genome in methylation assays, thus promoting the validation of standardized clinical DNA methylation biomarker values to progress toward clinical applications

Methylation profiles of miR34 gene family in Vietnamese patients suffering from breast and lung cancers.

The three genes encoding small non­coding microRNA (miR)34a, MIR34b and MIR34c act as tumor­suppressor genes. Their aberrant expressions regulated by DNA methylation have been frequently found in various types of cancer. In the present study, the DNA promoter methylation profiles of the MIR34 gene family were analyzed using the methylation specific polymerase chain reaction in order to clarify their association with breast and lung cancer, non­cancerous or normal adjacent tissues. The methylation frequency of MIR34a was significantly higher in breast cancer (49.37%) compared with normal adjacent tissues (30.38%). The methylation frequency of MIR34b/c was 59.49 and 62.03% in breast cancer and normal adjacent tissues, respectively. MIR34a methylation showed a significant concordance with that of MIR34b/c only in breast cancer tissue. MIR34a methylation was significantly associated with cancer and the invasive ductal carcinoma type of breast cancer (P=0.015 and P=0.02, respectively). Methylation frequency of MIR34a and MIR34b/c was 48.42 and 56.84% in lung cancer, and 47.22 and 51.39% in pulmonary diseases, respectively. No significant association was observed between the methylation status of MIR34a and MIR34b/c, and the clinicopathological features of lung cancer or with those of non­cancerous pulmonary diseases. Promoter methylation of MIR34a and MIR34b/c occurs frequently and concomitantly in breast and lung cancer, as well as in pulmonary diseases tissues, but not in breast normal tissues adjacent to tumor. These results of the present study emphasize the involvement of MIR34 methylation in human diseases, including cancer. Furthermore, MIR34a methylation may be a promising marker for a subtype of breast cancer.