Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing

BACKGROUND:Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. OBJECTIVE:Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. METHODS:We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. RESULTS:We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. CONCLUSION:The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing

Subfertility and risk of testicular cancer in the EPSAM case-control study

It has been suggested that subfertility and testicular cancer share genetic and environmental risk factors. We studied both subfertility and the strongest known testicular cancer susceptibility gene, the c-KIT ligand (KITLG), whose pathway is involved in spermatogenesis.The EPSAM case-control study is comprised of testicular cancer patients from the Province of Turin, Italy, diagnosed between 1997 and 2008. The present analysis included 245 cases and 436 controls from EPSAM, who were aged 20 years or older at diagnosis/recruitment. The EPSAM questionnaire collected information on factors such as number of children, age at first attempt to conceive, duration of attempt to conceive, use of assisted reproduction techniques, physician-assigned diagnosis of infertility, number of siblings, and self-reported cryptorchidism. Genotyping of the KITLG single nucleotide polymorphism (SNP) rs995030 was performed on the saliva samples of 202 cases and 329 controls.Testicular cancer was associated with the number of children fathered 5 years before diagnosis (odds ratio (OR) per additional child: 0.78, 95% confidence interval (CI): 0.58-1.04) and sibship size (OR per additional sibling: 0.76, 95% CI: 0.66-0.88). When considering the reproductive history until 1 year before diagnosis, attempting to conceive for at least 12 months or fathering a child using assisted reproduction techniques was not associated with the risk of testicular cancer, nor was age at first attempt to conceive or physician-assigned diagnosis of infertility. The SNP rs995030 was strongly associated with risk of testicular cancer (per allele OR: 1.83; 95%CI: 1.26-2.64), but it did not modify the association between number of children and the risk of testicular cancer.This study supports the repeatedly reported inverse association between number of children and risk of testicular cancer, but it does not find evidence of an association for other indicators of subfertility

Case–control study of HLA-G promoter methylation status, HPV infection and cervical neoplasia in Curitiba, Brazil: a pilot analysis

BACKGROUND: The causal association between persistent human papillomavirus (HPV) infection and cervical cancer has been established, but the mechanisms that favor HPV persistence in cervical cells are still unknown. The diminished capability of the immune system to control and resolve HPV infection is one of several hypotheses. The tolerogenic protein HLA-G has shown aberrant expression in a variety of cancers, which has been suggested as a mechanism for tumor escape from immunosurveillance. In the present study we evaluate the role of epigenetic modification (promoter de-methylation) of the HLA-G gene on susceptibility to HPV infection and development of high-grade cervical lesions. METHODS: A case–control study was carried out in Curitiba, Brazil, between February and June 2010. A total of 789 women aged 15–47 years were recruited: 510 controls with normal cervical cytology, and 279 cases with histologically confirmed cervical intraepithelial neoplasia grade 2 (CIN2, N = 150) or grade 3 (CIN3, N = 129). All women were administered a questionnaire by interview, which collected information on demographic and lifestyle factors, and a cervical sample was collected. HPV DNA detection was performed by GP5+/GP6+ primer-mediated PCR. HPV-positive samples were genotyped by multiplex PCR. A pilot analysis of HLA-G promoter methylation was carried out in a subset of the study population (96 cases and 76 controls) by pyrosequencing. HLA-G methylation and HPV infection status of cases and controls were compared, and confounding factors were computed by t Student and non-parametric Wilcoxon tests. Comparison of HLA-G methylation between cases and controls was assessed by the Bonferroni correction. The association of HLA-G methylation with CIN2/3 was evaluated by logistic regression. RESULTS: HPV prevalence was 19.6% in controls and 94.3% in CIN2/3 cases. HPV16, 31, 33, 35 and 18 were the most prevalent types. Methylation analysis of seven CpGs in the HLA-G promoter did not reveal any spontaneous de-methylation events in CIN2/3 cases (mean proportion of methylation: 75.8%) with respect to controls (mean 73.7%; odds ratio 1.01, 95% confidence interval 0.96, 1.07). CONCLUSIONS: This study did not support the hypothesis that spontaneous de-methylation events in the HLA-G promoter play a primary role in promoting escape from immunosurveillance in the development of precancerous cervical lesions