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.

Simultaneous CXCL12 and ESR1 CpG island hypermethylation correlates with poor prognosis in sporadic breast cancer.

BACKGROUND: CXCL12 is a chemokine that is constitutively expressed in many organs and tissues. CXCL12 promoter hypermethylation has been detected in primary breast tumours and contributes to their metastatic potential. It has been shown that the oestrogen receptor alpha (ESR1) gene can also be silenced by DNA methylation. In this study, we used methylation-specific PCR (MSP) to analyse the methylation status in two regions of the CXCL12 promoter and ESR1 in tumour cell lines and in primary breast tumour samples, and correlated our results with clinicopathological data. METHODS: First, we analysed CXCL12 expression in breast tumour cell lines by RT-PCR. We also used 5-aza-2'-deoxycytidine (5-aza-CdR) treatment and DNA bisulphite sequencing to study the promoter methylation for a specific region of CXCL12 in breast tumour cell lines. We evaluated CXCL12 and ESR1 methylation in primary tumour samples by methylation-specific PCR (MSP). Finally, promoter hypermethylation of these genes was analysed using Fisher's exact test and correlated with clinicopathological data using the Chi square test, Kaplan-Meier survival analysis and Cox regression analysis. RESULTS: CXCL12 promoter hypermethylation in the first region (island 2) and second region (island 4) was correlated with lack of expression of the gene in tumour cell lines. In the primary tumours, island 2 was hypermethylated in 14.5% of the samples and island 4 was hypermethylated in 54% of the samples. The ESR1 promoter was hypermethylated in 41% of breast tumour samples. In addition, the levels of ER alpha protein expression diminished with increased frequency of ESR1 methylation (p < 0.0001). This study also demonstrated that CXCL12 island 4 and ESR1 methylation occur simultaneously at a high frequency (p = 0.0220). CONCLUSIONS: This is the first study showing a simultaneous involvement of epigenetic regulation for both CXCL12 and ESR1 genes in Brazilian women. The methylation status of both genes was significantly correlated with histologically advanced disease, the presence of metastases and death. Therefore, the methylation pattern of these genes could be used as a molecular marker for the prediction of breast cancer outcome.

High Amounts of Total and Extracellular Vesicle-Derived Soluble HLA-G are Associated with HLA-G 14-bp Deletion Variant in Women with Embryo Implantation Failure.

PROBLEM: Human leukocyte antigen-G (HLA-G) expression is related to 14-bp insertion/deletion polymorphism at the 3'UTR of the HLA-G gene. Soluble forms of HLA-G are released as free molecules or via extracellular vesicles (EVs). Due to the crucial role of HLA-G during pregnancy, we analyzed the 14-bp polymorphism and the two secreted forms in implantation failure women (IF) and in fertile women (FW). METHOD OF STUDY: For the genetic analysis, 49 IF and 34 FW were genotyped. For sHLA-G quantification, serum samples from 35 IF and 23 FW were available. ExoQuick(™) kit was used for EVs precipitation. The total soluble HLA-G (sHLA-Gtot ) and vesicular sHLA-GEV were quantified by ELISA. The EVs size and concentration were determined by nanoparticle tracking analysis (NTA). RESULTS: An increased proportion of IF presented high levels of sHLA-Gtot (P = 0.02) and vesicular sHLA-GEV (P = 0.0003) compared to FW. The 14-bp deletion allele is more frequent in IF (P = 0.0002) and associated with high levels of sHLA-Gtot and vesicular sHLA-GEV . CONCLUSION: The high expression of sHLA-Gtot and sHLA-GEV , together with the presence of the 14-bp deletion allele, might be involved in implantation failure.

Considerations on regulatory sequences of the distal promoter region of the HLA-G gene.

Gene expression in eukaryotic cells is accomplished via association of transcription factors, some of which directly bind to DNA regulatory sequences. HLA-G codes for an immunoregulatory protein with tissue-specific expression, its unique promoter regulatory region is responsible for this feature. The aim of the present study was to explore motif composition as well as identify haplotypes in the HLA-G 5' distal promoter region. The sample was composed by 176 euro-descendents individuals genotyped by Sequence Based Typing of HLA-G distal promoter, encompassing 16 SNPs. Haplotypes were inferred by the expectation maximization algorithm. Only haplotypes with frequency higher than 1% were aligned to check for similarities and differences and thirteen haplotypes remained. For a better understanding of the nucleotide diversity of the analyzed region our approach was to split the whole sequence into two regions. Two contrasting haplotype groups were found in both regions, allowing us to suggest the existence of different transcription factors capable of binding cis elements while the intra-group variations suggest the intensity modulation of binding with regulatory factors.

Analysis of HLA-G polymorphisms in couples with implantation failure.

PROBLEM: HLA-G expression is related as an immune modulator of fetal-maternal tolerance, and its levels was correlated with pregnancy outcome. In a case-control study, we investigate the association between the genetic variability of the HLA-G gene and serum levels of soluble HLA-G in cases of embryo implantation failure. METHOD OF STUDY: Forty couples with at least two unsuccessful fresh embryo transfers (implantation failure; IF) and 83 fertile couples with at least two successful pregnancies was genotyped by sequencing-based typing. HLA-G alleles were defined by nucleotide sequence variations at exon 2, 3, and 4, and the quantification of soluble HLA-G (sHLA-G) was performed by ELISA. RESULTS: There was a significant difference between the HLA-G allelic distributions between IF couples and the control couples. The HLA-G*01:03:01 allele was increased in the IF couples. There were no significant differences in the serum levels of sHLA-G in the IF and control groups. CONCLUSION: The results suggest that the distribution of HLA-G products may play a significant role in the modulation of maternal-fetal immune response.