Anca Botezatu

Quantitative analysis of the relationship between microRNA‑124a, -34b and -203 gene methylation and cervical oncogenesis

Cervical cancer is a leading cause of mortality in women. Molecular and epidemiological data have unequivocally confirmed that high-risk human papillomaviruses (HPVs) are a major etiological agent of this malignancy, as host epigenetic alterations are induced in response to viral infection. The present study evaluated the methylation status of CpG islands surrounding miR-124a, miR-34b and miR-203 in 29 cervical cancer precursor lesions, 31 cervical tumors and 30 normal control samples, with the aim of identifying potential markers of cervical cancer. Direct quantitative methylation-specific PCR (qMSP) was used to evaluate the degree of methylation in the samples. HPV DNA was detected and genotyped using the Linear Array HPV Genotyping Test. Data were statistically analyzed using the Kruskal-Wallis test. Differences in miRNA hypermethylation between the tumor and control samples were highly significant for all the genes tested (p<0.0001). Significant results were also obtained regarding the hypermethylation of miR-124a and miR-203 in the precursor lesions compared to the control samples. Among the 29 patients with precursor lesions, 68.97% (20/29) presented high risk (hr)-HPV genotypes and 31.03% (9/29) were diagnosed with low risk (lr)-HPV. Significant results (p=0.0266) were obtained for miR-124a (hr-HPV group, mean 41.32; lr-HPV group, mean 6.74), revealing a strong association between the methylation process and the hr-HPV genotype. Borderline results (p=0.058) were obtained for miR-203 (hr-HPV group, mean 44.05; lr-HPV group, mean 3.33). These results confirm the involvement of epigenetic alterations in cervical oncogenesis. The lr-HPV precursor lesions had a methylation percent pattern similar to that of the normal samples, while the results for the hr-HPV precursor lesions and tumors indicate a possible involvement of the hr-HPV genotype in the miRNA methylation process.

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and promoter methylation in cervical oncogenic lesions and cancer.

The aim of this study was to investigate the role of methylenetetrahydrofolate reductase (MTHFR) polymorphisms and MTHFR methylation pattern in cervical lesions development among women from Romania, a country with high prevalence of human papillomavirus (HPV) cervical infections. To achieve this goal, blood samples and cervical cytology specimens (n = 77)/tumour tissue specimens (n = 23) were investigated. As control, blood and negative cytological smears (n = 50) were used. A statistically significant association was found between T allele of C677T polymorphism and cervical lesions, heterozygote women presenting a threefold increased risk (normal/cervical lesions and tumours: wild homozygote 34/41 (0.68/0.41), heterozygote 14/51 (0.28/0.51), mutant homozygote 2/8 (0.04/0.08); OR = 3.081, P = 0.0035). Using χ square test for the control group, the HPV‐negative and HPV‐positive patients with cervix lesions, a significant correlation between viral infection and T allele of C677T polymorphism (P = 0.0287) was found. The MTHFR promoter was methylated in all HGSIL and tumour samples, significant differences being noted between HPV‐positive samples, control group and cases of cervical dysplastic lesions without HPV DNA (P < 0. 0001) and between samples from patients with high‐risk (hr)HPV versus low‐risk (lr)HPV (P = 0.0026). No correlations between polymorphisms and methylation were observed. In Romania, individuals carrying T allele are susceptible for cervical lesions. MTHFR promoter methylation is associated with cervical severity lesions and with hrHPV.

Methylation pattern of methylene tetrahydrofolate reductase and small nuclear ribonucleoprotein polypeptide N promoters in oligoasthenospermia: a case-control study.

Alterations in DNA methylation patterns in several genes may lead to abnormal male sexual development and infertility. This study investigated the promoter methylation status of MTHFR and SNRPN in infertile men from Romania by quantitative methylation-specific PCR in order to investigate possible correlations with sperm abnormalities. The study groups included patients (n=27) with a median age of 31 years (range 26-41 years) as well as controls (n=11) with a median age of 30 years (range 24-37 years) recruited from couples seeking advice for infertility. DNA was isolated from sperm samples and promoter methylation was assessed using direct. Significant trends were detected for both genes that indicate a tendency towards promoter hypermethylation in spermatozoa with low motility (MTHFR P=0.0032, r=0.23; SNRPN P=0.0003, r=0.32) and poor morphology (MTHFR P=0.0012, r=0.27; SNRPN P=0.0003, r=0.33) but no trend was found in cases of low sperm count (MTHFR r=0.007; SNRPN r=0.06). The data indicate that the methylation patterns of the promoters of MTHFR and SNRPN are associated with changes in sperm motility and morphology, which could lead to male infertility. A large number of studies are now focused on the causes of male infertility. Among these are epigenetic modifications, which are important contributors to reproductive pathology in the male by providing dynamic changes of the phenotype according to the environmental and metabolic factors. The most known epigenetic modification is DNA methylation and alterations in this pattern in several genes could induce male infertility. The present study aims to investigate the promoter methylation status of the genes for methylene tetrahydrofolate reductase (MTHFR) and small nuclear ribonucleoprotein polypeptide N (SNRPN) in infertile males from Romania, in order to establish a correlation with sperm parameters. MTHFR is an enzyme involved in the folate pathway and in de novo nucleotide biosynthesis but also a good example for gene-environment interaction in phenotype development. SNRPN is involved in both somatic cell expression and inheritance of the imprint and the methylation pattern of its gene seems to correlate not only with imprinted disorders but also with infertility. Our study includes patients (n=27, median age 31 years, range 26-41 years) recruited from men seeking advice for couple infertility and control group (n=11, median age 30.5 years, range 24-37 years). The data we obtained indicated significant correlations between hypermethylation of the investigated genes and sperm motility and morphology. No significant correlation between DNA methylation and sperm number was found. Our data suggest that methylation pattern of MTHFR and SNRPN is linked with sperm anomalies of motility and morphology and therefore male infertility.

Assessment of Gene Expression Profiles in Peripheral Occlusive Arterial Disease

BACKGROUND Molecular events responsible for the onset and progression of peripheral occlusive arterial disease (POAD) are incompletely understood. Gene expression profiling may point out relevant features of the disease. METHODS Tissue samples were collected as operatory waste from a total of 36 patients with (n = 18) and without (n = 18) POAD. The tissues were histologically evaluated, and the patients with POAD were classified according to Leriche-Fontaine (LF) classification: 11% with stage IIB, 22% with stage III, and 67% with stage IV. Total RNA was isolated from all samples and hybridized onto Agilent 4×44K Oligo microarray slides. The bioinformatic analysis identified genes differentially expressed between control and pathologic tissues. Ten genes with a fold change ≥ 2 (1 with a fold change ≥ 1.8) were selected for quantitative polymerase chain reaction validation (GPC3, CFD, GDF10, ITLN1, TSPAN8, MMP28, NNMT, SERPINA5, LUM, and FDXR). C-reactive protein (CRP) was assessed with a specific assay, while nicotinamide N-methyltransferase (NNMT) was evaluated in the patient serum by enzyme-linked immunosorbent assay. RESULTS A multiple regression analysis showed that the level of CRP in the serum is correlated with the POAD LF stages (r(2) = 0.22, P = 0.046) and that serum NNMT is higher in IV LF POAD patients (P = 0.005). The mRNA gene expression of LUM is correlated with the LF stage (r(2) = 0.45, P = 0.009), and the mRNA level of ITLN1 is correlated with the ankle-brachial index (r(2) = 0.42, P = 0.008). CONCLUSIONS Our analysis shows that NNMT, ITLN1, LUM, CFD, and TSPAN8 in combination with other known markers, such as CRP, could be evaluated as a panel of biomarkers of POAD.

Type-specific human papillomavirus detection in cervical smears in Romania.

Anton G, Peltecu G, Socolov D, Cornitescu F, Bleotu C, Sgarbura Z, Teleman S, Iliescu D, Botezatu A, Goia CD, Huica I, Anton A‐C. Type‐specific human papillomavirus detection in cervical smears in Romania. APMIS 2010.

miR-29a associates with viro-immunological markers of HIV infection in treatment experienced patients.

MicroRNAs (miRNAs) are small, non‐coding RNA species essential for the post‐translational regulation of gene expression. Several miRNA have been proposed to contribute to Human immunodeficiency virus‐1 (HIV‐1) infection establishment, progression and latency. Among them, miR‐29a seems to be of particular interest. The aim of this study was to investigate the association between miR‐29a expression and immunologic and virologic markers of HIV infection progression in long‐term antiretroviral‐treated individuals. In a homogenous group of 165 young adults, with chronic HIV infection, parenterally acquired during childhood, the expression level of miR‐29a was found to be inversely correlated with HIV viral load and the degree of immunosuppression, expressed by both CD4 cell count and the CD4/CD8 ratio. There was a significant difference in miR‐29a expression according to the patients response to treatment, with the lowest levels expressed by patients with treatment failure, defined as detectable viremia and CD4 < 350 cells/mm3. No significant correlation was found between miRNA level and the nadir CD4 count or zenith HIV viral load. This study establishes the association between miR‐29a expression and markers of HIV infection in long‐term survivors, treatment‐experienced patients, suggesting its potential use as an indicator for the on‐treatment disease evolution. J. Med. Virol. 88:2132–2137, 2016.

LINC01101 and LINC00277 expression levels as novel factors in HPV‐induced cervical neoplasia

Recently long non‐coding RNAs were identified as new factors involved in gene expression regulation. To gain insight into expression pattern of these factors related to E7 HPV18 oncogene, this study uses HeLa cell culture transfected with E7‐siRNA. Gene expression profile was investigated using microarray analysis. After analysing the microarray results, we identified 15,387 RNA species differentially expressed in E7‐siRNA‐transfected cells compared with controls (fold change >2). The expression profiles of lncRNA species highlighted 731 lncRNAs and 203 lincRNAs. We selected two lincRNAs (LINC01101 and LINC00277) and we evaluated the expression profile in HPV‐induced neoplasia. Both lincRNAs investigated display a significantly reduced pattern of expression in cervical lesions and cancer, associated with clinical parameters. A connection between HPV presence and lincRNAs was noted. hrHPV‐positive samples exhibit significantly reduced LINC01101 and LINC00277 expression level (P < 0.05). These results provide new insights into involvement of lncRNA in HPV‐induced cervical cancer, enriching our understanding of their potential role in this pathology.

Comparative molecular approaches in Prader-Willi syndrome diagnosis.

Prader-Willi and Angelman syndromes are two distinct neurogenetic disorders caused by chromosomal deletions, uniparental disomy or loss of the imprinted gene expression in the 15q11-q13 region. PWS results from the lack of the paternally expressed gene contribution in the region. The aim of our study was to compare a new molecular approach based on the quantification of the expression of non-imprinted bi-allelic gene (NIPA1 and OCA2) with in house MS-PCR and the MS-MLPA test. Blood samples were collected from 12 patients, clinical criteria positives for Prader-Willi syndrome. DNA and RNA samples were isolated from white blood cells. Epigenetic changes at SNRPN gene locus were evaluated by MS-PCR technique. The expression levels of two non-imprinted genes (NIPA1 and OCA2) were evaluated in qReal-Time PCR, in order to identify type 1 and type 2 deletions. SALSA MS-MLPA kit ME028 was used to detect copy number changes and to analyze CpG islands methylation of the 15q11 region. MS-MLPA test confirmed that 8/12 patients presented different types of deletion at the SNRPN gene level (promoter, introns, and exons) and 4/8 displayed type 1 or type 2 deletion. In children with 15q11-13 deletions, the decreased level of NIPA1and OCA2 gene expression is related to chromosomal abnormality in the investigated area. The deletions were confirmed by MS-MLPA analysis, thus recommending NIPA1 and OCA2 gene expression as an alternate method to investigate deletions.

Human Papillomaviruses Oncoproteins

One of the most common sexually transmitted diseases is human papillomavirus (HPV) infection that affects around 80% of sexually active women. Although most of the women clear HPV infection, individuals with inadequate immune responses develop persistent infections which lead to premalignant lesions such as high-grade cervical intraepithelial neoplasia (CIN-III), with high potential to progress to cervical carcinoma. Based on molecular and epidemiological data, high risk papillomaviruses (hrHPVs) are now recognized as etiologic agents of cervical carcinoma, although some additional events are required [1, 2].

Correlation Between Chlamydia trachomatis IgG and Pelvic Adherence Syndrome

Chlamydia trachomatis genital tract infections are prevalent worldwide (Stamm 2008), with 92 million new chlamydia cases occured every year: 3–4 million new cases occur every year in the US, 5 million in Western Europe, and 16 million in Sub-Saharan Africa (World Health Organization 2001; Weinstock et al., 2004). Chlamydia prevalence has been reported to range from 3%–7% among asymptomatic populations in men, and in women range from 3.0% in the general population to 9.5% among university students (World Health Organization 2001; Stamm 2008; Patel et al.,. 2008; Forhan et al., 2009; Imai et al., 2010; Satterwhite et al., 2010).