Methylenetetrahydrofolate (MTHFR) and DNMT3b play imperative roles in DNA synthesis and de novo methylation. GSTM1 is involved in detoxification of carcinogens. Mitochondrial DNA deletion has been associated with lower motility in human sperm. We analysed if polymorphisms in MTHFR (C677T and A1298C) and DNMT3b (C46359T) are associated with non-obstructive male infertility. We also analysed if folate, vitamin B₁₂, homocysteine (Hcy), 8′-hydroxy-2′-deoxygnanosine (8-OHdG) levels, dietary folate intake and mtDNA deletion (4977 bp) affects fertility, such interactions are modified by deletion and methylation of GSTM1. In this case–control study, we included 179 oligoasthenoteratozoospermia patients and 200 fertile men. Single-nucleotide polymorphism analysis was performed by PCR-restriction fragment length polymorphism. The MTHFR (C677T and A1298C) and DNMT3b (C46359T) frequencies did not differ significantly in two groups. GSTM1 in association with mtDNA 4977 deletion is significantly associated with infertility. Plasma folate and vitamin B₁₂ levels are decreased and total Hcy is elevated in infertile men. GSTM1 methylation status was investigated by methylation-specific PCR. Methylation is significantly correlated with GSTM1 reduced/loss of expression in infertile men. Infertile men have significantly higher 8-OHdG levels. Dietary folate intake is not linked with GSTM1 methylation. Low folate intake in association with CT + TT genotypes (C677T) has significant protective effect on GSTM1 methylation. Results indicate that micronutrients, 8-OHdG levels, mtDNA deletion and GSTM1 promoter methylation are frequent alterations in infertility.

Granulosa cell tumors (GCTs) are relatively rare and are subtypes of the sex-cord stromal neoplasms. Methylation induced silencing in the promoters of genes such as tumor suppressor genes, DNA repair genes and pro-apoptotic genes is recognised as a critical factor in cancer development. We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 5 gene promoters in 25 GCTs cases by methylation specific PCR and RT-PCR. In addition, the compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were also included as the control. Frequencies of methylation in GCTs were 7/25 (28 % for FHIT), 6/25 (24% for FNACF), 3/25 (12% for Cyclin D2), 1/25 (4% for BRCA2) and 14/25 (56%) in RUNX3 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed that overall promoter methylation was higher in more advanced stage of the disease. Promoter methylation was associated with gene silencing in GCT cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression. These results may have implications in better understanding the underlying epigenetic mechanisms in GCT development, provide prognostic indicators, and identify important gene targets for treatment.

The cytokinesis-block micronucleus cytome (CBMNCyt) assay is a widely used technique for measuring DNA damage in human populations. The formation of micronuclei (MN) in dividing cells can result from chromosome breakage due to unrepaired or mis-repaired DNA lesions or chromosome malsegregation due to mitotic malfunction. The sensitivity of the MN assay to polymorphisms in various genes involved in DNA repair, activation/deactivation of carcinogens/chemicals/drugs/alcohol, folate metabolism pathway and micronutrient transport has been extensively reported in the literature. MN frequency is also an important index for determining DNA repair efficiency phenotype (including mis-repair), response to environmental exposure and identifying various dietary factors required for optimal genome stability. The aim of the present study is to review the reported in vivo associations between genotype and MN frequency in humans taking into considerations the presence of interactions with nutrients levels and/or exposure to genotoxins. One hundred and eleven publications linking MN frequency in peripheral blood lymphocytes to gene polymorphism were retrieved from PubMed. After applying exclusion criteria, only 37 studies were evaluated in the present review. Polymorphisms in XRCC1 (Arg280His), ERCC2 (Lys751Gln), CYP2E1 (c1/c2) and MTR (A2756G) were consistently associated with the MN formation. These results contribute substantial evidence to the hypothesis that genotype may influence MN frequency in human cells.

Glutathione S-transferases (GSTs) are an important group of isoenzymes that play an essential role in the detoxification of carcinogens. Polymorphism at exon 5 of the GST π family decreases the catalytic activity and affects the detoxification ability of the enzyme, GSTP1. GSTP1 promoter hypermethylation and loss of expression are frequently observed in various types of carcinoma. We hypothesized that somatic epigenetic modification in homozygous mutants increases the degree to which breast cancer risk is affected by lifestyle factors and dietary habits. The present study used tumor biopsies and blood samples from 215 breast cancer patients and 215 blood samples from healthy donors. GSTP1 polymorphism was studied using PCR-restriction fragment length polymorphism, methylation using methylation‑specific PCR and loss of expression using immunohistochemistry and western blotting. No significant increase was observed in the breast cancer risk of individuals with the mutant (Val) allele [odds ratio (OR), 1.48; 95% confidence interval (CI), 0.97‑2.26 for heterozygotes; OR, 1.42; 95% CI, 0.86‑2.42 homozygous mutants]. GSTP1 promoter hypermethylation was detected in one-third of tumor biopsies (74/215) and was found to be associated with a loss of expression. Genotype and tumor methylation associations were not observed. Estrogen (ER) and progesterone (PR) receptor‑positive tumors had a higher methylation frequency. GSTP1 polymorphism was not associated with increased promoter hypermethylation. The results suggest that GSTP1 methylation is a major event in breast carcinogenesis and may act as a tumor‑specific biomarker.

The Y chromosome-specific gene SRY is one of the key genes involved in human sex determination. The SRY gene encodes a testis-specific transcription factor that plays a key role in sexual differentiation and development in males and is located on the distal region of the short arm of the Y chromosome. Mutations in SRY gene result in XY sex reversal and pure gonadal dysgenesis. SRY expression initiates a network of gene activity that transforms the undifferentiated gonad, genital ridge into testis. Mutations in the SRY gene have been considered to account for only 10–15% of 46,XY gonadal dysgenesis cases, whereas the majority of the remaining cases may have mutation(s) in the SRY regulatory elements or other genes involved in the sex differentiation pathway. Patients both with gonadal dysgenesis and Y-chromosome presence are at high risk of developing gonadoblastoma. Using PCR, single strand conformational polymorphism (SSCP) and automated DNA sequencing, we analysed the mutations in the SRY gene in three 46,XY sex reversal patients. Two patients demonstrated nucleotide substitution (A→G) within the open reading frame just outside and upstream of the conserved DNA-binding motif called the high-mobility group (HMG) box, replacing glutamine at codon 57 with arginine. Altered SSCP patterns were also observed in these patients. Histological examination of gonads in patient 1 revealed the formation of gonadoblastoma. Patient 3 demonstrated A→T substitution which replaces serine at codon 143 with cysteine, just outside but downstream of the HMG box. Results suggest the involvement of SRY gene in sex reversal which further supports the relationship between SRY alterations, gonadal dysgenesis and/or primary infertility.