Different DNA methylation pattern of HPV16, HPV18 and HPV51 genomes in asymptomatic HPV infection as compared to cervical neoplasia.

Epigenetic alterations of human papillomavirus (HPV) genome play an important role in virus life cycle and carcinogenic progression. The aim of the current study was to investigate the correlation between the grade of cervical pathology and DNA methylation status within the L1 gene and the long control region (LCR) of HPV16, HPV18 and HPV51. HPV genomes were analyzed using bisulfite DNA modification procedure with the subsequent amplification of target DNA regions and sequencing. A collection of 202 cervical specimens was analyzed: 157 HPV16-positive specimens, 21 HPV18-positive specimens and 24 HPV51-positive specimens. This study revealed that methylation of CpG was significantly more prevalent in L1 gene as compared to LCR region of all three studied HPV types and the degree of DNA methylation level correlated with the severity of cervical neoplasia. An increased DNA methylation level of HPV16 promoter region in case of cervical cancer was determined.

Studies on the prevalence of oncogenic HPV types among Lithuanian women with cervical pathology.

Human papillomavirus (HPV) is the main cause of cervical cancer. Therefore, the detection of oncogenic HPV types is important in predicting the risk of cervical cancer. The aim of the current study was to estimate the prevalence of 16 carcinogenic and potentially carcinogenic HPV types in the study group of Lithuanian women with various grades of cervical pathology in comparison to healthy women. A total of 824 cervical specimens were investigated for HPV DNA: 547 specimens of women with abnormal cytology and 277 specimens of healthy women. Cytological diagnosis was confirmed by histology. For the detection of HPV infection, HPV DNA was amplified by PCR using three different primer systems. HPV DNA was detected in 67.6% of specimens collected from women with abnormal cytology and 24.2% of specimens collected from healthy women. The frequency of HPV-positive specimens correlated with the severity of cervical pathology: it ranged from 50.0% in the subgroup of atypical squamous cells to 80.6% in cervical cancer. In cases confirmed by histology the frequency of HPV-positive specimens ranged from 68.6% in the subgroup of cervical intraepithelial neoplasia grade 1 to 89.2% in cervical intraepithelial neoplasia grade 3 or carcinoma in situ. HPV DNA-positive samples were further investigated for the presence of 16 HPV types by multiplex PCR. The most common HPV type was HPV 16 (detected in 42.3% of HPV-positive specimens) followed by HPV 31 (10.1%), HPV 33 (8.2%), and HPV 56 (5.7%). In contrast, the frequency of HPV 18 was lower as compared to other countries.

Intra- and interindividual epigenetic variation in human germ cells.

Epigenetics represents a secondary inheritance system that has been poorly investigated in human biology. The objective of this study was to perform a comprehensive analysis of DNA methylation variation between and within the germlines of normal males. First, methylated cytosines were mapped using bisulphite modification-based sequencing in the promoter regions of the following disease genes: presenilins (PSEN1 and PSEN2), breast cancer (BRCA1 and BRCA2), myotonic dystrophy (DM1), and Huntington disease (HD). Major epigenetic variation was detected within samples, since the majority of sperm cells of the same individual exhibited unique DNA methylation profiles. In the interindividual analysis, 41 of 61 pairwise comparisons revealed distinct DNA methylation profiles (P=.036 to 6.8 x 10(-14)). Second, a microarray-based epigenetic profiling of the same sperm samples was performed using a 12,198-feature CpG island microarray. The microarray analysis has identified numerous DNA methylation-variable positions in the germ cell genome. The largest degree of variation was detected within the promoter CpG islands and pericentromeric satellites among the single-copy DNA fragments and repetitive elements, respectively. A number of genes, such as EED, CTNNA2, CALM1, CDH13, and STMN2, exhibited age-related DNA methylation changes. Finally, allele-specific methylation patterns in CDH13 were detected. This study provides evidence for significant epigenetic variability in human germ cells, which warrants further research to determine whether such epigenetic patterns can be efficiently transmitted across generations and what impact inherited epigenetic individuality may have on phenotypic outcomes in health and disease.

Structure-based ligand binding sites of protein p14.5, a member of protein family YER057c/YIL051c/YjgF.

Seventeen mutants with one, two or three amino acids substitutions of human protein p14.5, homologue to well-known tumor antigen from goat liver UK114 and a member of proteins YER057c/YIL051c/YjgF family, have been used for structure-functional relation studies and ligand binding analysis using cross-linking by triacryloyl-hexahydro-s-triazine (TAT), size exclusion chromatography, free fatty acid and 8-anilino-1-naphthalenesulfonic acid (ANS) binding assays. Amino acids having the most significant impact on the ligand binding activity have been determined: R107, N93, Y21 and F89. Arginine 107 has been identified as the most accessible amino acid in the cleft. Trimeric structure of protein p14.5 has been confirmed as being essential for stoichiometric small ligand binding activity and oligomeric structure of p14. Ligand binding activity may be related with the biological functions of these proteins, which still are not understood well.

DNA methylation differences in human p14.5 gene promoter region in normal and proliferating cells.

Methylation status of cytosines and its changes during cell proliferation was analyzed in the 5'-flanking region of the human p14.5 gene, which encodes a member of the YER057c/YIL051c/YjgF protein family. We describe evidence of dramatic DNA methylation differences revealed in the study, and present detailed mapping of methylated cytosines (metC) at the 5'-flanking region of the p14.5 gene in several human normal tissues and tumor cells lines. DNA methylation profiles demonstrated aberrant distribution of metC positions with the different degree of methylation along all analyzed 5'-flanking regions of the p14.5 gene in cancer cells. We investigated DNA methylation changes in p14.5 5'-flanking region during cell differentiation by using DNA samples of freshly isolated monocytes and macrophages. According to our data, cellular differentiation processes from monocytes to macrophages are related to the elevated degree of DNA methylation of the p14.5 gene at the putative binding motifs for several transcription factors. The present findings indicate that some cytosines in the promoter region may have some significance in the degree of expression of the p14.5 gene during cell proliferation and cancerogenesis.

Search for somatic DNA variation in the brain: investigation of the serotonin 2A receptor gene.

Somatic DNA variation represents one of the most interesting but also one of the least investigated genetic phenomena. In addition to the classical case of DNA hypermutability at the V(D)J region, there is an increasing body of experimental evidence suggesting that genes other than immunoglobulin in tissues other than lymphocytes also exhibit nonuniformity of DNA sequence, which opens new opportunities for explaining various features of multicellular organisms. Identification of somatic DNA mutability, however, is not a trivial task and numerous confounding factors have to be taken into account. In this work we investigated putative DNA variation in the serotonin 2A receptor gene (HTR2A). A series of real-time PCR-based experiments was performed on DNA samples (n = 8) from human brain and peripheral leukocytes. Amplification of the target DNA sequences was carefully matched to that of the control plasmid containing the insert of HTR2A. Sequencing of nearly 500 clones containing a total of 150,000 nucleotides did not show any evidence for somatic DNA variation in the brain and peripheral leukocytes. It is argued in this article that although intraindividual DNA mutability may be a more common phenomenon than is generally accepted, some of the earlier claims of genetic nonidentity on the brain cells may be premature.

Identification of differentially expressed genes in yeast Saccharomyces cerevisiae cells with inactivated Mmf1p and Hmf1p, members of proteins family YERO57c/YJGF.

We used differential display analysis of mRNA to investigate the differences between gene expression in wild-type (wt) yeast Saccharomyces cerevisiae cells and mutated ones with disrupted activity of genes MMF1 and HMF1, members of the YERO57c/YJGF family. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to determine the differences in the degree of expression of 14 specific transcripts in normal and mutated yeast cells. Obtained data demonstrate that disruption of genes encoding proteins Mmf1p, Hmf1p (or both of them) result in the correlative variation of expression level of the target 12 genes both in the cells with changed phenotype (mmf1 and mmf1 hmf1) and in the cells retaining w.t. shape and growth rate (wt cells, hmf1). Metabolic processes and cellular pathways have been indicated for Mmf1p and Hmf1p based on the different profiles of the expression of 14 genes in mmf1, hmf1 yeast S. cerevisiae cells.

Epigenetic studies of genomic retroelements in major psychosis.

This work is dedicated to the exploration of the role of epigenetic (epiG) factors in major psychosis. One of the key functions of epigenetic modification of the genome of eukaryotic cells is to suppress transcriptional activity of the retroelements. Examples of retroelements are endogenous retroviral sequences (ERVs), Alu's, and LINEs, among others, which as a rule are hypermethylated. There is evidence from schizophrenia (SCH) and other human complex diseases that some of the genomic retroelements become transcribed in the affected tissues. Our goal was to screen DNA samples from post-mortem brain tissues of individuals who were affected with major psychiatric illness for retroelements that were located in the hypomethylated fraction of the genomic DNA. Over 100 Alu sequences were cloned, sequenced, and mapped to the human genome. A substantial portion of the cloned Alu's are located close to or within the genes that may be interesting targets for further genetic, transcription, and epigenetic studies.

Monozygotic twins exhibit numerous epigenetic differences: clues to twin discordance?

The goal of this pilot study was to explore the putative molecular mechanisms underlying the phenotypic discordance of monozygotic (MZ) twins. Thus, patterns of epigenetic DNA modification were investigated in the 5'-regulatory region of the dopamine D2 receptor gene (DRD2) in two pairs of monozygotic twins, one concordant and one discordant for schizophrenia. The bisulfite DNA modification-based approach was used to fine-map methylated cytosines in DRD2 in genomic DNA extracted from lymphocytes. Numerous DNA methylation differences were identified in the analyzed region both within and between the pairs of MZ twins. "Epigenetic distances" between MZ twins were calculated and used for the comparison of twin DRD2 methylation profiles. It was detected that the affected twin from the pair discordant for schizophrenia was epigenetically "closer" to the affected concordant twins than to his unaffected MZ co-twin. Although the epigenetic analysis was conducted for only several hundred base pairs of DRD2, the fact that numerous studies identified nonuniform methylation patterns across the clones of bisulfite-modified DNA from the same individual, as well as nonuniform patterns across different individuals, argues for the universality of intra- and interindividual epigenetic variation. Epigenetic studies should provide insight into the molecular causes of differential susceptibility to a disease in genetically identical organisms that may generalize to singletons.

The genetic diversity of barley cultivars from the Baltic States and Belarus, as determined by RAMPs.

A Random Amplified Microsatellite Polymorphism (RAMP) analysis was carried out on 30 barley cultivars from the Baltic states and Belarus. Seven primer combinations produced 60 polymorphic DNA fragments ranging in size from 54 b to 400 b. A Genetic Distance coefficient (GDxy) matrix was generated and a dendrogram constructed using cluster analysis of the unweighted pair-group method of arithmetic averages (UPGMA). The genetic distance between cultivars ranged from 0.067 to 0.714. The results were compared with available pedigree information. The dendrogram did not indicate any clear pattern of division among the barley cultivars based on geographic origin.

Major psychosis and chromosome 22: genetics meets epigenetics.

Elucidation of genetic factors in schizophrenia and bipolar disorder remains a challenging task to psychiatric researchers. As a rule, data from genetic linkage and association studies are quite controversial. In this article, we further explore the possibility that in addition to DNA sequences variation, a putative epigenetic dysregulation of brain genes plays an important role in the etiopathogenesis of major psychosis. We provide an epigenetic interpretation of unclear genetic findings specifically pertaining to chromosome 22 in schizophrenia and bipolar disorder. It is suggested that epigenetic strategies, when applied in conjunction with traditional genetic ones, may significantly expedite the uncovering of the molecular causes of major psychosis.