Risk-associated coding synonymous SNPs in type 2 diabetes and neurodegenerative diseases: genetic silence and the underrated association with splicing regulation and epigenetics.

Single nucleotide polymorphisms (SNPs) are tentatively critical with regard to disease predisposition, but coding synonymous SNPs (sSNPs) are generally considered "neutral". Nevertheless, sSNPs in serine/arginine-rich (SR) and splice-site (SS) exonic splicing enhancers (ESEs) or in exonic CpG methylation targets, could be decisive for splicing, particularly in aging-related conditions, where mis-splicing is frequently observed. We presently identified 33 genes T2D-related and 28 related to neurodegenerative diseases, by investigating the impact of the corresponding coding sSNPs on splicing and using gene ontology data and computational tools. Potentially critical (prominent) sSNPs comply with the following criteria: changing the splicing potential of prominent SR-ESEs or of significant SS-ESEs by >1.5 units (Δscore), or formation/deletion of ESEs with maximum splicing score. We also noted the formation/disruption of CpGs (tentative methylation sites of epigenetic sSNPs). All disease association studies involving sSNPs are also reported. Only 21/670 coding SNPs, mostly epigenetic, reported in 33 T2D-related genes, were found to be prominent coding synonymous. No prominent sSNPs have been recorded in three key T2D-related genes (GCGR, PPARGC1A, IGF1). Similarly, 20/366 coding synonymous were identified in ND related genes, mostly epigenetic. Meta-analysis showed that 17 of the above prominent sSNPs were previously investigated in association with various pathological conditions. Three out of four sSNPs (all epigenetic) were associated with T2D and one with NDs (branch site sSNP). Five were associated with other or related pathological conditions. None of the four sSNPs introducing new ESEs was found to be disease-associated. sSNPs introducing smaller Δscore changes (<1.5) in key proteins (INSR, IRS1, DISC1) were also correlated to pathological conditions. This data reveals that genetic variation in splicing-regulatory and particularly CpG sites might be related to disease predisposition and that in-silico analysis is useful for identifying sSNPs, which might be falsely identified as silent or synonymous.

Drugs of abuse: epigenetic mechanisms in toxicity and addiction.

The abuse of substances such as ethanol, cocaine, amphetamines and heroin is associated with toxic effects on almost every system of the organism. Furthermore, the transition from occasional-recreational use to chronic abuse and addiction is a serious psychiatric disorder with only few chances for effective and definitive treatment since most individuals relapse, even after long periods of abstinence. It is therefore of utmost importance to elucidate the mechanisms by which these substances exert their toxicity and mediate addiction, in order to develop new, efficient therapeutic strategies with a long-term outcome, which are currently lacking. We already know that in a great number of these mechanisms, altered gene function is involved. But, with the new field of epigenetics, there is increasing evidence that changes in the epigenome are responsible for the altered gene function. The advances in the field of epigenetics towards elucidation of the mechanisms underlying toxicity and addiction for ethanol, cocaine, amphetamines and heroin are currently presented and discussed in this review.

Epigenetically-targeted therapies for the treatment of hematological malignancies.

Epigenetic modifications, which are heritable changes in gene expression not involving DNA sequence alterations, are important early events in the multi -step process of tumorigenesis. Among them, DNA methylation and histone acetylation are the most extensively studied. Although they are, by definition, somatically heritable, epigenetic modifications of DNA and histones are also reversible. This characteristic difference from genetic alterations makes them interesting targets for therapeutic intervention. The huge amount of knowledge gathered in the field of epigenetics the last decade, was followed by the development of novel therapies: old drugs finding new identity and new targets and an increasing list of novel compounds for the treatment of malignant diseases. Hematological malignancies offer a broad spectrum of diseases where epigenetic therapies are shown to be active, providing encouraging results. Some of the more recent reports on this field of therapeutic interventions are reviewed below.

Frequent presence of incomplete HPV16 E7 ORFs in lung carcinomas: memories of viral infection.

BACKGROUND: HPV16 E6/E7 oncoproteins are critical for cervical carcinogenesis. The corresponding oncogenes are also detected in head and neck cancer, but in lung cancer their presence is strongly debated. PCR-based detection protocols amplify different target sequences. OBJECTIVES: To examine the frequency of different length HPV16 E7 segments in lung carcinomas. STUDY DESIGN: We designed four different amplification schemes for the detection of overlapping segments of the HPV16 E7 ORF, all suitable for specific HPV detection in cervical carcinoma. In two schemes, the entire E7 ORF was targeted while in the remaining schemes internal, smaller sequences were targeted. In total, 76 specimens were used; 29 lung carcinoma specimens, 16 non-cancerous lung tissue specimens from the same patients and 31 bronchial washings from different lung cancer patients. RESULTS: Amplification of the entire HPV16 E7 ORF, using two protocols, demonstrated the absence of the specific HPV16 E7 sequences (74 samples either tested negative by the first PCR protocol or false positive by the second, based on sequencing or AvaII or PvuII digestion). However, both schemes targeting smaller E7 segments revealed the frequent presence of HPV16 E7 sequences in lung carcinoma specimens (14/23 positive by either scheme). CONCLUSIONS: HPV16 E7 sequences are frequently observed in lung carcinomas. Decreasing the size of PCR-target sequences increases the detection frequency, possibly indicating the presence of incomplete viral ORFs. Restriction endonuclease analysis is critical for verifying the reliability of the detection of these sequences.

DNA repair enables sex identification in genetic material from human teeth.

BACKGROUND: The purpose of this study was to test the effectiveness of a DNA repair protocol in improving genetic testing in compromised samples, frequently encountered in Forensic Medicine. METHODS: In order to stretch the experiment conditions to the limits, as far as quality of samples and DNA is concerned, we tried the repair protocol on ten ancient human teeth obtained from an equal number of skeletons from a burial site in Lerna, Middle Helladic Greece (2100-1700 BC). For these samples, sex was previously determined morphologically, serving as a reference to compare our molecular data with. The samples were analysed using the DNA amelogenin sex test assay prior and after DNA polymerase repair. For every individual, two molecular sex determinations were obtained by visualising PCR products on an agarose gel. RESULTS: DNA repair enabled genetic testing in these samples. Successful amplification of the amelogenin gene was obtained only from the repaired DNA in eight out of ten samples. Prior to the repair treatment, none of these samples yielded any PCR products, thus attesting to the authenticity of the amplified sequence. The concordance between morphological and molecular analysis was in reasonable agreement (71%). CONCLUSIONS: These results reveal the impact of the repair process in studying single copy genes from low quality DNA. This protocol could facilitate molecular analysis in compromised samples, encountered in forensic medicine, as well as enable genetic studies in ancient remnants.

In situ hybridization and reverse transcription-polymerase chain reaction for cyclin D1 mRNA in the diagnosis of mantle cell lymphoma in paraffin-embedded tissues.

Mantle cell lymphoma (MCL) is characterized by the chromosomal translocation t(11;14), which involves rearrangement of the bcl-1 proto-oncogene to the immunoglobulin heavy chain gene and results in overexpression of cyclin D1 mRNA. In this study, we evaluated the diagnostic relevance of three methods that may be helpful in the diagnosis of MCL: in situ hybridization (ISH) and a stringent reverse transcriptase-polymerase chain reaction (RT-PCR) protocol for cyclin D1 mRNA, and immunohistochemistry for cyclin D1 protein. The study group included 37 paraffin-embedded specimens (25 from lymph nodes and 12 from extranodal tissues) from 30 patients. MCL diagnosis was performed according to the Revised European-American Classification of Lymphoid Neoplasms. Twenty-nine patients with non-MCL lymphoproliferative disorders comprised the control group. Biotin-labeled ISH was performed in 28 cases of MCL, 24 (86%) of which were found to be positive. As shown by ISH in extranodal tissues, cyclin D1 mRNA was present not only in neoplastic lymphoid cells, but in other cell types as well. For this reason, RT-PCR results were considered reliable for MCL diagnosis only on informative material (from tissues that do not normally express cyclin D1); this method was evaluated as positive in 16 of 18 (89%) MCL cases. Cyclin D1 immunopositivity was present in 20 of 29 (69%) MCL cases. No members of the control group were found to express cyclin D1 mRNA by either ISH or RT-PCR under the stringent conditions used. In conclusion, stringent RT-PCR for cyclin D1 expression can be helpful in MCL diagnosis in paraffin-embedded material from lymph nodes. ISH is a sensitive method for cyclin D1 mRNA detection; its sensitivity is superior to that of cyclin D1 immunohistochemistry and similar to that of the stringent RT-PCR used. ISH is very specific as well, clearly more specific than RT-PCR, because it allows the correlation of molecular findings with morphology. This method can be applied on all types of paraffin-embedded tissues and provides an accurate tool for MCL diagnosis.

Genetic heterogeneity of HIV-1 in Greece.

The aim of this study was to detect and determine the genetic variation of HIV-1 in Greece and to analyze the phylogenetic relationships and transmission dynamics of identified variants. Eighty-six blood samples from HIV-1 seroconverted patients of different risk groups were collected from the AIDS clinic, AHEPA Hospital, Thessaloniki, Greece. Retroviral DNA was extracted from uncultured peripheral blood mononuclear cells. HIV-1 DNA sequences encoding a 500-bp fragment of the gp120 C2-C3 region were amplified from each study subject, and they were genetically subtyped by heteroduplex mobility assay and DNA sequencing. Genetic distances and phylogenetic relationships of DNA sequences were estimated using PHYLIP software. Our results revealed that 82 out of 86 (95.3%) subjects carried subtype B sequences, while four (4.7%) carried subtype A sequences. Subtype A in Greek individuals not having traveled abroad was documented. An average of intrasubtype B genetic divergence of 15% was noted. Our findings demonstrate the presence of at least two genetic subtypes of HIV-1 in northern Greece--subtype B and subtype A. The predominant subtype is subtype B, which was transmitted into Greece by multiple sources. Our observations lend support to the argument that the distribution of HIV-1 subtypes is determined by founder effects or other processes rather than any tropism for particular cell types or mode of transmission.

Increased frequency of the rare PstI allele (P2) in a population of CAD patients in northern Greece.

Restriction fragment length polymorphisms (RFLPs) at the apolipoprotein AI-CIII-AIV gene cluster and their association with coronary artery disease (CAD) and lipid levels were studied in a Northern Greek population. Ninety-five patients with CAD and fifty-four normal controls, angiographically proven, were included in this study. Using genomic hybridization techniques, three polymorphic restriction sites were identified at this locus: the PstI at the 3' end of the apoAI gene, the SacI at the 3' non-coding region of the apoCIII gene and the PvuII at the intergenic region between the apoCIII-AIV genes. The rare allele (P2) arising from the absence of the PstI restriction site was observed with a significantly higher frequency (p < 0.01) in patients compared to normals (0.11 vs 0.02). In contrast, the rare allele for the SacI polymorphic site had a similar distribution among patients and controls (0.12 vs 0.16). The same was observed for the PvuII RFLP (0.04 vs 0.05). Correlation of lipid and apolipoprotein AI levels with the three RFLPs revealed no significant association, although apo AI and HDL were lower in patients with the P2 allele. Thus, in this Greek population, only the PstI polymorphism, among the polymorphic restriction sites examined, appears to be associated with CAD.

HLA class II DNA-RFLP typing in 102 individuals from Northern Greece.

The serological identification of HLA class II alleles is often doubtful. Since accurate HLA typing is essential for the matching of donor-recipient pairs in allogeneic transplantation, an effort was made to establish DNA restriction fragment length polymorphism (RFLP) typing and to assess the correlation between the serological and RFLP techniques in the population of Northern Greece. One hundred and two healthy individuals (204 HLA-DR alleles) from Northern Greece were HLA-DR, DQ typed with both the microcytotoxicity and the Taq I RFLP method, using three exon-specific probes. DNA-RFLP typing revealed (1) concordant results with serology in 69.9% (142/204) of the alleles and (2) at least one HLA-DR allele discrepant to serology in 30.4% (62/204) of the alleles. Incorrect serological DR types (weak reactions or inability to distinguish between two alleles with a common epitope) were identified in 54 alleles (26.5%), while 3.9% (8/204) of serological "blank" alleles turned out to be definable alleles by RFPL. Of the individuals tested, 10.8% (11/102) were DR-homozygous by RFLP. This comparison of results obtained by serology and RFLP demonstrated the necessity of the clinical application of DNA typing, especially for organ transplantation where accurate HLA typing has an important influence on graft survival.

Synergistic interactions between transcription factors control expression of the apolipoprotein AI gene in liver cells.

The gene coding for apolipoprotein AI (apoAI), a plasma protein involved in the transport of cholesterol and other lipids in the plasma, is expressed predominantly in liver and intestine. Previous work in our laboratory has shown that different cis-acting elements in the 5'-flanking region of the human apoAI gene control its expression in human hepatoma (HepG2) and colon carcinoma (Caco-2) cells. Hepatocyte-specific expression is mediated by elements within the -256 to -41 DNA region relative to the apoAI gene transcription start site (+1). In this study it was found that the -222 to -110 apoAI gene region is necessary and sufficient for expression in HepG2 cells. It was also found that this DNA region functions as a powerful hepatocyte-specific transcriptional enhancer. Gel retardation and DNase I protection experiments showed that HepG2 cells contain proteins that bind to specific sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within this enhancer. Site-directed mutagenesis that prevents binding of these proteins to individual or different combinations of these sites followed by functional analysis of these mutants in HepG2 cells revealed that protein binding to any one of these sites in the absence of binding to the others was not sufficient for expression. Binding to any two of these sites in any combination was sufficient for only low levels of expression. Binding to all three sites was essential for maximal expression. These results indicate that the transcriptional activity of the apoAI gene in liver cells is dependent on synergistic interactions between transcription factors bound to its enhancer.

Cellular and tissue distribution of a single-strand-specific nuclease.

1. Specific antibodies which were raised against a single-strand-specific nuclease isolated from rat liver microsomes were used for characterizing this enzyme and determining its cellular and tissue distribution. 2. The single-strand-specific nuclease does not show any homology with other known nucleolytic enzymes. 3. It is mainly localized in microsomes and cytosol; traces of it are also found in nuclei, but it could not be detected in mitochondria. 4. Using the same specific antibodies we attempted to detect this nuclease in some other tissues which exhibit high metabolic rates, namely kidneys, heart and spleen. 5. Thus, with the aid of immunological techniques we were able to determine that at least part of the total poly(U) nucleolytic activity observed in kidney and heart is due to a nuclease immunologically identical to the enzyme under study. Kidneys were the best source for this enzyme.

Single-strand-specific nuclease from rat liver endoplasmic reticulum: characterization and mode of action.

1. The characteristics and mode of action of a single-strand-specific nuclease isolated from rat liver endoplasmic reticulum are investigated with respect to its DNA and RNA substrates. 2. The RNase activity of the enzyme is slightly influenced by the presence of divalent cations but the DNase activity is enhanced by divalent cations particularly Mn2+. 3. Activity is partially inhibited by the presence of EGTA; this effect is reversed most efficiently by the addition of Mn2+. 4. The enzyme exhibits small pH dependence between pH 6-9 and maximum activity is observed at pH 7-7.5 for both DNase and RNase activities. 5. Sulfhydryl group reagents do not affect its action but histidyl group reagents exert a small but definite effect. 6. The enzyme degrades DNA and RNA endonucleolytically producing fragments which possess 3'-OH and 5'-phosphate termini. 7. Monomers are not produced even after prolonged degradation. 8. The end product of poly(U)degradation ranges between two and four building blocks but the DNA product is longer probably due to considerable percentage of secondary structure.

The DNase activity of an endoplasmic reticulum nuclease and its effect on DNA synthesis in vitro.

An endoplasmic reticulum nuclease which was isolated previously in this laboratory from rat liver ( Kouidou et al. (1981) Eur.J. Bioch . 120, 9-14) was found to degrade linear and circular single stranded DNA but not double stranded DNA. The DNA fragments resulting from this cleavage were longer than 20 nucleotides. In addition the nuclease was found to improve the efficiency of DNA template used by DNA polymerase I in DNA synthesis in vitro. The results were the same whether incubation of the template with the nuclease was prior to addition of DNA polymerase I or simultaneously with polymerization. When nuclease was added after the completion of polymerization by DNA polymerase I it was ineffective unless the product was denatured. These data further corroborate the observation that double stranded DNA is not cleaved by this enzyme.

Endoplasmic reticulum nuclease. Purification and specificity.

An endonuclease, which was originally identified for its RNA polymerase inhibitory activity, was isolated from rat liver endoplasmic reticulum. The enzyme yields on gel chromatography four active fractions of different molecular weights (Mr 5.3 X 10(4), 9 X 10(4), 1.55 X 10(5) and Sephacryl S-200 fraction at V0). Each fraction contains polypeptide chains which give a single band on sodium dodecylsulphate electrophoresis (Mr 5.4 X 10(4). This indicates that the enzyme is an oligomeric protein and each of its subunits exhibits the same or very similar molecular weights. Deoxyribonucleoside and ribonucleoside triphosphates can bind to the endoplasmic reticulum nuclease. Binding is enhanced in the presence of divalent cations particularly Mg2+. The enzyme exhibits mainly RNase activity but can also degrade denatured DNA and DNA . RNA hybrids which contain breaks in one of the two strands. Poly(A) and mainly poly(U) are most susceptible to its nucleolytic activity whereas poly(C) is completely resistant.

Nucleic acid interaction with VERO cells. A temperature barrier in the interaction pattern.

The interaction of VERO cell monolayers with spin (nitroxide)-(labeled polynucleotides (1(N)n) was examined by electron spin resonance (ESR) spectroscopy at various temperatures. Nitroxide labels covalently linked to (A)n, (dUfl)n, (U)n and (A)n . (U)n were used to monitor the interaction. The VERO cells were grown on small quartz plates with a cell viability of 95% or better and then used directly for the ESR studies. The ESR results indicated that the interaction between VERO cells and spin-labeled nucleic acids is temperature dependent. No temperature dependence was found when VERO cells were in contact with nitroxide radicals which were free in solution or covalently bound to Sepharose 4B. The temperature dependence established with nitroxide-labeled nucleic acids indicates that a temperature barrier must exist between 20 and 26 degrees C for the interaction between nucleic acids and VERO cells; namely, at 26 degrees C or above spin-labeled nucleic acids interact significantly with a VERO cell surface; whereas, at 20 degrees C the ESR signal reports no interaction. It is concluded that a temperature-dependent phase transition of membrane components or cell surface products active at 26 degrees C or above play a key role in the nucleic acid cell surface interaction process.

Dependence of interferon induction on nucleic acid conformation.

UV and circular dichroism characteristics of duplex analogs belonging to the (A)n-(U)n and (I)n-(C)n series were determined to assign qualitatively the nature of conformational differences caused by 5-pyrimidine and c7 purine substitutions in such duplexes. Evidence is presented which shows that 5-pyrimidine substitution by bromine or methyl changes the duplex conformation of both series in a similar way, if at all. A c7 substitution in the purine ring affects the duplex conformation of the members in the same series similarly, but the conformational change appears to be different for the two series. To wit, it is proposed that in the duplexes the effect of the change (A)n leads to (c7A)n is an increase of the positive base tilt, whereas the change (I)n leads to (c7I)n causes a decrease where (c7A)n is poly (7-deazaadenylic acid) and (c7I)n is poly(7-deazainosinic acid), respectively. Poly(5-bromocytidylic acid) (br5C)n proved to be useful as a sensor strand for the intepretation of the spectroscopic data. The circular dichroism findings correlate well with observations made earlier on the interferon inducing ability for such duplexes, namely, duplexes based on the (c7A)n are inactive as interferon inducers, whereas duplexes based on (c7I)n are potent inducers. Furthermore, a 5-pyrimidine substitution does not substantially affect the interferon inducing ability, unless the thermal stability of the analog becomes critical, as in the case of (A)n-(br5U)n. Thus, this study provides the first evidence to link the interferon-inducing ability of a nucleic acid to a defined physical parameter of double helix, and reinforces the concept that interferon induction is dependent on the recognition of a particular spatial and steric organization of a double-stranded RNA.