Association of CYP1A1 MspI polymorphism in the esophageal cancer risk: a meta-analysis in the Chinese population.

BACKGROUND: Although many epidemiologic studies have investigated the CYP1A1 MspI gene polymorphisms and their associations with esophageal cancer (EC), definite conclusions cannot be drawn. To clarify the effects of CYP1A1 MspI polymorphisms on the risk of EC, a meta-analysis was performed in Chinese population. METHODS: Related studies were identified from PubMed, Springer Link, Ovid, Chinese Wanfang Data Knowledge Service Platform, Chinese National Knowledge Infrastructure (CNKI), and Chinese Biology Medicine (CBM) till October 2014. Pooled ORs and 95% CIs were used to assess the strength of the associations. RESULTS: A total of 13 studies including 1,519 EC cases and 1,962 controls were involved in this meta-analysis. Overall, significant association was found between CYP1A1 MspI polymorphism and EC risk when all studies in the Chinese population pooled into this meta-analysis (C vs. T: OR = 1.25, 95% CI = 1.04 to 1.51; CC + CT vs. TT: OR = 1.35, 95% CI = 1.06 to 1.72; CC vs. TT + CT: OR = 1.35, 95% CI = 1.03 to 1.76). When we performed stratified analyses by geographical locations, histopathology type, and source of control, significantly increased risks were found in North China (C vs. T: OR = 1.38, 95% CI = 1.12 to 1.70; CC vs. TT: OR = 1.72, 95% CI = 1.16 to 2.56; CC + CT vs. TT: OR = 1.52, 95% CI = 1.14 to 2.02; CC vs. TT + CT: OR = 1.55, 95% CI = 1.17 to 2.06), in the population-based studies (C vs. T: OR = 1.22, 95% CI = 1.05 to 1.42; CC vs. TT: OR = 1.38, 95% CI = 1.02 to 1.88; CC + CT vs. TT: OR = 1.36, 95% CI = 1.10 to 1.69; CC vs. TT + CT: OR = 1.43, 95% CI = 1.13 to 1.81) and ESCC (C vs. T: OR = 1.17, 95% CI = 1.04 to 1.32; CC + CT vs. TT: OR = 1.28, 95% CI = 1.08 to 1.52). CONCLUSIONS: This meta-analysis provides the evidence that CYP1A1 MspI polymorphism may contribute to the EC development in the Chinese population.

No association between MspI allele of the ADRA2A polymorphism and ADHD: meta-analysis of family-based studies.

There is evidence for a genetic contribution to attention-deficit hyperactivity disorder (ADHD), although no candidate genes have attained genome-wide significance to date. Given that the noradrenergic system has been implicated in ADHD, the gene for the α2-adrenergic receptor (ADRA2A) has been hypothesized to contribute to the pathogenesis of ADHD. The present investigation reports results from a meta-analysis of family-based studies that did not find a significant association between the MspI polymorphism of the ADRA2A gene and ADHD.

CYP1A1 MspI polymorphism and acute myeloid leukemia risk: meta-analyses based on 5018 subjects.

BACKGROUND: Evidence indicates that CYP1A1 MspI polymorphism might be a possible risk factor for several malignancies. A growing body of literature has been devoted to the association of CYP1A1 MspI polymorphism with acute myeloid leukemia (AML). However, the results remain conflicting. The aim of the present study was to derive a more precise estimation of the relationship. METHODS: Meta-analyses assessing the association of CYP1A1 MspI variation with AML were conducted and subgroup analyses on ethnicity and age groups were further performed. Eligible studies were identified for the period up to May 2012. RESULTS: A total of ten case-control studies including 1330 cases and 3688 controls were selected for analysis. The overall data failed to indicate a significant association of CYP1A1 MspI polymorphism with AML risk (C vs T: OR = 1.13; 95%CI = 0.87-1.48; CC vs TT: OR = 1.72; 95%CI = 0.99-3.01; CC + TC vs TT: OR = 1.16; 95%CI = 0.86-1.55). In subgroup analysis stratified by ethnicity, significant AML risk was shown among Asians (CC + TC vs TT: OR = 1.33; 95%CI = 1.09-1.62) but not Caucasians or mixed races. In subgroup analysis regarding age groups, no associations were observed in either the childhood AML or the adult AML subgroups. CONCLUSION: The results of the present study suggested that CYP1A1 MspI polymorphism might be a risk factor for AML among Asians. Further investigations are needed to confirm the conclusions.

A simple method for high-throughput quantification of genome-wide DNA methylation by fluorescence polarization.

To rapidly determine DNA methylation levels from a large number of biological or clinical samples, we have developed an accurate and sensitive method for high-throughput quantification of global methylation of 5'-Cm ( 5) CGG-3' sites in the genome, visualized by fluorescence polarization (FP) based measurement of DNA methylation (FPDM). In FPDM, the methyl-sensitive HpaII and methyl-insensitive MspI restriction enzymes were employed to achieve DNA cleavage, followed by incorporation of fluorescent dCMP into the enzyme-cleavage products through polymerase chain extension, yielding an FP-ratio between the HpaII- and MspI-restricted preparations as a measure of methylation. FPDM provided stable estimates of methylation level of submicrograms of lambda or human DNA, and of a 255-bp DNA segment containing a single HpaII/MspI restriction site in accord with, and more accurate than, determination by gel electrophoresis. FPDM was also applied to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA-methyltransferase inhibitor 5-aza-dC.

Targeted gene engineering in Clostridium cellulolyticum H10 without methylation.

Genetic engineering of Clostridium cellulolyticum has been developed slowly compared with that of other clostridial species, and one of the major reasons might be the restriction and modification (RM) system which degrades foreign DNA. Here, a putative MspI endonuclease gene, ccel2866, was inactivated by a ClosTron-based gene disruption method. The resulting C. cellulolyticum mutant H10ΔmspI lost the MspI endonuclease activity and can accept unmethylated DNA efficiently. Following that, an oxygen-independent green fluorescence protein gene was introduced into H10ΔmspI without methylation, generating a convenient reporter system to evaluate the expression of heterologous protein in C. cellulolyticum by green fluorescence. To further demonstrate the efficiency of the H10ΔmspI, double mutants H10ΔmspIΔldh and H10ΔmspIΔack were constructed by disrupting lactate dehydrogenase gene ccel2485 and acetate kinase gene ccel2136 in H10ΔmspI, respectively, without DNA methylation, and the stability of the double mutation was confirmed after the 100th generation. The mutant H10ΔmspI constructed here can be used as a platform for further targeted gene manipulation conveniently and efficiently. It will greatly facilitate the metabolic engineering of C. cellulolyticum aiming at faster cellulose degradation and higher biofuel production at the molecular level.

Genotype identification of human cystic echinococcosis in Isfahan, central Iran.

Echinococcosis/hydatidosis is one of the most important zoonotic diseases commonly found in different regions of Iran with a major economic and public health importance. In the current study, Echinococcus granulosus isolates were collected from hospitalized patients in Isfahan, central Iran. The genotypes of 30 samples were determined by polymerase chain reaction amplification of internal transcribed spacer-1 region of ribosomal DNA, followed by restriction fragment length polymorphism (RFLP) with two restriction enzymes namely AluI and MspI. As expected, each isolate yielded an approximately 1-kbp DNA fragment on the electrophoresis gel. According to RFLP results for both enzymes, all isolates had an equal pattern indicating the G1 genotype. Our findings confirmed that G1 is the dominant genotype of cystic echinococcosis in human in central Iran, with predilection to different organs including liver, lung, and brain, and warrants the importance of sheep dog cycle in public health.

T-RFLP-based mcrA gene analysis of methanogenic archaea in association with oral infections and evidence of a novel Methanobrevibacter phylotype.

INTRODUCTION: Increasing evidence suggests a role for methanogenic archaea (methanogens) in human health and disease via syntrophic interactions with bacteria. Here we assessed the prevalence and distribution of methanogens and possible associations with bacteria in oral biofilms. METHODS: Forty-four periodontal and 32 endodontic samples from necrotic teeth with radiographic evidence of apical periodontitis were analysed. Terminal restriction fragment length polymorphism analysis based on the mcrA gene, specific to methanogens, was applied. The prevalence and amounts of methanogens in endodontic samples were compared with those of Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema spp. and Synergistes spp. based on real-time quantitative polymerase chain reactions. RESULTS: Besides dominance of the mcrA gene corresponding to Methanobrevibacter oralis, one mcrA gene type, for which no cultivated member has been reported previously, was identified in five periodontal samples and one endodontic sample. Rates of non-synonymous vs. synonymous nucleotide substitutions suggest that this mcrA gene type codes for a functionally active methyl-coenzyme M reductase. Methanobrevibacter smithii, the prominent methanogen in the human gut system, was not detected. Mean proportions of methanogens were comparable to Synergistes spp. ranging from 0.5 to 1.0% of the total microbial community. Treponema spp. dominated with a mean proportion of 10%, while the mean proportions of the other endodontic pathogens were below 0.1%. A positive association between methanogens and Synergistes spp. was found. CONCLUSION: Our data provide evidence of a novel, as yet uncultured methanogenic phylotype in association with oral infections, and indicate possible interactions between methanogens and Synergistes spp., the nature of which deserves further investigation.

A HindIII polymorphism of fibronectin gene is associated with nephrolithiasis.

OBJECTIVES: To evaluate the association between fibronectin gene (FN1) polymorphisms and calcium oxalate nephrolithiasis as a genetic risk factor. METHODS: Genomic DNA of 143 patients with calcium oxalate nephrolithiasis and 154 healthy controls were screened for polymorphisms (HaeIII b, MspI, and HindIII) of the FN1 gene, using polymerase chain reaction-restriction fragments length polymorphism method. Allele and genotype frequencies were compared between the groups. RESULTS: Although the observed differences between distribution of genotypes of AA, AB, and BB (for HaeIII b), as well as CC, CD, and DD (MspI) were not significant, FF genotype for HindIII showed significant difference when compared with both EF and EE + EF genotype (P = .00202 and P = .00203, respectively). CONCLUSIONS: The results of our study revealed that HindIII polymorphism of the FN1 gene is highly associated with calcium oxalate stone disease. This association makes FN a good candidate for further studies about the etiology of stone disease, and in the future it could be a candidate marker for evaluating the genetic risks in patients with nephrolithiasis.

Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae).

The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment.

Evaluation of hot saline solution and restriction endonuclease techniques in cytogenetic studies of Cycloneda sanguinea L. (Coccinellidae)

The goal of the present study was to determine if simple methods, especially hot saline solution (HSS) and MspI and HaeIII restriction endonucleases, which do not require special equipments, may be helpful in studies of genetic variability in the lady beetle, Cycloneda sanguinea. The HSS method extracted the heterochromatin region, suggesting that it is composed mostly of DNA rich in A-T base pairs. However, the X and y chromosomes were resistant to HSS banding. These bands facilitated the identification of each chromosome. In this study, we used the restriction endonucleases with different G-C base target sequences: MspI C/GGC and HaeIII GG/CC. The use of restriction enzyme MspI did not show an effect on the autosomal chromosomes. On the other hand, the sex pair showed a pale staining, to help in the recognition of these chromosomes. HaeIII produced characteristic bands which were identified all along the chromosomes, facilitating the identification of each chromosome. Based on these results, we can consider the heterochromatin being heterogeneous. The findings obtained here, using different chromosomal banding techniques, may be useful in the identification of intraspecific chomosome variability, specifically in Coccinellidae (Coleoptera) chromosomes, even without special equipment.

A novel strategy to extract specific phylogenetic sequence information from community T-RFLP.

Cultivation-independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. Semi-automated terminal restriction fragment length polymorphism (T-RFLP) analyses yield high-resolution genetic profiles of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring. A serious limitation of T-RFLP analyses has been the inability to reliably affiliate observed terminal restriction fragments (T-RF) to phylogenetic groups. In the study presented here, we were able to overcome this limitation of T-RFLP. With a combination of adapter ligation, fragment size selection, and re-amplification with adapter site specific PCR, we were able to isolate a T-RF-fraction of a narrow size-range containing a T-RF that was significantly more abundant in heavy metal amended soils. Cloning the size-selected T-RF fraction allowed for the efficient isolation of clones containing this specific T-RF. Sequence determination and phylogenetic inference in RDP-II affiliated the sequence to unclassified cyanobacteria. Specific primer design and PCR amplification from bulk soil DNA allowed for independent confirmation of the results from bacterial T-RFLP and T-RF cloning. Our results show that specific T-RFs can be efficiently isolated and identified, and that the adapter ligation approach holds great potential for genetic profiling and for identification of community components of interest.

[The tissue-specific methylation of human-specific endogenous retroviral long terminal repeats]. / Tkanespetsifichnoe metilirovanie spetsifichnykh dlia cheloveka dlinnykh kontsevykh povtorov éndogennykh retrovirusov.

A possible involvement of retroelements in the epigenetic regulation of human gene expression was considered by the example of methylation of long terminal repeats (LTRs) of the human endogenous retrovirus family K (HERV-K). The methylation status of six HERV-K LTRs was determined in various gene-enriched regions of the human genome. The methylation of four LTRs was shown to be tissue-specific. Our results correlated with published data on the tissue-specific changes in the expression level of human genes adjacent to the LTRs under study. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

An asymmetric complex of restriction endonuclease MspI on its palindromic DNA recognition site.

Most well-known restriction endonucleases recognize palindromic DNA sequences and are classified as Type IIP. Due to the recognition and cleavage symmetry, Type IIP enzymes are usually found to act as homodimers in forming 2-fold symmetric enzyme-DNA complexes. Here we report an asymmetric complex of the Type IIP restriction enzyme MspI in complex with its cognate recognition sequence. Unlike any other Type IIP enzyme reported to date, an MspI monomer and not a dimer binds to a palindromic DNA sequence. The enzyme makes specific contacts with all 4 base pairs in the recognition sequence, by six direct and five water-mediated hydrogen bonds and numerous van der Waal contacts. This MspI-DNA structure represents the first example of asymmetric recognition of a palindromic DNA sequence by two different structural motifs in one polypeptide. A few possible pathways are discussed for MspI to cut both strands of DNA, either as a monomer or dimer.

Standardization of MspI and HaeIII restriction karyotypes in cattle.

Cattle chromosomes were digested with two restriction endonucleases, MspI and HaeIII. The banding pattern induced by each enzyme allowed the identification and pairing of all individual chromosomes and consequently the elaboration of the karyotype. This method is rapid and technically easy, and proved to be of great utility in cattle cytogenetic studies.

Clinical features and skewed X-chromosome inactivation in female carriers of X-linked recessive spinal and bulbar muscular atrophy.

In X-linked recessive disorders, a few female gene carriers become symptomatic. Recent evidence implicates skewed X-chromosome inactivation in such female carriers. We studied the clinical features of eight female gene carriers of X-linked recessive spinal and bulbar muscular atrophy (SBMA), and evaluated the relationship between phenotype and genotype from the viewpoint of X-chromosome inactivation. Seven of eight cases were symptomatic, showing mild muscle weakness, frequent muscle cramps, slight elevation of the serum creatinine kinase level, or neurogenic changes on the electromyogram. Only one carrier was asymptomatic clinically. For the estimation of X-chromosome inactivation, the methylation status of the androgen receptor (AR) gene was determined by polymerase chain reaction-based assay. Highly skewed inactivation of the affected AR gene was found in the asymptomatic carrier, while symptomatic carriers had a random or lower inactivation pattern of the affected AR gene. These findings suggest that most female carriers of SBMA show some clinical abnormalities, and highly skewed inactivation of the affected X-chromosome seems to closely relate with escape of the manifestation in female carriers of SBMA.

Linkage and association studies of the relationship between endometriosis and genes encoding the detoxification enzymes GSTM1, GSTT1 and CYP1A1.

An association between endometriosis and the glutathione S-transferase (GST) M1 null mutation has been reported in French and Slavic populations. We aimed to replicate this association of endometriosis in a UK population, and to test for association with the GSTT1 null mutation or the cytochrome P450 (CYP) 1A1 MspI polymorphism. We genotyped 148 women each with endometriosis (sporadic cases, n = 91; familial cases, n = 57), a population control of 95 male blood donors, and a control group of 53 women with a normal pelvis at hysterectomy. No significant differences were found between cases and controls in the frequencies of the GSTM1 and GSTT1 null mutations, or the CYP1A1 MspI polymorphism. However, the combination of the GSTM1 null genotype and the CYP1A1 MspI polymorphism was associated with a small increased risk of endometriosis, and this warrants further investigation. We also tested for linkage to the chromosome 1p13 region, to which GSTM1 has been mapped, in 52 sister-pairs with stage III-IV disease using three highly polymorphic microsatellite markers. However, there was no evidence of linkage, suggesting that this region may not be implicated in disease susceptibility.

CYP1A1 polymorphisms and lung cancer risk: a meta-analysis.

To examine the risk of lung cancer associated with the MspI-restriction fragment length polymorphism and Exon7-Val polymorphisms of CYP1A1, a meta-analysis of published case-control studies was undertaken using a random effects model. The principal outcome measure was the odds ratio for the risk of lung cancer, using homozygosity of the 'wild-type allele' as the reference group. Fifteen reports detailing the relationship between the lung cancer and the MspI and Ile-Val polymorphisms of CYP1A1 were identified. The odds ratio of lung cancer associated with the MspI combined variant and homozygous genotypes were 1.09 (0.94-1.25) and 1.27 (0.91-1.77), respectively. The odds ratio of lung cancer associated with the Ile-Val combined variant and homozygous genotypes were 1.16 (0.92-1.48) and 1.62 (0.93-2.82), respectively. The hypothesis that the modulation of carcinogen metabolism is under genetic control is a plausible and attractive mechanism for explaining inter-individual susceptibility of lung cancer. However, the results from this analysis provide little support for the role of variation in the CYP1A1 gene defined by either polymorphisms represents as lung cancer risk factor. Additional well-designed studies based on sample sizes commensurate with the detection of small genotypic risks may allow a more definitive conclusion.

Copy number related transgene expression and mosaic somatic expression in hemizygous and homozygous transgenic tilapia (Oreochromis niloticus).

Three lines of transgenic tilapia (Oreochromis niloticus) fish were generated with a construct containing a lacZ reporter gene spliced to a 4.7 kb 5' regulatory region of a carp beta actin gene. All these three lines contain different copy numbers of transgenes and the levels of lacZ expression were found to be related to transgene copy number. Mosaic patterns of somatic lacZ expression were observed in these three lines which differed between lines but were consistent within a line. We also observed that expression of the reporter gene in homozygous transgenic fish was approximately two-fold greater than in the hemizygous transgenics. Analysis of expression of the reporter gene on a tissue-to-tissue basis demonstrated that lacZ expression of the reporter gene in stably transformed fish occured with variable intensity in different organs and tissues and was also sometimes variable in different cells of the same tissue in Gland G2 generations of the transgenic lines.

Relaxation of IGF2 imprinting in Wilms tumours associated with specific changes in IGF2 methylation.

Relaxation of IGF2 imprinting occurs in Wilms tumours and many other cancers, but the mechanism of loss of imprinting (LOI) remains unknown. To investigate the role of altered DNA methylation in LOI, we examined the pattern of methylation of the human insulin-IGF2 region in Wilms tumours and the normal kidney. The analysis included regions homologous to three 'differentially methylated regions' of the mouse Igf2 gene (dmrs 0, 1 and 2). In tumours displaying normal IGF2 imprinting, and in the normal kidney, maternal allele-specific DNA methylation was identified spanning exons 2 and 3. This region is homologous to dmr 0, a site of maternal-specific differential methylation in the mouse. In Wilms tumours with relaxed imprinting or 11p15.5 LOH this region was unmethylated. No other differential methylation was identified. In particular, two sites of paternal methylation in the mouse (dmrs 1 and 2), and all three imprinted IGF2 promoters were not methylated in the kidney or in Wilms tumours. We postulate that LOI in Wilms tumours is associated with loss of maternal allele-specific methylation from a region located upstream of the imprinted IGF2 promoters. This region may contain cis acting sequences that coordinately influence imprinting.