Microsomal epoxide hydrolase polymorphisms and lung cancer risk: a quantitative review

To investigate the role of microsomal epoxide hydrolase (mEH) polymorphisms in the aetiology of lung cancer and to assess the interaction between mEH polymorphisms and smoking, we performed a meta-analysis of seven published studies, which included 2078 cases and 3081 controls, and a pooled analysis of eight studies (four published and four unpublished at that time) with a total of 986 cases and 1633 controls. The combined metaanalysis odds ratios (ORs) were 0.98 (95% confidence interval [CI] = 0.72-1.35) for polymorphism at amino acid 113 in exon 3 (His/His versus Tyr/Tyr genotype) and 1.00 (95% CI= 0.71-1.41) for polymorphism at amino acid 139 in exon 4 (Arg/Arg versus His/ His genotype). In the pooled analysis, we observed a significant decrease in lung cancer risk (OR = 0.70, 95% CI = 0.51-0.96) for exon 3 His/His genotype after adjustment for age, sex, smoking and centre. The protective effect of exon 3 polymorphism seems stronger for adenocarcinoma of the lung than for other histological types. The OR for high predicted mEH activity, compared with low activity, was 1.54 (95% CI = 0.77-3.07) in the meta analysis and 1.18 (95% CI = 0.92-1.52) in the pooled analysis. We did not find a consistent modification of the carcinogenic effect of smoking according to mEH polymorphism, although the risk of lung cancer decreased among never smokers with high mEH activity and among heavy smokers with the exon 3 His/His genotype. In conclusion, this study suggests a possible effect of mEH polymorphisms at exon 3 in modulating lung cancer. If present, this effect may vary among different populations, possibly because of interaction with genetic or environmental factors.     

Microsomal epoxide hydrolase polymorphisms and lung cancer risk: a quantitative review

To investigate the role of microsomal epoxide hydrolase (mEH) polymorphisms in the aetiology of lung cancer and to assess the interaction between mEH polymorphisms and smoking, we performed a meta-analysis of seven published studies, which included 2078 cases and 3081 controls, and a pooled analysis of eight studies (four published and four unpublished at that time) with a total of 986 cases and 1633 controls. The combined metaanalysis odds ratios (ORs) were 0.98 (95% confidence interval [CI] = 0.72-1.35) for polymorphism at amino acid 113 in exon 3 (His/His versus Tyr/Tyr genotype) and 1.00 (95% CI= 0.71-1.41) for polymorphism at amino acid 139 in exon 4 (Arg/Arg versus His/ His genotype). In the pooled analysis, we observed a significant decrease in lung cancer risk (OR = 0.70, 95% CI = 0.51-0.96) for exon 3 His/His genotype after adjustment for age, sex, smoking and centre. The protective effect of exon 3 polymorphism seems stronger for adenocarcinoma of the lung than for other histological types. The OR for high predicted mEH activity, compared with low activity, was 1.54 (95% CI = 0.77-3.07) in the meta analysis and 1.18 (95% CI = 0.92-1.52) in the pooled analysis. We did not find a consistent modification of the carcinogenic effect of smoking according to mEH polymorphism, although the risk of lung cancer decreased among never smokers with high mEH activity and among heavy smokers with the exon 3 His/His genotype. In conclusion, this study suggests a possible effect of mEH polymorphisms at exon 3 in modulating lung cancer. If present, this effect may vary among different populations, possibly because of interaction with genetic or environmental factors.     

Meta-Analysis of Microsomal Epoxide Hydrolase Gene Polymorphism and Risk of Hepatocellular Carcinoma

Background

Hepatocarcinogenesis is a complex process that may be influenced by many factors, including polymorphism in microsomal epoxide hydrolase (mEH). Previous work suggests an association between the Tyr113His and His139Arg mEH polymorphisms and susceptibility to hepatocellular carcinoma (HCC), but the results have been inconsistent.

Methods

PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between Tyr113His and His139Arg mEH polymorphism and susceptibility to HCC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated.

Results

Eleven studies were included in the meta-analysis, involving 1,696 HCC cases and 3,600 controls. The 113His- mEH allele was significantly associated with increased risk of HCC based on allelic contrast (OR = 1.35, 95% CI = 1.04–1.75, p = 0.02), homozygote comparison (OR = 1.65, 95% CI = 1.07–2.54, p = 0.02) and a recessive genetic model (OR = 1.54, 95% CI = 1.21–1.96, p<0.001), while individuals carrying the Arg139Arg mEH genotype had no association with increased or decreased risk of HCC.

Conclusion

The 113His- allele polymorphism in mEH may be a risk factor for hepatocarcinogenesis, while the mEH 139Arg- allele may not be a risk or protective factor. There is substantial evidence that mEH polymorphisms interact synergistically with other genes and the environment to modulate risk of HCC. Further large and well-designed studies are needed to confirm these conclusions.     

The clinical relevance and prognostic significance of microsomal epoxide hydrolase gene polymorphisms and their susceptibility to acquired aplastic anemia: an Egyptian study

Background: Microsomal epoxide hydrolase enzyme (mEPHX) is involved in xenobiotics detoxification. Two variants of mEPHX, Tyr113His and His139Arg, have been described. Both may lead to acquired aplastic anemia (AA).

Objectives: Assessing mEPHX genetic polymorphisms and detecting their impact on susceptibility and prognosis in Egyptian AA patients.

Participants and methods: mEPHX 113 and 139 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 100 patients with AA and 100 control subjects.

Results: Both mEPHX Tyr113His and His139Arg gene polymorphisms were associated with increased risk of developing AA, and have a significant impact of bad prognosis (p value?<?0.01).

Conclusions: These mEPHX gene polymorphisms can be considered as risk factors and predictive molecular markers for prognosis in AA patients.     

Systematic Review and Meta-Analysis of the Relationship between EPHX1 Polymorphisms and Colorectal Cancer Risk

Background

Microsomal epoxide hydrolase (EPHX1) plays an important role in both the activation and detoxification of PAHs, which are carcinogens found in cooked meat and tobacco smoking. Polymorphisms at exons 3 and 4 of the EPHX1 gene have been reported to be associated with variations in EPHX1 activity. The aim of this study is to quantitatively summarize the relationship between EPHX1 polymorphisms and colorectal cancer (CRC) risk.

Methods

Two investigators independently searched the Medline, Embase, CNKI, and Chinese Biomedicine Databases for studies published before June 2012. Summary odds ratios (ORs) and 95% confidence intervals (CIs) for EPHX1 Tyr113His (rs1051740) and His139Arg (rs2234922) polymorphisms and CRC were calculated in a fixed-effects model and a random-effects model when appropriate.

Results

This meta-analysis yielded 14 case-control studies, which included 13 studies for Tyr113His (6395 cases and 7893 controls) and 13 studies for His139Arg polymorphisms (5375 cases and 6962 controls). Overall, the pooled results indicated that EPHX1 Tyr113His polymorphism was not associated with CRC risk; while the His139Arg polymorphism was significantly associated with decreased CRC risk (Arg/His vs. His/His, OR = 0.90, 95%CI = 0.83–0.98; dominant model, OR = 0.92, 95%CI = 0.85–0.99). The statistically significant association between EPHX1 His139Arg polymorphism and CRC was observed among Caucasians and population-based case-control studies. This association showed little heterogeneity and remained consistently strong when analyses were limited to studies in which genotype frequencies were in Hardy–Weinberg equilibrium, or limited to studies with matched controls. When cumulative meta-analyses of the two associations were conducted by studies’ publication time, the results were persistent and robust.

Conclusion

This meta-analysis suggests that EPHX1 Tyr113His polymorphism may be not associated with CRC development; while the EPHX1 His139Arg polymorphism may have a potential protective effect on CRC.     

Systematic Review and Meta-Analysis of the Relationship between EPHX1 Polymorphisms and the Risk of Head and Neck Cancer

Aim

To evaluate the association between the EPHX1 Tyr113His and His139Arg polymorphisms in the EPHX1 gene and the risk of head and neck cancer.

Materials and Methods

Studies on the association of EPHX1 Tyr113His and His139Arg polymorphisms with HNC performed up until June 1st, 2014, were identified using a predefined search strategy. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the strength of these associations.

Results

In this meta-analysis, 10 case-control studies, which included 9 studies of Tyr113His (1890 cases and 1894 controls) and 10 studies of His139Arg polymorphisms (1982 cases and 2024 controls), were considered eligible for inclusion. Overall, the pooled results indicated that the EPHX1 Tyr113His polymorphism was significantly associated with increased HNC risk (Tyr/His vs. Tyr/Tyr, OR = 1.26, 95%1.02–1.57;His/His+ Tyr/His vs. Tyr/Tyr, OR = 1.29, 95% I = 1.03–1.61). However, no significant association was found between the His139Arg polymorphism and HNC risk. In the subgroup analysis, a statistically significant association between the EPHX1 Tyr113His polymorphism and HNC was observed in population-based case-control studies (PCC), which involved less than 500 participants and genotype frequencies in HWE. This association showed minimal heterogeneity after excluding studies that were determined to contribute to heterogeneity. After categorizing the studies by publication time, a sensitivity analysis and cumulative meta-analysis of the two associations were conducted, and the results of the two analyses were consistent.

Conclusion

Our meta-analysis suggests that EPHX1 Tyr113His polymorphism may be a risk factor for HNC, while the EPHX1 His139Arg polymorphism has no association with HNC risk.     

Solution 1H nuclear magnetic resonance determination of the distal pocket structure of cyanomet complexes of genetically engineered sperm whale myoglobin His64 (E7)-->Val, Thr67 (E10)-->Arg. The role of distal hydrogen bonding by Arg67 (E10) in modulating ligand tilt.

Sequence-specific 2D methodology has been used to assign the 1H NMR signals for all active site residues in the paramagnetic cyano-met complexes of sperm whale synthetic double mutant His64[E7]-->Val/Thr67[E10]-->Arg (VR-met-MbCN) and triple mutant His64[E7]-->Val/Thr67[E10]-->Arg/Arg45[CD3]-->Asn (VRN-metMbCN). The resulting dipolar shifts for noncoordinated proximal side residues were used to quantitatively determine the orientation of the paramagnetic susceptibility tensor in the molecular framework for the two mutants, which were found indistinguishable but distinct from those of both wild-type and the His64[E7]-->Val single point mutant (V-metMbCN). The observed dipolar shifts for the E helix backbone protons and Phe43[CD1], together with steady-state nuclear Overhauser effect between the E helix and the heme, were analyzed to show that both the E helix and Phe43[CD1] move slightly closer to the iron to minimize the vacancy resulting from the His64[E7]-->Val substitution, as found in V-metMbCN (Rajarathnam, K., J. Qin, G.N. LaMar, M. L. Chiu, and S. G. Sligar. 1993. Biochemistry. 32:5670-5680). The dipolar shifts of the mutated Val64[E7] and Arg67[E10] allow the determination of their orientations relative to the heme, and the latter residue is shown to insert into the pocket and provide a hydrogen bond to the coordinated ligand, as found in the naturally occurring ValE7/ArgE10 genetic variant, Aplysia limacina Mb. The oxy-complex of both A. limacina Mb and VR-Mb, VRN-Mb have been proposed to be stabilized by this hydrogen bonding interaction (Travaglini Allocatelli, C. et al. 1993. Biochemistry. 32:6041-6049). The magnitude of the tilt of the major magnetic axes from the heme normal in VR-metMbCN and VRN-metMbCN, which is related to the tilt of the ligand, is the same as in wild-type or V-metMbCN, but the direction of tilt is altered from that in V-metMbCN. It is concluded that the change in the direction of the ligand tilt in both the double and triple mutants, as compared to WT metMbCN and V-metMbCN single mutant, is due to the attractive hydrogen-bonding between ArgE10 and the bound cyanide.     

Putative EPHX1 enzyme activity is related with risk of lung and upper aerodigestive tract cancers: a comprehensive meta-analysis

Background

EPHX1 is a key enzyme in metabolizing some exogenous carcinogens such as products of cigarette-smoking. Two functional polymorphisms in the EPHX1 gene, Tyr113His and His139Arg can alter the enzyme activity, suggesting their possible association with carcinogenesis risk, particularly of some tobacco-related cancers.

Methodology/Principal Findings

A comprehensive systematic review and meta-analysis was performed of available studies on these two polymorphisms and cancer risk published up to November 2010, consisting of 84 studies (31144 cases and 42439 controls) for Tyr113His and 77 studies (28496 cases and 38506 controls) for His139Arg primarily focused on lung cancer, upper aerodigestive tract (UADT) cancers (including oral, pharynx, larynx and esophagus cancers), colorectal cancer or adenoma, bladder cancer and breast cancer. Results showed that Y113H low activity allele (H) was significantly associated with decreased risk of lung cancer (OR = 0.88, 95%CI = 0.80–0.96) and UADT cancers (OR = 0.86, 95%CI = 0.77–0.97) and H139R high activity allele (R) with increased risk of lung cancer (OR = 1.18, 95%CI = 1.04–1.33) but not of UADT cancers (OR = 1.05, 95%CI = 0.93–1.17). Pooled analysis of lung and UADT cancers revealed that low EPHX1 enzyme activity, predicted by the combination of Y113H and H139R showed decreased risk of these cancers (OR = 0.83, 95%CI = 0.75–0.93) whereas high EPHX1 activity increased risk of the cancers (OR = 1.20, 95%CI = 0.98–1.46). Furthermore, modest difference for the risk of lung and UADT cancers was found between cigarette smokers and nonsmokers both in single SNP analyses (low activity allele H: OR = 0.77/0.85 for smokers/nonsmokers; high activity allele R: OR = 1.20/1.09 for smokers/nonsmokers) and in combined double SNP analyses (putative low activity: OR = 0.73/0.88 for smokers/nonsmokers; putative high activity: OR = 1.02/0.93 for smokers/ nonsmokers).

Conclusions/Significance

Putative low EPHX1 enzyme activity may have a potential protective effect on tobacco-related carcinogenesis of lung and UADT cancers, whereas putative high EPHX1 activity may have a harmful effect. Moreover, cigarette-smoking status may influence the association of EPHX1 enzyme activity and the related cancer risk.     

Identification of active-site amino acid residues in the Chiba virus 3C-like protease

The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.     

Molecular diagnostics for myelin proteolipid protein gene mutations in Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a clinically heterogeneous, slowly progressive leukodystrophy. The recent detection of mutations in the myelin proteolipid protein (PLP) gene in several PMD patients offers the opportunity both to design DNA-based tests that would be useful in diagnosing a proportion of PMD cases and, in particular, to evaluate the diagnostic utility of single-strand conformation polymorphism (SSCP) analysis for this disease. A combination of SSCP analysis and direct sequencing of PCR-amplified DNA was used to screen for PLP mutations in 24 patients affected with leukodystrophies of unknown etiology. Two heretofore undescribed mutations in the PLP gene were identified, Asp202His in exon 4 and Gly73Arg in exon 3. The ease and efficiency of SSCP analysis in detecting new mutations support the utilization of this technique in screening for PLP mutations in patients with unexplained leukodystrophies.     

Alpha-1 antitrypsin gene polymorphism in Chronic Obstructive Pulmonary Disease (COPD)

Alpha-1-antitrypsin (AAT) plays an important role in the pathogenesis of emphysema, the pathological lesion underlying the majority of the manifestations of Chronic Obstructive Pulmonary Disease (COPD). In this study we tested the hypothesis that common AAT polymorphisms influence the risk of developing COPDs. We investigated PiM1 (Ala213Val), PiM2 (Arg101His), PiM3 (Glu376Asp), PiS (Glu264Val) and PiZ (Glu342Lys) SERPINA1 alleles in 100 COPD patients and 200 healthy controls. No significant differences were observed in allele frequencies between COPD patients and controls, neither did haplotype analysis show significant differences between the two groups. A cross-sectional study revealed no significant relationship between common SERPINA1 polymorphisms (PiM1, PiM2, PiM3) and the emphysematous type of COPD. In addition, FEV(1) annual decline, determined during a two-year follow up period, revealed no difference among carriers of the tested polymorphisms.     

Molecular characterization of genetic mutations in human lactate dehydrogenase (LDH) B (H) variant

Summary We have previously detected a single base substitution of G by A at the Arg codon CGC in exon 4 of the mutant lactate dehydrogenase (LDH) gene, an unstable LDH-B variant (case 1). Here, we use the polymerase chain reaction (PCR) to amplify genomic DNA of two cases (the original case 1 and a new patient, case 2). We were able to confirm that case 1 is homozygous for the mutation, causing a replacement of the conserved Arg by His at residue 173. The resulting LDH-B variant subunit is unstable in vivo. Whereas the mutation in exon 4 was not observed in case 2, a different single base substitution of A by C was detected at the Ser codon AGT in exon 3. This mutation causes a replacement of the conserved Ser by Arg at residue 131. Genomic analysis of the family of case 2 by mismatched PCR showed that the missense mutation was consistent with their biochemical phenotypes. The replacement results in a conformational change of the residues near the Ser, probably because the side chain of Arg is much more bulky than that of Ser. The change may affect the arrangement of the cofactor binding site and result in the loss of enzyme activity. The experimental observations are consistent with computer graphics analyses.     

Major mutation p.His281Tyr in Gene <Emphasis Type="Italic">GLB1</Emphasis> in patients with GM1-gangliosidosis in Ukraine

Mutation analysis of gene GLB1 in 26 patients from different regions of Ukraine diagnosed with GM1-gangliosidosis identified 15 pathogenic mutations. The most common was missense mutation p.His281Tyr (c.841C>T) in exon 8, which was found in at least one allele in 16 of 26 patients (61.5%), accounting for 20 of 52 (38.5%) mutant alleles. Mutation p.His281Tyr (c.841C>T) was described in rare cases in patients from Germany, Italy, and Portugal, while no alleles with such a replacement were found in other countries. This may indicate a much higher frequency of this mutation in Ukraine compared to other countries. Analysis of the information about the spatial distribution of the families with mutation p.His281Tyr (c.841C>T) revealed that most of the alleles with this mutation (14 of 20 or 70%) were found in patients from the western regions of the country, which may be due to the founder effect.     

A simple method for examination of polymorphisms of catalase exon 9: rs769217 in Hungarian microcytic anemia and beta-thalassemia patients

Catalase decreases the high, toxic concentrations of hydrogen peroxide but it lets the physiological, low concentrations in the cells mainly for signaling purposes. Its decreased activity may contribute to development of several pathological conditions. Catalase mutations occur frequently in exon 9, these were examined with different, complicated and costly methods. The aim of the current study was to evaluate a method for screening of polymorphisms in catalase exon 9. We used the slab gel electrophoresis of PCR amplicons without denaturation and silver staining for visualization of the DNA bands. We detected extra DNA bands in the 400-800 bp region of the catalase exon 9. Their single stranded nature was proved with nucleotide sequence analyses, comparison with the standard SSCP, staining with Sybr Green II and Sybr Green I, ethidium bromide, no digestion with RFLP (BstX I), and digestion with plant nuclease. We used this method for examination of polymorphisms of catalase exon 9 in microcytic anemia and beta-thalassemia patients. The lowest blood catalase activities were detected in microcytic anemia and beta-thalassemia patients with the TT genotypes of the C111T polymorphism. This method was sensitive for detection of G113A acatalasemia mutation, but poorly detected C37T and G5A acatalasemia mutations.     

Multiple analytical approaches reveal distinct gene-environment interactions in smokers and non smokers in lung cancer

Complex disease such as cancer results from interactions of multiple genetic and environmental factors. Studying these factors singularly cannot explain the underlying pathogenetic mechanism of the disease. Multi-analytical approach, including logistic regression (LR), classification and regression tree (CART) and multifactor dimensionality reduction (MDR), was applied in 188 lung cancer cases and 290 controls to explore high order interactions among xenobiotic metabolizing genes and environmental risk factors. Smoking was identified as the predominant risk factor by all three analytical approaches. Individually, CYP1A1*2A polymorphism was significantly associated with increased lung cancer risk (OR = 1.69;95%CI = 1.11–2.59,p = 0.01), whereas EPHX1 Tyr113His and SULT1A1 Arg213His conferred reduced risk (OR = 0.40;95%CI = 0.25–0.65,p<0.001 and OR = 0.51;95%CI = 0.33–0.78,p = 0.002 respectively). In smokers, EPHX1 Tyr113His and SULT1A1 Arg213His polymorphisms reduced the risk of lung cancer, whereas CYP1A1*2A, CYP1A1*2C and GSTP1 Ile105Val imparted increased risk in non-smokers only. While exploring non-linear interactions through CART analysis, smokers carrying the combination of EPHX1 113TC (Tyr/His), SULT1A1 213GG (Arg/Arg) or AA (His/His) and GSTM1 null genotypes showed the highest risk for lung cancer (OR = 3.73;95%CI = 1.33–10.55,p = 0.006), whereas combined effect of CYP1A1*2A 6235CC or TC, SULT1A1 213GG (Arg/Arg) and betel quid chewing showed maximum risk in non-smokers (OR = 2.93;95%CI = 1.15–7.51,p = 0.01). MDR analysis identified two distinct predictor models for the risk of lung cancer in smokers (tobacco chewing, EPHX1 Tyr113His, and SULT1A1 Arg213His) and non-smokers (CYP1A1*2A, GSTP1 Ile105Val and SULT1A1 Arg213His) with testing balance accuracy (TBA) of 0.6436 and 0.6677 respectively. Interaction entropy interpretations of MDR results showed non-additive interactions of tobacco chewing with SULT1A1 Arg213His and EPHX1 Tyr113His in smokers and SULT1A1 Arg213His with GSTP1 Ile105Val and CYP1A1*2C in nonsmokers. These results identified distinct gene-gene and gene environment interactions in smokers and non-smokers, which confirms the importance of multifactorial interaction in risk assessment of lung cancer.     

Polymorphisms in human soluble epoxide hydrolase: effects on enzyme activity, enzyme stability, and quaternary structure

Human soluble epoxide hydrolase (hsEH) has been shown to play a role in regulating blood pressure and inflammation. HsEH consists of an N-terminal phosphatase and a C-terminal epoxide hydrolase domain. In the present study, we examined the effects of polymorphisms in the hsEH gene on phosphatase activity, enzyme stability, and protein quaternary structure. The results showed that mutants Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the Arg103Cys/Arg287Gln (double mutant) have significantly lower phosphatase activity compared to the most frequent allele (MFA) of hsEH. In addition, the Lys55Arg, Arg103Cys, Cys154Tyr, Arg287Gln, and the double mutant have significantly lower kcat/Km values. The stabilities at 37 degrees C of purified Arg287Gln and Arg103Cys/Arg287Gln mutants were also significantly reduced compared to the MFA. HPLC size-exclusion studies showed that the MFA exists predominantly as a dimer. However, the Arg287Gln and Arg103Cys/Arg287Gln mutants show increased concentration of the monomer. We conclude that the Arg287Gln polymorphism disrupts putative intra- and inter-monomeric salt-bridges responsible for dimerization.     

Analysis of the transforming growth factor-beta1 (TGF-beta1) codon 25 gene polymorphism by LightCycler-analysis in patients with chronic hepatitis C infection

The polymorphism at position 25 of the gene encoding transforming growth factor-beta1 (TGF-beta1), which changes the amino acid sequence of the signal peptide sequence (arginine to proline), is causing a variation in TGF-beta1 production. The homozygous genotype (Arg25Arg) is associated with higher TGF-beta1 production than the heterozygous (Arg25Pro) genotype. Therefore, the possible involvement of this genetic variation in the TGF-beta1 gene for induction and progression of various diseases is under close investigation. At present, several labor-intensive established assays ranging from amplification refractory mutation system (ARMS)-PCR methodologies, sequence specific oligonucleotide probing (SSOP), restriction fragment length polymorphism (RFLP) analysis, 5' nuclease assays, and specialized fingerprinting protocols are applied to analyze the polymorphism in question. We developed a novel approach for analyzing this polymorphism in a LightCycler system and determined the allele frequency distributions between patients with different degrees of hepatic fibrosis induced by chronic hepatitis C virus infection. In patients with severe hepatic fibrosis (METAVIR-score 3-4), the Pro25 allele was twice as frequent compared to patients with mild fibrosis (METAVIR-score 0-2). However, we found no association of necroinflammatory activity and genotype distribution. This suggests that the stage of hepatic fibrosis, rather than the grade (inflammation), is influenced by the presence of proline at codon 25 in patients with chronic hepatitis C.     

Effects on sister chromatid exchange frequency of polymorphisms in DNA repair gene XRCC1 in smokers

The association between metabolic polymorphisms and cigarette smoking-induced cancers has been documented. However, the role of DNA repair polymorphism in carcinogenesis is less clear. To investigate if the polymorphisms of metabolic traits and DNA repair modulate smoking-related DNA damage, we used sister chromatid exchange (SCE) as a marker of genetic damage to explore the relationship of microsomal epoxide hydrolase (mEH), glutathione S-transferase M1 (GSTM1), and X-ray cross-complementing group 1 (XRCC1) and cigarette smoking-induced SCE. Sixty-one workers without significant exposure to mutagens were recruited. Questionnaires were completed to obtain detailed occupational, smoking, and medical histories. SCE frequency in peripheral lymphocytes was determined using a standard cytogenetic assay and GSTM1, mEH (exons 3 and 4), XRCC1 (codon 399) genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP). Smokers had higher SCE frequency than non-smokers (8.4 versus 7.1, P<0.05). Among workers who had smoked equal to or greater than 10 cigarettes each day, those with XRCC1 Arg/Gln+Gln/Gln had higher SCE frequency than those with XRCC1 Arg/Arg after adjusting for potential confounders (9.0 versus 7.9, P<0.05). The interaction of XRCC1 and cigarettes smoked per day on SCE frequency was also observed (P=0.02). There was no significant interaction between cigarettes smoked per day with GSTM1 and mEH on SCE frequency. Our results support previous epidemiological studies that XRCC1 may play a role in cigarette smoking-induced lung cancer.     

New mutant gene (transthyretin Arg 58) in cases with hereditary polyneuropathy detected by non-isotope method of single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) was analyzed to detect a mutation in the transthyretin (TTR) gene from the mother and son showing polyneuropathy with carpal tunnel syndrome. DNA segments containing TTR coding sequence were amplified by polymerase chain reaction, heat denatured and electrophoresed on a neutral polyacrylamide gel. The single-stranded DNA fragments in the gel were transferred to a nylon membrane and hybridized with biotinylated TTR cDNA probe, followed with chemiluminescent DNA detection. The mobility shift was found in the fragments of exon 3 from the patients' DNA. Sequencing analyses of the exon 3 confirmed a T----G base change, resulting in a Leu 58----Arg substitution. TTR Arg 58 is the first mutant TTR gene that has been detected by SSCP analysis. The rapid and sensitive detection of new mutations at various sites on the TTR gene is hereafter possible by the present method in the facilities for non-radioactive experiments.     

A novel mutant (transthyretin Ile-50) related to amyloid polyneuropathy. Single-strand conformation polymorphism as a new genetic marker.

DNA sequence polymorphisms in transthyretin (TTR) genes were investigated by single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products. The amplified DNA fragments that encode each exon of the normal TTR gene showed two bands, representing the two complementary single strands of DNA. In one patient with amyloid polyneuropathy, the exon 3 DNA showed a unique, aberrant migration pattern. Direct sequencing analysis of the amplified exon 3 revealed a single base change (G-to-T), resulting in a novel amino acid substitution (Ser-50----Ile). We also present the SSCP patterns for five known Japanese TTR variants.