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Background

Glutathione S-transferase omega-1 and 2 genes (GSTO1, GSTO2), residing within an Alzheimer and Parkinson disease (AD and PD) linkage region, have diverse functions including mitigation of oxidative stress and may underlie the pathophysiology of both diseases. GSTO polymorphisms were previously reported to associate with risk and age-at-onset of these diseases, although inconsistent follow-up study designs make interpretation of results difficult. We assessed two previously reported SNPs, GSTO1 rs4925 and GSTO2 rs156697, in AD (3,493 ADs vs. 4,617 controls) and PD (678 PDs vs. 712 controls) for association with disease risk (case-controls), age-at-diagnosis (cases) and brain gene expression levels (autopsied subjects).

Results

We found that rs156697 minor allele associates with significantly increased risk (odds ratio = 1.14, p = 0.038) in the older ADs with age-at-diagnosis > 80 years. The minor allele of GSTO1 rs4925 associates with decreased risk in familial PD (odds ratio = 0.78, p = 0.034). There was no other association with disease risk or age-at-diagnosis. The minor alleles of both GSTO SNPs associate with lower brain levels of GSTO2 (p = 4.7 × 10-11-1.9 × 10-27), but not GSTO1. Pathway analysis of significant genes in our brain expression GWAS, identified significant enrichment for glutathione metabolism genes (p = 0.003).

Conclusion

These results suggest that GSTO locus variants may lower brain GSTO2 levels and consequently confer AD risk in older age. Other glutathione metabolism genes should be assessed for their effects on AD and other chronic, neurologic diseases.

We previously reported a linkage region on chromosome 10q for age-at-onset (AAO) of Alzheimer (AD) and Parkinson (PD) diseases. Glutathione S-transferase, Omega-1 (GSTO1) and the adjacent gene GSTO2, located in this linkage region, were then reported to associate with AAO of AD and PD. To examine whether GSTO1 and GSTO2 (hereafter referred to as GSTO1h) are responsible for the linkage evidence, we identified 39 families in AD that lead to our previous linkage and association findings. The evidence of linkage and association was markedly diminished after removing these 39 families from the analyses, thus providing support that GSTO1h drives the original linkage results. The maximum average AAO delayed by GSTO1h SNP 7-1 (rs4825, A nucleotide) was 6.8 (± 4.41) years for AD and 8.6(± 5.71) for PD, respectively. This is comparable to the magnitude of AAO difference by APOE-4 in these same AD and PD families. These findings suggest the presence of genetic heterogeneity for GSTO1h’s effect on AAO, and support GSTO1h’s role in modifying AAO in these two disorders.

Background: Glutathione S-transferase Omega has been shown to be associated with Parkinson disease.

Results: Drosophila GSTO1 regulates mitochondrial ATP synthase activity in parkin mutants.

Conclusion: Drosophila GSTO1 plays a protective role in a Drosophila model of Parkinson disease.

Significance: These findings may lead to a better understanding of the molecular mechanism of neuroprotection due to GSTO in Parkinson disease.

A loss-of-function mutation in the gene parkin causes a common neurodegenerative disease that may be caused by mitochondrial dysfunction. Glutathione S-transferase Omega (GSTO) is involved in cell defense mechanisms, but little is known about the role of GSTO in the progression of Parkinson disease. Here, we report that restoration of Drosophila GSTO1 (DmGSTO1), which is down-regulated in parkin mutants, alleviates some of the parkin pathogenic phenotypes and that the loss of DmGSTO1 function enhances parkin mutant phenotypes. We further identified the ATP synthase β subunit as a novel in vivo target of DmGSTO1. We found that glutathionylation of the ATP synthase β subunit is rescued by DmGSTO1 and that the expression of DmGSTO1 partially restores the activity and assembly of the mitochondrial F1F0-ATP synthase in parkin mutants. Our results suggest a novel mechanism for the protective role of DmGSTO1 in parkin mutants, through the regulation of ATP synthase activity, and provide insight into potential therapies for Parkinson disease neurodegeneration.

Background

The major contribution to oxidant related lung damage in COPD is from the oxidant/antioxidant imbalance and possibly impaired antioxidant defence. Glutathione (GSH) is one of the most important antioxidants in human lung and lung secretions, but the mechanisms participating in its homeostasis are partly unclear. Glutathione-S-transferase omega (GSTO) is a recently characterized cysteine containing enzyme with the capability to bind and release GSH in vitro. GSTO has not been investigated in human lung or lung diseases.

Methods

GSTO1-1 was investigated by immunohistochemistry and Western blot analysis in 72 lung tissue specimens and 40 sputum specimens from non-smokers, smokers and COPD, in bronchoalveolar lavage fluid and in plasma from healthy non-smokers and smokers. It was also examined in human monocytes and bronchial epithelial cells and their culture mediums in vitro.

Results

GSTO1-1 was mainly expressed in alveolar macrophages, but it was also found in airway and alveolar epithelium and in extracellular fluids including sputum supernatants, bronchoalveolar lavage fluid, plasma and cell culture mediums. The levels of GSTO1-1 were significantly lower in the sputum supernatants (p = 0.023) and lung homogenates (p = 0.003) of COPD patients than in non-smokers.

Conclusion

GSTO1-1 is abundant in the alveolar macrophages, but it is also present in extracellular fluids and in airway secretions, the levels being decreased in COPD. The clinical significance of GSTO1-1 and its role in regulating GSH homeostasis in airway secretions, however, needs further investigations.

Background

Huntington disease (HD) is a fully penetrant, autosomal dominantly inherited disorder associated with abnormal expansions of a stretch of perfect CAG repeats in the 5' part of the IT15 gene. The number of repeat units is highly predictive for the age at onset (AO) of the disorder. But AO is only modestly correlated with repeat length when intermediate HD expansions are considered. Recently, suggestive association has been reported between a single nucleotide polymorphism (SNP; rs1801131, also known as A1298C) in the methyltetrahydrofolate reductase (MTHFR) gene and AO of HD. 5,10-MTHFR is a key enzyme in the folate metabolism, diverting metabolites toward methylation reactions or nucleotide synthesis. Using part of a previously established study cohort plus additional patients and appropriate statistical methods, we reinvestigated two polymorphisms in the MTHFR gene, C677T and A1298C, as well as their association with AO in 167 HD patients.

Results

There was no statistically significant impact on AO for HD patients, neither of MTHFR SNPs nor of the combinations thereof.

Conclusion

Contrary to previously described evidence the A1298C polymorphism in the MTHFR gene does not appear to modulate AO of HD patients.

Background

Glutathione S-transferases (GSTs) is a genetic factor for many diseases and exhibits great diversities among various populations. We assessed association of the genotypes of Glutathione S-transferases Omega-1 (GSTO1) A140D with ethnicity in China.

Results

Peripheral blood samples were obtained from 1314 individuals from 14 ethnic groups. Polymorphisms of GSTO1 A140D were measured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Logistic regression was employed to adjustment for regional factor. The frequency of GSTO1 140A allele was 15.49% in the total 14 ethnic populations. Compared to Han ethnic group, two ethnic populations were more likely to have AA or CA genotype [odds ratio (OR): 1.77, 95% confidence interval (95% CI): 1.05–2.98 for Uygur and OR: 1.78, 95% CI: 1.18–2.69 for Hui]. However, there were no statistically significant differences across 14 ethnic groups when region factor was adjusted. In Han ethnicity, region was significantly associated with AA or CA genotype. Han individuals who resided in North-west of China were more likely to have these genotypes than those in South of China (OR: 1.63, 95% CI: 1.21–2.20).

Conclusion

The prevalence of the GSTO1 140A varied significantly among different regional populations in China, which showed that geography played a more important role in the population differentiation for this allele than the ethnicity/race.

Background

Individual variability in arsenic metabolism may underlie individual susceptibility toward arsenic-induced skin lesions and skin cancer. Metabolism of arsenic proceeds through sequential reduction and oxidative methylation being mediated by the following genes: purine nucleoside phosphorylase (PNP), arsenic (+3) methyltransferase (As3MT), glutathione S-transferase omega 1 (GSTO1), and omega 2 (GSTO2). PNP functions as arsenate reductase; As3MT methylates inorganic arsenic and its metabolites; and both GSTO1 and GSTO2 reduce the metabolites. Alteration in functions of these gene products may lead to arsenic-specific disease manifestations.

Objectives

To find any probable association between arsenicism and the exonic single nucleotide polymorphisms (SNPs) of the above-mentioned arsenic-metabolizing genes, we screened all the exons in those genes in an arsenic-exposed population.

Methods

Using polymerase chain reaction restriction fragment length polymorphism analysis, we screened the exons in 25 cases (individuals with arsenic-induced skin lesions) and 25 controls (individuals without arsenic-induced skin lesions), both groups drinking similar arsenic-contaminated water. The exonic SNPs identified were further genotyped in a total of 428 genetically unrelated individuals (229 cases and 199 controls) for association study.

Results

Among four candidate genes, PNP, As3MT, GSTO1, and GSTO2, we found that distribution of three exonic polymorphisms, His20His, Gly51Ser, and Pro57Pro of PNP, was associated with arsenicism. Genotypes having the minor alleles were significantly overrepresented in the case group: odds ratio (OR) = 1.69 [95% confidence interval (CI), 1.08–2.66] for His20His; OR = 1.66 [95% CI, 1.04–2.64] for Gly51Ser; and OR = 1.67 [95% CI, 1.05–2.66] for Pro57Pro.

Conclusions

The results indicate that the three PNP variants render individuals susceptible toward developing arsenic-induced skin lesions.

The susceptibility to arsenic-induced diseases differs greatly between individuals, possibly due to interindividual variations in As metabolism that affect retention and distribution of toxic metabolites. To elucidate the role of genetic factors in As metabolism, we studied how polymorphisms in six genes affected the urinary metabolite pattern in a group of indigenous women (n = 147) in northern Argentina who were exposed to approximately 200 μg/L As in drinking water. These women had low urinary percentages of monomethylated As (MMA) and high percentages of dimethylated As (DMA). MMA has been associated with adverse health effects, and DMA has the lowest body retention of the metabolites. The genes studied were arsenic(+III)methyltransferase (AS3MT), glutathione S-transferase omega 1 (GSTO1), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), methylenetetrahydrofolate reductase (MTHFR), and glutathione S-transferases mu 1 (GSTM1) and theta 1 (GSTT1). We found three intronic polymorphisms in AS3MT (G12390C, C14215T, and G35991A) associated with a lower percentage of MMA (%MMA) and a higher percentage of DMA (%DMA) in urine. The variant homozygotes showed approximately half the %MMA compared with wild-type homozygotes. These polymorphisms were in strong linkage, with high allelic frequencies (72–76%) compared with other populations. We also saw minor effects of other polymorphisms in the multivariate regression analysis with effect modification for the deletion genotypes for GSTM1 (affecting %MMA) and GSTT1 (affecting %MMA and %DMA). For pregnant women, effect modification was seen for the folate-metabolizing genes MTR and MTHFR. In conclusion, these findings indicate that polymorphisms in AS3MT—and possibly GSTM1, GSTT1, MTR, and MTHFR—are responsible for a large part of the interindividual variation in As metabolism and susceptibility.

Purpose

To investigate whether genetic polymorphisms of glutathione S-transferases (GSTM1, GSTT1, and GSTO2) in relation to the work place contribute to the development of cataract.

Methods

The present case-control study consisted of 186 patients (108 females, 78 males) with cataract and 195 gender-matched healthy controls (111 females, 84 males) were randomly selected from unrelated volunteers in the same clinic. The GSTM1, GSTT1, and GSTO2 genotypes were determined using polymerase chain reaction (PCR) based methods.

Results

The null genotype of GSTM1 increased the risk of cataract (OR=1.51, 95%CI: 1.01–2.26, p=0.045). The prevalence of GSTT1 and GSTO2 genotypes was similar between cases and controls. There was significant difference between cases and controls for work place (χ2=4.16, df=1, p=0.041). Genetic polymorphisms (GSTM1, GSTO2) and work place that were significant by p<0.3 in the univariate analysis were included in the analysis for investigating the additive effects of the genotypes and work place on risk of cataract. Statistical analysis showed that the risk of cataract increased as a function of number of putative high risk factors (χ2=8.001, p=0.005).

Conclusions

This finding suggests that the polymorphisms of GSTM1 and GSTO2 and also work place may act additively for developing cataract.

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches.

Author Summary

Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal CAG expansion in the Huntingtin gene (HTT), resulting in an expanded polyglutamine track in the HTT protein. Longer CAG expansions correlate with an earlier more severe manifestation of the disease that produces choreic movement, behavioural and psychiatric disturbances, and dementia. Although the causative gene is widely expressed, neuropathology is characterized by striatal and cortical atrophy. HTT interacts with proteins involved in transcription, cell signaling, and transport. The pathogenic role of mutant HTT is not fully understood. This study shows that CAG expanded HTT RNA also contributes to neuronal toxicity. Mutant HTT RNA gives rise to small CAG-repeated RNAs (sCAGs) with neurotoxic activity. These short RNAs interfere with cell functions by silencing the expression of genes that are fully or partially complementary, through a mechanism similar to that of microRNAs. These findings suggest that a small RNA–dependent mechanism may contribute to HD neuronal cell loss. The exhaustive identification of the target genes modulated by sCAGs may lead to a better understanding of HD pathology, allowing the development of new therapeutic strategies.

Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC50 = 21 nM) and in situ (IC50 = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in the detoxification of chemo-therapeutic agents

S-(Phenacyl)glutathione reductase (SPG-R) plays a significant role in the biotransformation of reactive α-haloketones to non-toxic acetophenones. Comparison of the apparent subunit size, amino-acid composition, and catalysis of the reduction of S-(phenacyl)glutathiones indicated that a previously described rat SPG-R (Kitada et al. (1985) J. Biol. Chem. 260,11749-11754) is homologous to the omega-class glutathione transferase GSTO1-1. The available data show that the SPG-R reaction is catalyzed by GSTO1-1 and not by other GSTs, including the closely related GSTO2-2 isoenzyme. In the proposed reaction mechanism, the active-site cysteine residue of GSTO1-1 reacts with the S-(phenacyl)glutathione substrate to give an acetophenone and a mixed disulfide with the active-site cysteine; a second thiol substrate (e.g., glutathione or 2-mercaptoethanol) reacts with the active-site disulfide to regenerate the catalytically active enzyme and to form a mixed disulfide. A new spectrophotometric assay was developed that allows the rapid determination of SPG-R activity and specific measurement of GSTO1-1 in the presence of other GSTs. This is the first specific reaction attributed to GSTO1-1, and these results demonstrate the catalytic diversity of GSTO1-1, which, in addition to SPG-R activity, catalyzes the reduction of dehydroascorbate and monomethylarsonate (V) and also possesses thioltransferase and GST activity.

Background

The Inflammasomes are multi-protein complexes that regulate caspase-1 activation and the production of the pro-inflammatory cytokine IL-1β. Previous studies identified a class of diarylsulfonylurea containing compounds called Cytokine Release Inhibitory Drugs (CRIDs) that inhibited the post-translational processing of IL-1β. Further work identified Glutathione S-Transferase Omega 1 (GSTO1) as a possible target of these CRIDs. This study aimed to investigate the mechanism of the inhibitory activity of the CRID CP-456,773 (termed CRID3) in light of recent advances in the area of inflammasome activation, and to clarify the potential role of GSTO1 in the regulation of IL-1β production.

Methodology and Results

In murine bone marrow derived macrophages, CRID3 inhibited IL-1β secretion and caspase 1 processing in response to stimulation of NLRP3 and AIM2 but not NLRC4. CRID3 also prevented AIM2 dependent pyroptosis in contrast to the NLRP3 inhibitors glyburide and parthenolide, which do not inhibit AIM2 activation. Confocal microscopy and Western blotting assays indicated that CRID3 inhibited the formation of ASC complexes or ‘specks’ in response to NLRP3 and AIM2 stimulation. Co-immunoprecipitation assays show that GSTO1 interacted with ASC.

Significance

These results identify CRID3 as a novel inhibitor of the NLRP3 and AIM2 inflammasomes and provide an insight into the mechanism of action of this small molecule. In addition GSTO1 may be a component of the inflammasome that is required for ASC complex formation.

Variation in individual susceptibility to arsenic-induced disease may be partially explained by genetic differences in arsenic metabolism. Mounting epidemiological evidence and in vitro studies suggest that methylated arsenic metabolites, particularly monomethylarsonic (MMA3), are more acutely toxic than inorganic arsenic; thus, MMA3 may be the primary toxic arsenic species. To test the role of genetic variation in arsenic metabolism, polymorphisms in genes involved in one-carbon metabolism [methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), cystathionine-β-synthase (CBS), thymidylate synthase (TYMS), dihydrofolate reductase (DHFR), serine hydroxymethyltransferase 1 (SHMT1] and glutathione biosynthesis [glutathione S-transferase omega 1 (GSTO1)] were examined in an arsenic exposed population to determine their influence in urinary arsenic metabolite patterns. In 142 subjects in Cordoba Province, Argentina, variant genotypes for CBS rs234709 and rs4920037 SNPs compared with wild-type homozygotes were associated with 24% and 26% increases, respectively, in the mean proportion of arsenic excreted as monomethylarsonic acid (%MMA). This difference is within the range of differences in %MMA seen between people with arsenic-related disease and those without such disease in other studies. Small inverse associations with CBS rs234709 and rs4920037 variants were also found for the mean levels of the proportion of arsenic excreted as dimethylarsinous acid (%DMA). No other genetic associations were found. These findings are the first to suggest that CBS polymorphisms may influence arsenic metabolism in humans and susceptibility to arsenic-related disease.

Individual variability in human arsenic metabolism has been reported frequently in the literature. This variability could be an underlying determinant of individual susceptibility to arsenic-induced disease in humans. Recent analysis revealing familial aggregation of arsenic metabolic profiles suggests that genetic factors could underlie interindividual variation in arsenic metabolism. We screened two genes responsible for arsenic metabolism, human purine nucleoside phosphorylase (hNP), which functions as an arsenate reductase converting arsenate to arsenite, and human glutathione S-transferase omega 1-1 (hGSTO1-1), which functions as a monomethylarsonic acid (MMA) reductase, converting MMA(V) to MMA(III), to develop a comprehensive catalog of commonly occurring genetic polymorphisms in these genes. This catalog was generated by DNA sequencing of 22 individuals of European ancestry (EA) and 24 individuals of indigenous American (IA) ancestry. In (Italic)hNP(/Italic), 48 polymorphic sites were observed, including 6 that occurred in exons, of which 1 was nonsynonymous (G51S). One intronic polymorphism occurred in a known enhancer region. In hGSTO1-1, 33 polymorphisms were observed. Six polymorphisms occurred in exons, of which 4 were nonsynonymous. In contrast to hNP, in which the IA group was more polymorphic than the EA group, in hGSTO1-1 the EA group was more polymorphic than the IA group, which had only 1 polymorphism with a frequency > 10%. Populations representing genetic admixture between the EA and IA groups, such as Mexican Hispanics, could vary in the extent of polymorphism in these genes based upon the extent of admixture. These data provide a framework in which to conduct genetic association studies of these two genes in relevant populations, thereby allowing hNP and hGSTO1-1 to be evaluated as potential susceptibility genes in human arsenicism.

At least seven adult-onset neurodegenerative diseases, including Huntington’s disease (HD), are caused by genes containing expanded CAG triplets within their coding regions. The expanded CAG repeats give rise to extended stretches of polyglutamines (Qn) in the proteins expressed by the affected genes. Generally, n ≥40 in affected individuals and ≤36 in clinically unaffected individuals. The expansion has been proposed to confer a “toxic gain of function” to the mutated protein. Poly-Q domains have recently been shown to be excellent substrates of tissue transglutaminase. We investigated the effects of expression of glutathione S-transferase constructs containing poly-Q inserts of various lengths (GSTQn where n = 0, 10, 62 or 81) on the activity of some key metabolic enzymes in the host Escherischia coil-an organism not known to have transglutaminase activity. E. coil carrying the GSTQ62 construct exhibited statistically significant decreases in the specific activities of α-ketoglutarate dehydrogenase complex (KGDHC) and pyruvate dehydrogenase complex (PDHC). Previous work has shown that KGDHC and PDHC activities are reduced in the brains of Alzheimer’s disease (AD) patients. Our results suggest that KGDHC and PDHC may be particularly susceptible to the effects of a number of disparate insults, including those associated with AD and HD.

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by an expansion of CAG repeats in the IT15 gene. The age-at-onset (AAO) of HD is inversely related to the CAG repeat length and the minimum length thought to cause HD is 36. Accurate estimation of the AAO distribution based on CAG repeat length is important for genetic counseling and the design of clinical trials. In the Cooperative Huntington’s Observational Research Trial (COHORT) study, the CAG repeat length is known for the proband participants. However, whether a family member shares the huntingtin gene status (CAG expanded or not) with the proband is unknown. In this work, we use the expectation-maximization (EM) algorithm to handle the missing huntingtin gene information in first-degree family members in COHORT, assuming that a family member has the same CAG length as the proband if the family member carries a huntingtin gene mutation. We perform simulation studies to examine performance of the proposed method and apply the methods to analyze COHORT proband and family combined data. Our analyses reveal that the estimated cumulative risk of HD symptom onset obtained from the combined data is slightly lower than the risk estimated from the proband data alone.

Huntington disease (HD) is a dominantly transmitted neurodegenerative disorder that arises from expansion of a CAG trinucleotide repeat on chromosome 4p16.3. CAG repeat allele lengths are defined as fully penetrant at ≥ 40, reduced penetrance at 36–39, high normal at 27–35, and normal at ≤ 26. Fathers, but not mothers, with high normal alleles are at risk of transmitting potentially penetrant HD alleles (≥ 36) to offspring. We estimated the conditional probability of an offspring inheriting an expanded penetrant allele given a father with a high normal allele by applying probability definitions and rules to estimates of HD incidence, paternal birth rate, frequency of de novo HD, and frequency of high normal alleles in the general population. The estimated probability that a male high normal allele carrier will have an offspring with an expanded penetrant allele ranges from 1/6241 to 1/951. These estimates may be useful in genetic counseling for male high normal allele carriers.

Objectives

To identify and validate the biological significance of new genes/ proteins involved in the development of allergic airway disease in a murine asthma model.

Methods

Gene microarrays were used to identify genes with at least a 2-fold increase in gene expression in lungs of two separate mouse strains with high and low allergic susceptibility, respectively. Validation of mRNA data was obtained by western blotting and immunohistochemistry, followed by functional analysis of one of the identified genes in mice with targeted disruption of specific gene expression.

Results

Expression of two antioxidant enzymes, glutathione peroxidase-2 (Gpx-2) and glutathione-S-transferase Omega (GSTO) 1-1 was increased in both mouse strains after induction of allergic airway disease and localized in lung epithelial cells. Mice with targeted disruption of the Gpx-2 gene showed significantly enhanced airway inflammation compared to sensitized and challenged wild-type mice.

Conclusion

Our data indicate that genes encoding the antioxidants Gpx-2 and GSTO 1-1 are common inflammatory genes expressed upon induction of allergic airway inflammation, independently of allergic susceptibility. Furthermore, we provide evidence to illustrate the importance of a single antioxidant enzyme, Gpx-2, in protection from allergen-induced disease.

Objective: The ε4 allele of the apolipoprotein E (ApoE) gene has been defined as a critical factor for early onset neurodegeneration in Pick's, Parkinson's, and Alzheimer's disease. Unexpectedly, the ε4 allele appeared to delay the age of onset in Huntington's disease (HD) patients. Furthermore, sex specific effects were reported on earlier age of onset due to the ApoE ε2ε3 genotype in males with HD. The age of onset of HD is known to be negatively correlated with increasing lengths of pathogenetic CAG expansions in the huntingtin gene.

Methods: In order to examine the effects of CAG block lengths, we have correlated ApoE genotypes with the age of onset in 145 patients symptomatic for HD with psychiatric and somatic symptoms (depression, psychosis, dementia, choreic, and other movement disorders) harbouring only modestly expanded huntingtin alleles (41–45 CAGs).

Results: The negative correlation between age of onset and CAG block length was established in our HD cohort. Statistically significant effects of the ε4 allele were not obvious regarding clinical characteristics including age of onset, nor were any sex differences for the ε2ε3 genotype observed.

Conclusion: The ApoE genotype does not affect the course of HD significantly.

Background

Cerebral ischemia involves a series of reactions which ultimately influence the final volume of a brain infarction. We hypothesize that polymorphisms in genes encoding proteins involved in these reactions could act as modifiers of the cerebral response to ischemia and impact the resultant stroke volume. The final volume of a cerebral infarct is important as it correlates with the morbidity and mortality associated with non-lacunar ischemic strokes.

Methods

The proteins encoded by the methylenetetrahydrofolate reductase (MTHFR) and glutathione S-transferase omega-1 (GSTO-1) genes are, through oxidative mechanisms, key participants in the cerebral response to ischemia. On the basis of these biological activities, they were selected as candidate genes for further investigation. We analyzed the C677T polymorphism in the MTHFR gene and the C419A polymorphism in the GSTO-1 gene in 128 patients with non-lacunar ischemic strokes.

Results

We found no significant association of either the MTHFR (p = 0.72) or GSTO-1 (p = 0.58) polymorphisms with cerebral infarct volume.

Conclusion

Our study shows no major gene effect of either the MTHFR or GSTO-1 genes as a modifier of ischemic stroke volume. However, given the relatively small sample size, a minor gene effect is not excluded by this investigation.

Huntington’s disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant inheritance. The disease is caused by a CAG trinucleotide repeat expansion located in the first exon of the HD gene. The CAG repeat is highly polymorphic and varies from 6 to 37 repeats on chromosomes of unaffected individuals and from more than 30 to 180 repeats on chromosomes of HD patients. In this study, we show that the number of CAG repeats in the HD gene can be determined by restriction of the DNA with the endonuclease EcoP15I and subsequent analysis of the restriction fragment pattern by electrophoresis through non-denaturing polyacrylamide gels using the ALFexpress DNA Analysis System. CAG repeat numbers in the normal (30 and 35 repeats) as well as in the pathological range (81 repeats) could be accurately counted using this assay. Our results suggest that this high-resolution method can be used for the exact length determination of CAG repeats in HD genes as well as in genes affected in related CAG repeat disorders.

Background

There is a wide variation in susceptibility to health effects of arsenic, which, in part, may be due to differences in arsenic metabolism. Arsenic is metabolized by reduction and methylation reactions, catalyzed by reductases and methyltransferases.

Objectives

Our goal in this study was to elucidate the influence of various demographic and genetic factors on the metabolism of arsenic.

Methods

We studied 415 individuals from Hungary, Romania, and Slovakia by measuring arsenic metabolites in urine using liquid chromatography with hydride generation and inductively coupled plasma mass spectrometry (HPLC-HG-ICPMS). We performed genotyping of arsenic (+III) methyltransferase (AS3MT), glutathione S-transferase omega 1 (GSTO1), and methylene-tetrahydrofolate reductase (MTHFR).

Results

The results show that the M287T (T→C) polymorphism in the AS3MT gene, the A222V (C→T) polymorphism in the MTHFR gene, body mass index, and sex are major factors that influence arsenic metabolism in this population, with a median of 8.0 μg/L arsenic in urine. Females < 60 years of age had, in general, higher methylation efficiency than males, indicating an influence of sex steroids. That might also explain the observed better methylation in overweight or obese women, compared with normal weight men. The influence of the M287T (T→C) polymorphism in the AS3MT gene on the methylation capacity was much more pronounced in men than in women.

Conclusions

The factors investigated explained almost 20% of the variation seen in the metabolism of arsenic among men and only around 4% of the variation among women. The rest of the variation is probably explained by other methyltransferases backing up the methylation of arsenic.

To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2′-O-methyl phosphorothioate (CUG)n triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG)7, also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well.

Dynamic expansions of toxic polyglutamine (polyQ)-encoding CAG repeats in ubiquitously expressed, but otherwise unrelated, genes cause a number of late-onset progressive neurodegenerative disorders, including Huntington disease and the spinocerebellar ataxias. As polyQ toxicity in these disorders increases with repeat length, the intergenerational expansion of unstable CAG repeats leads to anticipation, an earlier age-at-onset in successive generations. Crucially, disease associated alleles are also somatically unstable and continue to expand throughout the lifetime of the individual. Interestingly, the inherited polyQ length mediating a specific age-at-onset of symptoms varies markedly between disorders. It is widely assumed that these inter-locus differences in polyQ toxicity are mediated by protein context effects. Previously, we demonstrated that the tendency of expanded CAG•CTG repeats to undergo further intergenerational expansion (their ‘expandability’) also differs between disorders and these effects are strongly correlated with the GC content of the genomic flanking DNA. Here we show that the inter-locus toxicity of the expanded polyQ tracts of these disorders also correlates with both the expandability of the underlying CAG repeat and the GC content of the genomic DNA flanking sequences. Inter-locus polyQ toxicity does not correlate with properties of the mRNA or protein sequences, with polyQ location within the gene or protein, or steady state transcript levels in the brain. These data suggest that the observed inter-locus differences in polyQ toxicity are not mediated solely by protein context effects, but that genomic context is also important, an effect that may be mediated by modifying the rate at which somatic expansion of the DNA delivers proteins to their cytotoxic state.