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Clinical Chemistry 51: 944-951, 2005. First published April 1, 2005; 10.1373/clinchem.2004.045955
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(Clinical Chemistry. 2005;51:944-951.)
© 2005 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Glutathione S-Transferase P1 *C Allelic Variant Increases Susceptibility for Late-Onset Alzheimer Disease: Association Study and Relationship with Apolipoprotein E {epsilon}4 Allele

Sergio Bernardini1,a, Lorenza Bellincampi1, Sabrina Ballerini1, Giorgio Federici1,2, Roberta Iori1, Alberto Trequattrini3, Fabrizio Ciappi3, Francesca Baldinetti4, Paola Bossù4, Carlo Caltagirone4,5 and Gianfranco Spalletta4,5

1 Departments of Internal Medicine and Laboratory Medicine-PTV, and 5 Department of Neuroscience, University of Rome "Tor Vergata", Rome, Italy.
2 Bambino Gesù-Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Hospital, Rome, Italy.
3 ASL Città di Castello, Perugia, Italy.
4 Istituto di Ricovero e Cura a Carattere Scientifico Fondazione Santa Lucia, Rome, Italy.

aAddress correspondence to this author at: Department of Internal Medicine, University of Rome "Tor Vergata", Via Montpellier 1, 00133 Rome, Italy. Fax 39-06-20902357; e-mail Bernardini{at}Med.UniRoma2.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Oxidative stress and neuronal cell death have been implicated in the pathogenesis of Alzheimer disease (AD). Considering that the glutathione transferase (GST) supergene family encodes isoenzymes that appear to be critical in protection against oxidative stress, we aimed at determining the various GSTP1, GSTM1, and GSTT1 polymorphisms and ApoE genotypes to investigate their role as susceptibility genes for late-onset AD (LOAD).

Methods: We included 210 LOAD patients and 228 healthy controls matched for age, sex, and educational level in our case–control genetic association study. GSTM1 and GSTT1 genotypes were studied by conventional PCR, whereas GSTP1 and ApoE genotypes were determined by real-time PCR on the LightCycler.

Results: We found a significant association between LOAD and the GSTP1*C allelic variant [odds ratio (OR) = 1.9; P <0.05], but no association between the GSTM1 and GSTT1 deleted genotypes and LOAD. In addition, a preliminary result suggested that carriers of both the GSTP1*C and ApoE {epsilon}4 allelic variants were at increased risk of LOAD (OR = 19.98; P <0.0001).

Conclusion: The GSTP1*C allelic variant should be considered a candidate for LOAD, particularly in persons having the ApoE {epsilon}4 allelic variant, because the GSTP1 and ApoE gene products are implicated in oxidative stress and apoptosis processes leading to ß-amyloid-mediated neurodegeneration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Alzheimer disease (AD) 1 exhibits etiologic, pathologic, and clinical heterogeneity (1)(2)(3). Except for rare cases of familial autosomal-dominant AD with early onset produced by mutations of presenilin 1 and 2 and ß-amyloid precursor genes, the vast proportion of cases are sporadic with late onset (4). In this last case, a combination of hereditary and environmental factors could be a crucial pathogenetic mechanism (1)(5).

Researchers have recently focused on the role of oxidative stress and cell death in AD because its high oxygen uptake and relatively low degree of antioxidant defenses make the central nervous system sensitive to oxidative stress (6)(7)(8). Thus, the role of glutathione S-transferase (GST; EC 2.5.1.18) isoenzymes as risk factors for AD could be important; in particular, GSTs detoxify commonly encountered products generated by oxidative damage (9), and reduced GST activity has been reported in multiple brain regions and in ventricular cerebrospinal fluid in short postmortem interval AD patients (10). Relative to their functional role, GSTs belong to a large family of different enzymes that catalyze the S-conjugation of glutathione with a wide variety of electrophilic compounds, including reactive oxygen species and products of cellular metabolism. In the cell, GSTs detoxify secondary oxidation products generated after interaction of reactive oxygen species that escape the first line of defense (superoxide dismutase, catalase, and glutathione peroxidase) with cellular macromolecules such as DNA, lipids, and protein. Because secondary oxidation products represent highly reactive molecules, without adequate detoxification an extended chain reaction will occur that ultimately leads to degradation of cellular components and cell death (11).

The GSTs have well-established polymorphisms in human populations. The GSTM1-1 and GSTT1-1 classes of enzymes have frequently occurring phenotypes that derive from deletion of the respective genes, called "null phenotypes" (12)(13). Carriers of homozygous deletions in the GSTM1 and GSTT1 genes lack the respective enzymatic activities. Approximately 50% of the Caucasian population is homozygous for a deletion of the GSTM1 gene, and ~20% are homozygous for a deletion of the GSTT1 gene (14). The GSTP1-1 class of enzymes is also polymorphic, and 4 allelic variants have been described at the GSTP1 locus, located on chromosome 11q13 at the electrophilic "H" site: GSTP1*A, *B, *C, and *D (15)(16). In particular, the GSTP1*D allelic variant is very rare (17). Two sites in the cDNA sequence are variables and are characterized by an A->G transition at nucleotide 313 (point mutation in exon 5) and a C->T transition at nucleotide 341 (point mutation in exon 6). The resulting codon variants encode for the amino acids Ile105 or Val105 and Ala114 or Val114: the GSTP1*A allelic variant encodes for Ile105/Ala114, GSTP1*B encodes for Val105/Ala114, GSTP1*C encodes for Val105/Val114, and GSTP1*D encodes for Ile105/Val114. The proteins encoded by the different GSTP1 allelic variants have different abilities to bind and possibly metabolize electrophilic compounds and products of oxidative stress. In addition, the brain is the organ in which GSTP1-1 is most often produced, in particular in the blood–brain barrier, where it might modulate the potency of neurotoxins and products of oxidative stress (9).

Over the past decade, there has been considerable interest in the biological and clinical consequences of the reported GST polymorphisms. The vast majority of reports have focused on the role of the GST polymorphisms in carcinogenesis and drug resistance, but recently some studies have also focused on neurologic diseases (18). Indeed, various degrees of association between GSTP1 polymorphisms and susceptibility to some diseases, such as Parkinson disease and multiple sclerosis, have been reported (19)(20). Even if results on this topic are conflicting and the association between GSTP1 polymorphisms and diseases has not been definitively established, it is clear that it might be a ubiquitous "disease-modifying" agent, acting on both exogenous and endogenous factors. At present, there has been only 1 study published, with a small study population, describing a possible role of the GSTT1 deleted genotype and no role of the GSTM1 deleted genotype on the susceptibility for AD (18). Furthermore, there are no published data about the role of any other GST genotype polymorphisms, such as GSTP1, on the susceptibility for AD.

The aim of our study was to determine the GSTP1, GSTM1, and GSTT1 polymorphisms in AD patients to clarify their role as susceptibility genes. We considered the late-onset AD (LOAD) subtype only because it is becoming apparent that a combination of genetic and environmental factors may play an important role in the development of LOAD (1). Considering that the apolipoprotein E (ApoE) {epsilon}4 allelic variant is an important factor in AD pathogenesis, we also studied ApoE genotypes (21).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patient selection criteria
Patients ≥65 years of age who had been diagnosed as being ≥65 years of age at onset of AD were considered appropriate for enrollment. Age at onset was defined as age at the onset of cognitive symptoms severe enough to compromise patient functioning, and was assessed through an interview with the caregiver. All included persons were unrelated Caucasian patients and were consecutively recruited in 2 memory clinics located in central Italy. These patients were drug free and had undergone their first clinical examination for the diagnosis of AD and the treatment of dementia symptoms.

The nature and purposes of this study were presented to the patients and explained to their responsible caregivers and/or legal guardians. Written informed consent was obtained from the patients or their representative and from caregivers before initiation of detailed screening activities.

Inclusion criteria were (a) diagnostic evidence of late-onset (age of onset ≥65 years) probable AD consistent with both DSM-IV (22) and NINCDS-ADRDA(23) criteria, and a Mini Mental State Examination (MMSE) score (24) <24; (b) being healthy and able to walk independently or with a walker or cane; (c) vision and hearing (eyeglasses and/or hearing aid permissible) sufficient for compliance with testing procedures; (d) laboratory values within the appropriate reference intervals or considered to be clinically insignificant by the investigator (see exclusion criteria). Results of computed tomography or magnetic resonance imaging performed within the last 12 months had to be available; if not, the patients had one of these examinations done before their inclusion in the study.

Exclusion criteria included (a) lack of a "reliable" caregiver (defined as someone able to report to the clinic to ensure compliance to treatment and clinic visits and to contact the patient at least twice weekly, with one contact being a personal visit); (b) major medical illness, e.g., diabetes (not stabilized), obstructive pulmonary disease, or asthma; hematologic/oncologic disorders; vitamin B12 or folate deficiency as evidenced by blood concentrations below the lower limits of the reference intervals; pernicious anemia; clinically significant and unstable active gastrointestinal, renal, hepatic, endocrine, or cardiovascular system disease; newly treated hypothyroidism; liver function tests (alanine aminotransferase or aspartate aminotransferase) >3 times the upper limit of the reference interval; or creatinine concentrations >150 µmol/L; (c) comorbidity of primary psychiatric (i.e., psychiatric disorder onset before the AD onset) or neurologic disorders (e.g., schizophrenia, major depression, stroke, Parkinson disease, seizure disorder, or head injury with loss of consciousness within the past year); (d) known suspected history of alcoholism or drug abuse; (e) patients and/or caregivers unwilling or unable to fulfill the requirements of the study; (f) computed tomography or magnetic resonance imaging evidence of focal parenchymal abnormalities; (g) scan evidence of neoplasm; and (h) admission to a nursing home or institutionalization within the last 3 months.

On the basis of the inclusion and exclusion criteria, 210 patients were consecutively enrolled in the study; their sociodemographic characteristics as well as their clinical, behavioral, and cognitive examinations data are summarized in Table 1 .


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  		Table 1. Sociodemographic and clinical characteristics of LOAD patients and controls.

diagnostic, cognitive, and behavioral evaluations
Trained clinical neurologists interviewed patients, using the MMSE (24) and the DSM-IV(22) and NINCDS-ADRDA (23) criteria for diagnosis of AD. The Mental Deterioration Battery (25) was used to assess selective cognitive performances to confirm cognitive deficits required for AD diagnosis. Behavioral domains were assessed by use of the Neuropsychiatric Inventory-10 items (26).

The MMSE (24) is a commonly used neurocognitive test measuring, by means of 16 items, orientation, language, verbal memory, attention, visuospatial function, and mental control with scores ranging from 30 (no impairment) to 0 (maximum impairment).

The Mental Deterioration Battery is a standardized, validated neuropsychologic instrument. The battery comprises 7 neuropsychologic tests (from which 8 performance scores can be derived). Of the 8 total scores, 4 pertain to the elaboration of verbal stimuli and 4 to visuospatial material. The tests were selected to provide information about the functionality of different areas of cognition: language (phonologic verbal fluency and sentence construction), verbal memory (Rey’s 15-word immediate recall and delayed recall), visual memory (immediate visual memory), logical reasoning (Raven’s progressive matrices ’47), and constructional praxis (copying drawings and copying drawings with landmarks) (27).

The Neuropsychiatric Inventory-10 (26) is a valid, reliable inventory to assess 10 neuropsychiatric dimensions in patients with dementia or other neurologic disorders. An informant rates the frequency and severity of each of these dimensions, and the product obtained by multiplication of the 2 scores is used as the final codification. The score for each dimension ranges from 0 to 12 with a maximum total score of 120 in the 10-item version.

Before the beginning of the study, the interviewers were trained by didactic instruction, live interviews, and a review of diagnostic rating. The raters were trained until they demonstrated an interrater reliability of at least 0.80 (K coefficient) for each item of the used scales.

control individuals
The control group included 228 individuals who were neither related to one another nor to LOAD patients; in particular, we selected only those individuals who were in the same age range (65–96 years) as the patients, had a MMSE score ≥24, and did not satisfy the DSM-IV and the NINCDS-ADRDA criteria for diagnosis of AD, as clinically assessed and confirmed by the memory tests of the Mental Deterioration Battery and by a MMSE score ≥24. Indeed, the cognitive level of the general Italian population could be so defined, according to Measso et al. (28). In our study, the persons enrolled as controls were Caucasian, showed no neurologic signs or symptoms at a clinical neurology examination, and were recruited from the general population of the same geographic region as the patients (central Italy).

The number of controls was slightly higher than the number of patients to perfectly match all demographic variables between the two groups. The sociodemographic characteristics and MMSE scores of the controls are shown in Table 1Up .

laboratory methods
Genomic DNA was purified from 200 µL of human whole blood by use of a MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics GmbH) in an automated extractor from the same manufacturer (MagNA Pure LC).

We performed ApoE genotyping by real-time PCR on a LightCycler Instrument (Roche Diagnostics) with the commercially available LightCycler ApoE Mutation Detection Kit (Roche Diagnostics). We performed GSTP1 genotyping with hybridization probes in combination with the LightCycler DNA Master Hybridization Probes Kit (Roche Diagnostics). The sequences of the PCR primers and the hybridization probes are given in Table 2 (19)(29)(30).


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  		Table 2. PCR primers and hybridization probes for GSTP1.

The PCR conditions used were 4 mM MgCl2, 0.2 µM each hybridization probe, 10 pmol of each PCR primer, 2 µL of the LightCycler DNA Master Hybridization Mix, and 50 ng of genomic DNA in a final volume of 20 µL. The PCR conditions were the same for the amplification of both exons, although the reactions were performed in 2 distinct runs. The cycling program for exon 5 was carried out as follows: a preheating step at 95 °C for 90 s, followed by 45 cycles of denaturation at 95 °C for 5 s, annealing at 65 °C for 10 s, and extension at 72 °C for 25 s, with a maximum ramp rate of 20 °C/s. Fluorescence was measured at the end of the annealing step of each cycle to monitor amplification. After amplification was complete, a final melting curve was recorded by denaturation at 95 °C for 90 s followed by a continuous temperature increase from 45 to 85 °C in increments of 0.2 °C/s.

The cycling program for exon 6 was carried out as follows: a preheating step at 95 °C for 30 s, followed by 45 cycles of denaturation at 95 °C for 5 s, annealing at 58 °C for 20 s, and extension at 72 °C for 25 s, with a maximum ramp rate of 20 °C/s. Fluorescence was measured at the end of the annealing period of each cycle to monitor amplification. After amplification was complete, a final melting curve was recorded by denaturation at 95 °C for 90 s followed by a continuous temperature increase from 45 to 85 °C in increments of 0.2 °C/s (29).

The fluorescence emitted by the LC-Red 640 dye bound to the hybridization probe was measured continuously in channel 2 (F2) during the slow temperature ramp to monitor the dissociation of the fluorophore-labeled detection probes from the complementary single-stranded DNA. The fluorescence signal recorded in the channel was then converted to a melting peak by plotting the negative derivative of the fluorescence with respect to temperature vs the temperature (–dF/dT vs T). The resulting melting peak allowed differentiation among the homozygous as well as the heterozygous genotypes for each exon. By combining the results of the melting curve analyses for exons 5 and 6, we could determine the allelic make-up of the analyzed samples.

Because GSTP1*A/*C and *B/*D produced heterozygous patterns for both exons, the above method could not distinguish between them. We therefore used an amplification refractory mutation system (ARMS) assay to differentiate these patterns, as described previously by Hemmingsen et al. (17). This included a forward primer upstream of the codon 105 substitution (5'-ACCCCAGGGCTCTATGGGAA-3') and 2 reverse primers [primer A (Ala114 specific), 5'-TCACATAGTCATCCTTGCCGG-3'; and primer B (Val114 specific), 5'-TCACATAGTCATCCTTGCCGA-3']. For each DNA sample, we performed 2 PCRs amplifying a 998-bp fragment. PCRs were carried out in 50 µL containing forward primer, reverse primer A or B (0.25 µM each of the forward and reverse primers), Taq polymerase (1 U), 200 µM each of the deoxynucleotide triphosphates, 1x polymerase buffer [10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1 nL/µL Triton X-100, 1.5 mM MgCl2], and target DNA (~0.5 µg). Conditions were as follows: 94 °C for 4 min, 30 cycles of denaturation (94 °C for 1 min), primer annealing (62 °C for 1 min), and elongation (72 °C for 2 min). ARMS PCR products were then digested with BsmAI to determine the cis/trans configuration (which variant at position 105 is paired with which allele at position 114) of the Ile105->Val105-encoding allele resolved in 8% acrylamide gels.

To detect deletion of GSTM1 and GSTT1 genes, we performed a multiplex PCR using the ß-globin gene as an internal control. The primer pairs for each gene were as shown in Table 3 . The PCRs for the 480-bp (GSTT1), 268-bp (ß-globin), and 215-bp (GSTM1) fragments were carried out as follows: a preheating step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 60 s, annealing at 58 °C for 45 s, and extension at 72 °C for 60 s, with final extension at 72 °C for 7 min. The PCR was performed in a DNA Thermal Cycler (Perkin-Elmer 9700; PE Applied Biosystem), and the PCR products were electrophoresed in 2.5% agarose gels containing 0.001 g/L ethidium bromide.


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  		Table 3. PCR primers for GSTM1 and GSTT1 genotypes.

The genotyping was done after the inclusion of all patients and controls, and the researcher in charge of genotyping was unaware of sample status as being from patients or controls.

statistical analyses
The sociodemographic categorical variables were confirmed by the {chi}2 tests, and the Student t-test was used for continuous variables. The Hardy–Weinberg distributions of ApoE and GSTP1 genotypes were evaluated by {chi}2 tests. Genotype distributions between LOAD patients and controls were compared by {chi}2 tests. We used multivariate logistic regression analysis to assess, in the total group, the main effects of ApoE and GSTP1 genotypes on prediction of risk for LOAD. In this analysis, being a patient or control was considered a dependent variable, whereas the ApoE {epsilon}4 allelic variant and GSTP1 genotypes (possession of *A, *B, or *C allelic variants) were considered independent variables. We did not consider the GSTM1 and GSTT1 genotypes because their distributions were not significantly different between LOAD patients and controls, as assessed by the {chi}2 test. We performed an additional univariate logistic regression analysis to investigate the prediction of risk of developing LOAD separately in groups of patients having individual or both GSTP1*C and ApoE {epsilon}4 allelic variants. In this analysis, being a patient or control was considered a dependent variable, whereas carrying both or individual ApoE {epsilon}4 and GSTP1*C allelic variants was considered an independent variable.

All tests were 2-tailed, and statistical significance was defined as P <0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The sociodemographic data shown in Table 1Up indicate that there were no differences between LOAD patients and controls regarding age, educational level, and gender. The clinical data for the LOAD patients are also reported in Table 1Up .

The distributions of ApoE, GSTP1, GSTM1, and GSTT1 genotypes in LOAD patients and controls are shown in Table 4 . The ApoE allelic frequencies observed in controls were as follows: {epsilon}2 = 0.044; {epsilon}3 = 0.886; {epsilon}4 = 0.070; the observed genotypes were in Hardy–Weinberg equilibrium ({chi}2 = 1.07; df = 4; P = 0.90). The ApoE allelic frequencies observed in LOAD patients were as follows: {epsilon}2 = 0.038; {epsilon}3 = 0.690; {epsilon}4 = 0.271; the observed genotypes were in Hardy–Weinberg equilibrium ({chi}2 = 1.76; df = 4; P = 0.78). The GSTP1 allelic frequencies observed in controls were as follows: *A = 0.721; *B = 0.232; *C = 0.046; *D = 0; the observed genotypes were in Hardy–Weinberg equilibrium ({chi}2 = 1.93; df = 4; P = 0.75). The GSTP1 allelic frequencies observed in LOAD patients were as follow: *A = 0.688; *B = 0.224; *C = 0.088; *D = 0; the observed genotypes were in Hardy–Weinberg equilibrium ({chi}2 = 0.82; df = 4; P = 0.94).


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  		Table 4. Distribution of ApoE and GSTM1, -T1, and -P1 genotypes and allelic variants in LOAD patients and controls.

We found no association with the deletion genotypes of both GSTM1 and GSTT1, whereas when we considered allelic variants GSTP1*A, *B, and *C, we found a significant difference only in the distribution of the GSTP1*C allelic variant, with allele frequencies of 9.2% and 17.7% in control individuals and LOAD patients, respectively. [We did not consider the allelic variant of *D because none of our cases and controls had this variant, in accordance with previous literature (17).] Furthermore, as expected, we found a significant statistical difference with the distribution of the overall ApoE genotypes. In particular, the distributions of the ApoE {epsilon}4 allelic variant were 14.1% and 48% in controls and LOAD patients, respectively, and the difference was statistically significant.

The odds ratios for different allelic variants of GSTP1 (*A, *B, and *C) and ApoE ({epsilon}4) in the total group are shown in Table 5 . The odds ratios indicate that possession of the GSTP1*C allelic variant was associated with a statistically significant increase in LOAD risk. In addition, possession of the ApoE {epsilon}4 allelic variant was associated with a statistically significant increase in LOAD risk. In this first multivariate logistic regression analysis, we found a nonsignificant statistical interaction (P = 0.295) between GSTP1*C allelic variant and ApoE {epsilon}4 allelic variant possession. This means that the 2 allelic variants have each a separate action on the risk of AD, that is independent of the presence or absence of the other allelic variant. Additional analysis (Table 6 ) indicated that those patients who carried both the ApoE {epsilon}4 and GSTP1*C allelic variants had a statistically significant increased risk of developing LOAD; however, considering the small number of controls and patients having both the ApoE {epsilon}4 and GSTP1*C allelic variants, the results of this last analysis must be considered preliminary. In particular, those who carried the ApoE {epsilon}4 allelic variant and did not carry the GSTP1*C allelic variant included 80 patients (38%) and 30 controls (13%); those who carried the GSTP1*C allelic variant and did not carry the ApoE {epsilon}4 allelic variant included 16 patients (8%) and 19 controls (8%); and those who carried both the ApoE {epsilon}4 allelic and GSTP1*C allelic variants included 21 patients (10%) and 2 controls (1%).


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  		Table 5. Risk for LOAD of individual GSTP1*A, *B, and *C and ApoE {epsilon}4 allelic variants.


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  		Table 6. Risk of developing LOAD in groups of patients carrying individual or both ApoE {epsilon}4 and GSTP1*C allelic variants.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we found a significantly increased susceptibility for LOAD in carriers of GSTP1*C or ApoE {epsilon}4 allelic variants considered separately. These risk factors in the total group of participants were independent of each other. In addition, when we considered separately groups who carried one or both of the genetic risk factors analyzed, we found preliminary evidence that individuals carrying both the ApoE {epsilon}4 and GSTP1*C allelic variants had an increased risk of developing LOAD. Nevertheless, these results should be interpreted with caution considering the limited number of participants carrying both alleles: 21 patients (10%) and 2 controls (1%).

The evidence of an association between increased risk of LOAD and the GSTP1*C allelic variant supports the hypothesis that GSTP1 could be involved in AD neurodegeneration. Although no functional data are available to explain the mechanism by which the GSTP1-1 isoforms could be linked to LOAD neurodegeneration, a hypothesis can be made. The GSTP1*C allelic variant might have (a) an influence in modulating the stability of the GSTP1-1/C-Jun N-terminal kinase (JNK) complex and consequently JNK activity, which is involved in the activation of apoptosis; and (b) a different efficiency in conjugating products of oxidative stress.

Different models suggest a role for apoptosis in the pathogenesis of AD. One possible mechanism is linked to the ability of ß-amyloid aggregates, formed intracellularly in neurons, to generate free radicals, leading to lipid peroxidation, oxidative stress, and apoptosis (31)(32). On the other hand, these aggregates are released into the extracellular space by a partially apoptotic mechanism that is restricted to the distal compartment of the neurons, beginning the process leading to senile plaque accumulation (33). It is well established that ß-amyloid induces apoptosis via mechanisms involving the JNK pathway, induction of CD95 ligand, and the release of Smac via AP-1/Bim activation from mitochondria (34)(35). In this context, monomeric GSTP1-1 could form a complex with JNK. This association is inversely correlated with JNK activity. The inhibition of JNK activity by monomeric GSTP1-1 is reversed by oxidative stress, which causes oligomerization of GSTP1-1 and, consequently, dissociation of the GSTP1-1–JNK complex. Thus, GSTP1-1 could interfere with the apoptosis machinery (36). Even if the C-terminal region of GSTP1-1 seems to be essential for binding JNK (37), it is also possible that other sites, such as the active center of the enzyme, might influence this binding or disturb JNK activity. The *C allelic variant in the H site could thus interfere with the regulatory role of GSTP1-1 on JNK activity and ultimately in the modulation of apoptosis, depending from the redox status of the cell.

Taking into account the possibility that GSTP1 polymorphisms could differentially affect the efficiency of the enzyme in conjugating products of oxidative stress, it should be pointed out that the GSTP1*C allelic variant is characterized by the codon GTC at nucleotide 313 (exon 5) and GTG at nucleotide 341 (exon 6). The resulting codon variants encode the amino acids Val105 and Val114 at the electrophilic H site. In particular, residue 105 is situated in the central part of the H site, which is responsible for binding of electrophilic substrates, whereas residue 114 is located at the beginning of helix 5 outside the H site. The possible amino acid substitutions linked to GSTP1 gene polymorphisms are represented by 3 amino acids (Ala, Val, and Ile), all of which are included in the class of unsubstituted alkyl amino acid having a methyl R group (Ala), an isopropyl R group (Val), or an isobutyl R group (Ile) branched at the ß-carbon of the amino acid. Thus, the main difference is in size. Indeed, the increase in size from Ala to Ile to Val might differentially limit free access of the substrate to the H site, the efficient conjugation with glutathione, and stabilization of the complex. On the other hand, the change of amino acid residues from small to large in the H site might change the hydrophobicity of the H site, influencing the thermostability of the enzyme. However, which substitution is more stabilizing is still the subject of debate (38).

The individual products of oxidative stress that could be differentially detoxified by GSTP1*C-encoded enzymes rather than by one of the other isoforms are still unknown. However, considering that the oxidative stress measured by 4-hydroxynonenals seems to be correlated with synapse loss in the brains of AD patients (2) and that 4-hydroxynonenals are detoxified by GSTP1-1, it is conceivable that the *C allelic variant could be directly involved in this mechanism in LOAD.

Among the GSTs investigated in our study, the only association with increased risk of LOAD was for GSTP1. Indeed, we found no association between the GSTT1 and GSTM1 deleted genotypes and LOAD. There are very few data in the literature about these associations. Only 1 study has reported a significant difference in the frequency of the GSTT1 deleted genotype in both males and females affected by AD compared with controls. However, the results are limited by the small number of cases investigated (i.e., 43 patients with AD) (18).

Apart from the association between the possession of the GSTP1*C allelic variant and LOAD risk, which in the total group of patients was independent of the presence of the ApoE {epsilon}4 allelic variant, we found an increase in the risk of LOAD (odds ratio = 19.98) in those individuals who carried both the GSTP1*C and ApoE {epsilon}4 allelic variants. Such a result, which seems to suggest a "cooperation against the brain" between the polymorphic GSTP1 and ApoE genes, is difficult to attribute to a single mechanism, however. Indeed, the possible pathogenetic involvement of the GSTP1*C allelic variant in LOAD, described here for the first time, is still to be clarified, and the molecular mechanisms by which the ApoE {epsilon}4 allelic variant influences the occurrence of LOAD are not well established at present. However, there is evidence that the ApoE gene product, which is involved mainly in the repair and maintenance of neuronal membranes, is multifunctional, with potential roles in amyloid deposition and clearance, microtubule stability, oxidative stress, and other cellular processes, such as apoptosis (7). From these observations, it can be concluded that products of both the GSTP1 and ApoE genotypes may independently interfere with the mechanism by which ß-amyloid can induce apoptosis via different pathways.

In conclusion, our results on the role of the GSTP1 polymorphism in increasing the risk of LOAD indicate that the *C allelic variant might be considered a candidate gene for the disease and suggest that it could be an active risk factor in persons carrying the ApoE {epsilon}4 allelic variant, adding a new hypothesis to the universal discussion in "deciphering the genetic basis of Alzheimer’s disease" (39).


   Footnotes
 
1 Nonstandard abbreviations: AD, Alzheimer disease; GST, glutathione S-transferase; LOAD, late-onset Alzheimer disease; ApoE, apolipoprotein E; MMSE, Mini Mental State Examination; and JNK, C-Jun N-terminal kinase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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