| Objective: Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disorder, with an incidence of 1- 2 per 100 000. It is characterized by a selective loss of upper and lower motor neurons in the brain and spinal cord. Approximately the 90% of cases are sporadic (SALS),the others are the familial cases (FALS).FALS are clinically and pathologically indistinguishable from SALS, suggesting a common mechanism of motor neuron degeneration. Until now,the etiopathogenisis and exact mechanisms underlying ALS are still less clear. In 15-20% of familial cases, mutations have been found in the gene encoding Cu-Zn superoxide dismutase (SOD1). In both FALS and SALS there is evidence that oxidative stress contributes to motor neuron injury, several studies revealed a significant increase in markers of oxidative stress in CNS tissue in SALS cases, so oxidative stress may play an important role in the pathology of ALS. Thus it is worthwhile screening for defective function in components of the anti-oxidant defense system for potential associations with ALS.Recently, it is reported that the activation of Nrf2/ARE signaling pathway could induce a series of antioxidant enzyme, antioxidant protein, anti-inflammatory and antitoxic protein. The expression of those antioxidant enzymes in nervous system exhibited broad neuroprotection against oxidative stress. The classic antioxidant enzyme glutathione S-transferases (GSTs) and NADPH quinone oxidoreductase 1 (NQO1) were two kinds of a series of downstream protein of Nrf2/ARE pathway.There are seven isoforms of GST (Alpha, Mu, Omega, Pi, Sigma, Theta and Zeta) in mammalian cells. The GSTA1 and GSTO2 are two new identified subgroups of GSTs. There is polymorphism nearby the promoter of GSTA1 (C69T), and the variant allele is associated with decreased expression of GSTA1 proteins. In human, the GSTO2 is polymorphic with an N142D substitution in the coding region, it is reported that the variant would decrease the activity of GSTO2.The common genetic polymorphism of NQO1 is a C to T point mutation at base pair 609 of exon 6, which codes for a proline to serine substitution in NQO1 protein. This mutation results in a loss of NQO1 activity.This study was designed to investigate the association between polymorphisms of GSTA1,GSTO2 and NQO1and the risk of ALS. By this way, we hope to offer some evidence for the prevention and therapy of ALS at molecular level.Methods: This case-control study included 143 ALS patients and 210 healthy controls. Five milliliter of venous blood from each subject was drawn and genomic DNA was extracted. Single nucleotide polymorphisms (SNPs) of GSTA1,GSTO2 and NQO1 were genotyped by polymerase chain reaction- restrictive fragment length polymorphism (PCR-RFLP) analysis.Statistical analysis was performed using SPSS11.5 software package. Hardy-Weinberg analysis was performed by comparing the observed and expected genotype frequencies in each groups using Chi-square test. Between cases and controls, the t test was used to examine the difference of age. The comparison of genotype and allele frequency was performed by Chi-square test. The odds ratio (OR) and 95% confidence interval (CI) were calculated using an unconditional logistic regression model. P <0.05 was considered significant for all statistical analyses.Result:1 The distributions of GSTA1,GSTO2 and NQO1 genotypes among healthy controls were compatible with those expected genotype frequencies from Hardy-Weinberg equilibrium (P =0.74,P =0.42,P =0.10,respectively). The distributions of GSTA1,GSTO2 and NQO1 genotypes among patients cases were compatible with those expected genotype frequencies from Hardy-Weinberg equilibrium, too (P=0.78, P=0.10, P=0.82, respectively), suggesting that sample could represent groups.2 The frequencies of three genotypes (CC, CT and TT) of GSTA1 C69T in ALS group and healthy controls were 79.72%,18.88%,1.40% and 76.19%,21.90%,1.91% respectively. The distributions of genotype were not significantly different in the ALS group compared to those in the control group (P=0.44). The frequencies of allele (C and T) of GSTA1 C69T in ALS patients and healthy controls were 89.16%,10.84% and 87.14%,12.86% respectively. The frequencies of allele were not significantly different in the ALS group compared to the control group (P=0.42). Compared with the CC genotypes, the CT genotype did not significantly increased the risk of ALS.(OR=0.82, 95%CI=0.48～1.40),so did the TT and CT+TT genotype(OR=0.70, 95%CI=0.13～3.90; OR=0.81, 95%CI=0.49～1.37, respectively).3 The frequencies of three genotypes (NN, ND and DD) of GSTO2 N142D in ALS patients and healthy controls were 47.55%,46.85%,5.60% and 66.67%,30.95%,2.38% respectively. The distributions of genotype were significantly different in ALS group compared to the control group (P=0.001). The frequencies of the genotype ND and DD of the GSTO2 N142D were significantly higher in ALS patients (52.45%) than those in healthy controls (33.33%). The frequencies of allele (N and D) of GSTO2 N142D in ALS patients and healthy controls were 70.97%,29.03% and 82.14%,17.86% respectively. The frequencies of allele were significantly different in the ALS group compared to the control group (P=0.000). The frequencies of the D allele of the GSTO2 N142D were significantly higher in ALS group (29.03%) than those in healthy controls (17.86%). Compared with the NN genotypes, the NN+ND genotype significantly increased the risk of ALS(OR=2.21, 95%CI=1.43～3.41).When calculated respectively, both NN and ND genotype could significantly increased the risk of ALS(OR=2.12, 95%CI=1.36～3.32;OR=3.30, 95%CI=1.04～10.45, respectively).4 The frequencies of three genotypes (CC, CT and TT) of NQO1C609T in ALS patients and healthy controls were 27.97%,48.95%,23.08% and 22.86%,55.71%,21.43% respectively, but there was no statistic difference (P =0.42). The frequencies of allele (C and T) of NQO1C609T in ALS patients and healthy controls were 52.45%,47.55% and 50.71%,49.29% respectively. The distributions of genotype were not significantly different in the ALS group compared to the control group (P=0.65). Compared with the CC genotypes, the CT genotype,TT genotype and CT plus TT genotype did not significantly increased the risk of ALS: OR=0.72, 95%CI=0.43～1.20 ; OR=0.88, 95%CI= 0.48～1.63 ; OR=0.76, 95%CI=0.47～1.24, respectively .5 The distributions of GSTA1,GSTO2 and NQO1 genotypes among each stratified subgroup according to clinical stageFor GSTA1 gene, stratification analysis showed no significant difference between the bulbar onset group and the spinal onset group in genotype distributions according to the disease onset location (P=0.78). Stratification analysis showed no significant difference between the≦36 months group and >36 months group in genotype distributions according to the symptom duration (P=0.21). Stratification analysis by age at onset and gender, there was no significant difference yet (P=0.90; P=0.73, respectively). (The CC genotype relative to the CT and TT genotypes).For GSTO2 gene, stratification analysis by disease onset location,symptom duration and age at onset, there was no significant difference in genotype distributions (P =0.77,P =0.23,P =0.54, respectively); stratification analysis showed no significant difference between the male group and female group in genotype distributions according to gender(P=0.24). ( the NN genotype relative to the ND and DD genotypes). For NQO1 gene, stratification analysis by disease onset location,symptom duration, age at onset and gender, there was no significant difference in genotype distributions (P =0.81,P =0.27,P =0.99,P =0.31, respectively).Conclusions:1 The risk of ALS were significantly increased in subjects with GSTO2 N142D ND genotype especially GSTO2N142D DD genotype. Therefore, ND genotype and DD genotype may be a potential risk factor for SALS.2 The GSTA1 C69T SNP and NQO1C609T SNP may have no association with susceptibility to the SALS.3 In the ALS patients, GSTA1 gene,GSTO2 gene and NQO1 gene stratification analysis showed no significant difference in genotype distributions according to disease onset location,symptom duration,age at onset and gender.
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