After informed consent was obtained, blood samples were drawn from 75 patients [Russian in origin, 32 males and 43 females, aged 28-72 years (55.5 +- 10.9 years)] with appar­ently sporadic MND. All patients were seen at the Depart­ment of Fundamental and Clinical Neurology of the Russian State Medical University, Moscow, Russia, and fulfilled the Revised El Escorial criteria for ALS/MND [29]. We subdi­vided our patients (as shown in Table 1) according to the classification of Norris et al. [30] and assessed progression rates with the Norris ALS Score [31] every 6 months up to the time of death. We defined patients with rapid progression as those who lost more than 10 points of the Norris ALS Score every 6 months, patients with moderate progression as those who lost 5-10 points, and patients with slow progres­sion as those who lost less than 5 points (see Table 1). These progression rates significantly differed when their regression lines were compared (r = –0.96; p <0.001; r = –0.97; p <0.001; r = –0.98; p <0.001).

      The control group consisted of 105 randomly sampled individuals from Russia. They were unrelated, neurologically normal, and matched by age, sex, and ethnicity to members of the patient group. DNA was extracted from peripheral leukocytes by standard methods [32].

      CYP2E1 Genotyping. The presence of a 96 bp insertion in the promoter region of the CYP2E1 gene was detect by polymerase chain reaction (PCR) as previously described [12]. The following primers were used: 5'-GTG ATG GAA GCC TGA AGA ACA-3' and 5'-CTT TGG TGG GGT GAG AAC AG-3'. Polymerase chain reaction was carried out in a 20 μL reaction mixture containing 5 pM of each primer, 2 μL of 10X PCR buffer [500 mM Tris-HCl, pH 8.8, 150 mM (NH₄)₂SO₄, 50 mM MgCl₂, 2 mg/mL bovine serum albumin (BSA)], 1.0 mM of each dNTP, and 0.5 units of Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania) and under conditions of: initial denaturation at 95° C for 5 min., followed by 30 cycles of 1 min. at 95° C, 1 min. at 66° C and 1 min. at 72° C. The PCR products were resolved on an 8% polyacryl­amide gel stained with ethidium bromide. The length of the fragment for the allele with no insertion, designated CYP2E1*1C, and containing six repeats in the 5' flanking region was 633 bp. The fragment for the allele with an inser­tion designated CYP2E1*1D and containing eight repeats in the 5' flanking region was 729 bp.

      CYP2D6 Genotyping. We used the method of Brown et al. [33], which distinguishes CYP2D6*4 alleles from all other CYP2D6 alleles by PCR and a restriction digest using the enzyme BstNI. This enzyme cuts at position 1934 in wild type but not in mutant alleles. The position of the 5' PCR primer was changed to include a non polymorphic BstNI restriction site at position 1772. The amplification mixture contained 5 pM of each primer (5'-GGT GTT CCT CGC GCG CTA TG-3' and 5'-CTC GGT CTC TCG CTC CGC AC-3'), 2 μL of 10X PCR buffer, 1.0 mM of each dNTP, and 0.5 units of Taq DNA polymerase (MBI Fermentas). Conditions for PCR were: initial denaturation at 95° C for 5 min., followed by 30 cycles of 1 min. at 95° C, 1 min. at 65° C and 1 min. at 72° C. Digests utilized 10 μL aliquots of PCR products (length 421 bp) and 1 unit of MvaI (isoschizomer of BstNI; MBI Fermen­tas), incubated overnight at 37° C. Digestion products were resolved electrophoretically on an 8% polyacrylamide gel and visualized by ethidium bromide staining and fluorescence under UV light. CYP2D6*4 homozygotes were identified as two bands of 77 and 344 bp, whereas non CYP2D6*4 homo­zygotes appeared as three bands of 77, 161, and 183 bp, respectively.

      GSTM1 and GSTT1 Genotyping. Homozygous dele­tions of the GSTM1 and GSTT1 genes were detected using a multiplex PCR technique, including primers for exon 4 of the GTP cyclohydrolase 1 gene (GCH1) as the internal control [34]. Polymerase chain reaction was performed in 20 μL of reaction mixture containing 2 μL of 10X PCR buffer, 1.0 mM of each dNTP, 0.5 units of Taq DNA polymerase (MBI Fermentas), and 5 pM of each primer (GSTM1 gene: 5'-GAA CTC CCT GAA AAG CTA AGC-3' and 5'-GTT GGG GCT CAA ATA TAC GGT GG-3'; GSTT1 gene: 5'-TTC CTT ACT GGT CCT CAC ATC TC-3' and 5'-TCA CCG GAT CAG GCC AGC A-3'; exon 4 of the GCH1 gene 5'-GTC CTT TTT GTT TTA TGA GGA AGG C-3' and 5'-GGT GAT GCA CTC TTA TAA TCT CAG C-3'). The PCR con­ditions were: initial denaturation at 95° C for 5 min., followed by 30 cycles of 1 min. at 95° C, 1 min. at 63° C and 1 min. at 72° C. The PCR products were analyzed by electrophoresis on an 8% polyacrylamide gel and visualized by ethidium bro­mide staining and fluorescence under UV light. The presence or absence of the GSTT1 and GSTM1 genes was detected by the presence or absence of a band at 480 or 215 bp, respec­tively. A band at 297 bp (exon 4 of GCH1) signified success­ful amplification. This technique could not distinguish between heterozygous and homozygous positive genotypes, but could conclusively identify the null genotypes.

      GSTP1 Genotyping. The GSTP1 exon 5 Ile105Val polymorphism was detected using PCR followed by restric­tion analysis [35]. The primers used were: 5'-GTA GTT TGC CCA AGG TCA AG-3' and 5'-AGC CAA CCT GAG GGG TAA G-3'. The amplification mixture contained 2 μL of 10X PCR buffer, 1.0 mM of each dNTP, 5 pM of each primer, and 0.5 units of Taq DNA polymerase (MBI Fermentas). The PCR conditions were: initial denaturation at 95° C for 5 min., followed by 30 cycles of 1 min. at 95° C, 1 min. at 65° C and 1 min. at 72° C. Digestions made use of 10 μL aliquots of PCR products and 1.5 units of Alw26I (isoschizomer of BsmAI; MBI Fermentas), incubated overnight at 37° C. Restriction fragments were resolved electrophoretically on an 8% polyacrylamide gel and visualized by ethidium bromide staining and fluorescence under UV light. The length of the fragment for the GSTP1*A allele containing isoleucine at codon 105 was 320 bp, and that of the fragment for the GSTP1*B allele with valine at that codon was 222 bp.

      NAT2 Genotyping. The NAT2 gene polymorphism was detected by Baranova et al. [36]. The most common slow alleles, S1, S2, and S3 (NAT2*5, NAT2*6, and NAT2*7, respectively) and one wild type fast allele, F1 (NAT2*4), were identified. Polymerase chain reaction amplification was per­formed with primers 5'-GCT GGG TCT GGA AGC TCC TC-3' and 5'-TTG GGT GAT ACA TAC ACA AGG G-3' in 20 μL of PCR mixture containing 2 μL of 10X PCR buffer, 1.0 mM of each dNTP, 5 pM of each primer, and 0.5 units of Taq DNA polymerase (MBI Fermentas). The PCR included initial denaturation at 95° C for 5 min., followed by 35 cycles of 1 min. at 95° C, 1 min. at 57° C and 1 min. at 72° C. Ampli­fied products were restricted with the enzymes KpnI [to detect the NAT2*5 (S1) allele], TaqI [to detect the NAT2*6 (S2) allele] and BamHI [to detect the NAT2*7 (S3) allele]. Restric­tion fragments were resolved electrophoretically on an 8% polyacrylamide gel and visualized by ethidium bromide stain­ing and fluorescence under UV light.

      Statistical Analyses. We computed Hardy-Weinberg equilibrium for CYP2E1, CYP2D6, GSTP1 and NAT2 geno­types among patient and control groups, to determine whether the distribution of alleles was as expected (the GSTM1 and GSTT1 genotypes were coded as wild type or null, making direct calculation of the Hardy-Weinberg equilibria impos­sible). Differences in allele and genotype frequency between patients and controls were assessed with the two-sided ^{χ 2} test with Yate’s correction. The GraphPadInStat version 3 pro­gram was used for the analysis of 2x2, 2x3 and 2x4 contin­gency tables and relative risk (RR) calculations. The Bon­ferroni correction was introduced in the case of 2x3 and 2x4 contingency tables by correction of critical p values (http:// graphpad.com/instat/instat.htm). Non parametric rank-order correlations (gamma or Spearman correlations) were used to determine the associations between the genotypes of the ana­lyzed genes and clinical features (Statistica software version 6; http://www.statsoft.com).

Clinical characteristic		N (%)
Diagnosis	Cervical onset ALS	37 (49,3)
	Lumbar onset ALS	13 (17,3)
	Truncal onset ALS	5 (6,7)
	Diffuse onset ALS	4 (5,3)
	Progressive bulbar palsy	16 (21,3)
Variations of MN involvement	Classical upper and lower MN involvement	40 (53,3)
	Predominant lower MN involvement	23 (30,7)
	Predominant upper MN involvement	12 (16,0)
Progression rate	Rapid progression	41 (54,6)
	Moderate progression	17 (22,7)
	Slow progression	17 (22,7)
ALS – amyotrophic lateral sclerosis MN – motor neuron