Association between GSTM1, GSTT1, and GSTP1 variants and the risk of end stage renal disease

Figures & data

Figure 1. Agarose gel electrophoresis (2%) pattern of GSTM1 and GSTT1 multiplex PCR. From left to right lane 1 shows 100 base pairs DNA molecular weight marker; GSTT1 yields a product of 480 bp, whereas the amplification product of GSTM1 is 230 bp and β-globin gene (268 bp) as an internal control. Lanes 2 GSTT1/GSTM1 genotypes, lanes 3 GSTT1/GSTM1null genotypes, lanes 4 and 7 GSTT1null/GSTM1 genotypes, lanes 5, and 6 GSTT1null/GSTM1null genotypes.

Table 1. Primers and PCR conditions for all the GSTs variants.

Marker	Primer sequence	PCR condition	References
GST T1 & M1 & globin multiplex PCR	M1: 5´-TTC TGG ATT GTA GCA GAT CA-3´ M2: 5´- CGC CAT CTT GTG CTA CAT TGCCCG-3´ T1: 5´-TTC CTT ACT GGT CCT CAC ATC TC- 3´ T2: 5´-TCA CCG GAT CAT GGC CAG CA- 3´ B1 (β-globin): 5´- CAA CTT CAT CCA CGT TCA CC-3´ B2 (β-globin): 5´- GAA GAG CCA AGG ACA GGT AC-3´	Initial denaturation 94 °C for 5 min followed by 35 cycles at 94 °C for 1 min, annealing at 60 °C for 30 s, extension at 72 °C for 30 s with a final extension at 72 °C for 5 min	^21
GST P1	P1: 5´-GTA GTT TGC CCA AGG TCA AG-3´ P2: 5´-AGC CAC CTG AGG GGT AAG-3´	Initial denaturation 94 °C for 5 min followed by 35 cycles at 94 °C for 1 min, annealing at 61 andn JM, Sanchez P, Ruiz-Reguena E.°C for 30 s, extension at 72 °C for 1 min with a final extension at 72 °C for 5 min	^21

Figure 2. A total of 12% polyacrylamide gel pattern of GSTP1 polymorphism digested PCR products with ALW26I enzyme. From left to right lane 1 indicate lanes 1 homozygous Val/Val, lane 2 heterozygote Ile/Val, lane 3 wild type Ile/Ile, lane 4 PCR product, and lane 5 molecular marker.

Table 2. Characteristics and distribution of risk factors in patients with ESRD and control subjects in a population from western Iran.

Parameter	Patients with ESRD (n = 136)	Control subjects (n = 137)	p Values
Age	58.1 ± 13.3	55.7 ± 7.3	N.S^a
Sex (male/female)	89–47	87–50	N.S
TAC (mM)	8.9 ± 2.4	6.4 ± 2.3	<0.001
MDA (μM)	2.04 ± 0.4	1.1 ± 0.33	<0.001

Serum TAC and MDA concentrations compared between patients and controls using non-parametric two independent sample test Mann–Whitney and age by the two-tailed Student’s t-test and sex by the χ² test.

^a N.S: no significance.

Table 3. Distribution of GST M1 & T1 genotypes in patients with ESRD and control subjects.

GST M1 & T1 genotypes	Patients with ESRD (n = 136)	Control subjects (n = 137)
GST normal	17 (12.8%)	45 (32.8%)
GSTM1-null	46 (34.6%)	40 (29.2%)
GSTT1-null	21 (15.8%)	28 (20.4%)
GST-null	49 (36.8%)	24 (17.5%)
	χ^2= 22.6, df = 3, p < 0.001
GST normal	17 (20.2%)	45 (39.8%)
GSTM1-null + GSTT1-null	67 (79.8%)	68 (68.2%)
	χ^2= 8.57, df =1, p = 0.003
GST normal	17 (12.8%)	45 (32.8%)
GSTM1-null + GSTT1-null + GST-null	116 (87.2%)	92 (67.2%)
	χ^2= 15.4, df =1, p = <0.001
GST normal	17 (27.0%)	45 (52.9%)
GSTM1-null	46 (73.0%)	40 (47.1%)
	χ^2= 10.0, df = 1, p = 0.002

Distribution of GST M1 & T1 genotypes in patients with ESRD and control subjects were calculated using χ² test analysis.

GST-null = T1−-M1−, GSTM1-null = T1+-M1− and GSTT1-null = T1−-M1− GST normal + T1+ − M1+.

Table 4. Odds ratios of GST M1 & T1 genotypes in patients with ESRD after adjustment for sex and age.

GST M1 & T1 genotypes	Patients with ESRD (n = 133)	Control subjects (n = 137)
GST normal	Reference group (n = 17)	Reference group (n = 45)
GSTM1-null + GSTT1-null	2.61 (1.4–5.0, p = 0.004, n = 84)	n = 113
GST normal	Reference group (n = 17)	Reference group (n = 45)
GSTM1-null + GSTT1-null + GST-null	3.3 (1.8–6.2, p < 0.001, n = 116)	n = 92
GST normal	Reference group (n = 17)	Reference group (n = 45)
GST-null	1.8 (1.4–2.3, p < 0.001, n = 49)	n = 24
GST normal	Reference group (n = 17)	Reference group (n = 45)
GSTM1-null	3.04 (1.5–6.1, p = 0.002, n = 46)	n = 40
GST normal	Reference group (n = 17)	Reference group (n = 45)
GSTT1-null	1.41 (0.95–2.1, p = 0.091, n = 38)	n = 73

Odds ratios as estimates of relative risk for disease were calculate and 95% confidence intervals were obtained using regression binary logistic analysis.

GST-null = T1−-M1−, GSTM1-null = T1+-M1− and GSTT1-null = T1−-M1−, GST normal + T1+ −M1+.

Table 5. The relationship between GST T1 & M1 genotypes with TAC and MDA between ESRD patients and control subjects.

GST M1 & T1 genotypes	Patients with ESRD (n = 133)	Control subjects (n = 137)	p Values
GST normal	n = 17	n = 45
TAC	8.16 ± 2.43	6.43 ± 2.37	0.010
MDA	2.03 ± 0.47	1.16 ± 0.4	<0.001
GSTM1-null	n = 46	n = 40
TAC	9.42 ± 2.33	6.45 ± 2.21	<0.001
MDA	1.99 ± 0.41	1.11 ± 0.28	<0.001
GSTT1-null	n = 21	n = 28
TAC	9.46 ± 2.41	6.45 ± 2.77	<0.001
MDA	2.04 ± 0.36	1.02 ± 0.23	<0.001
GST-null	n = 49	n = 24
TAC	8.36 ± 2.36	6.46 ± 1.98	0.001
MDA	2.09 ± 0.39	1.14 ± 0.37	<0.001

The relationship between of GST M1 & T1 genotypes with TAC and MDA between patients with ESRD and control subjects was calculated using ANOVA test analysis.

Table 6. The distribution and odds ratios of GST P1 genotypes and alleles in patients with ESRD after adjustment for sex and age.

	Patients with ESRD (n = 133)	Control subjects (n = 137)
GST P1 genotypes
A/A	74 (55.6%)	70 (51.1%)
A/G	50 (37.6%) 0.84 (0.51–1.4, p = 0.51, n = 50)	56 (40.2%)
G/G	9 (6.8%) 0.88 (0.55–1.4, p = 0.593, n = 9)	11 (8.0%)
	χ^2=0.592, df = 2, p = 0.744
GST P1 alleles
A	198 (74.4%)	197 (71.9%)
G	68 (25.6%) 0.879 (0.6–1.3, p = 0.5, n = 68)	71 (28.1%)
	χ^2 = 0.443, df = 1, p = 0.506

The distribution of GST P1 genotypes and alleles in patients with ESRD and control subjects were derived using χ² test analysis. Odds ratios as estimates of relative risk for disease were calculate and 95% confidence intervals were obtained using χ² regression binary logistic analysis.

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