Glutathione S-transferase M1, T1 and P1 gene polymorphisms and the risk of developing type 2 diabetes mellitus in Egyptian diabetic patients with and without diabetic vascular complications

Peer review under responsibility of Alexandria University Faculty of Medicine.

Available online 18 April 2014

Figures & data

Table 1 Primer sequences and cycler conditions for amplification of GSTM1 and GSTT1 genes.

Genes	Primers	Cycler conditions
GSTM1	Forward	GSTM1	Initial Denaturation	95 °C–5 min
	5′-GAACTCCCTGAAAAGCTAAAGC-3′	35 Cycles	Denaturation	94 °C–30 s
	Reverse		Annealing	55 °C–30 s
	5′-GTTGGGCTCAAATATACGGTGG-3′		Extension	72 °C–30 s
			Final elongation	72 °C–10 min
GSTT1	Forward	GSTT1	Initial Denaturation	94 °C–5 min
	5′-TTCCTTACTGGTCCTCACATCTC-3′	35 Cycles	Denaturation	94 °C–1 min
	Reverse		Annealing	59 °C–1 min
	5′-TCACGGGATCATGGCCAGCA-3′		Extension	72 °C–1 min
			Final elongation	72 °C–10 min
CYP1A1	Forward
(Exon-7)	5′-GAACTGCCACTTCAGCTGTCT-3′
	Reverse
	5′-CAGCTGCATTTGGAAGTGCTC-3′

Table 2 Some demographical and clinical characteristics, anthropometric and radiological data, as well as biochemical parameters among the studied groups.

Items		Controls (n = 51) mean/median ± SD/Min–Max	Diabetic patients(n = 54)mean/median ± SD/Min–Max	p-value
Gender
Male		n = 16 (31.4%)	n = 20 (37.0%)	χ^2p = 0.541
Female		n = 35 (68.6%)	n = 34 (63.0%)
Age	(years)	43.37 ± 11.07	49.96 ± 8.99	^tp1 = 0.001^*
BMI	(kg/m^2)	27.39 ± 2.8	30.56 ± 3.97	^tp1 < 0.001^*
CIMT	(cm)	0.44 (0.3–0.62)	0.58 (0.40–2.30)	^MWp1 < 0.001^*
Duration of disease	(years)	–	10.5 ± 5.49	–
Whole blood HbA1c	(%)	4.84 ± 0.38	8.52 ± 2.25	^tp1 < 0.001^*
Serum (Fasting)
Glucose	(mg/dL)	90 (74–99)	168 (78–448)	^MWp1 < 0.001^*
Creatinine	(mg/dL)	0.9 (0.6–1.2)	1.0 (0.7–5.1)	^MWp1 < 0.001^*
ALT	(U/L)	15 ± 7	26 ± 12	^tp1 < 0.001^*
Total cholesterol	(mg/dL)	174 ± 22	207 ± 12	^tp1 < 0.001^*
HDL-cholesterol	(mg/dL)	55 ± 12	44 ± 13	^tp1 < 0.001^*
LDL-cholesterol	(mg/dL)	100 ± 19	130 ± 45	^tp1 < 0.001^*
Triglycerides	(mg/dL)	94 ± 31	172 ± 77	^tp1 < 0.001^*
Items	Control group(n = 51)	T2DM without vascular complications(n = 27)	T2DM with vascular complications(n = 27)	p2/p3/p4 values
Blood pressure
Systolic (mmHg)	115 (90–135)	130 (90–135)	140 (110–180)	^tp2 < 0.001^*
				^tp3 < 0.001^*
Diastolic (mmHg)	70 (60–85)	80 (60–85)	90 (60–110)	^tp4 < 0.001^*
Urine ACR
(mg/gm)	11.0(2.0–28.5)	15.7 (2.3–29.6)	60.0 (1.5–1749.0)	^t p2 = 0.153
				^tp3 < 0.001^*
				^tp4 < 0.001^*

p1-value describes the statistical difference between control group and whole diabetic cases.

p2-value describes the statistical difference in urinary ACR between control group and diabetic cases without vascular complications.

p3-value describes the statistical difference in urinary ACR between control group and diabetic cases with vascular complications.

p4-value describes the statistical difference in urinary ACR between diabetic cases without and with vascular complications.

Abbreviations: ACR, Albumin to creatinine ratio; ALT, Alanine aminotransferase; BMI, body mass index; CIMT, Carotid intima media thickness, HbA1c, Glycated hemoglobin, HDL-C, high density lipoprotein cholesterol, LDL-C, Low density lipoprotein cholesterol.

^∗A p-value less than 0.001 was considered highly significant.

* A p-value less than 0.05 was considered statistically significant.

Table 3 The agreement of GSTP1 genotypes with Hardy Weinberg (HW) equilibrium.

Genotype	Observed		HW expected		x^2	p-value
	Number	%	Number	%
Control group
Wild (AA)	38	74.5	37.1	72.7	1.002	0.316
Heterozygous mutant (AG)	11	21.6	12.8	25.1
Homozygous mutant (GG)	2	3.9	1.1	2.2
Diabetic group
Wild (AA)	29	53.7	30.4	56.3	0.995	0.318
Heterozygous mutant (AG)	23	42.6	20.3	37.6
Homozygous mutant (GG)	2	3.7	3.4	6.1
Total subjects
Wild (AA)	67	63.8	67.2	64.0	0.014	0.902
Heterozygous mutant (AG)	34	32.4	33.6	32.0
Homozygous mutant (GG)	4	3.8	4.2	4.0

A p-value less than 0.05 was considered statistically significant.

Table 4 Distribution of GSTM1, GSTT1, and GSTP1 gene polymorphisms among the studied groups.

GSTs genepolymorphisms	Control group (n = 51)		Total number of diabetic cases (n = 54)		Diabetic cases with vascular complications (n = 27)		Diabetic cases without vascular complications (n = 27)		p-value
	Number	%	Number	%	Number	%	Number	%
GSTM1 gene									χ^2p1 = 0.631 χ^2p2 = 0.859
Present	25	49.0	29	53.7	14	51.9	15	55.6
Deleted	26	51.0	25	46.3	13	48.1	12	44.4
GSTT1 gene									χ^2p1 = 0.832 χ^2p2 = 0.507
Present	33	64.7	36	66.7	16	59.3	20	74.1
Deleted	18	35.3	18	33.3	11	40.7	7	25.9
GSTP1 gene									^MCp1 = 0.053 ^MCp2 = 0.023^*
Wild (AA)	38	74.5	29	53.7	18	66.7	11	40.7
Heterozygous mutant (AG)	11	21.6	23	42.6	9	33.3	14	51.9
Homozygous mutant (GG)	2	3.9	2	3.7	0	0	2	7.4

p1: Statistical differences between control group and total number of diabetic cases.

p2: Statistical difference between control and each of diabetic cases with and without vascular complications of T2DM.

Abbreviations: MC, Monte Carlo test; χ², Chi square test.

* A p-value less than 0.05 was considered statistically significant.

Table 5 Frequency of diabetic patients with the three GST gene polymorphisms according to each vascular complication.

Diabetic complications	GSTM1				GSTT1				GSTP1				p-value
	Present		Deleted		Present		Deleted		Wild type		Heterozygous
	No.	%	No.	%	No.	%	No.	%	No.	%	No.	%
CAD													χ^2p1 = 0.082 χ^2p2 = 0.581 χ^2p3 = 1.000
Positive	5	35.7	9	69.2	9	56.3	5	45.5	9	50.0	5	55.6
Negative	9	64.3	4	30.8	7	43.8	6	54.5	9	50.0	4	44.4
PAD													^FEp1 = 1.000
Positive	3	21.4	2	15.4	5	31.3	0	0.0	3	16.7	2	22.2	^FEp2 = 0.060
Negative	11	78.6	11	84.6	11	68.6	11	100.0	15	83.3	7	77.8	^FEp3 = 1.000
CVD Positive Negative													–
	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0	0	0.0
	14	100.0	13	100.0	16	100.0	11	100.0	18	100.0	9	100.0
Retinopathy Positive Negative													χ^2p1 = 0.568 χ^2p2 = 0.034^* χ^2p3 = 0.695
	8	57.1	6	46.2	5	68.8	3	27.3	10	55.6	4	44.4
	6	42.9	7	53.8	11	31.3	8	72.7	8	44.4	5	55.6
Neuropathy Positive Negative													^FEp1 = 0.326 ^FEp2 = 0.019^* ^FEp3 = 0.093
	13	92.9	10	76.9	0	100.0	7	63.6	17	94.4	6	66.7
	1	7.1	3	23.1	16	0.0	4	36.4	1	5.6	3	33.3
Nephropathy Positive Negative													^FEp1 = 0.695 ^FEp2 = 0.692 ^FEp3 = 0.193
	10	71.4	8	61.5	10	62.5	8	72.7	10	55.6	8	88.9
	4	28.6	5	38.5	6	37.5	3	27.3	8	44.4	1	11.1

P1: statistical differences of GSTM1according to each vascular complication.

P2: statistical differences of GSTT1 according to each vascular complication.

P3: statistical differences of GSTP1 according to each vascular complication.

Abbreviations: CAD, Coronary artery disease; χ², Chi square test; FE, Fisher Exact test; PAD, peripheral arterial disease; CVD, cerebrovascular disease.

* A p-value less than 0.05 was considered statistically significant.

Table 6 The frequency of diabetic patients with GSTM1, GSTT1, and GSTP1 gene polymorphisms according to the presence of one or more vascular complications of diabetes.

GSTs gene polymorphisms	Number of diabetic microvascular and macrovascular complications										p-value
	1		2		3		4		5
	No.	%	No.	%	No.	%	No.	%	No.	%
GSTM1
Present	3	21.4	3	21.4	3	21.4	4	28.6	1	7.1	0.921
Deleted	4	30.8	1	7.7	4	30.8	3	23.1	1	7.1
GSTT1
Present	2	12.5	3	18.8	3	18.8	6	37.5	2	12.5	0.132
Deleted	5	45.5	1	9.1	4	36.4	1	9.1	0	0.0
GSTP1
A allele	11	78.6	7	87.5	12	85.7	13	92.9	2	50.0	0.393
G allele	3	21.4	1	12.5	2	14.3	1	7.1	2	50.0

A p-value less than 0.05 was considered statistically significant.

Monte Carlo test was used for comparing between numbers of complications categories.

Table 7 Distribution of combinations of glutathione S-transferase genotypes among diabetic patients and control group and the risk of developing diabetes mellitus.

Combined GST polymorphisms	Control group (n = 51)		Diabetic patients (n = 54)		p-value	OR	95% Confidence interval
	No.	%	No.	%
GSTM1 and GSTT1
Both present ®	16	31.4	22	40.7
Both deleted	26	51.0	21	38.9	χ^2p = 0.226	0.587	0.247–1.392
One present + one deleted	9	17.6	11	20.4	χ^2p = 0.832	0.889	0.298–2.647
GSTM1 and GSTP1
Present M1 + wild (AA) P1 ®	16	31.4	14	25.9
Present M1 + hetero mutant (AG) P1	9	17.6	12	22.2	χ^2p = 0.461	1.523	0.495–4.685
Present M1 + homo mutant (GG) P1	0	0	2	3.7	^FEp = 0.484	-	-
Deleted M1 + wild (AA) P1	22	43.1	15	27.8	χ^2p = 0.615	0.779	0.294–2.060
Deleted M1 + hetero mutant (AG) P1	2	3.9	11	20.4	χ^2p = 0.021^*	6.285	1.184–33.347
Deleted M1 + homo mutant (GG) P1	2	3.9	0	0.0	^FEp = 0.492	-	-
Present M1 + AG and GG-P1	9	17.6	14	25.9	χ^2p = 0.305	1.778	0.590–5.354
Deleted M1 + AG and GG- P1	4	7.8	11	20.4	χ^2p = 0.090	3.142	0.814–12.131
GSTT1 and GSTP1
Present T1 + wild (AA) P1 ®	24	47.1	23	42.6
Present T1 + hetero mutant (AG) P1	7	13.7	11	20.4	χ^2p = 0.379	1.639	0.542–4.960
Present T1 + homo mutant (GG) P1	1	2.0	2	3.7	^FEp = 0.552	2.087	0.176–24.616
Deleted T1 + Wild (AA) P1	14	27.5	6	11.1	^FEp = 0.152	0.447	0.146–1.362
Deleted T1 + hetero mutant (AG) P1	4	7.8	12	22.2	χ^2p = 0.070	3.130	0.881–11.123
Deleted T1 + homo mutant (GG) P1	1	2.0	0	0.0	^FEp = 1.000	-
Present T1 + AG and GG-P1	5	15.7	13	24.1	χ^2p = 0.091	2.713	0.834–8.823
Deleted T1 + AG an GG- P1	5	9.8	12	22.2	χ^2p = 0.124	2.504	0.762–8.230

Abbreviations: FE, Fisher exact test; OR, Odds ratio; ®, reference group; χ²: Chi square test; Hetero, heterozygous; Homo, homozygous.

* A p-value less than 0.05 was considered statistically significant.