Novel association between superoxide dismutase polymorphism and progression of nocturnal enuresis

Figures & data

Table 1. primers used in this study for the detection of SNP in SOD1 and SOD2 genes.

Gene	RS #	Primers	Sequence (5’ − 3’)	Fragment size	Annealing temp
SOD1	rs1041740 (C/T)	FO	CGCGGTTTCTAAAGATCCAG	704
		RO	CCGCAGTCACTCTTACTTTGG
		R	AAAAGAGAGCCTGTCTACTTGGTG	C allele: 421	60.2°C
		R	GTAAAAGAGAGCCTGTCTACTTGGTA	T allele: 421	59.4°C
	rs17880135 (G/T)	FO	CCTGAGAGAATGCTTTTCAATG	481
		R	GTTGTTTTAACCACTTACTGTGAACC	G allele: 252	56.6°C
		R	GTTGTTTTAACCACTTACTGTGAAAC	T allele: 252
SOD2	rs4880 (T/C)	FO	GCACTAGCAGCATGTTGAGC	709
		R	GAGCCCAGATACCCCAAAA	T allele: 356	57.3°C
		R	GAGCCCAGATACCCCAAAG	C allele: 356

Table 2. Comparison of age and gender among the studied groups. χ2: chi-square test.

		Control group (n = 47)	Cases Group (n = 53)	Test of significance	P-value
Age (years) mean±SD		13.06 ± 2.05	13.29 ± 2.01	t=0.56	0.567
Gender	Males n (%)	20 (42.6%)	28 (52.8%)	X^2=1.05	0.305
	Females n (%)	27 (57.4%)	25 (47.2%)

Table 3. Comparison of biochemical parameters within the studied groups. Z: Mann Whitney U test. Parameters are described as median (min-max) (interquartile range). P is significant when < 0.05. *Statistically significant.

Parameters	Control Group (n=47)	Cases Group (n=53)	Test of significance	P value
SOD (U/mg protein)	60.37 (4.81–82.65) (48.88–69.47)	42.83 (−18.31, 140.86) (27.63–72.96)	Z=2.01	P=0.04*
MDA (nmol/mL)	3.43 (0.25–9.18) (2.16–5.62)	4.55 (0.69–22.51) (2.78–7.37)	Z=1.59	P=0.112
PC (nmol/mg protein)	5.15 (1.34–9.25) (2.94–6.86)	8.39 (2.17–19.56) (6.86–11.15)	Z=5.79	P<0.001*
NO (mg/L)	1.284 (0.69–5.67) (1.11–1.54)	1.26 (0.57–4.15) (0.857–1.78)	Z=0.580	P=0.562
CAT(KU/L)	30.59 (5.71–96.05) (21.81–45.22)	31.98 (2.71–101.87) (25.09–46.09)	Z=0.364	P=0.716
IL −8 (pg/mL)	25.71 (5.38–95.25) (13.24–38.48)	32.52 (6.36–112.46) (23.81–42.0)	Z=1.27	P=0.204

Figure 1. Gel electrophoresis (2%) for PCR product of SOD1 rs1041740. Lane M indicates DNA marker (50 bp). Specific 421 bp band illustrates C or T alleles; Lanes (C1, C2 and C3) represent CC homozygous genotyping, where T allele absents in control group. Lanes (P1, P2, P3, P4 and P5) represent CT heterozygous genotyping; where both C and T alleles were detected at 421 bp in cases group; lane P6 represents TT homozygous genotyping where T allele appears in cases group.

Figure 2. There were no significant differences in SOD1 rs1041740 genotypes between studied groups.

Table 4. Comparison of SOD1 rs1041740 genotypes between the studied groups. Data was expressed as frequency and percentage. χ2: Chi-Square test, Z: Mann Whitney U test, FET: Fisher’s exact test. Parameters are described as numbers (Percentage). P is significant when < 0.05.

Genotype	Cases group n (%)	Control group n (%)	Test of significance	Odds ratio (95% CI)	P value
CT	47 (88.7%)	38 (86.4%)	χ^2=0.119	1.24 (0.369–4.15)	P=0.730
CC	3 (5.7%)	0	FET=2.57	Undefined	P=0.11
TT	3 (5.7%)	6 (13.6%)	χ^2=1.82	0.380 (0.089–v1.62)	P=0.178

Figure 3. Gel electrophoresis (2%) for the PCR product of SOD1 rs17880135; Lane M indicates DNA marker (50 bp). Specific 256 bp bands illustrate G or T alleles; Lanes (C1, C2 and C3) represent GG homozygous genotyping where T allele absents in control group. Lane (P1, P2, P4, P5 and P6) represent GT heterozygous genotyping, where G and T alleles appear at 256 bp in cases group. Lane P3 represents TT homozygous genotyping where T allele appears in cases group.

Figure 4. Significant difference in SOD1 rs17880135 was found between studied groups.

Table 5. Comparison of SOD1 rs17880135 genotypes between the studied groups. χ2: Chi-Square test, Z: Mann Whitney U test. Parameters are described as numbers (Percentage). P is significant when < 0.05. * Statistically significant.

Genotype	Cases group n (%)	Control group n (%)	Test of significance	Odds ratio (95% CI)	P value
GT	48 (90.6%)	6 (14%)	χ^2=56.6	59.2 (16.76–209.11)	p<0.001*
TT	1 (1.9%)	2 (4.7%)	χ^2=0.599	0.394 (0.035–4.50	p=0.439
GG	4 (7.5%)	35 (81.4%)	χ^2=53.67	0.019 (0.0052–0.07)	p<0.001*

Figure 5. Gel electrophoresis (2%) for PCR product of SOD2 rs4880. Lane M indicates DNA marker (50 bp). Specific 356 bp band illustrates T or C allele. Lanes (C1, C2 and C3) represent TT homozygous genotyping where C allele absents in control group. Lanes (P1, P2) represent CC homozygous genotyping where C allele appears at lanes P1 and P2 at 356 bp in cases group while lanes (P3, P4, P5 and P6) represent TC heterozygous genotyping where T and C alleles appear at 356 bp in cases group.

Figure 6. Significant higher TT allele genotype in SOD2 rs4880 between studied groups.

Table 6. Comparison of SOD2 rs4880 genotypes between the studied groups. χ2: Chi-Square test, parameters are described as number (percentage).

Genotype	Cases group n (%)	Control group n (%)	Test of significance	Odds ratio (95% CI)	P value
CT	37 (71.2%)	38 (82.6%)	χ^2=1.78	0.519 (0.197–1.37)	p=0.181
CC	3 (5.8%)	5 (10.9%)	χ^2=0.847	0.502 (0.113–2.22)	p=0.357
TT	12 (23.1%)	3 (6.5%)	χ^2=5.16	4.3 (1.13–16.36)	p=0.02*

Table 7. Relation between SOD1 rs17880135 SNP genotypes and laboratory findings among cases. Z: Mann Whitney U test, *statistically significant. Parameters are described as median (min-max) (interquartile range).

Cases group	SOD1 rs17880135		test of significance	P value
	GT (N=49)	GG&TT (N=4)
SOD (U/mg protein)	42.83(−18.31, 140.86) (27.63–74.08)	51.0(17.82–72.42) (22.91–70.27)	Z=0.034	P=0.973
MDA (nmol/mL)	4.12(0.69–22.51) (4.12–7.12)	7.06(5.84–10.83) (6.04–9.98)	Z=1.85	P=0.064
PC (nmol/mg protein)	8.39(2.17–19.56) (6.86–11.03)	9.63(4.34–14.57) (5.10–13.89)	Z=0.370	P=0.711
NO (mg/L)	1.26(0.65–4.15) (0.857–1.78)	1.38(0.57–3.4) (0.737–2.94)	Z=0.236	P=0.814
CAT (kU/L)	32.54(2.71–101.87) (25.37–47.49)	28.02(12.13–31.16) (12.13–31.16)	Z=1.23	P=0.218
IL8 (pg/mL)	33.58(11.58–112.46) (27.55–43.55)	12.46(6.36–14.21) (6.36–14.21)	Z=2.58	P=0.01*

Table 8. Relation between SOD2 rs4880 SNP genotypes and laboratory findings among cases. Z: Mann Whitney U test, *statistically significant. Parameters are described as median (min-max) (interquartile range).

Cases group	SOD2 rs4880		test of significance	P value
	CT (N=37)	CC&TT (N=15)
SOD (U/mg protein)	41.10 (−18.31, 140.86) (20.94–65.91)	54.64 (13.88–122.54) (30.0–86.24)	Z=1.28	P=0.20
MDA (nmol/mL)	5.38 (1.23–22.51) (3.29–7.54)	3.11 (0.69–10.43) (1.95–6.65)	Z=1.97	P=0.049*
PC (nmol/mg protein)	8.07 (2.17–19.56) (6.86–11.74)	9.0 (4.34–14.57) (6.14–9.58)	Z=0.638	P=0.524
NO (mg/L)	1.32 (0.66–3.55) (1.02–2.01)	1.11 (0.57–4.15) (0.772–1.45)	Z=0.454	P=0.650
CAT (KU/L)	28.02 (9.86–101.87) (19.62–47.49)	33.95 (2.71–74.51) (30.59–47.90)	Z=0.803	P=0.422
IL8 (pg/mL)	31.46 (6.36–112.46) (23.14–38.54)	71.76 (65.86–77.66) (65.86–77.66)	Z=2.09	P=0.037*