Endothelial nitric oxide synthase gene polymorphisms and the risk of vasculopathy in sickle cell disease

Figures & data

Figure 1 Genotyping of eNOS intron 4 polymorphism. Lanes 2, 3, and 7 show 420-bp bands, denoting the wild type (b) allele. Lanes 4 and 6 show 393-bp bands denoting homomutant (a) allele. Lanes 1, 5, and 8 show two bands at 427 and 393 bp denoting heteromutant type (ba). M is a marker ladder.

Figure 2 Genotyping of eNOS T-786C polymorphism in the promoter region. Lanes 1, 3, and 7 show 140 bp bands, denoting wild type (T) allele. Lanes 5, 6, and 8 show 90 bp bands, denoting homomutant type (C) allele. Lanes 2 and 4 show bands at 140 and 90 bp, denoting heteromutant type (TC). M is a marker ladder. NB: Bands at 50 and 40 bp could not be seen.

Table 1 Distribution of eNOS genotypes among SCD patients and the controls

	Case (n = 100)	Control (n = 80)	Chi-square	P value	Odds ratio	95% Confidence interval
eNOS 786T> C
Wild (T/T)	39 (39%)	33 (41.3%)	0.094	0.759	1.098	0.603–2.000
Hetero (T/C)	49 (49%)	38 (47.5%)	0.040	0.841	1.062	0.590–1.913
Homo (C/C)	12 (12%)	9 (11.3%)	0.024	0.876	1.076	0.429–2.697
eNOS 4a/b
Wild (4b/4b)	52 (52%)	50 (62.5%)	1.995	0.158	1.538	0.845–2.800
Heterozygous (4a/4b)	49 (49%)	38 (47.5%)	1.060	0.303	1.412	0.731–2.725
Homozygous (4a/4a)	16 (16%)	10 (12.5%)	0.441	0.507	1.333	0.569–3.124

Odds ratio and P-value were calculated by Pearson's chi-square test.

SCD, sickle cell disease; eNOS, endothelial nitric oxide synthase.

Table 2 Distribution of eNOS haplotypes among SCD patients and controls

			Patients		Controls
Haplotype	eNOS 786T>C eNOS 4a/b	Number	%	Number	%	Chi-square	P value	Odds ratio	95% Confidence Interval
Haplotype 1	T-4b	25	25	23	28.8	0.320	0.572	0.826	0.426–1.603
Haplotype 2	T-4a	14	14	10	12.5	0.087	0.769	1.140	0.477–2.722
Haplotype 3	C-4b	27	27	27	33.8	0.964	0.326	0.726	0.383–1.377
Haplotype 4	C-4a	34	34	20	25.0	1.714	0.190	1.545	0.804–2.971

Odds ratio and P-value was calculated by Pearson's chi-square test.

SCD, sickle cell disease; eNOS, endothelial nitric oxide synthase.

Table 3 Comparison between SCD patients carrying various eNOS genotypes according to gender

	Female (n = 47)	Male (n = 53)	Chi-square	P value	Odds ratio	95% Confidence interval
eNOS 786T> C
Wild type (T/T)	24 (51.1%)	15 (28.3%)	5.425	0.020*	2.643	1.156–6.045
Heterozygous (T/C)	18 (38.3%)	31 (58.5%)	4.064	0.044*	2.270	1.017–5.066
Homozygous (C/C)	5 (10.6%)	7 (13.2%)	0.156	0.693	1.278	0.377–4.336
eNOS 4a/b
Wild type (4b/4b)	31 (66.0%)	21 (39.6%)	6.921	0.009*	2.952	1.305–6.681
Heterozygous (4a/4b)	10 (21.2%)	22 (41.5%)	4.686	0.030*	2.626	1.082–6.374
Homozygous (4a/4a)	6 (12.8%)	10 (18.9%)	0.690	0.406	1.589	0.530–4.768

*Statistically significant.

Odds ratio and P-value was calculated by Pearson's chi-square test.

SCD, sickle cell disease; eNOS, endothelial nitric oxide synthase.

Table 4 Comparison between various eNOS genotypes according to presence and absence of SCD manifestations (VOC, ACS, and PHTN)

The eNOS genotypes			Chi-square	P-value	Odds ratio	95% confidence interval
ecNOS 786T>C	ACS– (n = 84)	ACS+ (n = 16)
CC	5 (5.95%)	7 (43.75%)	18.183	0.000*	12.289	3.221–46.878
TC	44 (52.38%)	5 (31.25%)	2.4010	0.121	0.4130	0.132–1.293
TT	35 (41.66%)	4 (25.00%)	1.5690	0.210	2.1930	0.638–7.200
eNOS 4a/b	ACS– (n = 84)	ACS+ (n = 16)
4a/4a	12 (14.28%)	4 (25.00%)	1.1480	0.284	2.0000	0.553–7.237
4a/4b	25 (29.76%)	7 (43.75%)	1.2090	0.272	1.8360	0.615–5475
4b/4b	47 (55.95%)	5 (31.25%)	3.2860	0.070	2.7950	0.892–8.751
ecNOS 786T>C	VOC– (n = 31)	VOC+ (n = 69)
CC	3 (25%)	9 (13.04%)	0.230	0.632	1.4000	0.352–5.573
TC	14 (28.6%)	35 (50.07%)	0.265	0.607	1.2500	0.534–2.926
TT	14 (35.9%)	25 (36.23%)	0.717	0.397	1.4490	0.613–3.429
eNOS 4a/b	VOC– (n = 31)	VOC+(n = 69)
4a/4a	3 (9.67%)	13 (18.84%)	1.336	0.248	2.1670	0.570–8.232
4a/4b	5 (16.12%)	27 (39.13%)	5.201	0.230	3.3430	1.144–9.768
4b/4b	23 (74.19%)	29 (42.02%)	8.866	0.003*	3.9660	1.556–10.109
ecNOS 786T>C	PHTN– (n = 68)	PHTN+ (n = 32)
CC	8 (11.76%)	4 (12.5%)	0.11	0.916	1.0710	0.289–3.859
TC	29 (42.64%)	20 (62.5%)	3.432	0.064	2.2410	0.947–5.308
TT	31 (45.58)	8 (25.0%)	3.877	0.490	2.5140	0.990–6.381
eNOS 4a/b	PHTN– (n = 68)	PHTN+ (n = 32)
4a/4a	10 (14.70%)	6 (18.75%)	0.265	0.607	1.3380	0.44–4.073
4a/4b	18 (26.47)	14 (43.75%)	2.986	0.084	2.1600	0.894–5.220
4b/4b	40 (58.82)	12 (37.5%)	3.960	0.046*	2.3810	1.004–5.647

*Statistically significant.

Odds ratio and P-value were calculated by Pearson's chi-square test.

SCD, sickle cell disease; eNOS, endothelial nitric oxide synthase; ACS+, have acute chest syndrome; ACS–, not have acute chest syndrome; VOC+, have vaso-oclusive crisis; VOC–, do not have vaso-oclusive crisis; PHTN+, have pulmonary hypertension; PHTN–, do not have pulmonary hypertension.

Table 5 Comparison between various eNOS haplotypes (eNOS 786T>C – eNOS 4a/b) according to presence and absence of SCD manifestations (VOC, ACS, and PHTN)

	Haplotype 1 T-4b	Haplotype 2 T-4a	Haplotype 3 C-4b	Haplotype 4 C-4a
VOC
VOC +	15 (21.7%)	10 (14.5%)	14 (20.3%)	30 (43.5%)
VOC−	10 (32.3%)	4 (12.9%)	13 (41.9%)	4 (12.9%)
Chi-square	1.262	0.045	5.085	8.911
P-value	0.261	0.832	0.024	0.003*
Odds ratio	0.583	1.144	0.352	5.192
95% Confidence interval	0.227–1.502	329–3.976	0.140–0.888	1.639–16.446
ACS
ACS+	2 (12.5%)	2 (12.5%)	3 (18.8%)	9 (56.3%)
ACS–	23 (27.4%)	12 (14.3%)	24 (28.6%)	25 (29.8%)
Chi-square	1.587	0.036	0.658	4.202
P-value	0.208	0.850	0.417	0.040*
Odds ratio	0.379	0.857	0.577	3.034
95% Confidence interval	0.080–1.798	0.173–4.257	0.151–2.207	1.017–9.051
PHTN
PHTN+	4 (12.5%)	4 (12.5%)	8 (25.0%)	16 (50.0%)
PHTN–	21 (30.9%)	10 (14.7%)	19 (27.9%)	18 (26.5%)
Chi-square	3.922	0.088	0.096	5.369
P-value	0.048	0.767	0.757	0.021*
Odds ratio	0.320	0.829	0.860	2.778
95% Confidence interval	0.100–1.027	0.239–2.875	0.329–2.244	1.155–6.682

*Statistically significant.

Odds ratio and P-value were calculated by Pearson's chi-square test.

SCD, sickle cell disease; eNOS, endothelial nitric oxide synthase; ACS+, have acute chest syndrome; ACS–, not have acute chest syndrome; VOC+, have vaso-oclusive crisis; VOC–, do not have vaso-oclusive crisis; PHTN+, have pulmonary hypertension; PHTN–, do not have pulmonary hypertension.

Table 6 Comparison between eNOS (eNOS 786T>C – eNOS 4a/b) genotypes according to number of VOC and A/C ratio

	eNOS 786 T>C			eNOS 4a/b
	CC	TC	TT	Aa	Ab	Bb
VOC
Mean ± SD	2.89 ± 2.03	1.97 ± 1.07	1.68 ± 0.99	2.31 ± 1.25	2.19 ± 1.11	1.66 ± 1.31
Median	2	2	1	2	2	1
25–75% Quentin	1–5	1–2	1–1	1–3	1–3	1–2
P-value	0.163			0.011*
A/C ratio
Mean ± SD	0.32 ± 0.26	0.57 ± 0.57	0.48 ± 0.39	0.41 ± 0.51	0.62 ± 0.82	0.46 ± 0.41
Median	0.20	0.32	0.44	0.17	0.32	0.35
25–75% Quentin	0.9–0.56	0.11–0.72	0.17–0.66	0.09–0.6	0.16–0.77	0.11–0.66
P-value	0.55			0.66

*Statistically significant.

P-value was calculated by the Kruskal–Wallis test.

SCD, sickle cell disease; eNOS, endothelial nitric oxide synthase; VOC, vaso-oclusive crisis; A/C ratio, albumin/creatinine ratio; SD, standard deviation.

Table 7 Comparison between eNOS (eNOS 786T>C – eNOS 4a/b) haplotypes according to number of VOC and A/C ratios

	eNOS haplotype
	Haplotype 1 T-4b	Haplotype 2 T-4a	Haplotype 3 C-4b	Haplotype 4 C-4a
VOC
Mean ± SD	1.4 ± 0.63	2.1 ± 1.29	1.93 ± 1.77	2.27 ± 1.11
Median	1	2	1	2
25–75% Quentin	1–2	1–3	1–2	1–3
P-value				0.02*
A/C ratio
Mean ± SD	0.56 ± 0.42	0.37 ± 0.32	0.37 ± 0.38	0.63 ± 0.84
Median	0.6	0.28	0.3	0.31
25–75% Quentin	0.19–0.81	0.17–0.51	0.11–0.4	0.1–0.78
P-value				0.496

*Statistically significant.

P-value was calculated by the Kruskal–Wallis test.

SCD, sickle cell disease; eNOS, endothelial nitric oxide synthase; VOC, vaso-oclusive crisis; A/C ratio, albumin/creatinine ratio; SD, standard deviation.