A meta-analysis on the relationship of eNOS 4b/a polymorphism and diabetic nephropathy susceptibility

Figures & data

Figure 1. Selection process of references for the meta-analysis.

Table 1. Characteristics of the studies included in the meta-analysis.

Study ID	Country (Ethnicity)	Type of DM	M/F	Age (yr)^a	BMI	DM duration (yr)^a	Genotyping method	Source of DM/DN	Statue of non-DM	Selection criteria for DN
Wang, 2005	China (Asian)	2	50/49	63.9	–	>5	PCR	Population	Healthy	UAER >20 µg/min
Miao, 2003–1	①China (Asian)	2	–	61.0 ± 14.5	–	>10	PCR	Hospital	–	AER >30 mg/d
Miao, 2003–2	②Malaysia (Asian)	2	–	61.0 ± 14.5	–	>10	PCR	Hospital	–	AER >30 mg/d
Miao, 2003–3	③India (Indian)	2	–	61.0 ± 14.5	–	>10	PCR	Hospital	–	AER >30 mg/d
Dong, 2005	China (Asian)	2	26/44	63.1 ± 7.3	–	15.3 ± 3.1	PCR-RFLP	Hospital	Healthy	UAER >20 µg/min
Luo, 2003	China (Asian)	2	29/20	59.0 ± 10.5	22.4 ± 3.0	9.9 ± 6.4	PCR	Hospital	Healthy	UAER >20 µg/min
Shestakova, 2006	Russia (Caucasian)	1	30/33	25.7 ± 6.5	22.6 ± 2.4	12.6 ± 2.8	PCR	–	Healthy	Persistent macroalbuminuria (AER >300 mg/24 h) with or without antihypertensive therapy in the absence of the urinary tract infection
Li, 2001	China (Asian)	2	30/49	63.1 ± 7.3	24.1 ± 2.2	15.3 ± 3.1	PCR-RFLP	Hospital	Healthy	UAER 20–200 µg/min
Rippin, 2003	United Kingdom (Caucasian)	1	–	–	–	–	PCR	–	–	Overt proteinuria (AER ≥300 mg/24 h)
Sun, 2001	China (Asian)	2	–	59.8 ± 9.5	–	5.3 ± 6.2	PCR-RFLP	Hospital	unclear	Microalbuminuria (UAER 20–199 µg/min), macroalbuminuria (UAER >200 µg/min) or ESRD
Rahimi, 2013	Iran (Caucasian)	2	–	56.0 ± 8.6	27.3 ± 4.5	9.1 ± 5.8	PCR-RFLP	Hospital	Healthy	Microalbuminuria (albumin to creatinine ratio, ACR 30–299 mg/g) and macroalbuminuria (ACR ≥300 mg/g)
Mohseni, 2011	Iran (Caucasian)	2	–	53.1 ± 10.4	27.0 ± 4.0	–	PCR	Hospital	–	Microalbuminuria (AER >30 mg/24 h)
Shoukry, 2012	Egypt (African)	2	108/92	55.3 ± 5.8	27.4 ± 3.2	14.5 ± 4.3	PCR-RFLP	Hospital	–	ACR ≥300 mg/g
Ezzidi, 2008	Tunisia (African)	2	–	59.6 ± 10.8	27.4 ± 5.0	13.5 ± 6.3	PCR-RFLP	Hospital	Healthy	AER >30 mg/24 h and/or high plasma creatinine levels (>176 µmol/L)
Degen, 2001–1	Germany (Caucasian)	①1	–	–	–	>10	–	–	–	Persistent microalbuminuria (AER ≥ 30 mg/24 h)
Degen, 2001–2	Germany (Caucasian)	②2	–	–	–	>10	–	–	–	Persistent microalbuminuria (AER ≥30 mg/24 h)
Santos, 2011	Portugal, Spain, Italy, and Germany (Caucasian)^b	2	–	60.4 ± 9.7	28.7 ± 5.0	15.0 ± 9.1	PCR or PCR-RFLP	–	Healthy	Microalbuminuria (UAER 20–199 µg/min or 17–174 mg/L), macroalbuminuria (UAER 200 µg/min or >174 mg/L) or ESRD
Neugebauer, 2000	Japan (Asian)	2	29/10	59.0 ± 8.6	23.9 ± 2.3	15.2 ± 4.5	–	Hospital	Healthy	The ratio of urinary albumin to urinary creatinine between 20 and 200 mg/g
Dong 2007–1	①China (Asian)	2	–	60.0 ± 12.3	–	>10	PCR	Hospital	–	AER ≥30 ml/24 h
Dong, 2007–2	②Singapore (Asian)	2	–	60.0 ± 12.3	–	>10	PCR	Hospital	–	AER ≥30 ml/24 h
Shimizu, 2002	Japan (Asian)	2	–	64.1 ± 9.7	22.3 ± 2.8	17.7 ± 9.4	PCR-RFLP	–	–	Dialysis due to diabetic nephropathy or overt proteinuria with an elevated serum creatinine level (≥1.5 mg/dl) or overt proteinuria (ACR ≥300 mg/g)
Ahluwalia, 2008	India (Indian)	2	68/127	60.0± 6.2	28.0 ± 4.0	16.5 ± 6.4	PCR-RFLP	Hospital	–	Urinary albumin >500 mg/L or ACR >300 µg/mg
Ksiazek, 2003	Poland (Caucasian)	2	86/92	57.9 ± 8.2	29.1 ± 4.2	8.7 ± 3.1	PCR	–	Healthy	On the basis of a questionnaire, medical records and the measurement of albumin excretion
Mollsten, 2006–1	①Finnish (Caucasian)	1	–	38.7 ± 9.2	–	27.3 (5–47)	PCR	Hospital	–	Microalbuminuria (incipient nephropathy, UAER 20–200 µg/min) or macroalbuminuria (overt nephropathy, UAER >200 µg/min)
Mollsten, 2006–2	②Swedish (Caucasian)	1	76/74	48.7 ± 10.2	–	35.0 (11–65)	PCR	Hospital	–	Microalbuminuria (incipient nephropathy, UAER 20–200 µg/min) or macroalbuminuria (overt nephropathy, UAER >200 µg/min)
Fujita, 2000	Japan (Asian)	2	61/41	61.0 ± 12.0	23.1 ± 4.0	–	PCR	–	–	UAER >200 µg/min
Lin, 2002	China (Asian)	2	–	59.9 ± 11.2	–	11.7 ± 7.0	PCR	–	Healthy	UAER >200 µg/min or quantitative urine protein >500 mg/d or qualitative urine protein greater than (++)
Zhang, 2005	China (Asian)	2	–	54.0 ± 15.0 M^c	21.0 ± 7.0	52.0 ± 8.2 M^c	PCR	–	Healthy	UAER ≥30 mg/d
Ma, 2003	China (Asian)	2	81/86	61.5 ± 8.5	24.8 ± 3.4	11.3 ± 5.2	PCR-RFLP	Hospital	Healthy	Microalbuminuria (AER 30–300 mg/24 h), macroalbuminuria (AER >300 mg/24 h) or ESRD (AER >300 mg/24 h, increased creatinine or dialysis)
Guo, 2011	China (Asian)	2	–	64.3 ± 9.7	24.5 ± 4.0	15.2 ± 6.5	PCR	Hospital	Healthy	AER ≥30 mg/24 h
Xin, 2004	China (Asian)	2	56/74	62.9 ± 9.4	24.5 ± 3.6	11.4 ± 6.0	PCR	–	–	Microalbuminuria (UAER 20–200 µg/min), macroalbuminuria (UAER >200 µg/min and urine protein >500 mg/d)

Notes: ^aGiven as mean ± SD or as mean (min – max).

^bThis study did not give separate data for every ethnicity, so it was considered as one study.

^cM represented month that different from year.

yr, year; PCR, polymerase chain reaction; PCR-RFLP, polymerase chain reaction-restricted fragment length polymorphisms; –, not reported.

Table 2. Distribution of NOS3 genotypes and p values in χ² test of HWE in the eligible studies.

			Case			DM control			Non-DM control			HWE (p values)
Study ID	Country (Ethnicity)	Type of DM	4b/b(%)	4a/b(%)	4a/a(%)	4b/b(%)	4a/b(%)	4a/a(%)	4b/b(%)	4a/b(%)	4a/a(%)	DN	DM	non-DM
Wang, 2005	China (Asian)	2	73 (0.74)	26 (0.26)	0 (0)	82 (0.78)	23 (0.22)	0 (0)	87 (0.87)	13 (0.13)	0 (0)	0.13	0.21	0.49
Miao, 2003	① China (Asian)	2	66 (0.78)	14 (0.16)	3 (0.04)	56 (0.86)	9 (0.14)	0 (0)	–	–	–	0.06	0.55	–
	② Malaysia (Asian)	2	45 (0.88)	5 (0.10)	0 (0)	17 (0.85)	3 (0.15)	0 (0)	–	–	–	0.71	0.72	–
	③ India (Indian)	2	18 (0.69)	7 (0.27)	1 (0.04)	7 (0.64)	4 (0.36)	0 (0)	–	–	–	0.76	0.46	–
Dong, 2005	China (Asian)	2	43 (0.61)	27 (0.39)	0 (0)	53 (0.83)	11 (0.17)	0 (0)	62 (0.73)	22 (0.26)	1 (0.01)	0.05	0.45	0.54
Luo, 2003	China (Asian)	2	20 (0.41)	2 (0.04)	27 (0.55)	23 (0.66)	4 (0.11)	8 (0.23)	34 (0.92)	1 (0.03)	2 (0.05)	0.00	0.00	0.00
Shestakova, 2006	Russia (Caucasian)	1	14 (0.22)	48 (0.76)	1 (0.02)	34 (0.52)	31 (0.47)	1 (0.02)	–	–	–	0.00	0.04	–
Li, 2001	China (Asian)	2	50 (0.63)	29 (0.37)	0 (0)	53 (0.83)	11 (0.17)	0 (0)	62 (0.74)	22 (0.26)	0 (0)	0.05	0.45	0.17
Rippin, 2003	United Kingdom (Caucasian)	1	344 (0.74)	108 (0.23)	12 (0.03)	297 (0.75)	90 (0.23)	9 (0.02)	–	–	–	0.32	0.49	–
Sun, 2001	China (Asian)	2	90 (0.68)	38 (0.29)	4 (0.03)	311 (0.78)	82 (0.21)	6 (0.02)	93 (0.82)	18 (0.16)	2 (0.02)	1.00	0.82	0.32
Rahimi, 2013	Iran (Caucasian)	2	86 (0.71)	32 (0.26)	3 (0.02)	124 (0.72)	46 (0.27)	3 (0.02)	73 (0.72)	28 (0.28)	0 (0)	0.99	0.59	0.11
Mohseni, 2011	Iran (Caucasian)	2	16 (0.80)	4 (0.20)	0 (0)	157 (0.71)	54 (0.25)	9 (0.04)	80 (0.83)	16 (0.17)	0 (0)	0.62	0.13	0.37
Shoukry, 2012	Egypt (African)	2	124 (0.62)	64 (0.32)	12 (0.06)	131 (0.66)	60 (0.30)	9 (0.05)	–	–	–	0.34	0.53	–
Ezzidi, 2008	Tunisia (African)	2	314 (0.62)	162 (0.32)	29 (0.06)	234 (0.59)	143 (0.36)	21 (0.05)	511 (0.68)	217 (0.29)	20 (0.03)	0.19	0.89	0.59
Degen, 2001	Germany (Caucasian)	①1	87 (0.67)	41 (0.32)	2 (0.02)	136 (0.70)	55 (0.28)	3 (0.02)	–	–	–	0.25	0.33	–
	Germany (Caucasian)	②2	142 (0.72)	53 (0.27)	2 (0.01)	161 (0.74)	50 (0.23)	6 (0.03)	–	–	–	0.22	0.38	–
Santos, 2011	Portugal, Spain, Italy, Germany (Caucasian)^c	2	237 (0.68)	99 (0.28)	11 (0.03)	168 (0.72)	59 (0.25)	5 (0.02)	67 (0.67)	30 (0.30)	3 (0.03)	0.87	0.95	0.87
Neugebauer, 2000	Japan (Asian)	2	27 (0.69)	9 (0.23)	3 (0.08)	71 (0.87)	10 (0.12)	1 (0.01)	133 (0.86)	22 (0.14)	0 (0)	0.11	0.36	0.34
Dong, 2007	① China (Asian)	2	42 (0.93)	3 (0.07)	0 (0)	30 (0.71)	12 (0.29)	0 (0)	–	–	–	0.82	0.28	–
	② Singapore (Asian)	2	66 (0.78)	14 (0.16)	3 (0.04)	56 (0.86)	9 (0.14)	0 (0)	–	–	–	0.63	0.55	–
Shimizu, 2002	Japan (Asian)	2	180 (0.78)	44 (0.19)	6 (0.03)	156 (0.77)	44 (0.22)	3(0.01)	–	–	–	0.11	0.96	–
Ahluwalia, 2008	India (Indian)	2	146 (0.75)	28 (0.14)	21 (0.11)	189 (0.74)	61 (0.24)	5 (0.02)	–	–	–	0.00	0.98	–
Ksiazek, 2003	Poland (Caucasian)	2	105 (0.59)	58 (0.33)	15 (0.08)	147 (0.63)	66 (0.28)	19 (0.08)	252 (0.76)	74 (0.22)	4 (0.01)	0.10	0.01	0.58
Mollsten, 2006	① Finnish (Caucasian)	1	553 (0.70)	208 (0.26)	32 (0.04)	242 (0.68)	99 (0.28)	15 (0.04)	–	–	–	0.03	0.24	–
	② Swedish (Caucasian)	1	103 (0.69)	40 (0.27)	7 (0.05)	147 (0.75)	46 (0.23)	4 (0.02)	–	–	–	0.24	0.86	–
Fujita, 2000	Japan (Asian)	2	81 (0.79)	21 (0.21)	0 (0)	55 (0.85)	10 (0.15)	0 (0)	–	–	–	0.25	0.50	–
Lin, 2002	China (Asian)	2	71 (0.90)	8 (0.10)	0 (0)	41 (0.85)	6 (0.13)	1 (0.02)	64 (0.91)	6 (0.09)	0 (0)	0.64	1.59	0.71
Zhang, 2005	China (Asian)	2	159 (0.88)	21 (0.12)	0 (0)	131 (0.85)	21 (0.14)	2 (0.01)	145 (0.87)	20 (0.12)	1 (0.01)	0.41	0.21	0.73
Ma, 2003	China (Asian)	2	127 (0.76)	39 (0.23)	1 (0.01)	105 (0.80)	25 (0.19)	2 (0.02)	82 (0.82)	18 (0.18)	0 (0)	0.28	0.72	0.32
Guo, 2011	China (Asian)	2	56 (0.81)	7 (0.10)	6 (0.09)	70 (0.96)	0 (0)	3 (0.04)	60 (0.95)	1 (0.02)	2 (0.03)	0.00	0.00	0.00
Xin, 2004	China (Asian)	2	110 (0.85)	16 (0.12)	4 (0.03)	128 (0.94)	8 (0.06)	0 (0)	–	–	–	0.00	0.72	–

Table 3. Quality indicators from Newcastle-Ottawa scale (NOs).

	Selection				Comparability		Exposure
Study ID	1	2	3	4	5.1	5.2	6	7	8	Total stars
Wang, 2005	☆	☆	☆	☆	☆	☆	☆	☆	–	8
Miao, 2003	☆	–	–	–	☆	☆	☆	☆	–	5
Dong, 2005	☆	–	☆	☆	–	–	☆	☆	–	5
Luo, 2003	☆	–	☆	–	☆	☆	☆	☆	–	6
Shestakova, 2006	☆☆	–	–	–	–	–	☆	☆	–	3
Li, 2001	☆	–	–	☆	☆	☆	☆	☆	–	6
Rippin, 2003	☆	–	–	–	–	–	☆	☆	–	3
Sun, 2001	☆	–	☆	–	–	–	☆	☆	–	4
Rahimi, 2013	☆	–	☆	–	–	–	☆	☆	–	4
Mohseni, 2011	☆	–	☆	–	☆	–	☆	☆	–	5
Shoukry, 2012	☆	–	☆	–	☆	–	☆	☆	–	5
Ezzidi, 2008	☆	–	☆	–	☆	–	☆	☆	–	5
Degen, 2001	☆	–	–	–	–	–	–	–	–	1
Santos, 2011	☆	☆	☆	–	☆	–	☆	☆	–	6
Neugebauer, 2000	☆	☆	–	–	☆	–	–	–	–	3
Dong, 2007	☆	–	☆	–	–	–	☆	☆	–	4
Shimizu, 2002	☆	☆	☆	–	☆	–	☆	☆	–	6
Ahluwalia, 2008	☆	–	☆	–	☆	–	☆	☆	–	5
Ksiazek, 2003	–	–	☆	–	☆	–	☆	–	–	3
Mollsten, 2006	☆	–	☆	–	☆	–	☆	☆	–	5
Fujita, 2000	☆	–	–	–	☆	–	☆	☆	–	4
Lin, 2002	☆	–	☆	☆	☆	☆	☆	☆	–	7
Zhang, 2005	☆	–	☆	–	–	–	☆	☆	–	4
Ma, 2003	☆	–	☆	☆	☆	☆	☆	☆	–	7
Guo, 2011	☆	–	☆	–	☆	–	☆	☆	–	5
Xin, 2004	☆	–	–	–	☆	–	☆	☆	–	4

Notes: 1, Is the case definition adequate?; 2, representativeness of the cases; 3, selection of controls; 4, definition of controls; 5.1, study controls for the most important factor; 5.2, study controls for additional factor(s); 6, ascertainment of exposure; 7, Same method of ascertainment for cases and controls; and 8, non-response rate.

Table 4. Random effects odds ratios and heterogeneity results for the association of eNOS 4b/a gene polymorphisms and DN.

		Ethnicity according to “nine geographic race”^a				Type of DM^a			HWE^b
DM control	ALL	Asian	Indian	Caucasian	African	All	T1DM	T2DM	All	in HWE	not in HWE
Number of studies	31	17	2	10	2	31	5	26	31	27	4
Pre-allele model
I^2 (%)	57.7	64.4	62.6	9.0	22.1	57.7	48.9	59.8	57.7	48.8	76.4
PHeterogeneity	0.000	0.000	0.102	0.360	0.257	0.000	0.098	0.000	0.000	0.003	0.005
OR	1.26	1.51	0.61	1.10	1.00	1.26	1.16	1.29	1.17	1.12	1.64
95% CI	1.10–1.45	1.13–2.01	0.06–6.07	0.98–1.24	0.80–1.24	1.10–1.45	0.93–1.45	1.09–1.54	1.08–1.27	1.03–1.22	1.29–2.08
Dominant model
I^2 (%)	53.6	57.7	0.0	34.2	27.7	53.6	67.3	51.9	53.6	44.1	72.7
PHeterogeneity	0.000	0.001	0.788	0.134	0.240	0.000	0.016	0.001	0.000	0.007	0.012
OR	1.27	1.54	0.95	1.15	0.96	1.27	1.25	1.28	1.17	1.12	1.82
95% CI	1.09–1.48	1.16–2.06	0.63–1.43	0.97–1.36	0.73–1.27	1.09–1.48	0.91–1.73	1.08–1.53	1.07–1.28	1.02–1.23	1.34–2.49
Recessive model
I^2 (%)	21.5	10.3	0.0	0.0	0.0	21.5	0.0	30.8	21.5	18.7	44.6
PHeterogeneity	0.167	0.346	0.392	0.901	0.692	0.167	0.802	0.094	0.167	0.144	0.217
OR	1.50	2.32	5.32	1.08	1.17	1.50	1.13	1.65	1.48	1.42	1.74
95% CI	1.12–2.02	1.30–4.15	2.05–13.77	0.77–1.52	0.72–1.89	1.12–2.02	0.72–1.78	1.12–2.42	1.18–1.86	1.05–2.88	1.10–1.83
Additive model
I^2 (%)	19.7	10.5	0.0	0.0	0.0	19.7	0.0	28.1	19.7	30.4	18.9
PHeterogeneity	0.188	0.344	0.396	0.861	0.566	0.188	0.705	0.118	0.188	0.214	0.230
OR	1.52	2.38	4.80	1.12	1.13	1.52	1.16	1.66	1.49	1.41	1.84
95% CI	1.13–2.04	1.33–4.29	1.34–12.49	0.80–1.58	0.69–1.85	1.13–2.04	0.74–1.82	1.14–2.42	1.18–1.87	1.09–1.83	1.10–3.09
Co-dominant model
I^2 (%)	48.9	48.7	0.0	27.4	5.1	48.9	64.8	47.2	48.9	39.6	74.8
PHeterogeneity	0.001	0.013	0.814	0.192	0.305	0.001	0.023	0.004	0.001	0.019	0.008
OR	1.14	1.29	0.54	1.14	0.91	1.14	1.22	1.12	1.09	1.62	1.05
95% CI	0.98–1.33	0.97–1.71	0.34–0.87	0.97–1.35	0.72–1.16	0.98–1.33	0.88–1.68	0.94–1.34	0.99–1.20	0.95–1.17	1.15–2.28
Number of studies	15	10	0	4	1	15	0	15	15	13	2
Pre-allele model
I^2 (%)	72.1	73.1	–	75.0	–	72.1	–	72.1	72.1	46.3	78.5
PHeterogeneity	0.000	0.000	–	0.007	–	0.000	–	0.000	0.000	0.034	0.031
OR	1.74	2.09	–	1.37	1.34	1.74	–	1.74	1.59	1.46	8.97
95% CI	1.33–2.28	1.36–3.22	–	0.82–2.29	1.10–1.64	1.33–2.28	–	1.33–2.28	1.41–1.80	1.29–1.66	4.48–17.95
Dominant model
I^2 (%)	58.4	55.5	–	67.1	–	58.4	–	58.4	58.4	32.8	44.2
PHeterogeneity	0.002	0.017	–	0.028	–	0.002	–	0.002	0.002	0.120	0.181
OR	1.67	2.02	–	1.33	1.31	1.67	–	1.67	1.55	1.46	8.82
95% CI	1.30–2.16	1.39–2.93	–	0.80–2.20	1.03–1.66	1.30–2.16	–	1.30–2.16	1.35–1.78	1.26–1.68	3.53–22.01
Recessive model
I^2 (%)	47.5	50.7	–	61.4	–	47.5	–	47.5	47.5	31.4	67.6
PHeterogeneity	0.040	0.058	–	0.075	–	0.040	–	0.040	0.040	0.167	0.079
OR	3.13	3.22	–	3.32	2.22	3.13	–	3.13	3.16	2.52	9.39
95% CI	1.55–6.32	0.94–11.02	–	0.76–14.49	1.24–3.97	1.55–6.32	–	1.55–6.32	2.15–4.65	1.66–3.85	3.22–27.37
Additive model
I^2 (%)	49.8	49.5	–	67.5	–	49.8	–	49.8	49.8	37.1	66.2
PHeterogeneity	0.030	0.065	–	0.046	–	0.030	–	0.030	0.030	0.122	0.085
OR	3.41	3.59	–	3.57	2.36	3.41	–	3.41	3.41	2.72	10.08
95%CI	1.66–7.02	1.06–12.14	–	0.71–18.04	1.31–4.24	1.66–7.02	–	1.66–7.02	2.31–5.02	1.78–4.16	3.44–29.53
Co-dominant model
I^2 (%)	9.2	0.0	–	29.4	–	9.2	–	9.2	9.2	8.0	0.0
PHeterogeneity	0.350	0.667	–	0.236	–	0.350	–	0.350	0.350	0.366	0.352
OR	1.35	1.65	–	1.19	1.16	1.35	–	1.35	1.34	1.32	4.06
95% CI	1.15–1.60	1.28–2.11	–	0.85–1.67	0.90–1.48	1.15–1.60	–	1.15–1.60	1.16–1.55	1.14–1.53	0.87–18.96

Notes: ^aCalculated by the REM in the meta-analysis.

^bCalculated by the FEM in the sensitivity analysis.

Figure 2. Meta-analysis for eNOS 4b/a polymorphism in DN (pre-allele model: 4a vs. 4b) compared with DM patients.

Figure 3. Meta-analysis for eNOS 4b/a polymorphism in DN (dominant model: 4aa + 4ab vs. 4bb) compared with DM patients.

Figure 4. Meta-analysis for eNOS 4b/a polymorphism in DN (recessive model: 4aa vs. 4bb + 4ab) compared with DM patients.

Figure 5. Meta-analysis for eNOS 4b/a polymorphism in DN (additive model: 4aa vs. 4bb) compared with DM patients.

Figure 6. Meta-analysis for eNOS 4b/a polymorphism in DN (co-dominant model: 4ab vs. 4aa + 4bb) compared with DM patients.

Figure 7. Funnel plot for articles associated with eNOS polymorphism and DN compared with DM patient under dominant model.