Intronic Variants in OCT1 are Associated with All-Cause and Cardiovascular Mortality in Metformin Users with Type 2 Diabetes

Figures & data

Figure 1 Flow chart of subjects per investigated group.

Abbreviations: T2DM, type 2 diabetes mellitus; T1DM, type 1 diabetes mellitus.

Table 1 Baseline Characteristics of the Study Participants

	All (n=3049)	ND (n=1820)	T2DM (n=1220)	p-values^a	MUT (n=73)	NMUT (n=1147)	p-values^b
Age (years)	62.7 (10.6)	60.9 (11.2)	65.5 (9.0)	<0.001	65.1 (7.2)	65.5 (9.1)	0.718
Male sex (%)	70.0	70.0	70.1	0.973	72.6	69.9	0.621
Body mass index (kg/m^2)	27.5 (4.0)	26.9 (3.8)	28.3 (4.2)	<0.001	29.2 (4.6)	28.2 (4.1)	0.041
Hypertension (%)	72.6	66.7	81.2	<0.001	83.6	81.1	0.302
Smoking (%)	19.4	21.3	16.7	0.076	19.2	16.6	0.080
Systolic blood pressure (mm Hg)	141 (23)	138 (22)	145 (24)	<0.001	153 (23)	144 (23)	0.002
LDL cholesterol (mmol/L)	3.02 (0.89)	3.07 (0.90)	2.95 (0.88)	<0.001	2.70 (0.88)	2.97 (0.88)	0.013
HDL cholesterol (mmol/L)	1.00 (0.28)	10.40 (0.29)	0.94 (0.25)	<0.001	0.91 (0.21)	0.94 (0.25)	0.243
Triglycerides (mmol/L)	1.94 (1.39)	1.81 (1.39)	2.13 (1.37)	<0.001	2.28 (1.25)	2.44 (1.37)	0.318
Total cholesterol (mmol/L)	5.40 (1.14)	5.5 (1.12)	5.3 (1.18)	0.010	5.02 (1.05)	5.4 (1.18)	0.017
HbA1c	6.3 (1.24)	5.7 (0.44)	7.2 (1.48)	<0.001	8.0 (1.40)	7.1 (1.46)	<0.001
NT-pro-BNP (ng/L)	912 (2069)	735 (1753)	1177 (2450)	<0.001	852 (1437)	1198 (2499)	0.246
Cystatin c (mg/L)	1.01 (1.99)	0.96 (0.38)	1.06 (0.43)	<0.001	0.95 (0.31)	1.07 (0.44)	0.020
C-reactive protein (mg/L)	10.07 (19.93)	8.44 (17.09)	12.5 (23.38)	<0.001	9.13 (16.15)	12.76 (23.75)	0.207
Sodium (mmol/l),	141 (2)	141 (2)	140 (3)	<0.001	140 (2)	140 (3)	0.151
Cortisol (nmol/L)	22 (8)	21 (8)	22 (7)	0.003	22 (7)	22 (7)	0.920
Atrial fibrillation (%)	12.11	10.95	13.87	0.016	11.27	14.03	0.514
Left-ventricular hypertrophy (%)	8.17	6.98	10.00	0.004	13.70	9.76	0.268
STA-CAD (%)				<0.001^c
Class1	47.23	45.16	50.08		50.68	50.04
Class2	19.55	18.08	21.72		24.66	21.53
Class3	12.23	11.43	13.52		13.70	13.51

Notes: Data are given as mean (SD) if not otherwise stated. ^ap-value stating differences between non-diabetics and individuals with T2DM. ^bp-value stating differences between individuals with T2DM with and without metformin therapy. ^cOnly overall test available.

Abbreviations: All, all individuals; ND, non-diabetic individuals; T2DM, individuals with type 2 diabetes mellitus; MUT, metformin users with T2DM; NMUT, non-metformin users with T2DM; SD, standard deviation; LDL, low-density lipoprotein; HDL, high-density lipoprotein; HbA1c, haemoglobin A1c; NT-pro-BNP, N-terminal prohormone of brain natriuretic peptide; STA-CAD, coronary artery disease stage.

Table 2 Mortality Data of the Investigated Subgroups

	All		ND		MUT		NMUT		p-value^a
	n	% Male	n	% Male	n	% Male	n	% Male
ACD	891	73.4	399	73.4	24	83.3	463	73.0	0.201
CVD	555	72.8	226	73.9	18	77.8	307	71.7	0.683
Non CVD	336	74.4	173	72.8	6	100	156	75.6	0.189
Survival	2158	68.63	1421	69.0	49	67.4	684	67.8

Note: ^aThe given p-value states the difference of individuals with T2DM with and without metformin therapy.

Abbreviations: All, all individuals; ND, non-diabetic individuals; MUT, metformin users with T2DM; NMUT, non-metformin users with T2DM; T2DM, type 2 diabetes mellitus; ACD, all-cause death; CVD, cardiovascular death; Non CVD, death other than cardiovascular death.

Table 3 Mortality Data per Genotype in All Individuals and Individuals with T2DM Using Metformin

SNPs	Alleles	n	All	n=3049		n	MUT	n=73
			ACD	CVD	% of ACD/n		ACD	CVD	% of ACD/n
rs461473	GG	2385	691	431	62/18	60	18	14	78/23
G/A	GA/AA	664	200	124	62/19	13	6	4	67/31
rs609468	CC	1811	521	331	64/18	48	12	9	75/19
C/T	CT/TT	1238	370	224	61/18	25	12	9	75/36
rs622591	CC	1813	521	331	64/18	48	12	9	75/19
C/T	CT/TT	1236	370	224	61/18	25	12	9	75/36
rs3777392	CC	2403	702	435	62/18	57	17	12	71/21
C/T	CT/TT	646	189	120	63/19	16	7	6	80/38
rs9295125 G/T	GG	1049	295	188	64/18	26	11	8	73/31
rs3818678 G/C	GT/TT GC/CC	2000	596	367	62/18	47	13	10	77/21
rs456598	GG	2326	686	419	61/18	60	17	13	76/27
G/A	GA/AA	723	205	136	66/19	13	8	6	75/46

Abbreviations: All, all individuals; SNPs, single-nucleotide polymorphisms; MUT, metformin users with T2DM; T2DM, type 2 diabetes mellitus; ACD, all-cause death; CVD, cardiovascular death.

Table 4 Association of Intronic OCT1 SNPs with All-Cause and Cardiovascular Death in Metformin Users with T2DM (MUT)

rs Number		All-Cause Death		Cardiovascular Death
Major/Minor		Unadjusted	Adjusted	Unadjusted	Adjusted
Allele	n	73	73	73	73
rs461473	p-value	0.006	0.006	0.021	0.018
G/A	HR	3.31	11.46	3.16	28.86
	CI	1.41–7.78	1.99–66.06	1.19–8.39	1.76–473.31
rs609468	p-value	0.053	0.057	0.118	0.031
C/T	HR	1.65	2.27	0.16	4.43
	CI	0.99–2.75	0.98–5.25	0.88–2.98	1.15–17.11
rs622591	p-value	0.053	0.057	0.118	0.031
C/T	HR	1.65	2.27	0.16	4.43
	CI	0.99–2.75	0.98–5.25	0.88–2.98	1.15–17.11
rs3777392	p-value	0.147	0.698	0.047	0.066
C/T	HR	1.65	1.32	2.08	8.67
	CI	0.84–3.25	0.32–5.37	1.01–4.28	0.86–87.05
rs9295125	p-value	0.082	0.290	0.204	0.183
G/T	HR	0.57	0.62	0.63	0.47
rs3818678 G/C	CI	0.31–1.07	0.25–1.51	0.31–1.29	0.16–1.43
rs456598	p-value	0.095	0.308	0.157	0.867
G/A	HR	2.12	2.30	2.17	0.84
	CI	0.88–5.13	0.46–11.41	0.74–6.35	0.12–5.99

Note: The statistically tested allele is the minor allele.

Abbreviations: HR, hazard ratio; CI, 95% confidence interval.

Table 5 Association of Intronic OCT1 SNPs with All-Cause and Cardiovascular Death in Non-Metformin Users with T2DM (NMUT)

rs Number		All-Cause Death		Cardiovascular Death
Major/Minor		Unadjusted	Adjusted	Unadjusted	Adjusted
Allele	n	1147	1147	1147	1147
rs461473	p-value	0.866	0.973	0.467	0.635
G/A	HR	0.98	1.00	0.91	0.94
	CI	0.80–1.21	0.82–1.24	0.70–1.18	0.72–1.22
rs609468	p-value	0.898	0.793	0.440	0.246
C/T	HR	1.01	0.98	0.93	0.89
	CI	0.87–1.18	0.84–1.15	0.76–1.12	0.73–1.08
rs622591	p-value	0.869	0.805	0.459	0.252
C/T	HR	1.01	0.98	0.93	0.89
	CI	0.87–1.18	0.84–1.15	0.77–1.13	0.73–1.09
rs3777392	p-value	0.796	0.922	0.457	0.673
C/T	HR	0.97	1.01	0.91	0.95
	CI	0.80–1.18	0.83–1.23	0.71–1.17	0.73–1.22
rs9295125	p-value	0.094	0.039	0.068	0.038
G/T	HR	1.12	1.15	1.16	1.19
rs3818678 G/C	CI	0.98–1.28	1.01–1.32	0.99–1.36	1.01–1.41
rs456598	p-value	0.637	0.496	0.843	0.780
G/A	HR	0.95	0.93	0.98	0.97
	CI	0.79–1.16	0.76–1.14	0.77–1.24	0.76–1.23

Note: The statistically tested allele is the minor allele.

Abbreviations: HR, hazard ratio; CI, 95% confidence interval.

Table 6 Association of Intronic OCT1 SNPs with All-Cause and Cardiovascular Death in Non-Diabetic Patients (ND)

rs Number		All-Cause Death		Cardiovascular Death
Major/Minor		Unadjusted	Adjusted	Unadjusted	Adjusted
Allele	n	1820	1820	1820	1820
rs461473	p-value	0.991	0.813	0.539	0.534
G/A	HR	1.00	1.03	1.09	1.09
	CI	0.82–1.22	0.83–1.26	0.84–1.40	0.83–1.42
rs609468	p-value	0.458	0.715	0.861	0.935
C/T	HR	0.94	0.97	0.98	1.01
	CI	0.79–1.11	0.82–1.15	0.79–1.22	0.81–1.26
rs622591	p-value	0.379	0.612	0.794	0.908
C/T	HR	0.93	0.96	0.97	1.00
	CI	0.78–1.10	0.81–1.14	0.78–1.21	0.80–1.25
rs3777392	p-value	0.180	0.376	0.660	0.975
C/T	HR	0.85	0.90	0.94	1.00
	CI	0.68–1.08	0.71–1.14	0.70–1.26	0.75–1.35
rs9295125	p-value	0.118	0.074	0.876	0.837
G/T	HR	1.12	1.14	1.01	1.02
rs3818678 G/C	CI	0.97–1.28	0.99–1.31	0.84–1.22	0.85–1.23
rs456598	p-value	0.303	0.595	0.669	0.413
G/A	HR	0.89	0.94	1.06	1.13
	CI	0.71–1.11	0.75–1.18	0.80–1.41	0.85–1.50

Note: The statistically tested allele is the minor allele.

Abbreviations: HR, hazard ratio; CI, 95% confidence interval.

Figure 2 Hazard ratios for cardiovascular death per OCT1 genotype in diabetic metformin users. Squares: hazard ratios calculated with an additive cox proportional hazards regression model adjusted for confounders, error bars: 95% confidence interval, different shades of grey represent different intronic OCT1 SNPs or SNP combinations. Homozygosity for the major allele is set as 1.

Table 7 Association of Intronic OCT1 SNPs with All-Cause and Cardiovascular Death in Metformin Users with T2DM (MUT) After Adjustment for Glycemic Control

rs Number	Major/Minor	All-Cause Death			Cardiovascular Death
	Allele	p-value	HR	CI	p-value	HR	CI
rs461473	G/A	0.009	14.38	1.94–106.78	0.061	11.25	0.89–141.61
rs609468	C/T	0.046	2.59	1.02–6.61	0.012	13.17	1.78–97.63
rs622591	C/T	0.046	2.59	1.02–6.61	0.012	13.17	1.78–97.63
rs3777392	C/T	0.781	1.23	0.29–5.11	0.070	9.90	0.83–118.41
rs9295125 rs3818678	G/T G/C	0.171	0.49	0.18–1.36	0.077	0.30	0.08–1.14
rs456598	G/A	0.264	2.85	0.45–17.94	0.845	0.80	0.08–17.68

Note: The statistically tested allele is the minor allele. n = 73.

Abbreviations: HR, hazard ratio; CI, 95% confidence interval.