Plasma homocysteine levels and genetic variants of homocysteine metabolism related genes confer no independent risk of vascular events in a German population

Objective: An elevation of total plasma homocysteine (Hcys) levels has become generally accepted as risk factor for arterial and venous vascular disease. However, elevated Hcys levels may rather constitute a secondary phenomenon of disease than the cause.

Patients and Methods: In the present study we investigated fasting total plasma Hcys levels and nine polymorphisms involved in Hcys metabolism together with carotid intima media thickness (IMT) as intermediate phenotype of atherosclerosis in 714 individuals of 187 families: 187 consecutive patients ascertained by clinical vascular disease and with proven carotid artery sclerosis (f/m: 53/134; age in years in average; standard deviation: 64.3; 8.8), 139 spouses (f/m: 112/27, age: 61.5; 8.8) and 388 children (f/m: 177/211, age: 36.2; 8.1).

Results: The patients had higher Hcys levels than the spouses and children as analyzed by ANOVA (table 1). However, logistic regression analysis with stratification for the classical risk factors (other parameters of table 1) revealed no significant difference in Hcys levels between the groups (not shown), but significant differences in gender, IMT and prevalence of hypertension and diabetes. Regarding all subjects, IMT was significantly influenced by age, gender, presence of arterial hypertension (p<0.001 each), presence of diabetes mellitus (p<0.002)and pack years (p=0.001), but not by Hcys levels (p=0.70). Homozygosity for the T allele of the polymorphism methylenetetrahydrofolate reductase c.677C>T was significantly associated with higher Hcys levels (p=0.003 in ANOVA analysis), but not with IMT. The other polymorphisms involved in homocysteine metabolism showed no significant effects (table 2).

Conclusion: Classical risk factors were associated with vascular disease and an increase in IMT. Though plasma Hcys levels were higher in patients as opposed to spouses and children, plasma Hcys levels and polymorphisms of Hcys metabolism did not contribute significant independent risks for increased IMT or vascular events when data were stratified. These results suggest that elevated Hcys levels rather constitute a secondary than causative phenomenon of carotid artery sclerosis. The clinical impact of elevated Hcys levels might have been over-estimated in the past.

Table 1. Personal and clinical data

	all (n=714)	patients (n=187)	spouses (n=139)	offsprings (n=388)	ANOVA (F) or Chi2; df=2 three subgroups	ANOVA (F) or Chi2; df=2 patients vs. spouses
age in years	48.5±15.8 (13–85)	64.3±8.8 (39–85)	61.5±8.8 (38–85)	36.2±8.1 (13–61)	F=903; p<0.001	F=8.29; p=0.004
female gender	0.48	0.28	0.81	0.52	Chi2=88.3; p<0.001	Chi2=87.0; p<0.001
hypertension	0.37	0.75	0.45	0.16	Chi2=196; p<0.001	Chi2=29.8; p<0.001
RR sys	138±19.4 (86–216)	148±19.7 (95–216)	144±19.7 (86–209)	131±16.0 (90–176)	F=67.6; p<0.001	F=1177; p<0.083
RR dia	78.5±10.2 (53–112)	81.6±10.3 (55–112)	81.1±10.5 (53–107)	78.5±10.2 (53–112)	F=25.1; p<0.001	F=26.8; p<0.619
Lp (a)	34.0±39.2 (8–245)	40.0±45.4 (0–254)	30.3±32.8 (9–176)	32.4±37.9 (8–253)	F=3.1; p<0.046	F=4.47; p<0.035
pack years	13.3±18.0 (0–123)	25.0±25.0 (0–99)	9.0±16.7 (0–123)	9.5±11.2 (0–60)	F=53.7; p<0.001	F=39.0; p<0.001
diabetes	0.13	0.37	0.12	0.02	Chi2=130; p<0.001	Chi2=23.3; p<0.001
IMT	0.76±0.20 (0.44–1.67)	0.96±0.20 (0.57–1.67)	0.79±0.15 (0.53–1.36)	0.65±0.11 (0.44–1.15)	F=269; p<0.001	F=59.9; p<0.001
Hcys	13.3±4.3 (4–33)	15.2±0.50 (4–33)	13.3±4.0 (6–33)	12.4±3.7 (6–30)	F=30.3; p<0.001	F=13.8; p<0.001
Gender, prevalence of hypertension and prevalence of diabetes: Chi2-analysisOthers: ANOVA. Levels following±depict the standard deviation, range in brackets

Table 2. Polymorphisms

	all (n=714)	patients (n=187)	spouses (n=139)	offsprings (n=388)	comparison of all three groups; df=4	comparison of patients versus spouses; df=2
CBS c.833T>C TT;CT;CC	0.98;0.02;0.00	0.98;0.02;0.00	1.00;0.00;0.00	0.98;0.02;0.00	Chi2=2.66; p=0.27	Chi2=2.29; p=0.18
CBS 844ins68 del/del; del/ins; ins/ins	0.88;0.12;0.00	0.85;0.14;0.01	0.88;0.12;0.00	0.89;0.11;0.00	Chi2=3.77; p=0.44	Chi2=0.96; p=0.62
DHFR c.594+59 del19bp del/del; del/ins; ins/ins	0.21;0.46;0.33	0.17;0.48;0.35	0.19;0.50;0.31	0.24;0.43;0.33	Chi2=4.04; p=0.40	Chi2=0.38; p=0.83
GST-O1 c.428C>A AA;AC;CC	0.08;0.45;0.48	0.07;0.41;0.52	0.08;0.45;0.47	0.08;0.52;0.40	Chi2=6.48; p=0.17	Chi2=0.66; p=0.72
MTHFR c.677C>T CC;CT;TT	0.47;0.45;0.08	0.45;0.43;0.12	0.46;0.48;0.06	0.48;0.45;0.08	Chi2=3.90; p=0.42	Chi2=3.08; p=0.21
MTHFR c.1298A>C AA;AC;CC	0.48;0.43;0.09	0.52;0.39;0.09	0.48;0.44;0.08	0.46;0.44;0.10	Chi2=1.82; p=0.77	Chi2=0.61; p=0.74
MTR c.2576A>G AA;AG;GG	0.63;0.33;0.04	0.63;0.34;0.04	0.64;0.30;0.06	0.63;0.33;0.04	Chi2=1.04; p=0.91	Chi2=0.89; p=0.64
RFC1 c.80G>A GG;AG;AA	0.34;0.47;0.19	0.34;0.45;0.21	0.38;0.42;0.20	0.33;0.49;0.18	Chi2=2.05; 0.73	Chi2=0.36; p=0.84
Tc2 c.776C>G CC;CG;GG	0.28;0.45;0.27	0.26;0.47;0.27	0.29;0.52;0.29	0.28;0.45;0.27	Chi2=0.61; p=0.96	Chi2=0.56; p=0.76
The relative prevalence of the different genotypes are given for all individuals and for the subgroups patients, spouses and offsprings as well as the results of Chi2 testing of all groups and of patients and spouses.