Frequent Mutation in the ABCC6 Gene (R1141X) Is Associated With a Strong Increase in the Prevalence of Coronary Artery Disease
Background— Pseudoxanthoma elasticum (PXE) is an inborn disorder of the connective tissue with specific skin, ocular, and cardiovascular disease (CVD) manifestations. Recently, we and others have identified mutations in the gene coding for the ABCC6 transporter in PXE patients with ocular and skin involvement. In the Netherlands, as in the rest of Europe, a particular premature truncation variant ABCC6 (R1141X) was found in a large cohort of PXE patients. Given the association between CVD and PXE, we hypothesized that heterozygosity of this ABCC6 mutation could also confer an increased risk for CVD.
Methods and Results— To assess the relationship between the frequent R1141X mutation in the ABCC6 gene and the prevalence of premature coronary artery disease (CAD), we conducted a case-control study of 441 patients under the age of 50 years who had definite CAD and 1057 age- and sex-matched population-based controls who were free of coronary disease. Strikingly, the prevalence of the R1141X mutation was 4.2 times higher among patients than among controls (3.2% versus 0.8%; P<0.001). Consequently, among subjects with the R1141X mutation, the odds ratio for a coronary event was 4.23 (95% CI: 1.76 to 10.20, P= 0.001).
Conclusion— The presence of the R1141X mutation in the ABCC6 gene is associated with a sharply increased risk of premature CAD.
Received May 1, 2002; revision received June 17, 2002; accepted June 18, 2002.
Pseudoxanthoma elasticum (PXE) is an inborn disorder, the hallmark of which is dystrophic mineralization of elastic tissues of the skin, retina, and arterial walls.1–4⇓⇓⇓ Most PXE patients seem random, but autosomal recessive and autosomal dominant inheritance also is observed.5,6⇓ The frequency of PXE in the general population is unknown, particularly because it is likely that individuals with a mild clinical phenotype will escape diagnosis. Recently, we and others elucidated the molecular basis of PXE by demonstrating mutations in an ATP-binding cassette (ABC) transporter gene (ABCC6) as the cause for this disorder.7–12⇓⇓⇓⇓⇓ Cardiovascular manifestations of PXE include accelerated atherosclerosis, which results in myocardial infarction at a young age and is attributed to calcification of the internal elastic laminae of the coronary arteries. On several occasions we were struck by the fact that, in our patients suffering from premature cardiovascular disease, PXE was found to be concomitantly present. Whether carriership of a single ABCC6 gene mutation on one allele would also confer additional risk for coronary artery disease (CAD) was hitherto impossible to assess. This situation changed with the elucidation of the molecular basis of PXE. In parallel studies (Xiaofeng Hu, unpublished data), we found that the R1141X mutation is the most common mutation in Dutch PXE patients and families, and it seems as though this is the case for the rest of Europe as well. We therefore studied the prevalence of the R1141X mutation in the ABCC6 gene in patients with premature CAD and in a large population-based group of healthy controls to further delineate the role of this genetic variation as a risk factor for CAD.
Case and Control Population
Consecutive Dutch patients under the age of 50 years with CAD (n=441) referred between 1995 and 2001 to the Atherosclerosis Outpatient Clinic of the Academic Medical Center of the University of Amsterdam were included in the study. Patients qualified for inclusion after a myocardial infarction, surgical or percutaneous coronary revascularization, or a coronary angiogram with evidence of at least a 70% stenosis in a major epicardial artery. The Institutional Review Board approved the protocol. All patients gave informed consent.
Control subjects (n=1057) were selected from the participants of the Cardiovascular Disease Risk Factor Monitoring Project, a large project that screened for cardiovascular risk factors and was carried out in 3 Dutch towns (Amsterdam, Doetinchem, and Maastricht) between 1987 and 1991. All participants completed an informed consent form, agreeing to the use of stored blood samples for further scientific research. A detailed description of these examinations was published previously.13 Approximately 2 controls per case were selected, group matched for sex and age (within 5 years). All controls were Dutch and reported no history of myocardial infarction, percutaneous transluminal coronary angiography, or coronary artery bypass grafting in a self-administered questionnaire.
Genomic DNA was extracted according to standard protocols. The polymerase chain reaction primers used to amplify exon 24 were MRP6 ex 24F:AAGGTCTTCTCTGCCCTGGCTCTT and MRP6 ex 24R:CTTCCCTCTCCCATCCATCCTTCT.
After polymerase chain reaction (20 ng/μL DNA in 25 μL), the product and an internal control were digested with the restriction enzyme BsiY1. Mutated products remained uncut. The fragments obtained were separated on a 3% agarose gel and visualized after staining with ethidium bromide. The presence of the mutation was confirmed by direct sequencing.
In the CAD patients, plasma cholesterol and triglycerides were determined with commercially available enzymatic methods (Boehringer Mannheim, FRG, No. 237574, and Sera-PAK, No. 6639, respectively). To determine high-density lipoprotein cholesterol, the polyethylene glycol 6000 precipitation method was used. Low-density lipoprotein cholesterol was calculated by the Friedewald formula. The biochemical analysis for the controls has been described previously.13
Fisher’s exact test was applied to compare allele frequencies between groups, and exact 95% CIs were calculated for the odds ratio, with adjustment for matching criteria. Risk factors were compared between cases and controls and between carriers and noncarriers with the use of either Fisher’s exact test or t test, where appropriate.
The characteristics of the 441 cases and 1057 controls are presented in Table 1. As expected, the frequency of increased body mass index, dyslipidemia, smoking, hypertension, and diabetes was increased in cases versus controls. In cases, 14 of 441 (3.2%, 95% CI: 1.9 to 5.6) were carriers of the R1141X truncation variant, whereas 8 of 1057 controls (0.8%, 95% CI: 0.3 to 1.5) carried this ABCC6 mutation, yielding a statistically significant difference at a probability value <0.001 with an odds ratio corrected for age and sex of 4.23 (95% CI: 1.76 to 10.20).
We subsequently categorized the premature CAD patients in carriers (n=14) and noncarriers (n=427) of the R1141X variant of the ABCC6 gene (Table 2). The major risk factors for CAD were equally divided in both groups.
We demonstrate in a large case-control study that a strong association exists between a frequent mutation in the ABCC6 gene (R1141X) and the presence of premature CAD. Carriers of this mutation had an odds ratio of 4.2 for CAD when compared with noncarriers. In addition, we could not find a relation between this mutation and other major CAD risk factors, suggesting that this mutation in the ABCC6 transporter is operating through a novel pathway in atherogenesis.
PXE is characterized by deranged elastic fiber metabolism, resulting in fragmentation and calcification of elastic fibers, with resultant changes in the skin, eyes, gastrointestinal tract, and cardiovascular system. Cardiovascular manifestations in PXE include premature CAD, cerebrovascular disease, peripheral vascular disease, and renovascular hypertension. Calcium deposits in the elastic lamina of the arterial wall indeed resemble the other calcium deposits seen in PXE patients.
PXE-like elastic tissue disorders have also been documented in sickle cell disease, β-thalassemia and sickle thalassemia, Marfan’s syndrome, Ehlers-Danlos’ syndrome, and Paget’s disease.14 The pathology of these PXE-like syndromes is generally considered to be one of the manifestations of the underlying systemic illness. PXE, or at least a number of its clinical manifestations, could therefore also be considered as secondary to an underlying systemic disorder.15
Recently, mutations in the ABCC6 gene have been established as the cause of PXE. The exact biological function of ABCC6, however, is presently still unknown, as is the functional relationship of this transmembrane transporter to the pathogenesis of the PXE phenotype. ABCC6 messenger-RNA was reported to be highly expressed in the liver and kidney, in contrast to tissues characteristically affected by PXE.16
Whatever the specific pathophysiology of PXE, our study results seem to indicate that mutations in the ABCC6 gene are not rare in the general population and contribute to an increased propensity toward premature atherosclerotic vascular disease. If our data are subsequently confirmed in other cohorts, this might have implications for genetic screening in PXE kindreds and may require a more aggressive approach toward CAD prevention in these individuals.
Dr Kastelein is an Established Investigator of the Netherlands Heart Foundation (2000D039). Dr Boer was funded by the Netherlands Heart Foundation (98.067).
Ringpfeil F, Lebwohl MG, Christiano AM, et al. Pseudoxanthoma elasticum: mutations in the MRP6 gene encoding a transmembrane ATP-binding cassette (ABC) transporter. Proc Natl Acad Sci U S A. 2000; 97: 6001–6006.
Verschuren WMM, van Leer EM, Blokstra A, et al. Cardiovascular disease risk factors in the Netherlands. Neth J Cardiol. 1993; 6: 205–210.
Aessopos A, Farmakis D, Loukopoulos D. Elastic tissue abnormalities resembling pseudoxanthoma elasticum in β thalassemia and the sickling syndromes. Blood. 2002: 99: 30–35.