OBJECTIVE To test the hypothesis that vitamin D receptor polymorphism is associated with calcific aortic valve stenosis.
DESIGN The distribution of one polymorphism of the vitamin D receptor (BsmI B/b) was examined in 100 consecutive patients with calcific valvar aortic stenosis and compared with a control group of 100 patients (paired match for age, sex, and the presence of coronary artery disease from a total of 630 patients without calcified aortic valves). Polymerase chain reaction and restriction fragment length polymorphism were used to determine genotypes.
RESULTS There was a significant difference in vitamin D receptor allele and genotype frequencies between the two groups. The allele B had a higher prevalence in patients with calcific aortic stenosis (B = 0.56, b = 0.44) than in the control cohort (B = 0.40, b = 0.60) (p = 0.001).
CONCLUSIONS There is a significant association of vitamin D receptor polymorphism with calcific aortic valve stenosis. The B allele of the vitamin D receptor is more common in patients with calcific aortic valve stenosis. It now needs to be evaluated whether other genes that control calcium homeostasis are involved in the pathogenesis of this disorder.
- vitamin D receptor
- calcific aortic stenosis
- aortic valve
- genetic polymorphism
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Degenerative calcification of the aortic valve is the most common cause of valvar aortic stenosis and is becoming even more frequent with the increasing age of our population.1 2 Clinical factors associated with atherosclerosis are also associated with calcification of the aortic valve, and early lesions in calcific aortic valves show similarities to atherosclerosis.3 However, approximately 50% of patients with severe aortic stenosis have no coronary artery disease. Although calcification of the aortic valve is a disease of the elderly population, there is evidence that it is not simply a consequence of aging.4 Additional and as yet undefined factors are likely to have an important impact on degenerative calcification of the aortic valve.
The influence of genetic polymorphism of the vitamin D receptor on bone metabolism has been investigated in several studies in the past decade. BsmI polymorphism of the vitamin D receptor gene is a predictor of bone mineral mass.5-9 As bone metabolism is not just a function of bone cells but a complex interaction of hormones and organs—bone, intestine, kidney, thyroid, parathyroid, and so on—controlling calcium homeostasis, we postulated that alterations in the latter might influence calcification of extraosseous structures such as the aortic valve. The present case–control association study (matched for age, sex, and the prevalence of coronary artery disease) was designed to test the hypothesis that the BsmI vitamin D receptor polymorphism is associated with calcific aortic valve stenosis.
One hundred consecutive patients with calcific aortic stenosis in whom cardiac catheterisation was undertaken formed the case group. Calcific aortic stenosis was diagnosed when calcification of the aortic valve was visible by fluoroscopy, and if the aortic valve area was < 1.0 cm2 or the mean transvalvar gradient > 40 mm Hg, determined by invasive measurements.
A control group of 100 patients, matched for age, sex, and the presence of coronary artery disease, was formed from among 630 patients who underwent left heart catheterisation for suspected coronary artery disease and had no gradient across the aortic valve.
All patients gave written informed consent. The study was approved by the local ethics committee of the University Hospital of Aachen.
All patients underwent invasive examination. Retrograde left sided cardiac catheterisation was performed using fluid filled catheters. The mean gradient across the aortic valve was determined by simultaneous measurement of aortic and left ventricular pressures. Measurements were averaged over three beats. Cardiac output was determined by thermodilution (five measurements averaged). Aortic valve area was calculated using Gorlin's equation.10
Genomic DNA of patients was extracted from whole blood using the QIAmp blood kit 250 (Qiagen, Valencia, California, USA). Polymerase chain reaction (PCR) of genomic DNA segments was achieved using a standard PCR thermocycler (Hybaid PCR express, Hybaid, Ashford, Kent, UK). Details of the amplification protocol have been described previously.6 Genotyping was independently performed by two investigators.
Statistical analysis was done using the computer program Graph Pad Instat tm V2.05. Data are presented as mean (SD). The differences of the frequencies of alleles and genotypes between case patients and controls were analysed by the χ2 test and analysis of variance (ANOVA). A probability value of p < 0.05 was considered significant.
The case patients represent a normal cross section of elderly patients with degenerative calcific aortic valve stenosis. Their mean (SD) age was 69.4 (9.3) years. Forty eight per cent of the case patients had additional coronary artery disease. The diagnosis of coronary artery disease was not established in all cases before cardiac catheterisation. The mean gradient across the aortic valve was 59 (20) mm Hg, corresponding to a mean valve area of 0.8 (0.3) cm. Cardiac risk factors in patients with calcific aortic stenosis were as follow: 66% had arterial hypertension, 36% had hypercholesterolaemia, 31% had a positive history of coronary artery disease, 18% were current smokers, and 15% had type 2 diabetes mellitus.
The control group was matched for age, sex, and the presence of coronary artery disease. Hence the mean age, prevalence of coronary artery disease, and distribution of male and female subjects were identical to the case group. The distribution of cardiac risk factors (with the exclusion of hypercholesterolaemia) was similar in the control group and the case group. In the control group 64% had arterial hypertension, 63% had hypercholesterolaemia, 27% had a positive history of coronary artery disease, 25% were current smokers, and 11% had type 2 diabetes mellitus. Table 1 summarises the clinical characteristics of the case and control groups.
In the case group the allele frequency was 0.54 for the B allele and 0.46 for the b allele: 24 patients were homozygous for the B allele, 15 patients were homozygous for the b allele, and 61 patients were heterozygous. In the control group the allele frequency was 0.40 for the B allele and 0.60 for the b allele: 20 patients were homozygous for the B allele, 40 patients were homozygous for the b allele, and 40 patients were heterozygous. Thus the allele frequency of the B allele was 35% higher in the case group than in the control group. This was significant (allele frequency difference in the χ2analysis, p = 0.001; genotype differences in the ANOVA analysis, p = 0.023). Table 2 summarises allele and genotype frequencies of the two groups.
Aortic valve stenosis caused by degenerative calcification of the valvar structures has become the most common valvar pathomorphology requiring cardiac surgery.1 2 Clinical factors associated with atherosclerosis are also associated with calcification of the aortic valve, but 50% of patients with severe aortic stenosis do not have coronary artery disease, and there is evidence that calcific aortic stenosis is not simply a consequence of aging.3 4Other factors must be important for the pathogenesis of this disease. This case–control study showed an association between a polymorphism of the vitamin D receptor gene and the prevalence of calcific aortic stenosis: there was a significant association between the B allele of the vitamin D receptor and calcific aortic stenosis; and the B allele was more frequent in case patients than in controls.
The contribution of vitamin D receptor polymorphism to bone metabolism has often been evaluated during the last decade. Vitamin D receptor polymorphism seems to predict bone density or bone mineral mass. Bone mineral mass is less in individuals with the B allele than in those with the b allele.6-9 Furthermore, people with the B allele have more rapid bone loss with advancing age,7 whereas those with the b allele have greater calcium absorption11 and a better correlation between 1,25 dihydroxyvitamin D concentrations and the 45Ca absorption index.12 In addition to the lower bone mineral density, more rapid bone loss, and blunted calcium absorption, raised parathormone concentrations have been found in subjects with the BB genotype.13 However, other studies have produced conflicting data. While one study detected an influence of the vitamin D receptor genotype on bone metabolism only in the presence of a certain genotype of the oestrogen receptor,14 another found an inverse correlation between vitamin D receptor genotypes and bone mineral density, indicating that vitamin D receptor polymorphism could also be a marker of linkage disequilibrium with another relevant gene.15 Other studies were unable to detect any association between the vitamin D receptor genotypes and bone metabolism.16-19 However, there is a high level of confidence in published reports that vitamin D receptor polymorphism is associated with bone mineral density.20
In this study we found an association between the B allele—which seems to predispose gene carriers to blunted calcium absorption, more rapid bone loss, reduced bone mineral density, and raised parathormone secretion—and the prevalence of calcific aortic valve stenosis. There might be several hypotheses to explain the relation between genotype and the development of aortic stenosis. Calcification of the aortic valve is believed to be a process taking years or decades.1-4 Individuals with a slightly unfavourable bone mineral density might develop mechanisms to overcome this alteration of calcium homeostasis. Like parathormone, other hormones, proteins, or second messengers might trigger calcification of extraosseous structures like the aortic valve. Another possible explanation is the hypothesis that vitamin D receptor polymorphism is merely a marker of linkage disequilibrium with another gene involved in calcium metabolism; this as yet unknown gene might be important for osseous and extraosseous calcification. The aortic valve is likely to be one of the first extraosseous structures involved because of the high level of mechanical stress to which it is subjected.
We are aware that no firm pathophysiological conclusions can be drawn from a genetic association study. Nevertheless an interaction between genes controlling calcium metabolism or calcification and the development of calcific aortic stenosis is intriguing and requires further study.
In the light of recently suggested criteria that should be met by a high quality association study,21 this study had certain limitations. It is proposed that association studies should have large sample sizes, small p values, and report associations that make biological sense and alleles that affect the gene product in a physiologically meaningful way. Furthermore, they should include an initial study with a subsequent independent replication, and the association should be observed both in family based and population based studies. The author of these recommendations admitted that most studies do not meet all these criteria.21 Our study included a relatively small number of case and control patients, a limitation which is mitigated by the fact that case and control patients were carefully phenotyped and matched. Furthermore, the results of our study have not yet been analysed in a second replication study. Thus it is desirable that our results should be confirmed in a larger study in the near future. The difference in the allele frequencies between the case and control groups reported in this study was high at 0.14, and is similar to differences of alleles reported in other genetic association studies.22-24 Moreover the reported association makes biological sense and alleles affect the phenotype in a physiologically meaningful way.
The vitamin D receptor genotype is associated with the prevalence of calcific aortic stenosis. This is the first report on a genetic association with aortic valve stenosis. Further larger studies are required to prove the influence of the vitamin D receptor gene on the development of calcific aortic stenosis.
This study was supported by a grant of the START program of the University Hospital of Aachen.