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Purpose: This study sought to determine the association between BsmI polymorphism and bone mineral density, 25-hydroxyvitamin D, and parathyroid hormone levels in patients with epilepsy.
Methods: We recruited ambulatory young adults with epilepsy who were taking phenytoin. Data regarding demographics, basic laboratory studies, history of clinical epilepsy, parathyroid hormone, and vitamin D levels, as well as BsmI polymorphism of the vitamin D receptor (VDR) gene, were obtained. The bone mineral density (BMD) of the lumbar spine and left femur were measured using dual-energy x-ray absorptiometry.
Key Findings: Ninety-four patients were included in the study. BsmI polymorphism had a statistically significant lower T-score of the lumbar spine and left femoral neck than patients with wild-type VDR gene (p < 0.01 and p < 0.01, respectively). In addition, patients with BsmI polymorphism had a statistically significant lower z-score of the lumbar spine and left femoral neck than patients with wild-type VDR gene (p < 0.01 and p < 0.01, respectively). The 25-hydroxyvitamin D level in patients with wild-type genes was higher than in epileptic patients with BsmI polymorphism (p < 0.01 and p < 0.01, respectively). Parathyroid hormone level in patients with wild-type VDR gene or patients having BsmI polymorphism was not correlated with BMD at either site.
Significance: In patients with epilepsy taking phenytoin, having BsmI polymorphism was associated with lower BMD.
Epilepsy is a chronic condition that may affect individuals for years. The long-term use of antiepileptic drugs (AEDs) in patients who have seizures has been associated with alterations in bone mineral density (BMD). In comparison with the general population, patients with epilepsy have a six times greater risk of fracture, and the reduction in their bone density can be detected as early as 1–5 years after commencing treatment. The fracture risk of the epileptic population is approximately twice that of the general population, independent of seizure-related falls, whereas 35% of the fractures may be related to seizures (Petty et al., 2007). It is important to note that the fractures are associated with increased rates of morbidity and mortality (Germaine-Smith et al., 2011).
Previous studies on the bone health of patients with epilepsy have reported a significant variability in the prevalence of low BMD, ranging from 26% to 75% depending on ethnicity and/or country of origin; this may be due to genetic variability (Farhat et al., 2002; Pack et al., 2003; Lado et al., 2008; Phabphal et al., 2009). Moreover, the epileptic treatment is characterized by unpredictable efficacy and optimal dose response, and especially adverse drug reactions in individual patients. Treatment with phenytoin (PHT) was reported to be affected by genetic variability in drug metabolism >25 years ago (Löscher et al., 2009). For instance, reduced metabolism of PHT in people with certain cytochrome P450 (CYP)2C9 alleles was reported by Mamiya et al. (1998), and HLA-B*1502 was found to be associated with Stevens-Johnson syndrome/toxic epidermal necrosis induced by PHT (Hung et al., 2010). The principle of pharmacogenetics in the field of epilepsy is to test how the patient’s genotype might affect variation in response to AEDs among individuals—especially regarding the most important adverse reactions.
Vitamin D plays a central role in bone biology and mineral homeostasis. Vitamin D, by interacting with its receptors, plays an important role in calcium homeostasis by regulating bone cell growth and differentiation, intestinal calcium absorption, and parathyroid secretion. According to a report by Morrison et al. (1992), BsmI polymorphism has been associated with bone turnover, and bone density or fracture. This finding was confirmed by meta-analysis (Cooper & Umbach, 1996). Nevertheless, the studies of BsmI polymorphism of the vitamin D receptor (VDR) gene in patients with epilepsy who are taking PHT are limited. Only two studies have focused on BsmI polymorphism in patients with epilepsy (Tsukahara et al., 2002; Lambrinoudaki et al., 2011). However, the evidence pertaining to this issue is limited by confounding factors such as including several AEDs that affect BMD differently. Moreover, all of these studies consisted of a single measurement of BMD in the lumbar spine and small sample sizes.
This study was conducted with two objectives, affording us the opportunity to, first, determine the prevalence of low BMD in patients with a wild-type VDR gene and BsmI polymorphism and second, to evaluate the association between BsmI polymorphism and BMD, parathyroid hormone activity, and 25-hydroxyvitamin D level in 94 young among ambulatory adults with epilepsy who were taking PHT.
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The results of this study among young adults with epilepsy who were receiving phenytoin confirm that epileptic patients who had BsmI polymorphism have lower BMD than epileptic patients who have wild-type VDR gene. Epileptic patients with BsmI polymorphism were more likely to have BMD level below the expected range for age. The study also showed that epileptic patients with BsmI polymorphism had lower serum 25-hydroxyvitamin D level than epileptic patients with wild-type VDR gene. Moreover, serum parathyroid hormone level, serum calcium level, and serum phosphate level showed no statistically significant difference between epileptic patients with BsmI polymorphism and epileptic patients with wild-type VDR gene.
In 1992, Morrison et al. described for the first time a relationship between BsmI polymorphism and osteocalcin levels. In that work, the presence of the wild-type VDR gene was related to a higher BMD in the normal population and in twin pairs. In 1994, Morrison reported that BsmI polymorphisms were associated with lower BMD in postmenopausal women (Morrison et al., 1994). A great deal of attention was focused on this relationship, and these VDR polymorphisms have been the most extensively studied genetic markers. The frequency of the BsmI polymorphisms was found to be as high as 17% among Caucasians. In our study, we found a frequency of BsmI polymorphism of 28.7% in patients with epilepsy. By contrast, a previous study found a frequency of BsmI polymorphism of 23.7% in patients with systemic lupus erythematosus and only 17% in healthy subjects (Chaimuangraj et al., 2006).
The association between BMD and BsmI polymorphism has been the subject of many studies. Recent reports have identified BsmI polymorphism to be associated with reduced BMD and osteoporosis among postmenopausal woman (Chen et al., 2001), young adults (Kung et al., 1998), and prepubertal individuals (Saiz et al., 1997). In addition, BsmI polymorphism has been associated with a low BMD, and tends to be associated with an increased risk of fractures, independent of BMD level (Langdahl et al., 2000; Garnero et al., 2005). Of interest, a recent report by Creatsa et al. (2011) indicated that individuals with BsmI polymorphism may more responsive to oral alendronate than patients with wild-type gene. To date, only two studies have been conducted to evaluate the association between the BsmI polymorphism and BMD in patients with epilepsy. Tsukahara et al. (2002) examined the allelic variation in the VDR gene among 18 Japanese children with primary epilepsy who had taken various long-term AEDs. This study did not reveal any statistically significant association between the genotype by BsmI restriction fragment length polymorphism and low BMD. Lambrinoudaki et al. (2011) conducted a study to evaluate 73 long-term users of monotherapy AED and found a statistically significant association between BMD and the BsmI polymorphism. However, both of these studies had some limitations. First, they included epileptic patients taking CYP-inducer AEDs, CYP-inhibitor AEDs, or neither. Secondly, both studies had small sample sizes. Thirdly, BMD was measured at only the lumbar spine. To solve the problem in the design and analysis of these previous studies, it is necessary that: (1) the sample be large enough to have sufficient statistical power; (2) the sample population be a homogenous example of epileptic patients taking a single AED; and (3) the study design allow for the evaluation of BMD at femur and lumbar sites. The measurements of the regional skeleton such as the lumbar spine or the femur are often done clinically because of their importance in predicting fracture risk in later life. Our study was conducted to evaluate the BsmI polymorphism in 94 epileptic patients taking PHT as monotherapy and to measure the z-score and T-score at both the lumbar spine and femoral neck. The results of this study support the existence of a significant association between both T-score and z-score of BMD at both the lumbar spine and the femoral neck. Vitamin D is an important hormone in bone biology and mineral homeostasis. Its actions are mediated through the active metabolism of 1,25-hydroxyvitamin D3. Most of the biologic activities of 1,25-hydroxyvitamin D3 are mediated by a high-affinity receptor that acts as a ligand-activated transcription factor. The major steps involved in the control of gene transcription by the VDR include ligand binding, heterodimerization with the retinoid X receptor (RXR), binding of the heterodimer to vitamin D response elements (VDREs), and recruitment of other nuclear proteins into the transcriptional preinitiation complex (Valdivielso & Fernandez, 2006). Chromosome 12cen-q12 is the location of the VDR-encoding gene, in which at least 22 unique mutations have been reported. The BsmI polymorphism occurs in the intron between exons VIII and IX in the 3′ region of the hVDR gene and is in linkage with two of the other polymorphic sites (ApaI and TaqI). The mechanism underlying the association of BsmI VDR polymorphism and bone metabolism is uncertain. A possible mechanism may be the BsmI VDR polymorphism affecting calcium metabolism, and/or osteoblast and osteoclast function. In previous studies, AEDs, especially CYP enzyme inducer, have been shown to induce bone loss via the alteration of osteoclast differentiation and function (Dziak et al., 1988; Koide et al., 2009). Vitamin D receptor polymorphism, when present, can alter bone metabolism and, therefore, affect the susceptibility to the osteopenic effects of AEDs.
Studies on the association between BsmI polymorphism and 25-hydroxyvitamin D or parathyroid hormone are limited and also conflicting. Two studies have found that the BsmI polymorphisms are associated with differences in the 25-hydroxyvitamin; the baseline 1,25-hydroxyvitamin D levels were higher in premenopausal woman with the BsmI polymorphism than in those with the wild-type, which may have important implications in relation to the pathophysiology of osteoporosis (Howard et al., 1995; Laaksonen et al., 2002). Lambrinoudaki et al. (2011) reported that in an epileptic population, the BsmI polymorphism was associated with lower levels of serum 25-hydroxyvitamin D. This association was confirmed in our study. In addition, the effect of the BsmI polymorphism on PTH has been investigated in patients with chronic renal failure or dialysis. The findings of these studies were in the same direction; BsmI polymorphism may affect circulating levels of PTH as patients with wild-type VDR gene had a significantly lower serum PTH level (Fernández et al., 1997; Tagliabue et al., 1999). These findings were not supported by our study.
Nevertheless, the present study has some limitations. The study assessed the calcium intake via quantitative information regarding certain calcium sufficient food and estimated calcium intake by manual calculation. We obtained the information on calcium intake via an interview based on the patient’s recall of calcium intake over the previous 7 days. The dietary intake estimated from the recall technique over a short period of time may not be a good representation of usual or long-term calcium intake. Secondly, we included a few patients who had taken other AEDs before inclusion. However, one bone remodeling cycle takes approximately 3 months and it has been supported that BMD may return to normal after three remodeling cycles. Therefore, part treatment with other AED, but stopped at 6 years previously should not have had an impaired on BMD in our study. Thirdly, concerning the serum PHT levels and BMD, Lau et al. (1995) found that PHT biphasically increases [3H]-thymidine incorporation, cell number, alkaline phosphatase activity, and collagen synthesis in culture human bone cells, indicating that PHT stimulates both growth and differentiation. In theory, there is no reason to suppose that BsmI polymorphism should affect AED metabolism as the gene plays no role in the metabolism of PHT. However, we did not find any association between BsmI and serum PHT levels. In the future, we plan to evaluate whether gene polymorphisms have any effects on PHT metabolism and subsequent serum PHT concentration.
The strengths of this study were that it comprised a large epileptic population of young adult epileptic patients, allowing us to assess the association between VDR gene polymorphism and BMD, 25-hydroxyvitamin D, and parathyroid hormone level. Secondly, we had a homogenous sample population (epileptic patients taking a CYP-inducer AED). Thirdly, the measurement of 25-hydroxyvitamin D and parathyroid hormone as well as that of the VDR gene polymorphism and BMD used modern techniques and appropriate measurement sites (femur and lumbar spine). Furthermore, we excluded patients with potential confounding factors that are known to affect BMD such as low calcium intake, previous fracture 1 year before inclusion, smoking, alcohol consumption, abnormal menstruation, use of drugs including hormonal drugs, and recent weight change. Therefore, our study was able to evaluate the true association between VDR gene polymorphism and BMD in young adult patients with epilepsy.
In conclusion, to our knowledge, this is the first study to investigate the association between BsmI polymorphism and BMD, parathyroid hormone, and 25-hydroxyvitamin D level among young patients with epilepsy who are taking CYP-inducer AED, PHT as monotherapy. Our study confirmed an association between BMD and the BsmI polymorphism. The presence of BsmI polymorphism in patients with epilepsy was associated with BMD below the expected range for age. Therefore, patients with epilepsy taking CYP-inducer AEDs should have their BMDs monitored. In a future study, we will evaluate which AED medication is associated with the least reduction of BMD in outpatients with long-term use of AEDs and with BsmI polymorphism.