European Journal of Neurology 2011, 18: 9398

doi:10.1111/j.1468-1331.2010.03103.x

BsmI vitamin D receptors polymorphism and bone mineral density in men and premenopausal women on long-term antiepileptic therapy
I. Lambrinoudakia, G. Kaparosb, E. Armenia, A. Alexandrouc, C. Damaskosc, E. Logothetisb, M. Creatsaa, A. Antonioud, E. Kouskounib and N. Triantafylloue
a

2nd Department of Obstetrics and Gynecology, University of Athens, Aretaion Hospital; bHormonal Laboratory, University of Athens,

Aretaieion Hospital; c1st Department of Surgery, University of Athens, Laiko Hospital; dRadiology Department, University of Athens, Aretaieion Hospital; and eDepartment of Neurology, University of Athens, Aiginiteion Hospital, Athens, Greece

Keywords:

bone mineral density, BsmI, epilepsy, VDR

Received 14 December 2009 Accepted 25 March 2010

Background: Utilization of antiepileptic drugs (AEDs) has long been associated with bone deleterious eects. Furthermore, the BsmI restriction fragment polymorphism of the vitamin D receptor (VDR) has been associated with reduced bone mineral density (BMD), mostly in postmenopausal women. This study evaluates the association between bone metabolism of patients with epilepsy and the BsmI VDRs polymorphism in chronic users of AEDs. Methods: This study evaluated 73 long-term users of antiepileptic drug monotherapy, in a cross-sectional design. Fasting blood samples were obtained to estimate the circulating serum levels of calcium, magnesium, phosphorus, parathormone, 25hydroxyvitamin D as well as the VDRs genotype. Bone mineral density at the lumbar spine was measured with Dual Energy X-Ray Absorptiometry. Results: Bone mineral density was signicantly associated with the genotype of VDR (mean BMD: Bb genotype 1.056 0.126 g/cm2; BB genotype 1.059 0.113 g/cm2; bb genotype 1.179 0.120 g/cm2; P < 0.05). Additionally, the presence of at least one B allele was signicantly associated with lower bone mineral density (B allele present: BMD = 1.057 0.12 g/cm2, B allele absent: BMD = 1.179 0.119 g/ cm2; P < 0.01). Patients with at least one B allele had lower serum levels of 25hydroxyvitamin D when compared with bb patients (22.61 ng/ml vs. 33.27 ng/ml, P < 0.05), whilst they tended to have higher levels of parathyroid hormone. Discussion: Vitamin D receptor polymorphism is associated with lower bone mass in patients with epilepsy. This eect might be mediated through the vitamin D-parathormone pathway.

Introduction
Epilepsy is a common neurological disorder, knowing to aect approximately 50 million people of all ages worldwide [1]. It is estimated that 410 per 1000 people of the general population are in need of antiepileptic therapy at a certain time. Furthermore, there is an increased utilization of antiepileptic drugs (AEDs) for the treatment of several non-epileptic neurological conditions such as neuropathic pain syndromes, trigeminal neuralgia, migraine and essential tremor as well as psychiatric disorders [2].
Correspondence: E. Armeni, 8b, Nestou Street, Vrilissia, GR-15235 Athens, Greece (tel.: +30 210 6009726; fax: +30 210 7333310; e-mail: elenaarmeni@hotmail.com).

Accumulating evidence has linked epilepsy with osteoporosis. Osteoporosis in patients with epilepsy is a multifactorial condition, related to the type of epilepsy, falls, habitus, family history and to the presence of conditions aecting bone metabolism. Amongst other risk factors, AEDs-treatment may be one additional cause of osteoporosis [3,4]. Long-term AED therapy is an independent risk factor for reduced bone mineral density (BMD), at least in some patients [3,58]. In addition, AEDs users have an increased risk for fractures [5,8,9], which can be an important cause of morbidity and mortality. The fracture risk of the epileptic population is approximately twice that of the general population, independently of seizure-related falls, whilst 35% of fractures may be related to seizure [5,8,10].

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As the report from Morrison et al. [11], many candidate genes have been associated with bone turnover, bone density or fractures [12]. Amongst them, the vitamin D receptor (VDR) gene is by far the most extensively investigated, and more specically, the BsmI restriction fragment length polymorphism. Epileptic treatment, on the other hand, can be characterized by varying responses, unpredictable outcomes and adverse eects. Treatment with phenytoin was reported to be aected by genetic variability in drug metabolism more than 25 years ago [13]. The principle of pharmacogenetics in the eld of epilepsy is to test how a patients genotype might aect variation in response to AEDs amongst individuals, in terms of dierent adverse reactions or eectiveness of the drugs. Many drugmetabolizing enzymes, transporters, and targets have well-characterized functional variants. Therefore, genetic variation can have a signicant eect on the ecacy of AEDs, provoking alterations in pharmacokinetic and pharmacodynamic pathways [1315]. Even if many theories have been proposed, the exact mechanisms for the adverse eect of AEDs on bone health are not well known yet. Furthermore, no data exist as to the possible association between VDRs polymorphism and the AEDs induced bone disease. The aim of this study was therefore to evaluate the association between the VDRs polymorphism BsmI genotypes and the individual responses of bone metabolism after long-term utilization of AEDs.

thyroid ) liver renal ) parathyroid and gonadal function. Inclusion criteria were AED-monotherapy for a time period 1 year and regular menses for women. Exclusion criteria were intake of medications knowing to aect bone metabolism (e.g. bisphosphonates, calcium supplements, steroids, calcitonin, thiazides, glucocorticoids, or vitamin D), daily alcohol intake, smoking, nutritional deciency, thyroid liverrenal disorders, hypogonadism or hyperparathyroidism. All patients who fullled these criteria were invited to participate in the study. Written informed consent was obtained by all subjects. Institutional Review Board approval was obtained by the Ethics Committee of Aretaieion Hospital.
Protocol

Participants were evaluated in a cross-sectional design. A detailed medical history was recorded for every patient, which included category and dosage of the antiepileptic therapy. Anthropometric parameters were measured at 8:30 a.m. in light clothing, and the Body Mass Index (BMI) was computed. The measurements of height and weight were performed using an electronic scale with height rod, and BMI was estimated using the following equation: BMI = body weight (kg)/height2 (m). Subsequently, fasting venous blood samples were collected and stored at )80C until assessment. The BMD was evaluated the same morning.
Bone densitometry

Methods
Subjects

Consecutive ambulatory patients with epilepsy, regularly evaluated in the outpatient clinic of the tertiary care center of the Aiginiteion Hospital in Athens between January 2006 and December 2008, were included in the study. A total of 73 subjects on longterm anti-epileptic monotherapy were retrieved, of which 41 patients were on valproic acid, 23 were on oxcarbazepine and nine patients on levetiracetam; 31 subjects (42.47%) had generalized seizures, 12 (16.44%) had partial seizures and 30 patients (41.09%) had partial secondarily generalized seizures. Before recruitment, patients had to complete a questionnaire according to the presence of conditions knowing to aect bone turnover and BMD. The questionnaire evaluated their medical history, smoking, alcohol intake, dietary calcium intake, current and prior drug intake, bone fractures, daily exercise and the presence of regular menses. Subsequently, the subjects underwent a laboratory evaluation, which included

Bone mineral density of the lumbar spine was measured using a Norland-Excell Plus-XR-36 densitometer (Norland Medical Systems, Fort Atkinson, WI, USA) by Dual Energy X-Ray Absorptiometry (DXA). Within-subject coecient of variation was 1.1%. Using the Greek normative database [16], Z-scores (number of SD below age and sex-matched controls) were calculated. In young adults, bone density below the expected range for age was determined according to the International Society of Clinical Densitometry denition for this term, as a BMD Z-score at one site <)2.
Biochemical evaluation

Serum levels of total calcium, phosphate, magnesium, 25-hydroxyvitamin D3 and parathormone (PTHi) were evaluated using standard commercially available methodologies. According to our laboratory, the reference values range as follows: calcium 8.510.2 mg/dl; phosphorus 2.44.1 mg/dl; magnesium 1.72.2 mg/dl;

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25-hydroxyvitamin D3 30.074.0 ng/ml; PTHi 10 55 pg/ml.

VDR genotyping

Table 2 Mean levels of hormones, biochemical parameters and BMD according to the genotype of the vitamin D receptor Ballele Characteristics BMD Z-Score Serologic test results Serum Ca (mg/dl) Serum P (mg/dl) Serum Mg (mg/dl) 25(OH)Vit.D3 (ng/dl) PTHi (pg/dl) BB+Bb 1.06 0.12 )0.28 0.68 9.84 3.65 1.97 19.64 44.87 0.57 0.59 0.17 6.58 22.89 bb 1.18 0.12 0.33 0.72 10.09 3.62 1.99 22.5 33.46 0.75 0.56 0.22 4.28 14.02 P-value 0.012 0.021 0.39 0.73 0.69 0.028 0.052

Blood samples were drown by atraumatic venipuncture into trisodium circle tubes. Genomic DNA was isolated from leukocyte nucleids using High Pure PCR Template Preparation kit (Roche). Real-time PCR was performed in capillaries with a reaction volume of 10 ll, containing 2 ll of DNA (50100 ng), 0.3 lM sence primer VDR bF (TAG GGG GGA TTC TGA GGA ACT A) and antisense primer VDR bsm A (AGT TTT GTA CCC TGC CCG C), 1.4 ll 25 mM MgCl2, 1 ll H2O, 1 ll of Light Cycler DNA Master HybProbe (ready to use reactionmix for PCR containing Taq DNA polymerase, reaction buer, dNTP mix with dUTP instead of dTTP and 10 mM MgCl2), 0.6 lM Sensor b, 3 modied with uorescein (AGT ATT GGG AAT GCG CAG GCC-FL) and 0.6 lM Anchor b 5 labeled with Light Cycler Red 640 and 3 terminally blocked by phosphorylation (Red 640-TCT GTG GCC CCA GGA ACC CTG-pH). Sensor b covers the BsmI restriction site. The polymorphism was dened as BB (absence of restriction site on both alleles), Bb (heterozygous) and bb (presence of restriction site on both alleles).
Statistical analysis

BMD, bone mineral density; PTHi, parathormone. Bold indicates statistical signicant (P < 0.05) associations.

means SD. Non-parametric tests were used in cases of deviation from normal distribution. Adjustments for possible confounding factors were performed by multiple regression analysis. Statistical signicance was set at the 0.05 level.

Results
The baseline demographic and anthropometric characteristics of the study population are presented in Table 1. The mean levels of BMD, PTHi and 25hydroxyvitamin D3 did not dier regarding the sex of the patients or the utilization of each individual AED. Our analysis showed the following results (Table 2): BMD was associated with the VDR genotype, and specically, patients with the unfavorable B allele (homozygotes, heterozygotes) had lower BMD at statistically signicant levels (BMD: Bb genotype 1.056 0.126 g/cm2, BB genotype 1.059 0.113 g/ cm2, bb genotype 1.179 0.119 g/cm2, P < 0.05 for linear trend; Fig. 1). In addition, the presence of at least one B allele was associated with signicantly lower
160 000 140 000

Data analysis was performed using SPSS version 17.0. (SPSS, Chicago, IL, USA). Data on qualitative characteristics are expressed as percent values, whilst data on quantitative characteristics are expressed as
Table 1 Baseline demographic and anthropometric characteristics of the study population Characteristics Age (years) Weight (kg) BMI (kg/m2) Height (m) AEDs dosage (g/day) Duration of AEDs intake (years) BMD (g/cm2) Z-SCORE Serologic test results Serum Ca (mg/dl) Serum P (mg/dl) Serum Mg (mg/dl) 25(OH)Vit.D3 (ng/ml) PTHi (pg/ml) Mean 31.99 70.75 24.89 1.68 1610.96 8.27 1.07 )0.18 9.86 3.62 1.96 19.73 44.09 Minimum 16.00 48.00 17.63 1.50 500.00 1.50 0.81 )1.90 8.70 2.20 1.57 5.30 13.20 Maximum 54.00 105.00 36.51 1.88 3500.00 25.00 1.42 1.55 11.50 5.10 2.33 40.70 128.00 SD 8.82 14.16 4.00 0.09 562.87 6.67 0.13 0.77 0.64 0.58 0.18 6.29 23.13

P-value = 0.033

BMD L2-L4 (g/cm2)

120 000 100 000 80 000 60 000 40 000 20 000 00 000

bb

Bb

BB

VDR Genotypes
Figure 1 Mean levels of bone mineral density in lumbar spine (L2L4) according to vitamin D receptors genotype.

BMD, Bone mineral density; PTHi, parathormone; AEDs, antiepileptic drugs; 25(OH)Vit.D3 = 25-hydroxy-vitamin D3.

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BMD, and lower z-scores. Bone density below the expected range for age (4.1% of subjects) correlated positively with the presence of the BB genotype (P = 0.031). This association remained signicant after adjusting for the daily drug dosage, duration of therapy, sex, age and BMI. The presence of at least one B allele was associated with lower levels of serum 25hydroxyvitamin D3 (bb genotype: 22.5 4.28 ng/ml vs. 19.64 6.58 ng/ml, P = 0.028). Furthermore, patients having at least one B allele tended to have higher levels of PTHi compared to patients with bb genotype (P = 0.052). Regression analysis revealed that the presence of at least one unfavorable B allele was a signicant predictor of BMD, independently of age, sex, BMI, daily dosage and duration of therapy (b = )0.149; P = 0.001).

Discussion
The results of this study support the existence of a signicant association between BMD of the lumbar spine, measured by DXA, and VDR genotype in men and premenopausal women on AEDs. The presence of the BB genotype correlated positively with BMD below the expected range for age in this sample of young adults. The presence of the unfavorable B allele was an independent predictor of lower BMD. Vitamin D plays a central role in bone biology and mineral homeostasis. Its actions are mediated through the active metabolite 1,25 dihydroxyvitamin D3, produced after the hydroxylation of vitamin D3 to its active form in liver and kidney [17]. The eects of 1,25 dihydroxyvitamin D3 on bone mineral homeostasis are mediated by the nuclear VDR, a ligand-activated transcription factor [18]. Chromosome 12cen-q12 is the location of the VDR-encoding gene, in which at least 22 unique mutations have been reported. Restriction site polymorphisms in the VDR gene associated with BMD changes are mainly the FokI, the TaqI, the BsmI, the ApaI and the EcoRV [17,18]. 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) [19]. The frequency of the unfavorable BB genotype was found to be as high as 17% amongst Caucasians [18]. The association between BMD and the BsmI restriction fragment polymorphism of VDR has been subject of many studies. Recent reports have identied an association between the presence of the B allele and spinal BMD in postmenopausal women [20], which appears to be similar between black and white premenopausal women [21]. In addition, this polymorphism has been strongly associated with an increased risk of fractures, independent of BMD levels in

postmenopausal women [22], as well as with stress fractures amongst healthy men and women [23]. Other studies proposed that homozygotes for the B allele had a more than twofold increased risk of hip fracture compared with the wild type [24]. Furthermore, the BsmI polymorphism has been related to lumbar spine BMD in healthy adolescent girls [25]. On the contrary to the above, some studies have failed to corroborate these ndings [10,26]. Data relating VDR polymorphism with PTHi and 25-hydroxyvitamin D3 is relatively limited. One study suggests that baseline 1,25dihydroxyvitamin D levels are higher in premenopausal women with the BB genotype than in those with the wild type, a nding suggesting lower receptor activity of the BB genotype [27]. In addition, evaluating patients with chronic renal failure, another study proposed that the BsmI polymorphism may aect circulating levels of PTHi [28]. Beyond healthy subjects and osteoporotic postmenopausal women, the potential role of the BsmI restriction fragment polymorphism of VDR as a determinant of BMD has been evaluated in patients with thalassemia, thyroid disorders and renal failure. The homozygous BB genotype has been suggested as an additional risk factor for the development of bone disease in patients with homozygous beta thalassemia [29] and hyperthyroidism [30]. Furthermore, renal failure patients with the bb genotype, who underwent renal transplantation, have been considered as being protected to some extent against bone loss after the procedure [31]. AEDs-induced bone disease, however, has not been investigated yet, regarding the BsmI restriction fragment length polymorphism of VDR. The association between reduced BMD and utilization of AEDs remains controversial. The inducers of the CYP450 are most commonly associated with bone alterations, because they accelerate the hepatic microsomal metabolism of vitamin D to polar inactive metabolites. Decreased biologically active vitamin D leads to decreased absorption of calcium in the gut. This results to hypocalcemia and increased circulating PTH, and subsequently increased bone turnover [32]. Data, however, are still contradictory. Recent studies demonstrate diering results regarding the eect of carbamazepine, an inducer of the CYP450, on bones. Valproate on the other hand, an inhibitor of the CYP450, has only recently been associated with increased bone resorption [8,32,33]. Furthermore, Ensrud et al. [34] reported an independent association between the use of non-enzyme-inducing AEDs and the rate of bone loss in the hip ()0.53%/year; P = 0.04) whilst the use of enzyme-inducing AEDs had no signicant eect on hip bone loss ()0.46%/year;

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P = 0.31). Finally, the newer non-enzyme-inducing AEDs have not been extensively investigated yet. Beyond the use of AEDs, bone loss in the epileptic population could be related to the disease itself. Epilepsy is associated with recurrent falls, seizure-related injuries as well as with inactivity, which can predispose to bone loss. In addition, cerebral palsy, frequently associated with epilepsy, is related with osteopenia [4]. Our study shows a statistically signicant association between BMD and the BsmI polymorphism of VDR. Interestingly, all patients with bone density below the expected range for age were of the BB genotype and had lower circulating 25hydroxyvitamin D3 levels compared to wild type. To our knowledge, this is the rst study to investigate this association in an epileptic population. Only one study [35] tried to evaluate BMD in patients with pediatric epilepsy examining the presence of the B allele as well, but the BB genotype was not found amongst the study population. The mechanism underlying the association of BsmI VDR polymorphism and bone metabolism is yet unclear. The presence of polymorphic variants of the VDR gene can alter metabolic pathways and therefore, aect the susceptibility to the osteopenic eects of AEDs. Vitamin D receptor, which is found to be present rather in osteoblasts than in osteoclasts, might interact with AEDs and regulate thus the transcription of target genes involved in bone metabolism, such as the gene for osteocalcin, the induction of which is positively related with new bone formation [36]. Equally possible, this interaction may aect the gene of parathyroid hormone, which is normally down-regulated [37]. Our study bears certain limitations. The relative small sample size may have not provided us with enough statistical power to detect a clear association between markers of bone metabolism and bone disease in patients with epilepsy. Another limitation is the single measurement of BMD in lumbar spine. In addition, the number of patients with bone density below the expected range for age was low. This might be related to the fact that the subjects were slightly overweight that may have aected bone metabolism positively. Even though, the presence of a strong association between VDRs genotype and bone metabolism in patients with epilepsy is supportive of a synergistic role of this polymorphism in the development of bone disease. The impact of epilepsy on bone metabolism is considered as a type of secondary osteoporosis [3]. Physicians awareness of epilepsy-associated bone disease remains limited. Evidence suggests that bone pathology related to epilepsy can be prevented or treated. Even though, only few physicians recommend screening for bone disorders and even fewer prescribe calcium and

vitamin D supplements [10,32]. The results of the current study support the routine evaluation of patients with epilepsy for bone loss. Furthermore, the potential role of VDR polymorphism in the development of bone disease in patients with epilepsy should be evaluated by larger studies, as it might be helpful to ordinate patients as high risk and low risk, regarding the development of osteoporosis.

Acknowledgements
Funding for this study was provided by the Special Account for Research Grants by the National and Kapodistrian University of Athens, SARG 70/3/8259.

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