Neuroscience Letters 561 (2014) 74–79
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
The role of vitamin D levels and vitamin D receptor polymorphism on Parkinson’s disease in the Faroe Islands Maria Skaalum Petersen a,∗ , Sára Bech a , Debes Hammershaimb Christiansen b , Anna Vibeke Schmedes c , Jónrit Halling a a
Department of Occupational Medicine and Public Health, The Faroese Hospital System, Tórshavn, Faroe Islands Food and Veterinary Authority, National Reference Laboratory for Fish Diseases, Tórshavn, Faroe Islands c Department of Clinical Biochemistry, Lillebaelts Hospital, Vejle, Denmark b
h i g h l i g h t s • • • •
25(OH)D levels and VDR gene ApaI, BsmI and TaqI polymorphism were examined in a Faroese cohort. No association was found between 25(OH)D levels and PD. No association was found between the VDR polymorphisms. The VDR ApaI/AC genotype was significantly associated with 25(OH)D levels.
a r t i c l e
i n f o
Article history: Received 15 August 2013 Received in revised form 5 December 2013 Accepted 21 December 2013 Keywords: Parkinson’s disease Vitamin D receptor Polymorphism Faroe Islands
a b s t r a c t The role of vitamin D in Parkinson’s disease (PD) has been proposed and both low serum 25hydroxyvitamin D levels (25(OH)D) and vitamin D receptor polymorphisms (VDR) have been linked to PD. The aim of this study is to investigate the associations among 25(OH)D and three VDR polymorphisms and PD in the Faroese population where the prevalence of PD is high. We conducted a case–control study where 121 cases were studied for 25(OH)D levels and VDR polymorphisms against 235 randomly selected controls, matched by gender and age. No significant difference was observed in 25(OH)D levels between PD cases and controls (P = 0.49), although cases had slightly lower values than controls. As well, no differences were found in genotype frequencies between cases and controls in the VDR polymorphisms studied (ApaI, BsmI, TaqI) (P = 0.70, P = 0.56 and P = 0.54, respectively). However, we found that VDR ApaI/AC genotype was significantly associated with 25(OH)D levels (P = 0.01). Although our results indicate no association between PD and vitamin D polymorphisms and/or 25(OH)D levels, the study cannot exclude a weak association. However, the known doubling in PD prevalence in the Faroe Islands cannot be explained by the polymorphisms examined in the VDR gene or the 25(OH)D levels and has to be explored further. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Despite many years of research on Parkinson’s disease (PD), the etiology remains poorly understood. Many different etiologic factors seem to be capable of causing PD [23] and individual vulnerability may further complicate the Picture [14,17]. Vitamin D is an environmentally modifiable factor as it is largely determined by diet and sunlight exposure. The effect of vitamin D and genetic variants in the vitamin D receptor (VDR) gene on PD
∗ Corresponding author at: Department of Occupational Medicine and Public Health, The Faroese Hospital System, Sigmundargøta 5, Postbox 14, FO-110 Tórshavn, Faroe Islands. Tel.: +298 316696. E-mail address: [email protected] (M.S. Petersen). 0304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.053
has recently gained interest [4,6–8,15,16,19,22,27]. VDR and 1␣hydroxylase, which converts 25-hydroxyvitamin D (25(OH)D) to the active metabolite 1,25-dihydroxyvitamin D by binding to VDR, are most highly expressed in neurons of the substantia nigra where dopaminergic neurons are selectively lost in PD [8]. In a crosssectional study, significantly lower 25(OH)D levels were observed in Caucasian PD patients, compared to both healthy controls and patients with Alzheimer’s Disease [6]. A following study confirmed a high prevalence of vitamin D insufficiency in patients with recent onset of PD [7]. In a long-term Finnish cohort study measuring vitamin D levels before PD occurrence, PD incidence was three times higher in individuals in the lowest quartile of 25(OH)D concentrations than in those in the highest quartile [16]. Moreover, a higher prevalence of low 25(OH)D concentrations has been reported in Japanese patients with more severe PD, compared with those with
M.S. Petersen et al. / Neuroscience Letters 561 (2014) 74–79
milder PD [25–27]. Rodent studies have confirmed that the VDR is expressed in both developing and adult rat brain [22], and VDR knockout mice show muscular and motor impairment [3]. The effect of the VDR genotypes on PD, along with their possible importance in predisposing certain individuals to PD, has received little attention so far. However, some genetic studies have shown genotypes in the VDR gene that are associated with risk of PD [4,15,27]. One Korean case–control study found that the minor BsmI genotype was over-represented in PD patients, compared with controls [15]. Another recent Japanese study showed associations between the VDR FokI genotype and milder forms of PD [27]. Lv et al., however, did not find any difference in the genotype frequency of VDR TaqI comparing Chinese Han patients with control [19]. The prevalence of PD in the Faroese population is twice as high as expected [28,29], but this high prevalence remains unexplained. Due to the high prevalence of PD in the Faroes [28,29] and the proposed low vitamin D levels in the Faroese population [6], we find the Faroes a highly appropriate place to study the association between VDR polymorphisms and vitamin D levels and PD. 2. Materials and methods 2.1. Cases and controls
75
Germany. Before LC–MS/MS, serum was purified by protein precipitation and further purification of the supernatant by use of a phospholipid removal plate (OstroTM 96-well plate, Waters, Hedehusene, Denmark). The Department of Clinical Biochemistry, Lillebaelts Hospital participated with satisfactory results in the external quality control scheme from DEQAS Vitamin D External Quality Assessment Scheme, London, UK. 2.3. VDR SNP analysis The DNA was extracted from the blood samples using PUREGENETM genomic DNA purification kit (Gentra Systems, MN, USA) or on the QIAsymphony SP (Qiagen, Hilden, Germany) using the QIAsymphony DNA mini kit (Qiagen) according to the guidelines of the manufacturer. SNP genotyping of the three VDR gene SNP’s ApaI rs7975232, BsmI rs1544410, and TaqI rs731236 was performed with the Type-it Fast SNP Probe Kit (Qiagen) according to the manufacturer’s instructions using validated TaqMan® SNP genotyping primers and probe (Applied Biosystems, Foster City, CA, USA). SNP reaction mixture was set up on the ABI 7500 Fast real-time PCR system (Applied Biosystems) and PCR cycling conditions were as outlined in the Type-if Fast SNP Probe manual (Qiagen). Positive controls (homozygotes and heterozygotes) were used in each run for BsmI rs1544410, and TaqI rs731236. Furthermore exome results from ten PD cases confirmed the results from the Type-it Fast SNPProbe Kit.
The case–control study of Faroese PD patients conducted in 2005 has been previously described [9,24,29]. A total of 80 verified PD cases and 154 controls were included in the study. In 2005, the crude and age-adjusted prevalence of PD in the Faroese was 207 and 218 per 100,000 inhabitants, respectively. During 1995–2005, the average annual incidence was 21.1 per 100,000 persons for PD [29]. The case-recruitment period was May–June 2005, while the controls were collected in the period January–March 2005. In 2011 a new round of recruitment was performed. PD patients newly diagnosed since 2005 were invited to participate. New cases were identified via (1) the Chief Pharmacist in the Faroes (individuals being prescribed levodopa-containing drugs or dopa-agonists in a defined time frame), (2) the local neurology specialist, and (3) the Faroese Hospital System. All cases were examined clinically by the same neurologist (SB) as in 2005 using the United Kingdom Parkinson’s Disease Brain Bank [12] criteria to establish diagnoses and the Hoehn and Yahr [11] scale for clinical staging. From all consenting cases and controls, a blood sample was drawn and all individuals answered a questionnaire on life-style and environmental exposures in a face-to-face interview. In all, seventy-eight letters of invitation were sent to potential PD patients. Ten were deceased, four could not be contacted, four reported not having PD and ten declined to participate. Fifty individuals were examined and 41 were verified as having PD while three did not have PD, one had neuroleptic induced PD, one had essential tremor, one had multiple system atrophy, and three had progressive supranuclear palsy. Two controls for each patient were randomly retrieved from the Faroese Population Registry, using the date of birth and gender as matching parameters. A total of 102 invitation letters were sent. One person was deceased, two were not reached, 18 declined participation and 81 controls accepted to participate. The recruitment period for cases was September 2011 and for controls January 2012.
Comparison of genotype frequency of the VDR polymorphisms and allele frequency between cases and controls was performed by chi-square analyses. Vitamin D levels were normally distributed in the combined group (cases and controls) and the case group. Natural logarithmic transformation did not improve the normality distribution in the controls and thus the original scale was used. The relationship between VDR polymorphisms and 25(OH)D values was evaluated with linear regression analyses, adjusted for the matching factors of gender, age, and year of blood drawn as the first model, and then including current smoking, consumption of whale blubber, and genotype as possible confounders in the second model. Smoking and blubber consumption in adulthood was dichotomized: current smoking versus never smoking; whale blubber more than once per month versus once per month or less. To identify any interaction or effect measure modifiers between the (1) 25(OH)D levels and SNPs, (2) gender and current smoking in relation to 25(OH)D levels, and (3) disease status and current smoking in relation to 25(OH)D levels product terms for factors were constructed. Multivariate unconditional logistic regression analysis adjusting for potential confounders was performed to assess the relationship between risk of PD and 25(OH)D levels and between PD and genotypes. All the statistical analyses were repeated, stratified by year of blood drawn, as sample collection occurred on two occasions, 6 years apart. The analyses were carried out using the IBM SPSS statistics 21 (IBM, NY, USA). Two-sided P values less than 0.05 were used to formally consider statistical significance.
2.2. Analysis of serum vitamin D level
3. Results
25-Hydroxyvitamin D3 (25(OH)D) was determined by ultraperformance liquid chromatography, followed by tandem mass spectrometry (LC–MS/MS). Deuterated 25-hydroxyvitamin D3 was used as internal standard and calibration was performed by using a commercially available calibrator from Chromsystems, Munich,
A total of 121 patients and 235 controls are included. Baseline characteristics of the PD patients and controls are shown in Table 1. Only eight cases had early onset PD (1 per month in adulthoode Whale blubber consumption ≤1 per month in adulthoodf
Serum 25(OH)D, nmol/L, mean (SD)
Pa
39.3 (23.9) 41.1(24.8) 43.4 (23.2) 47.4 (26.3) 36.6 (24.1) 37.1 (22.9) 37.15 (23.42) 43.63 (25.52) 41.9 (25.10) 38.8 (23.4)
0.49 0.41 0.87 0.14 0.64
Abbreviation: PD, Parkinson’s disease; 25(OH)D, 25-hydroxyvitamin D; SD, standard deviation. a Linear regression adjusted for gender, age and year of blood drawn. b Occasional smokers included. c n = 47. d n = 137. e n = 231. f n = 111.
terms of gender and age distribution as expected due to matching on these variables. However, there was a significant difference between cases and controls in terms of current smoking and intake of blubber in adult life with cases reporting higher intake. However, stratifying according to sampling period, the difference in blubber intake was only significant in 2005 (P < 0.0001), not in 2011 (P = 0.12). No significant difference was observed in 25(OH)D levels between PD cases and controls (P = 0.49), although cases had slightly lower values than controls (Table 2). Gender was a predictor of vitamin D levels with significantly higher values in women (mean: 46.1 nmol/L versus 36.9 nmol/L, P < .001), as was year of blood drawn with significantly higher values in 2011 than 2005 (mean: 47.1 nmol/L versus 37.1 nmol/L, P < .001). All analyses were adjusted for the matching variables and year of blood drawn. Current smoking and consumption of whale blubber in adulthood were not significant predictors of vitamin D status (P = 0.14 and P = 0.64 respectively). Adjusted for age, gender, and year of blood drawn, smokers had lower vitamin D values than non-smokers, and higher values were observed in the group with higher blubber intake, although the difference was not statistically significant. No interaction was observed between gender and current smoking (P = 0.58) or current smoking and disease status (P = 0.58) in relation to 25(OH)D levels. A total of 70.8% cases and 63.8% controls had levels below 50 nmol/L. Nine per cent of cases and 8% of controls had values above 75 nmol/L. As all cases and all controls were collected in the same period of year, seasonal difference cannot be tested. However, season did not have significant influence on 25(OH)D levels when
combining the groups (P = 0.49), but significantly lower 25(OH)D values were observed in samples from cases and controls collected in 2005, compared with 2011 (mean: 34.44 nmol/L versus 48.95 nmol/L, P < 0.001 and 38.46 nmol/L versus 46.20 nmol/L, P = 0.03). The ApaI genotype was a significant predictor of vitamin D levels in adjusted analysis, but not BsmI or TaqI (Table 3). Adjusted for gender, age, and year of blood drawn, subjects with genotype AC had significantly higher 25(OH)D values than subjects with AA (P = 0.01) and CC genotype (P = 0.03). There was no difference in 25(OH)D levels between AA and CC genotype (P = 0.8). Stratifying according to affected status showed slightly stronger association in controls than cases, but with the same tendency, probably due to the smaller sample size in the PD group (Table 3). Multiple logistic regressions did not show any difference in PD cases and controls in genotype or allele frequency for the three SNPs (Table 4), neither did stratification according to gender (data not shown). The Faroese distribution of the ApaI, BsmI, and TaqI genotypes were not different from the HapMap-CEU genotype for cases, controls or the combined group (P = 0.11 to P = 0.56) [21]. 4. Discussion In this study, no statistical difference was found in the prevalence of low 25(OH)D levels in PD cases compared with controls, even though cases had slightly lower mean levels, and their blood draw was in the summer while the control blood samples were collected in the winter, hence higher values would be expected due
M.S. Petersen et al. / Neuroscience Letters 561 (2014) 74–79
77
Table 3 Vitamin D receptor polymorphisms and 25-hydroxyvitamin D levels in Parkinson’s disease cases and controls. SNP
Genotype
PD cases Serum 25(OH)D, ng/mL, mean (SD)
Pa
Controls Serum 25(OH)D, ng/mL, mean (SD)
Pa
Combined group Serum 25(OH)D, ng/m, mean (SD)
Pa
ApaI rs7975232
AA AC CC
35.0 (22.8) 41.3 (25.9) 40.2 (20.7)
0.09
38.5 (21.0) 45.3 (27.0) 35.4 (22.2)
0.05
37.2 (20.8) 43.9 (26.6) 36.8 (22.3)
0.01
BsmI rs1544410
AA AG GG
36.9 (23.2) 39.3 (23.9) 40.2 (24.6)
0.65
37.7 (21.3) 43.5 (26.2) 39.2 (24.0)
0.14
37.4 (21.8) 42.2 (25.5) 39.6 (24.1)
0.32
TaqI rs731236
CC CT TT
36.9 (23.2) 38.8 (24.1) 40.9 (24.4)
0.53
37.7 (21.3) 43.4 (26.0) 39.5 (24.3)
0.18
37.4 (21.8) 41.9 (25.4) 40.0 (24.3)
0.42
Abbreviation: PD, Parkinson’s disease; 25(OH)D, 25-hydroxyvitamin D; SD, standard deviation. a Linear regression adjusted for gender, age and year of blood drawn.
to seasonal variation. 25(OH)D levels were deficient (
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
The role of vitamin D levels and vitamin D receptor polymorphism on Parkinson’s disease in the Faroe Islands Maria Skaalum Petersen a,∗ , Sára Bech a , Debes Hammershaimb Christiansen b , Anna Vibeke Schmedes c , Jónrit Halling a a
Department of Occupational Medicine and Public Health, The Faroese Hospital System, Tórshavn, Faroe Islands Food and Veterinary Authority, National Reference Laboratory for Fish Diseases, Tórshavn, Faroe Islands c Department of Clinical Biochemistry, Lillebaelts Hospital, Vejle, Denmark b
h i g h l i g h t s • • • •
25(OH)D levels and VDR gene ApaI, BsmI and TaqI polymorphism were examined in a Faroese cohort. No association was found between 25(OH)D levels and PD. No association was found between the VDR polymorphisms. The VDR ApaI/AC genotype was significantly associated with 25(OH)D levels.
a r t i c l e
i n f o
Article history: Received 15 August 2013 Received in revised form 5 December 2013 Accepted 21 December 2013 Keywords: Parkinson’s disease Vitamin D receptor Polymorphism Faroe Islands
a b s t r a c t The role of vitamin D in Parkinson’s disease (PD) has been proposed and both low serum 25hydroxyvitamin D levels (25(OH)D) and vitamin D receptor polymorphisms (VDR) have been linked to PD. The aim of this study is to investigate the associations among 25(OH)D and three VDR polymorphisms and PD in the Faroese population where the prevalence of PD is high. We conducted a case–control study where 121 cases were studied for 25(OH)D levels and VDR polymorphisms against 235 randomly selected controls, matched by gender and age. No significant difference was observed in 25(OH)D levels between PD cases and controls (P = 0.49), although cases had slightly lower values than controls. As well, no differences were found in genotype frequencies between cases and controls in the VDR polymorphisms studied (ApaI, BsmI, TaqI) (P = 0.70, P = 0.56 and P = 0.54, respectively). However, we found that VDR ApaI/AC genotype was significantly associated with 25(OH)D levels (P = 0.01). Although our results indicate no association between PD and vitamin D polymorphisms and/or 25(OH)D levels, the study cannot exclude a weak association. However, the known doubling in PD prevalence in the Faroe Islands cannot be explained by the polymorphisms examined in the VDR gene or the 25(OH)D levels and has to be explored further. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Despite many years of research on Parkinson’s disease (PD), the etiology remains poorly understood. Many different etiologic factors seem to be capable of causing PD [23] and individual vulnerability may further complicate the Picture [14,17]. Vitamin D is an environmentally modifiable factor as it is largely determined by diet and sunlight exposure. The effect of vitamin D and genetic variants in the vitamin D receptor (VDR) gene on PD
∗ Corresponding author at: Department of Occupational Medicine and Public Health, The Faroese Hospital System, Sigmundargøta 5, Postbox 14, FO-110 Tórshavn, Faroe Islands. Tel.: +298 316696. E-mail address: [email protected] (M.S. Petersen). 0304-3940/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.12.053
has recently gained interest [4,6–8,15,16,19,22,27]. VDR and 1␣hydroxylase, which converts 25-hydroxyvitamin D (25(OH)D) to the active metabolite 1,25-dihydroxyvitamin D by binding to VDR, are most highly expressed in neurons of the substantia nigra where dopaminergic neurons are selectively lost in PD [8]. In a crosssectional study, significantly lower 25(OH)D levels were observed in Caucasian PD patients, compared to both healthy controls and patients with Alzheimer’s Disease [6]. A following study confirmed a high prevalence of vitamin D insufficiency in patients with recent onset of PD [7]. In a long-term Finnish cohort study measuring vitamin D levels before PD occurrence, PD incidence was three times higher in individuals in the lowest quartile of 25(OH)D concentrations than in those in the highest quartile [16]. Moreover, a higher prevalence of low 25(OH)D concentrations has been reported in Japanese patients with more severe PD, compared with those with
M.S. Petersen et al. / Neuroscience Letters 561 (2014) 74–79
milder PD [25–27]. Rodent studies have confirmed that the VDR is expressed in both developing and adult rat brain [22], and VDR knockout mice show muscular and motor impairment [3]. The effect of the VDR genotypes on PD, along with their possible importance in predisposing certain individuals to PD, has received little attention so far. However, some genetic studies have shown genotypes in the VDR gene that are associated with risk of PD [4,15,27]. One Korean case–control study found that the minor BsmI genotype was over-represented in PD patients, compared with controls [15]. Another recent Japanese study showed associations between the VDR FokI genotype and milder forms of PD [27]. Lv et al., however, did not find any difference in the genotype frequency of VDR TaqI comparing Chinese Han patients with control [19]. The prevalence of PD in the Faroese population is twice as high as expected [28,29], but this high prevalence remains unexplained. Due to the high prevalence of PD in the Faroes [28,29] and the proposed low vitamin D levels in the Faroese population [6], we find the Faroes a highly appropriate place to study the association between VDR polymorphisms and vitamin D levels and PD. 2. Materials and methods 2.1. Cases and controls
75
Germany. Before LC–MS/MS, serum was purified by protein precipitation and further purification of the supernatant by use of a phospholipid removal plate (OstroTM 96-well plate, Waters, Hedehusene, Denmark). The Department of Clinical Biochemistry, Lillebaelts Hospital participated with satisfactory results in the external quality control scheme from DEQAS Vitamin D External Quality Assessment Scheme, London, UK. 2.3. VDR SNP analysis The DNA was extracted from the blood samples using PUREGENETM genomic DNA purification kit (Gentra Systems, MN, USA) or on the QIAsymphony SP (Qiagen, Hilden, Germany) using the QIAsymphony DNA mini kit (Qiagen) according to the guidelines of the manufacturer. SNP genotyping of the three VDR gene SNP’s ApaI rs7975232, BsmI rs1544410, and TaqI rs731236 was performed with the Type-it Fast SNP Probe Kit (Qiagen) according to the manufacturer’s instructions using validated TaqMan® SNP genotyping primers and probe (Applied Biosystems, Foster City, CA, USA). SNP reaction mixture was set up on the ABI 7500 Fast real-time PCR system (Applied Biosystems) and PCR cycling conditions were as outlined in the Type-if Fast SNP Probe manual (Qiagen). Positive controls (homozygotes and heterozygotes) were used in each run for BsmI rs1544410, and TaqI rs731236. Furthermore exome results from ten PD cases confirmed the results from the Type-it Fast SNPProbe Kit.
The case–control study of Faroese PD patients conducted in 2005 has been previously described [9,24,29]. A total of 80 verified PD cases and 154 controls were included in the study. In 2005, the crude and age-adjusted prevalence of PD in the Faroese was 207 and 218 per 100,000 inhabitants, respectively. During 1995–2005, the average annual incidence was 21.1 per 100,000 persons for PD [29]. The case-recruitment period was May–June 2005, while the controls were collected in the period January–March 2005. In 2011 a new round of recruitment was performed. PD patients newly diagnosed since 2005 were invited to participate. New cases were identified via (1) the Chief Pharmacist in the Faroes (individuals being prescribed levodopa-containing drugs or dopa-agonists in a defined time frame), (2) the local neurology specialist, and (3) the Faroese Hospital System. All cases were examined clinically by the same neurologist (SB) as in 2005 using the United Kingdom Parkinson’s Disease Brain Bank [12] criteria to establish diagnoses and the Hoehn and Yahr [11] scale for clinical staging. From all consenting cases and controls, a blood sample was drawn and all individuals answered a questionnaire on life-style and environmental exposures in a face-to-face interview. In all, seventy-eight letters of invitation were sent to potential PD patients. Ten were deceased, four could not be contacted, four reported not having PD and ten declined to participate. Fifty individuals were examined and 41 were verified as having PD while three did not have PD, one had neuroleptic induced PD, one had essential tremor, one had multiple system atrophy, and three had progressive supranuclear palsy. Two controls for each patient were randomly retrieved from the Faroese Population Registry, using the date of birth and gender as matching parameters. A total of 102 invitation letters were sent. One person was deceased, two were not reached, 18 declined participation and 81 controls accepted to participate. The recruitment period for cases was September 2011 and for controls January 2012.
Comparison of genotype frequency of the VDR polymorphisms and allele frequency between cases and controls was performed by chi-square analyses. Vitamin D levels were normally distributed in the combined group (cases and controls) and the case group. Natural logarithmic transformation did not improve the normality distribution in the controls and thus the original scale was used. The relationship between VDR polymorphisms and 25(OH)D values was evaluated with linear regression analyses, adjusted for the matching factors of gender, age, and year of blood drawn as the first model, and then including current smoking, consumption of whale blubber, and genotype as possible confounders in the second model. Smoking and blubber consumption in adulthood was dichotomized: current smoking versus never smoking; whale blubber more than once per month versus once per month or less. To identify any interaction or effect measure modifiers between the (1) 25(OH)D levels and SNPs, (2) gender and current smoking in relation to 25(OH)D levels, and (3) disease status and current smoking in relation to 25(OH)D levels product terms for factors were constructed. Multivariate unconditional logistic regression analysis adjusting for potential confounders was performed to assess the relationship between risk of PD and 25(OH)D levels and between PD and genotypes. All the statistical analyses were repeated, stratified by year of blood drawn, as sample collection occurred on two occasions, 6 years apart. The analyses were carried out using the IBM SPSS statistics 21 (IBM, NY, USA). Two-sided P values less than 0.05 were used to formally consider statistical significance.
2.2. Analysis of serum vitamin D level
3. Results
25-Hydroxyvitamin D3 (25(OH)D) was determined by ultraperformance liquid chromatography, followed by tandem mass spectrometry (LC–MS/MS). Deuterated 25-hydroxyvitamin D3 was used as internal standard and calibration was performed by using a commercially available calibrator from Chromsystems, Munich,
A total of 121 patients and 235 controls are included. Baseline characteristics of the PD patients and controls are shown in Table 1. Only eight cases had early onset PD (1 per month in adulthoode Whale blubber consumption ≤1 per month in adulthoodf
Serum 25(OH)D, nmol/L, mean (SD)
Pa
39.3 (23.9) 41.1(24.8) 43.4 (23.2) 47.4 (26.3) 36.6 (24.1) 37.1 (22.9) 37.15 (23.42) 43.63 (25.52) 41.9 (25.10) 38.8 (23.4)
0.49 0.41 0.87 0.14 0.64
Abbreviation: PD, Parkinson’s disease; 25(OH)D, 25-hydroxyvitamin D; SD, standard deviation. a Linear regression adjusted for gender, age and year of blood drawn. b Occasional smokers included. c n = 47. d n = 137. e n = 231. f n = 111.
terms of gender and age distribution as expected due to matching on these variables. However, there was a significant difference between cases and controls in terms of current smoking and intake of blubber in adult life with cases reporting higher intake. However, stratifying according to sampling period, the difference in blubber intake was only significant in 2005 (P < 0.0001), not in 2011 (P = 0.12). No significant difference was observed in 25(OH)D levels between PD cases and controls (P = 0.49), although cases had slightly lower values than controls (Table 2). Gender was a predictor of vitamin D levels with significantly higher values in women (mean: 46.1 nmol/L versus 36.9 nmol/L, P < .001), as was year of blood drawn with significantly higher values in 2011 than 2005 (mean: 47.1 nmol/L versus 37.1 nmol/L, P < .001). All analyses were adjusted for the matching variables and year of blood drawn. Current smoking and consumption of whale blubber in adulthood were not significant predictors of vitamin D status (P = 0.14 and P = 0.64 respectively). Adjusted for age, gender, and year of blood drawn, smokers had lower vitamin D values than non-smokers, and higher values were observed in the group with higher blubber intake, although the difference was not statistically significant. No interaction was observed between gender and current smoking (P = 0.58) or current smoking and disease status (P = 0.58) in relation to 25(OH)D levels. A total of 70.8% cases and 63.8% controls had levels below 50 nmol/L. Nine per cent of cases and 8% of controls had values above 75 nmol/L. As all cases and all controls were collected in the same period of year, seasonal difference cannot be tested. However, season did not have significant influence on 25(OH)D levels when
combining the groups (P = 0.49), but significantly lower 25(OH)D values were observed in samples from cases and controls collected in 2005, compared with 2011 (mean: 34.44 nmol/L versus 48.95 nmol/L, P < 0.001 and 38.46 nmol/L versus 46.20 nmol/L, P = 0.03). The ApaI genotype was a significant predictor of vitamin D levels in adjusted analysis, but not BsmI or TaqI (Table 3). Adjusted for gender, age, and year of blood drawn, subjects with genotype AC had significantly higher 25(OH)D values than subjects with AA (P = 0.01) and CC genotype (P = 0.03). There was no difference in 25(OH)D levels between AA and CC genotype (P = 0.8). Stratifying according to affected status showed slightly stronger association in controls than cases, but with the same tendency, probably due to the smaller sample size in the PD group (Table 3). Multiple logistic regressions did not show any difference in PD cases and controls in genotype or allele frequency for the three SNPs (Table 4), neither did stratification according to gender (data not shown). The Faroese distribution of the ApaI, BsmI, and TaqI genotypes were not different from the HapMap-CEU genotype for cases, controls or the combined group (P = 0.11 to P = 0.56) [21]. 4. Discussion In this study, no statistical difference was found in the prevalence of low 25(OH)D levels in PD cases compared with controls, even though cases had slightly lower mean levels, and their blood draw was in the summer while the control blood samples were collected in the winter, hence higher values would be expected due
M.S. Petersen et al. / Neuroscience Letters 561 (2014) 74–79
77
Table 3 Vitamin D receptor polymorphisms and 25-hydroxyvitamin D levels in Parkinson’s disease cases and controls. SNP
Genotype
PD cases Serum 25(OH)D, ng/mL, mean (SD)
Pa
Controls Serum 25(OH)D, ng/mL, mean (SD)
Pa
Combined group Serum 25(OH)D, ng/m, mean (SD)
Pa
ApaI rs7975232
AA AC CC
35.0 (22.8) 41.3 (25.9) 40.2 (20.7)
0.09
38.5 (21.0) 45.3 (27.0) 35.4 (22.2)
0.05
37.2 (20.8) 43.9 (26.6) 36.8 (22.3)
0.01
BsmI rs1544410
AA AG GG
36.9 (23.2) 39.3 (23.9) 40.2 (24.6)
0.65
37.7 (21.3) 43.5 (26.2) 39.2 (24.0)
0.14
37.4 (21.8) 42.2 (25.5) 39.6 (24.1)
0.32
TaqI rs731236
CC CT TT
36.9 (23.2) 38.8 (24.1) 40.9 (24.4)
0.53
37.7 (21.3) 43.4 (26.0) 39.5 (24.3)
0.18
37.4 (21.8) 41.9 (25.4) 40.0 (24.3)
0.42
Abbreviation: PD, Parkinson’s disease; 25(OH)D, 25-hydroxyvitamin D; SD, standard deviation. a Linear regression adjusted for gender, age and year of blood drawn.
to seasonal variation. 25(OH)D levels were deficient (
