Vitamin D Receptor Polymorphisms in a Spanish Cohort of Parkinson's Disease Patients

Introduction

Vitamin D receptor (VDR) is a nuclear hormone receptor that modulates gene transcriptions in response to 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃), the active form of Vitamin D (Butler et al., 2011). In fact, 25-hydroxyvitamin D₃ (25[OH]D₃) is metabolized to 1,25(OH)₂D₃ by 1-α-hydroxylase (Yeshokumar et al., 2015). Both 1-α-hydroxylase and VDR are abundantly expressed in the human brain, particularly in the substantia nigra (Cui et al., 2013, Eyles et al., 2005). The activation of VDR by 1,25[OH]₂D₃ is critical for the transcription of vitamin D responsive genes. This ligand-binding process enables VDR to promote histone modifications via the recruitment of co-activator proteins and the activity of histone acetyltransferases (HATs), thus activating gene expression. Interestingly, the unbound VDR, generally located in the cytoplasm, can also bind DNA and interact with histone deacetylases (HDACs) through co-repressor proteins, leading to gene repression (Fetahu et al., 2014). This interaction between VDR and HDACs (specifically HDAC4 and HDAC6) is known to decrease cyclically in response to 1,25[OH]₂D₃ in MCF-10A cells (Malinen et al., 2011).

Importantly, an imbalance in HAT and HDAC activity has been observed in Parkinson's disease (PD). This imbalance is evidenced by the epigenetic alterations in fibroblasts from PD patients (Yakhine-Diop et al., 2019b), including increased levels of certain class I (HDAC2 and HDAC3) and II (HDAC4) HDAC proteins (Yakhine-Diop et al., 2019a), which correlate with decreased levels of acetylated proteins (Yakhine-Diop et al., 2019b) and peptides (Yakhine-Diop et al., 2018) in idiopathic PD.

The link between VDR and PD pathogenesis is supported by the observations of low levels of 25[OH]D₃ in PD patients' plasma (Ding et al., 2013) and polymorphisms in the VDR gene affecting receptor activity (Smolders et al., 2009). Reduced vitamin D levels correlate with postural instability (Peterson et al., 2013) and symptom severity (Suzuki et al., 2012) in PD patients. Interestingly, the administration of 1,25[OH]₂D₃ has a neuroprotective effect in animal models of PD, attenuating the inflammation-mediated neurodegeneration (Kim et al., 2006) and promoting neurogenesis (Cataldi et al., 2018). Furthermore, Vitamin D has been found to protect 6-Hydroxydopamine (6-OHDA)-injured rats by enhancing dopamine levels via increased endogenous glial cell line-derived neurotrophic factor expression in the substantia nigra (Smith et al., 2006, Wang et al., 2001). However, VDR gene variants can impair its function and the enzymatic regulation of vitamin D metabolism (Smolders et al., 2009).

Since then, evidences suggest that VDR polymorphisms play a role in PD pathogenesis (Butler et al., 2011), despite some inconsistencies in the data. For example, the FokI C allele is significantly represented in Hungarian PD patients (Torok et al., 2013) and is also associated with the milder form of PD in Japanese population (Suzuki et al., 2012). The VDR BsmI GG genotype in Korean PD patients (Kim et al., 2005) further reinforces the role of these polymorphisms. Contrarily, studies in Faroese (Petersen et al., 2014) and Taiwanese populations (Lin et al., 2014) did not find an association between VDR polymorphisms and PD risk. These differences observed across distinct PD populations could be attributed to the strength of linkage disequilibrium between adjacent genetic variants in the VDR gene (Fang et al., 2005).

The VDR gene, situated on the long arm of the chromosome 12, contains eight coding exons (2-9) and six isoforms (1f-1c) in its 5′ noncoding exon1 (Fig. 1) (Deuster et al., 2017). Among ∼57 single nucleotide polymorphisms (SNPs) identified in VDR gene (Fang et al., 2005), our research concentrated on the four well-known restriction fragment length polymorphisms (RFLPs): TaqI (rs731236), ApaI (rs7974232), BsmI (rs1544410), and FokI (rs10735810). In this study, we investigated these VDR polymorphisms in Spanish sporadic PD patients. Notably, the prevalence of the BsmI genotype was fivefold higher in PD patients compared to controls, a finding that was statistically significant. This suggests that the BsmI polymorphism could be a potential risk factor for PD within the Spanish population. Furthermore, our analysis revealed a significant association between the combination of BsmI and FokI polymorphisms and sporadic PD, indicating their combined relevance as a risk factor in this population.

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FIG. 1.

Gene structure of VDR and its four SNPs. White and black charts represent, respectively, the noncoding exon and coding exon. FokI polymorphism is located in exon 2 and provokes a cleavage in T (f) when C (F) is substituted. Both BsmI and ApaI are located in intron 8 and induce restriction site in G (b,a) when A(B) and T(A) are, respectively, substituted. TaqI is in exon 9 and cleaves in C(t) when T is substituted. In the case of absence of cleavage site in restriction enzyme assays, the respective allele is denoted with a capital letter (A for Apa-I, T for Taq-I, F for FokI, and B for BsmI), while the presence of a restriction site is indicated by a lowercase letter (a, t, f, b). SNPs, single nucleotide polymorphisms; VDR, vitamin D receptor.

Materials and Methods

Results

Discussion

PD is a neurological disorder characterized by the progressive onset of both nonmotor and motor symptoms (Tibar et al., 2018). Although nonmotor symptoms may precede motor symptoms (Kumar et al., 2022), the clinical diagnosis of PD primary relies on motor symptoms, such as tremors, rigidity, postural instability. Within our study population, 33.3% of patients exhibit tremors, 26% show rigidity, and 20% have mixed symptoms, while the rest remain unclassified (Table 1). Except for two individuals, the rest of PD patients have an asymmetrical onset of symptoms either on left or on the right. The average age of PD onset in Spanish patients is around 60 years old, which is consistent with the average age of disease onset in the Western world (Post et al., 2020). Nevertheless, 5.76% of individuals developed PD before the age of 40, with 3.84% of them being females. Patients beneath this age are considered young-onset PD patients (Post et al., 2020), which is a subset of early-onset PD (≤60 years). When the cut-off age is set at 50 years, the percentage of young-onset Parkinson's patients reached 23%.

Certain mutations in PARK genes have been associated with both sporadic and familial PD (Correia Guedes et al., 2020) and may be considered high risk factors for developing early-onset PD. However, genetic mutations account for only 10% of PD cases (Yakhine-Diop et al., 2016), suggesting that most cases results from a combination of genetic susceptibility and environmental factors (González-Polo et al., 2015).

Environmental factors, such as sunlight exposure and/or polymorphisms in VDR gene (Suzuki et al., 2012), can influence the regulation of vitamin D metabolism. Although the relationship between reduced sun exposure, vitamin D levels, and the severity of PD symptoms remains unclear (Peterson et al., 2013), it is evident that the level of 25[OH]D₃ is lower in PD patients than in healthy subjects (Gezen-Ak et al., 2017). In our study population, no association was found between ApaI, TaqI, or FokI polymorphisms and PD. The four SNPs were not associated with the age of onset of PD nor with the occurrence of symptoms, such as tremors, akinetic rigidity, or mixed symptoms.

Notably, 50% of patients in the early-onset PD group presented tremor, while only 34% exhibited tremor in the late-onset PD group. Interestingly, lower levels of 25[OH]D₃ have been reported in young PD patients with tremor symptoms compared to those with akinetic rigidity (Gezen-Ak et al., 2017). Unfortunately, our study did not measure vitamin D levels in patients' serum to establish an association with the four genetic variants of VDR gene. Gender differences in nonmotor symptoms, such as fatigue, depression, and sleep disorders, have been observed in PD patients (Haaxma et al., 2007). As previously reported (Haaxma et al., 2007), 64% of women experienced tremor, while only 31% of men did. Unlike to this report, the age of onset in females (57.35 years) was younger than in males (60.97 years). This age difference becomes particularly significant in the early-onset group, with women averaging 42.86 years and men 51.31 years.

In contrast to the other SNPs, BsmI was significantly associated with PD in this Spanish cohort (Table 3). This association was further strengthened when BsmI was combined with FokI. Our results suggest that the G allele is protective, with GGCT being the most frequently observed in the control group. In our study, the distribution of VDR BsmI genotypes in HC (AA 6.25%, AG 31.25%, GG 62.5%) was quite similar to that observed in healthy Korean (AA 1.3%, AG 26%, GG 72.7%). Surprisingly, the GG genotype was considered as a risk factor for PD in Korean population (Kim et al., 2005). However, in the Spanish population, the AG genotype for BsmI had the highest frequency. Therefore, the A allele was considered a genetic risk for PD and may double the incidence in men. Furthermore, the A allele was associated with an increased risk of vitamin D deficiency (Rahmadhani et al., 2017), which sustains the association of BsmI polymorphism with PD development.

Conclusions

BsmI genotypes and the A allele were significantly associated with PD, although not with the age of disease onset. In our study, the average age of Parkinson's patients is 60 years. Remarkably, two-thirds of the youngest patients, who presented with tremors, being women. This could be attributed to the fact that women were younger than men in this study. However, SNPs were not significantly associated with PD symptoms or gender. It is important to note that while the BsmI A allele increases the incidence of PD in men, the G allele appears to be protective.