The CYP24A1 gene variant rs2762943 is associated with low serum 1,25‐dihydroxyvitamin D levels in multiple sclerosis patients

Vitamin D is considered to play a role in multiple sclerosis (MS) etiopathogenesis. A polymorphism in the CYP24A1 gene, rs2762943, was recently identified that was associated with an increased MS risk. CYP24A1 encodes a protein involved in the catabolism of the active form of vitamin D. The immunological effects of carrying the rs2762943 risk allele were investigated, as well as its role as genetic modifier.


INTRODUC TI ON
Multiple sclerosis (MS) is a complex immune-mediated disorder of the central nervous system (CNS) wherein both a polygenic background and environmental factors contribute not only to MS risk but also to disease activity [1][2][3][4][5][6]. One of the environmental factors involved in MS etiopathogenesis is vitamin D status. In recent years, numerous studies have revealed that lower serum 25-hydroxyvitamin D (25OHD) levels are associated with an increased risk for MS [4,5].
Furthermore, vitamin D status is also associated with MS disease activity, and patients with lower serum 25OHD levels were shown to have higher disease activity, although findings seem to be stronger for radiological rather than clinical outcomes [7]. Similarly, vitamin D supplementation in relapsing-remitting MS (RRMS) patients significantly improved the development of new magnetic resonance imaging (MRI) lesions, although the study was negative on the proportion of patients with no evidence of disease activity (NEDA-3), which was the primary outcome [8].
In a recent study, by means of targeted resequencing in 524 MS patients and 546 healthy controls and subsequent genotyping in an independent cohort of 3450 MS patients and 1688 healthy controls, a variant located in the cytochrome P450 family 24 subfamily A member 1 (CYP24A1) gene, rs2762943, was identified that was associated with an increased risk for MS [9]. The CYP24A1 gene encodes a protein that initiates the degradation of the physiologically active

Quantification of serum levels of 25OHD, 1,25(OH) 2 D and calcium by chemiluminescence immunoassays
A schematic flowchart summarizing the main steps performed in the study design is represented in Figure 1.  Table 1 shows a summary of demographic and main clinical characteristics of these patients.
Briefly, peripheral blood was collected by standard venipuncture and allowed to clot spontaneously for 30 min. Serum was isolated by centrifugation and stored frozen at −80°C until used. Serum levels of F I G U R E 1 Flowchart showing the study design. The effect of the rs2762943 polymorphism located in the CYP24A1 gene was investigated in a cohort of 340 multiple sclerosis (MS) patients. In a subcohort of 167 patients with available serum samples, levels of 25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D (1,25(OH) 2 D) and calcium were measured by chemiluminescence immunoassays. The immunological effects of the rs2762943 polymorphism were investigated in three subgroups of MS patients with blood samples collected at the same time as the serum samples used to measure vitamin D levels: (i) immunophenotyping was performed by flow cytometry in peripheral blood mononuclear cells (PBMCs) from 15 MS patients to determine the expression of HLA class II and costimulatory molecules; (ii) levels of pro-inflammatory and anti-inflammatory cytokines were measured by Simoa (single-molecule array) assays in serum samples from 20 MS patients; (iii) levels of neurofilament light chain (NfL) were quantified by Simoa in serum samples from 49 MS patients. Finally, the role of rs2762943 as genetic modifier was investigated in the full cohort of 340 MS patients by analyzing the association between the polymorphism and the Expanded Disability Status Scale (EDSS), Multiple Sclerosis Severity Score (MSSS), annualized relapse rate (ARR) and age at disease onset.

Immunophenotyping of peripheral blood cells and flow cytometric analysis
Immunophenotyping was performed in peripheral blood mononuclear cells (PBMCs) from a subgroup of 15 MS patients, six risk allele carriers for rs2762943 and nine non-carriers, that were collected at the same time as the serum samples used to measure vitamin D levels ( Table S1)

Quantification of serum biomarker levels by singlemolecule array assays
Serum levels of C-X-C motif chemokine ligand 13 (CXCL13), interferon gamma (IFNG), interleukin 10 (IL-10) and granulocyte macrophage colony-stimulating factor (GM-CSF) were measured in 20 MS patients, 10 risk allele carriers for the rs2762943 polymorphism and 10 non-carriers (Table S1). Serum neurofilament light chain (NfL) levels were measured in 49 MS patients, 26 risk allele carriers and 23 non-carriers (Table S1). These analytes were chosen based on their reported association with MS pathology and disease activity [12][13][14][15]. Biomarker levels were quantified in the same serum samples used to determine the vitamin D status using commercially available immunoassay kits (CXCL13 cat#102635, IFNG cat#103337, IL-10 cat#101643, GM-CSF cat#102329 and NFL cat#103186; Quanterix) and run on the fully automated ultrasensitive Simoa HD-1 Analyzer (Quanterix). Samples were run in duplicate in accordance with manufacturers' instructions with appropriate standards and internal controls. Both the mean intra-assay coefficient of variation of duplicates and the mean inter-assay coefficient of variation were <11% for all assays.

CYP24A1 rs2762943 polymorphism and disease activity and disability progression measures
The role of rs2762943 as genetic modifier of MS was evaluated in the full cohort of 340 patients. Of these, 72 (21.2%) patients were risk allele carriers and 268 (78.8%) were non-carriers ( Table 2).

CYP24A1 rs2762943 polymorphism is associated with MS and vitamin D levels
The most recent large-scale genetic study of MS susceptibility was perused to test the association of the CYP24A1 rs2762943 gene variant [3]. The variant had an odds ratio (OR) of 1.47 for the minor allele T that reached genome-wide level of statistical significance at the study's discovery phase (p = 3.9 × 10 −8 ; Table S2).
rs2762943 was located underneath the association peak of a nearby variant also located in the CYP24A1 gene, rs2248137 (OR = 1.12; p = 7.8 × 10 −11 ; Figure S1). After controlling for the top variant, association of rs2762943 with MS remained nominally significant (OR = 1.28; p = 1.2 × 10 −3 ; Table S2). The two variants have a large allele frequency difference, but the two risk alleles are part of the same haplotype (D′ = 0.9713; Table S3), suggesting a putative common contribution to MS susceptibility. These results confirm the association of the CYP24A1 rs2762943 polymorphism identified in our previous targeted resequencing study [9] with MS risk.
Next, the Genome-Wide Association Study Catalog was searched to identify any association of the CYP24A1 rs2762943 polymorphism with other phenotypes and traits [17]. Genome-wide associations with metabolites and metabolite biomarkers (p < 5 × 10 −8 ) were observed, including vitamin D levels (Table S4), altogether suggesting that the variant is interrupting key metabolic mechanisms.

Expression of the MHC class II and co-stimulatory markers in blood cell populations is similar between risk allele carriers and non-carriers
Whether risk allele carriers for the rs2762943 polymorphism differed from non-carriers in terms of steady state myeloid and lymphoid cell activation profiles was next investigated. For this, the expression levels of MHC class II and co-stimulatory molecules on classical monocytes and B cells were assessed. As shown in Figure 3, the presence of the risk allele was not associated with changes in the expression levels for HLA-DR, CD40, CD80 and CD86. Similarly, in T cells, the risk allele was not associated with changes in the expression levels of HLA-DR.

Serum levels of the pro-inflammatory cytokines IFNG and GM-CSF are increased in risk allele carriers
As a next step, it was evaluated whether the presence of the risk allele for the rs2762943 polymorphism was associated with changes in the levels

Presence of the CYP24A1 rs2762943 risk allele has no impact on MS disease activity and disability progression measures but influences age at disease onset
Main demographic and clinical characteristics such as sex, MS phenotype, disease duration and percentage of patients who received DMT at any time during follow-up were comparable between risk allele carriers and non-carriers ( Table 2).
When the rs2762943 polymorphism was evaluated as genetic modifier of MS, it was observed that risk allele carriers were younger at disease onset compared to non-carriers (p = 0.04; Table 3).
Comparison of disability measures such as EDSS and MSSS both at 10 years from disease onset and at last follow-up did not reveal significant differences between risk allele carriers and non-carriers (Table 3). Similarly, ARR at 10 years from disease onset was comparable between the two groups of patients ( Table 3). Further stratification of the whole MS group into disease phenotypes (RR, SP and primary progressive) was not associated with significant differences for age at disease onset, inflammatory disease activity and disability measures between risk allele carriers and non-carriers (Table S5).
Based on the findings of increased levels of pro-inflammatory cytokines in risk allele carriers, and in view of the strong associations reported in the literature between NfL and radiological measures of disease activity such as the number of contrast-enhancing lesions and number of T2 lesions [19], serum levels of NfL were also measured in a subgroup of MS patients as a proxy of CNS radiological inflammation. Figure 4b shows the distribution of serum NfL levels in risk allele carriers and non-carriers, which were comparable amongst the two groups of patients.

DISCUSS ION
In the present study, the functional immunological consequences of carrying the risk allele for the rs2762943 polymorphism in the [17], was found to be associated with an increased risk for MS in a recent targeted resequencing study carried out by our group [9] and also in the largest genome-wide association study up to now conducted in MS [2]. Interestingly, rs2762943 remains as an independent signal for association with MS and is linked to vitamin D related traits [17].  [20,21].
In consequence, CYP24A1 limits the amount of the active form of vitamin D in target tissues by speeding up its catabolism and also by reducing the pool of 25OHD available for 1α-hydroxylation in the kidney [20,21]. Although it can hydroxylate both, 1,25(OH) 2 D is the preferred substrate for CYP24A1 [22].
Several studies have pointed to a role of CYP24A1 in MS.
Carriers of the risk minor allele for the CYP24A1 rs2248137 polymorphism were found to have significantly lower serum levels of 25OHD [23], and also risk allele carriers for another CYP24A1 polymorphism, rs2248359, had increased expression of CYP24A1 in frontal and temporal cortex but not in subcortical white matter from brain tissue of individuals without neurological history [24].
In a recent study, CYP24A1 expression was significantly increased in PBMCs from controls and MS patients after the addition of 25OHD in basal conditions, and also in PBMCs from MS patients after culturing cells with a number of inflammatory stimuli [25]. In a second part of the study, it was evaluated whether the presence of the risk allele for the rs2762943 polymorphism could act as a genetic modifier in MS. Low vitamin D is considered a moderate risk factor for MS susceptibility based on a number of observational studies demonstrating an association between low serum 25OHD levels and increased MS risk [28][29][30]. The role of vitamin D influencing disease prognosis in MS is more controversial. Higher 25OHD levels have been associated with lower relapse risk [31][32][33], lower risk of subsequent development of new T2 lesions and contrastenhancing lesions on brain MRI [21] and lower change in EDSS scores [16]. However, other studies have not reported an association of vitamin D levels with MS disease activity [7,34]. Furthermore, a number of randomized clinical trials indicate that vitamin D supplementation does not seem to have beneficial effects on ARR or EDSS scores in MS patients [35][36][37]. In our study, the presence of the risk allele for rs2762943 in MS patients had no effect on disease course, since it was not associated with increased inflammatory disease activity evaluated by the ARR or higher risk of disability progression indicating that the CYP24A1 rs2762943 gene variant seems to have more impact on MS susceptibility than on disease prognosis.

CON CLUS ION
Altogether, these findings indicate that the risk allele for the rs2762943 polymorphism in the CYP24A1 gene is associated with