Sequence analysis of four vitamin D family genes (VDR, CYP24A1, CYP27B1 and CYP2R1) in Vogt-Koyanagi-Harada (VKH) patients: identification of a potentially pathogenic variant in CYP2R1

Background VKH is a rare autoimmune disease. Decreased level of vitamin D has recently been found to be involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. This study was designed to screen the vitamin D pathway genes for pathogenic mutations, if any, in VKH patients. Methods Genomic DNA was extracted from blood samples collected from patients with VKH disease and healthy controls. Entire coding region, exon-intron junctions of four genes were sequenced in DNA from 39 Saudi VKH patients and 50 ethnically matched healthy individuals. All patients and controls were unrelated. Results Vitamin D levels in VKH patients were found either insufficient (21–29 ng/mL) or deficient (<20 ng/mL). Sequencing analysis of the VDR, CYP24A1, CYP27B1 and CYP2R1 detected twelve nucleotide changes in these genes in our cohort of 39 patients; 4 of which were non-coding, 6 were synonymous coding and 2 were non-synonymous coding sequence changes. All synonymous coding variants were benign polymorphisms with no apparent clinical significance. A non-synonymous coding sequence variant (c.2 T > C; p.1Met?) found in VDR is an initiation coding change and was detected in control individuals as well, while another variant (c.852G > A; p.284 M > I) found in CYP2R1 is predicted to be disease causing by mutationtaster software. This potentially pathogenic variant was found in 17 out of 39 VKH patients. Conclusions Screening of four Vitamin D pathway genes in 39 VKH patients shows that a potentially pathogenic sequence variant in CYP2R1 may cause VKH in a subset of patients. These findings support the previous observation that low vitamin D levels might play a role in VKH pathogenesis and mutations in genes involved in vitamin D anabolism and catabolism might be of importance in VKH pathobiology.


Background
Vogt-Koyanagi-Harada (VKH) syndrome is a rare multisystem autoimmune disease that affects melanin containing tissues, including the eye, inner ear, meninges and skin. The disease is characterized by bilateral uveitis associated with a varying degree of auditory, neurological and cutaneous manifestations [1][2][3]. VKH affects more frequently people with darker skin pigmentation. Asians, Native Americans, Middle Easterners and Hispanics are most frequently affected [4]. It predominates in patients aged between 20 and 50 years with a female:male ratio of 2:1.
The classic clinical course is characterized by severe bilateral granulomatous panuveitis, hypoacusis and meningitis in addition to cutaneous involvement with poliosis, vitiligo and alopecia. If not treated appropriately it results in severely decreased vision or even leads to blindness [2,5,6]. Although the exact cause of VKH disease remains unclear, studies have shown that an autoimmune response directed against melanocytes plays a major role in the onset of this disease [7,8]. Although a number of HLA and non-HLA genes have been shown to be associated with VKH [9][10][11][12] the genetic basis of VKH still remain illusive. Therefore, further studies on the association of autoimmune modulatory genes may yield informative data for the genetic background of VKH disease.
Vitamin D is produced in the skin or obtained from the diet [13]. Its receptor has been found in the immune cells, and some immune cells are able to produce Vitamin D3 [14][15][16][17]. The biologically active metabolite of Vitamin D3, 1,25(OH) 2 D 3 , has been shown to have immunomodulatory action alongside its role in the bone and calcium metabolism [18,19]. Vitamin D receptor (VDR) gene negative mice showed a significantly increased susceptibility to several autoimmune diseases, such as autoimmune encephalomyelitis [20,21], autoimmune uveitis [22] and allergic asthma [23]. Moreover, multiple studies found decreased levels of serum Vitamin D in several human autoimmune diseases, such as multiple sclerosis [24][25][26], rheumatoid arthritis [27,28], Behçet's disease [29], Graves disease [30] and systemic lupus erythematosus [31]. It has been reported that decreased 1,25(OH) 2 D 3 level may play a role in the development of VKH disease [32]. Vitamin D deficiency, compromising the immunoregulatory action leading to the autoimmune diseases like VKH could result from either environmental factors or defect in genes concern with Vitamin D metabolism pathway or both. We, therefore, entertained the hypothesis that genetic variation in the Vitamin D genes could be associated with VKH disease.
In Saudi Arabia, VKH is a common cause of uveitis [33] but no study has been conducted to explore the role of vitamin D pathway genes in VKH pathogenesis. In the present study, we screened Vitamin D metabolism pathway genes (VDR, CYP24A1, CYP27B1 and CYP2R1) to examine the possible involvement of variation in these genes with VKH disease in Saudi patients. We identified a novel missense variant in CYP2R1 in VKH patients which might be responsible for low vitamin D level in these patients. Overall, our results showed that a variation/polymorphisms in Vitamin D pathway genes tested here are not responsible for VKH in Saudi population. However, we detected a variant in CYP2R1 gene that may be pathogenic for VKH disease.

Subjects
All subjects were recruited from Magrabi Hospital Almadinah Almunawarah. We collected 39 VKH patients and 50 control individuals for this study. All affected and control individuals signed informed written consent prior to start of the study. In case of minor, consent was taken from parents. All patients were examined clinically by a senior ophthalmologist and diagnosed as VKH. Revised diagnostic criteria has been used for VKH diagnosis [3]. Systemic observations for vertigo, poliosis and alopecia, vitiligo, hearing impairment and tinnitus were recorded for all VKH patient. Ethical approval for the study was obtained from the IRB of the Center for Genetics and Inherited Diseases (CGID), Taibah University Almadinah Almunawarah. All experimental procedures were conducted in accordance with the tenets of the Declaration of Helsinki.
All patients were diagnosed using slit lamp biomicroscopy while cornea was found clear. Fundus examination was carried out using indirect ophthalmoscope and a 20× diopter aspheric lens.

Blood collection and gDNA extraction
In this study, we screened 39 VKH patients and 50 controls for 4 vitamin D pathway genes. Peripheral blood samples of 6 ml was collected from each of the patients and the controls in EDTA tubes. Extraction of genomic DNA was performed using Qiagen blood mini kit. DNA was quantified using Maestro spectrophotometer.

Vitamin D measurement
Serum levels of 25-hydroxyvitamin D3 (25OHD3) were measured in all 39 VKH patients and 50 controls by radioimmunoassay using the Wallac 1470 Gamma Counter (Wallac Inc, Gaithersburg, MD, USA). 25OHD3 level of >30 ng/mL was considered normal. Vitamin D deficiency was defined as a serum level of 25OHD of ≤20 ng/mL and insufficiency as a serum level between 21-29 ng/mL.

Sequencing of vitamin D pathway genes
gDNA was diluted to 20 ng/ul concentration and PCR amplification of coding regions of all four genes was performed using primers flanking exons following a protocol used earlier [34]. Primer sequences are available on request. Bidirectional sequencing of all fragments was carried out using BigDye (Applied Biosystems, Foster city, CA) chain termination chemistry. Fragments were then separated on AB 3500 genetic analyzer (Life Technologies). All sequenced fragments were analyzed using BioEdit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and compared to the reference sequences of corresponding genes from UCSC genome browser (http://genome.ucsc.edu/cgibin/hgGateway). In house controls were used and it was ensured that controls are healthy individuals without having any ocular disease(s) or previous ophthalmic surgeries.

Clinical description of subjects
In this study, 39 unrelated VKH patients (Table 1)  Recurrence was observed in four cases.
All patients and controls were screened for serum vitamin D levels. Most of the patients were found Vitamin D deficient (Table 1). Among 50 controls, 12 were found Vitamin D deficient, 23 were Vitamin D insufficient and 15 showed Vitamin D levels of more than 30 ng/mL.

Mutation detection
The full coding region, exon-intron junctions and the 5′ and 3′UTRs of VDR, CYP24A1, CYP27B1 and CYP2R1 were sequenced in all 39 patients. Controls were screened only for the variants detected in patients. We detected twelve nucleotide changes in both patients and controls (Table 1). Of all these, 4 were non-coding (g.64978G > T in VDR, g.2989C > T in CYP27B1, g.821C> T and g.15916 T > C in CYP24A1), 6 were synonymous coding p.284 M > I) in CYP2R1 is predicted to be a disease causing by MutationTaster software (Fig. 1). Multiple sequence alignment shows that the amino acid methionine at position p.284 M is evolutionarily conserved (Fig. 2).

Discussion
It is known that vitamin D plays an important role in melanin production and its deficiency has been shown to be associated with skin depigmentation [35]. In VKH patients, melanocytes tends to disappear from the outer Recently polymorphisms in Vitamin D receptor (VDR) and 7-dehydrocholesterol reductase (DHCR7) genes have been associated with Behçet's disease [36][37][38].
VDR encodes vitamin D receptor which shows high binding affinity for vitamin D3. Vitamin D3 binding activates VDR and ligand-activated VDR performs its function of gene expression by chromatin modification and the transcription regulation. DHCR7 coverts 7-dehydrocholesterol to cholesterol and, thus, reduces the substrate for vitamin D3 synthesis [47]. DHCR7 mutations have been shown to control vitamin D levels in serum [48,49]. Similarly, low vitamin D serum level has been associated with VKH disease. Both Behçet's disease and VKH manifest intraocular inflammation (Uveitis) which strengthens the hypothesis that variations in vitamin D pathway genes may cause VKH as well. The current study is based on this hypothesis and hence, we screened four vitamin D family genes (VDR, CYP24A1, CYP27B1 and CYP2R1) in 39 VKH patients to detect possible mutations underlying VKH in Saudi population. We identified various population polymorphisms in all these genes (Table 1). However, we identified a novel homozygous missense mutation (c.852G > A; p.284 M > I) in CYP2R1 gene in 17 VKH patients (Fig. 1). This variant in not present in the homozygous state in 50 control individuals. This mutation changes a conserved amino acid methionine to Isoleucine. In silico analysis predicted that this mutation is probably pathogenic. I-Mutant software (used for prediction of protein stability upon single point mutation) predicted the mutant protein as less stable or with decreased stability [50]. Moreover, we used DUET (predicting effects of mutations on protein stability via an integrated computational approach), SDM (predicting effects of mutations on protein stability and malfunction) and mCSM (predicting the effect of mutation in protein using graph based signatures) for prediction of effect of mutation of the protein and found that this mutation is indeed destabilizing [51][52][53].
Failure to detect pathogenic variants in other vitamin D genes such as CYP24A1, CYP27B1 and VDR does not rule out the possibility that other relevant vitamin D gene mutations could cause VKH in Saudi patients.
Deep sequencing in a large number of samples would be required to find if any other Vitamin D pathway gene mutations are associated with VKH disease. Also, the non-genetic factors causing Vitamin D deficiency in these patients should be explored.

Conclusions
These findings support the previous observation that low vitamin D levels might play a role in VKH pathogenesis and mutations in genes involved in vitamin D anabolism and catabolism might be of importance in VKH pathobiology. In conclusion, our study for the first time reports a potentially causative role of CYP2R1 mutation in VKH disease. Studies on larger cohort of patients are needed to confirm this observation.