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ORIGINAL ARTICLE
Year : 2022 | Volume
: 42
| Issue : 1 | Page : 60-67
Analyses of serum 25-hydroxyvitamin D levels and single nucleotide polymorphisms in the vitamin D receptor gene for acne vulgaris: a case–control study
Mohammed Hassan M1, Olfat G Shaker2, Talal A Abd el-Raheem1, Sara M Yasen1
1 Department of Dermatology, Faculty of Medicine, Fayoum University, Fayoum, Egypt
2 Department of Biochemistry and Molecular Biology, Faculty of Medicine, Cairo University, Giza, Egypt
Correspondence Address:
Olfat G Shaker
Professor of Biochemistry and Molecular Biology, Faculty of Medicine, Cairo University, Kasr AL Ainy Street 11562, Giza
Egypt
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ejdv.ejdv_14_21
Background Acne vulgaris (AV) is one of the diseases that are widespread in Egypt. It is a disease that mainly affects the pilosebaceous units of the face, neck, and others.
Aim To analyze and assess the role of 25-hydroxyvitamin D levels in serum as well as different vitamin D receptor (VDR) genes (Taq-1, Apa-1, Fok-1, and Cdx-2 polymorphisms) among Egyptian patients having AV.
Patients and methods A total of 300 patients with acne and another 300 healthy controls were included in the study. Patients were subdivided into four different groups: group 1 received cholecalciferol 8000 IU/day for 3 months; group 2 applied 1–2 g of topical vitamin D analog for 3 months, group 3 had no treatment, and lastly, group 4 included healthy controls. For all patients, skin manifestations were assessed. Serum 25(OH)3 D was measured in all participants before and after treatment. Different VDR gene polymorphisms were measured.
Results Patients with acne showed a statistically significant decrease in serum 25(OH) D3 concentration than controls, with P value of 0.001. Regarding VDR polymorphisms (rs731236 for Taq-1, rs7975232 for Apa-1, rs2228570 for Fok-1, and rs11568820 Cdx-2 polymorphisms), the results revealed that there was a statistically significant difference between patients with acne and controls regarding Taq-1 and Apa-1 genotypes, with P values of 0.001 and 0.026, respectively, whereas regarding Fok-1 and Cdx-2 genotypes, the results revealed that there were no statistically significant differences between the studied patients and the controls (P=0.690 and 0.113, respectively).
Conclusion Vitamin D and VDR polymorphisms (Taq-1, Apa-1, Fok-1, and Cdx-2) play important roles in AV.
Keywords: acne vulgaris, vitamin D receptor polymorphism, vitamin D
Acne is a very common skin disease that affects teenagers, with a prevalence of 35–90%, and it is also reported that it leads to severe physical and psychological stress [1],[2]. In Egypt, acne vulgaris (AV) is one of the diseases that are widespread in young males and females [3].
Vitamin D (VD) and vitamin D receptor (VDR) polymorphisms play important roles in the pathogenesis of AV, which when detected early can be considered in the treatment of the disease [4],[5],[6],[7].
Genetic alterations in VDR can occur owing to the many effects mediated by 1, 25 (OH)2 D, which lead to defects in gene induction, affecting calcium metabolism and immune function [8],[9].
AimThe aim was to assess the role of VD in the treatment of AV and to study its relation to VDR Apa-1, Taq-1, Fok-1, and Cdx-2 polymorphisms.
Patients and methodsA total of 300 patients with AV were included in the study, fulfilling the rules of the Declaration of Helsinki 1975. This study was an interventional randomized control trial. It was conducted at the Dermatology Outpatient Clinic, Fayoum University Hospitals, Egypt, between December 2018 and June 2020. Ethical committee approval was taken from Fayoum University as well as written consent from each participant before the start of the study.
Moreover, 300 healthy controls were also included. Full history taking and essential investigations were performed on all participants.
The sample was calculated by using the computer software SPSS (Chicago, Illinois: SPSS Inc.) ‘random sample of cases’ using equation (1). The calculated prevalence of AV was 54.2% of the total number of all reported population, and the power of the test was 70% at 95% confidence interval. The sample size was calculated to be 300 cases [10].
Exclusion criteria of the study included patients with dermatologic diseases other than AV, pregnant and lactating women, patients with history of drugs that cause acneiform eruptions, patients taking systemic steroids, calcium, patients taking bisphosphonates, patients taking immunosuppressive agents, patients with any hormonal disturbance (e.g. polycystic ovary syndrome), patients with sarcoidosis, patients with renal disease, patients with malignancy, patients who received treatment for acne within the past 6 months, and patients receiving or had received phototherapy during the past one year. Global Acne Grading System (GAGS) was used to assess the severity of acne [8],[9].
Patient groups were classified according to the treatment they received. Group 1 (100 patients) was treated with systemic VD supplementation as cholecalciferol 6000–8000 IU/day for 3 months (Devarol, Memphis, Tennessee, USA). Group 2 (100 patients) was treated with topical VD analog, 1–2 g once/night for 3 months (Calcipoheal; APIC, Baltimore, MD, USA). Group 3 (100 patients) was not subjected to any treatment. There were no adverse effects for the treatment as there was no hypervitaminosis for VD administration.
From all participants, 3-ml peripheral blood sample was withdrawn from each participant by venipuncture in a dry sterile vacutainer tube which was taken in a plain tube for serum separation that was used in detecting all markers for AV and for measurement of 25(OH)3 D levels. Besides, 2-ml whole blood was taken in an EDTA tube for DNA extraction, and then genotyping of the studied SNP (rs731236 for Taq-1, rs7975232 for Apa-1, rs2228570 for Fok-1, and rs11568820 Cdx-2) polymorphisms was done using real-time PCR.
Genotyping of Taq-1 (rs731236), Apa-1 (rs7975232), Fok-1 (rs2228570), and Cdx-2 (rs11568820)
Predesigned primer/probe sets for the four genotypes were used (Applied Biosystems, Waltham, Massachusetts, USA). Probes were synthesized with reporter dye FAM or VIC covalently linked at the 5/end and a quencher dye MGB linked to the 3/end of the probe (Applied Biosystems).
Total serum 25-hydroxyvitamin D (vitamin D) ELISA kit was provided by Sunred Biocompany (Shanghai, China).
Human 25(OH) D level in the sample was measured as follows: purified human 25(OH) D antibody was coated to microtiter plate wells, making solid-phase antibody, and then 25(OH) D was added to wells and combined with 25(OH)D-antibody with labeled enzyme. Then, after complete washing, a substrate was added. Reaction was terminated by the addition of a sulfuric acid solution, and the color change was measured spectrophotometrically at a wavelength of 450 nm. The concentration of 25(OH) D in the samples was then determined.
The standard serum values of 25(OH) D are interpreted as follows [8]: severe deficiency: 0–10 ng/ml, deficiency: 10–20 ng/ml, suboptimal: 20–30 ng/ml, optimal: 30–50 ng/ml, high: 50–100 ng/ml, and toxic: more than 100 ng/ml.
Statistical analysis
Data analysis was performed using the statistical package for the social science (SPSS 17.0), Windows 8.1. For quantitative parametric data, an independent Student t test was used to compare measures of two independent groups. Moreover, one-way analysis of variance test was used for comparing more than two-independent groups with Bonferroni post-hoc to test significance at P value less than 0.05. However, for quantitative nonparametric data, the Kruskal–Wallis test and Mann–Whitney test were used to compare more than two-independent groups. For measuring the correlation between qualitative data, bivariate Pearson correlation test was used to find out the association between different groups with a two-tailed to test the significance. Sensitivity and specificity tests were generated for testing a new test with receiver operating characteristic curve). P value less than 0.05 was considered as a cutoff value for significance.
ResultsThere were 300 patients with AV, with a mean age of 21.10±3.93 years. Overall, 21.6% of the patients were males, whereas the rest were females. The study included 300 healthy controls, with a mean age of 23.15±7.56 years. Overall, 30% of the healthy controls were males, whereas 70% were females. The results showed that there was no statistically significant difference between the two groups with respect to age and sex, with P values of 0.070 and 0.448, respectively.
The 300 patients with AV were subdivided into three groups:
Group 1 (100 patients) was treated with systemic VD supplementation.Group 2 (100 patients) was treated with topical VD analog.Group 3 (100 patients) did not receive any treatment.The study flow diagram is illustrated in [Figure 1]. First, 25-hydroxyvitamin D (25 (OH)2 D) levels in serum were measured for all patients before and after treatment. On analyzing the patients before treaments, the results revealed that there was a statistically significant decrease in VD levels for group 1, with a mean of 10.34±3.8, compared with the control group (26.25±4.5), with P value of 0.001. There was also a statistically significant decrease in the VD level in serum among group 2, with a P value of 0.005 (8.19±2.5). Regarding group 3, the level of VD also showed a significant decrease, with a P value of 0.001 (10.59±6.3) ([Figure 2]).
Figure 2 Vitamin D level in serum before treatment among Egyptian groups.After patient groups were given treatments, we started to compared the levels of VD before treatments to analyze the change in the levels. The results showed that in both groups that underwent tratements, the level of VD in serum increased (14.57±4.3 and 10.93±3.0 for groups 1 and 2, respectively), showing a statistically significant difference, with P value of 0.004.
For all groups, the acne score was calculated to detect the severity of the disease where global score was used ([Table 1]).
According to the treatment and after follow-up, the results revealed that the disease global score improved. We repeated the VD test one more time 2 weeks after the experiment, and it was reported that the level of VD increased in the serum.
Based on the severity of the disease and patients undertaking treatment, there was a huge change in the severity of the disease after treatment for groups 1 and 2, respectively. For group 1, 5% of the patients changed from severe to moderate acne, whereas other 2.5% changed from moderate to mild. As for group 2, almost 20% of the patients score changed to mild acne after treatment.
According to the skin type, all patients in this study are either skin type 3 or 4.71% of all studies were classified to skin type 3, whereas the rest are skin type 4.
Then, we started to analyze different VDR genes among the patient groups and compared them with controls.
The results revealed that there was a statistically significant difference between patients with acne and controls regarding Taq-1 and Apa-1 genotypes, with P values of 0.001 and 0.026, respectively ([Table 2] and [Table 3]).
Table 2 Taq-1 genotype and allele frequency analyses among the studied groups
Table 3 Apa-1 genotype and alleles frequency analyses among the studied groupsHowever, regarding Fok-1 and Cdx-2 genotypes, the results revealed that there was no statistically significant difference between the studied patients and the controls, with P values of 0.690 and 0.113, respectively ([Table 4] and [Table 5]).
Table 4 Fok-1 genotype and allele frequency analysis among the studied groups
Table 5 Cdx-2 genotype and allele frequency analyses among the studied groupsWhen comparing the results of genotypes with respect to the acne scores, the severity of the disease, and baseline serum 25(OH) D3 concentration in the patient group, the results revealed the following:
Taq-1 genotypes
A total of 30 patients with AA genotype had serum 25(OH) D3 concentration between 6.98 and 35.87 (mean±SD: 22.58±8.41), 255 patients with AG genotype had serum 25(OH) D3 concentration between 4.66 and 33.56 (mean±SD: 11.91±7.08), and 45 patients with GG genotype had serum 25(OH) D3 concentration between 5.72 and 12.41 (mean±SD: 7.72±1.90). There was a highly statistically significant difference, with P value less than 0.001, between Taq-1 genotypes regarding baseline serum 25(OH) D3 concentration in the patient group.
Apa-1 genotypes
Among patients with severe acne, five (4.8%) patients had AC genotype and five (33.3%) patients had CC genotype. Among patients with moderate acne, 55 (30.6%) patients had AA genotype and 20 (19%) patients had AC genotype.
Among patients with mild acne, 125 (69.4%) patients had AA genotype, 80 (76.2%) patients had AC genotype, and 10 (66.7%) patients had CC genotype. There was a statistically significant difference, with P value less than 0.05, between Apa-1 genotypes regarding disease severity in the patient group. There was no statistically significant difference between Apa-1 genotypes regarding baseline serum 25(OH) D3 concentration in the patient group, where P value was more than 0.05.
Cdx-2 genotypes
A total of 85 patients with CC genotype had baseline GAGS between 4 and 24 (median: 10), 200 patients with CT genotype had baseline GAGS between 6 and 34 (median: 15), and 15 patients with TT genotype had baseline GAGS between 12 and 20 (median: 20). There was a statistically significant difference, with P value of 0.013, between Cdx-2 genotypes regarding baseline severity of the disease in the patient group. There was no statistically significant difference between Cdx-2 genotypes regarding baseline serum 25(OH) D3 concentration in the patient group, where P value was 0.326.
Finally, we compared the concentration level of baseline serum 25(OH) D3 among the studied groups ([Table 6]).
Table 6 Comparison between both patient and control groups regarding baseline serum 25(OH) D3 concentrationReceiver operating characteristic curve analysis for serum 25(OH) D3 concentration
The receiver operating characteristic curve analysis of serum 25(OH) D3 concentration in patients with AV is determined in [Table 7]. The calculated sensitivity, specificity, and diagnostic accuracies for the studied parameters to discriminate patients with AV from healthy controls are shown in [Table 7]. For discrimination of acne group versus control group, regarding 25(OH) D3 concentration in serum, the area under the curve was 0.92, with a sensitivity of 95% and specificity of 98.8% ([Figure 3]).
Figure 3 ROC curve of serum 25(OH) D3 concentration of the acne vulgaris patient group. ROC, receiver operating characteristic.In the study, there were no adverse effects for the patients who were treated with systemic VD supplementation, whereas for the other group for patients who were treated with topical VD analog, 30% of the patients had erythema and 60% got burning sensations.
The limitation of the current study was that the patients are recruited from only one center; multicenter plans can be included in future studies. VDR can be measured as well by ELISA to indicate the association with the results. Other genotypes that are related to AV can be measured as well.
DiscussionIn general, according to the WHO, AV is considered as the most common skin disease, with a prevalence of 35–90% in teenagers. It was reported as well in 2017 that the disease has significant physical and psychological effects and was classified as a chronic disease [1],[2],[11].
To our knowledge, the present study is unique to evaluate the most commonly studied four different combinations of VDR polymorphisms: rs731236 for Taq-1, rs7975232 for Apa-1, rs2228570 for Fok-1, and rs11568820 Cdx-2 polymorphisms in patients with acne. It is also the first study to assess the effect of VD supplementations on the treatment of AV.
In this study, A-allele and AA genotype of Taq-1 polymorphism can be considered protective against acne development owing to their higher frequencies in controls than the patients. Despite the fact that Taq-I polymorphisms appear to be nonfunctional, they can be used as a marker for functional allele detection due to the linkage disequilibrium.
Moreover, the mutant C-allele of Apa-1 polymorphism may have a protective role in occurrence of acne owing to the higher frequency of its combined variant in controls than the patients. Although Apa-1 polymorphism appears to be ineffective, it can be used as a marker for functional allele detection owing to linkage disequilibrium.
Despite there being no previous studies to compare our results regarding genotyping combined together, in 2012, a Cdx-2 polymorphism was evaluated among patients with acne, which showed that the mutant TT genotype was suggested to be protective against development of acne. A dysfunction of Cdx-2 is a key contributor in the pathogenesis of IBD via alteration in TNF-α levels [12].Taq-1 and Apa-1 polymorphisms included in the study and their relation with AV disease were previously reviewed in 2018, where the genetic variations within the VDR gene that may cause significant VDR dysfunction were explained [13],[14].
Low serum 25(OH) D3 levels have been reported in many other inflammatory diseases such as systemic lupus erythematosus, cardiovascular disease, rheumatoid arthritis, multiple sclerosis, metabolic disorders, inflammatory bowel disease, and psoriasis as well [14],[15].
As for the baseline serum 25(OH) D3 concentration, our results agree with Swelam et al. [13], where there was a statistically significant negative correlation between acne score and baseline serum 25(OH) D3 concentration in patients with acne.
Moreover, in agreement with our study, a study conducted in 2020 reported that VD levels in patients with acne were significantly low as compared with the controls [16].
ConclusionVD and its derivatives play their biological roles through the VDR. It can be concluded that low serum 25(OH) D3 levels in AV can be one of the biomarkers for the detection of the disease, which in return are involved in its pathogenesis. It can also be concluded that VDR polymorphisms (rs731236 for Taq-1, rs7975232 for Apa-1, rs2228570 for Fok-1, and rs11568820 Cdx-2) can be used as effective biomarkers for early detection and later on treatment of AV among Egyptian patients.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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