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Association between VNN1 gene polymorphism and the risk for psoriasis

Doss, Reham W.a; Rashed, Laila A.c; Emad El-Din Arafa, Ahmedb

Journal of the Egyptian Women’s Dermatologic Society: May 2018 - Volume 15 - Issue 2 - p 106–110
doi: 10.1097/01.EWX.0000532096.34561.9f
Original articles
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Background Psoriasis is a chronic inflammatory hyperproliferative disease. The vanin gene family [vanin-1 (VNN1), VNN2, VNN3] encodes for pantetheinase enzymes that catalyzes the conversion of pantetheine into pantothenic acid and cysteamine. These metabolites are also major players in inflammatory and oxidative responses.

Objective To detect VNN1 tissue level and VNN1 (T26I) gene polymorphism, and the possible association with psoriasis.

Patients and methods This case control study included 60 patients with chronic plaque psoriasis and 60 age-matched and sex-matched healthy controls. Reverse transcriptase-PCR was used to detect the VNN1 expression and the VNN1 gene (T26I) polymorphism.

Results VNN1 tissue levels were significantly higher in psoriasis patients compared with controls (P<0.001). There was no statistical difference in VNN1 (T62I) TT, TI, II genotype between patients and controls (P>0.05 in all).

Conclusion Although the VNN1 was overexpressed in patients, it is unlikely that this overexpression is a consequence of the studied polymorphism. The high level of VNN1 may contribute to the development of psoriasis.

Departments of aDermatology

bPublic Health, Faculty of Medicine, Beni-Suef University, Beni Suef

cBiochemistry Department, Faculty of Medicine, Cairo University, Cairo, Egypt

Correspondence to Reham W. Doss, Dermatology Department, Faculty of Medicine, Beni-Suef University, Mohammed Hassan Street, Beni Suef 62111, Egypt Tel: +20 82 231 8610; fax: +20 82 231 8605; e-mail: rehamdoss@yahoo.com

Received October 17, 2017

Accepted March 9, 2018

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Introduction

Psoriasis is a chronic inflammatory skin condition affecting 2–3% of the world population 1.

Over the past decade, numerous reports have provided evidence concerning the role of genetic polymorphisms in pathogenesis of psoriasis 2.

The human vanin gene family, which consists of three genes, vanin-1(VNN1), vanin-2(VNN2), and vanin-3(VNN3), is located on chromosome 6q22–24 3. Vanins were identified as enzymes that are involved in the coenzyme A synthesis. Their enzymatic activity catalyzes the conversion of pantetheine into pantothenic acid (vitamin B5, a coenzyme A precursor) and into the antioxidant cysteamine 3. Pantothenic acid and cysteamine have crucial roles in both inflammatory and oxidative responses 4.

Elevated VNN1 levels were detected in autoimmune diseases such as systemic sclerosis and inflammatory bowel disease (IBD) 5,6.

VNN1 and VNN3 were found to be induced by proinflammatory cytokines TH1/TH17, cytokines that are known to be involved in the pathogenesis of psoriasis 7,8.

Pantothenic acid, is also involved in epidermal lipid synthesis 9. Lipids are important for skin barrier function, as they are part of the lamellar bodies formed by keratinocytes in the stratum spinosum 10. Psoriasis is characterized by disturbed skin barrier function 11, so the change of level of pantothenic acid and consequently coenzyme A could contribute to the disturbed skin barrier function noticed in the skin of psoriasis patients.

In this study, we aimed to investigate a hypothesized association between the VNN1 expression levels and the presence of psoriasis.

Among various single-nucleotide polymorphisms (SNPs) identified for the VNN1 gene, we chose to test for the T26I (rs2294757) due to its location in the coding region, previously demonstrated to be functional in-silico due to its location in a splicing regulation site at the coding region of VNN112.

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Patients and methods

Patients

In this case control study, 60 patients with psoriasis vulgaris (30 males and 30 females) and a similar number of age, BMI, sex matched controls were included. All the patients were recruited from the Dermatology Outpatient Clinic, Beni-Suef University Hospital. A written informed consent was taken before data collection. Patient confidentiality was maintained, and all personal data were concealed. The study was approved by the local research Ethics Committee of the Faculty of Medicine, Beni-Suef University.

Patients with other types of psoriasis were not included in the study. The patients included in the study did not receive any local or systemic treatments for at least a month before samples were taken.

In order to exclude an interaction between coexisting diseases (hypertension, diabetes, and cardiovascular disease) and psoriasis in terms of VNN1 polymorphism, psoriasis patients suffering from such disease(s) were excluded from the study; patients with symptoms suggestive of IBD were also excluded from the study.

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Methods

All study population was subjected to medical history taking, skin examination, and routine investigations.

The psoriasis area and severity index (PASI) score was calculated to each patient to assess psoriasis severity 13.

BMI was calculated from the recorded height and weight: weight in kilograms divided by height in meters squared.

Venous blood samples were collected from the study patients after overnight fasting; cholesterol, triglycerides, high-density lipoproteins (HDLs), and low-density lipoproteins were estimated by an automated clinical chemistry analyzer.

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Skin biopsy

Punch skin biopsies (4 mm) were obtained from the back of each control patient and from psoriasis lesions in the patients.

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Detection of the VNN1 expression using reverse transcription-quantitative qPCR

The RNeasy Kit by (Qiagen, Valencia, California, USA) was used for extraction of RNA from tissue homogenate.

The SuperScript III First-Strand System (Waltham, Massachusetts, USA) was used to synthesize complementary DNA (cDNA).

Briefly, 1 μg of RNA was added to 50 ng/μl random primers, 50 μmol/l oligo (dT) 20 and 10 mmol/l dNTP in 10 μl total volume, then incubated for 5 min at 56°C, and later cooled 3 min on ice.

The above mentioned mixture was added to the reverse transcriptase master mixture (25 mol/l MgCl2, 1 μl of SuperScript III RT, 0.1 mol/l DTT and 2 μl RT buffer), incubated for 10 min at 25°C and then for 50 min at 50°C. Real-time PCR amplification and analysis were performed using an Applied Biosystem with software version 3.1 (StepOne; Foster, California, USA). The reaction contained SYBR Green Master Mix (Applied Biosystems) and gene-specific primer pairs which are shown in Table 1 and were designed with Gene Runner Software (Hasting Software Inc., Hasting, New York, USA) from RNA sequences from the gene bank. Primers were annealed at 60°. cDNA (2 μl), each primer (900 nmol/l), and 2× SYBR Green PCR Master Mix were subjected to qRT-PCR. Amplification done at 50° for 2 min, then at 95°for 10 min followed by denaturation for 40 cycles and later, annealing and extension for 10 min at 60°.

Table 1

Table 1

The v 1.7 sequence detection software [from PE Biosystems (Foster City, California, USA)] was used for calculation of the real-time assays data. The relative expression of the studied gene mRNA was calculated using the comparative Ct method. All values were normalized to ribosomal phosphoprotein P0 (RPLP0) reference gene in the same sample and the values were reported as fold change over background levels detected in the diseased groups.

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Detection of VNN1 gene (T26I) polymorphism using restriction fragment length polymorphism

Genomic DNA was isolated from homogenized tissue samples by means of a DNA extraction kit supplied by (Qiagen Inc., Dusseldorf, Germany). The DNA was kept till analysis at −20°C.

Briefly, VNN1 T26I polymorphism (rs2294757), was detected using the end-point fuorescent measurement by the Applied Biosystems TaqMan MGB.

Probes: [mutant/wild type]; VIC/FAM-VIC-CAGCTGCA[A/G]TGAAAGT.

The PCR mixture constituents are genomic DNA (50 ng), from each primer (10 pmol), PCR buffer, dNTP (2.5 mmol/l from each), and Taq polymerase (1 U) (Fermentas, Ontario, Burlington, Canada).

The mixture was subjected to initial denaturation, annealing and extention.

The existence of PCR product was identified on a ethidium bromide stained agarose gel. 10 μl of the product was digested using Hinf I enzyme (Promega, Madison, Wisconsin, USA) for 12 h at 37°C.

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Statistical analysis

Mean and SD were used to present quantitative data. To compare means of two groups, Student’s t-test was used. Frequencies and percentages were used to introduce qualitative data. χ2-Test was used for comparisons between the two groups. Odds ratios and their 95% confidence intervals were used to compare between the groups. The significance level was set at a P value of less than 0.05. Statistical analysis was performed with the statistical package for the social sciences (SPSS) 16.0 for Windows (SPSS Inc., Chicago, Illinois, USA).

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Results

The patient’s age was from 18 to 65 and the mean age was 45.8±13.14 years. The disease onset ranged between 0.5 and 40 years (10.51±10.26 years) and the plaque duration ranged from 6 to 24 months (5.6±6.1 months) while their PASI score range was from 3 to 31.2, with a mean of 13.73±8.17.

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Vanin-1 tissue expression

Our results showed that the mean expression of VNN1 mRNA in the lesional skin of patients was significantly higher than that of controls: 3.3±2.3 versus 1.3±0.4 pg/µg, respectively (P<0.001) (Fig. 1).

Figure 1

Figure 1

On the other hand, no statistically significant relation was found between the tissue expression of VNN1 mRNA in the lesional skin and either patients’ age (r=0.13, P>0.05), disease duration (r=0.04, P>0.05), plaque duration (r=−0.17, P>0.05), or PASI score (r=−0.17, P>0.05).

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Vanin-1 DNA polymorphism

Precisely, 24 (40%) psoriasis patients had the wild T/T genotype; 28 (46.7%) patients had a homozygous VNN1 genotype (I/I genotype), a total of 8 (13.3%) of 60 were heterozygous, having T/I genotype (Table 2).

Table 2

Table 2

Likewise, 32 (53.3%) of 60 control patients had the T/T genotype (normal wild), and 26 (43.3%) controls had the I/I genotype; only two (3.4%) had the T/I genotype (heterozygote). The likelihood ratio indicated that the mutant genotypes did not constitute a risk factor for the development of psoriasis (P>0.05) (Table 2).

On analysis of allele frequency using Hardy–Weinberg equilibrium, both groups exhibited similar frequencies for both alleles (P=0.36). The dominant T allele frequency was 47% in patients compared to 53% in controls. Similarly, the recessive I allele frequency was 53% in patients compared to 45% in healthy controls.

Our results also showed that there were no statistically significant differences among the three genotypes regarding patients’ age, sex, and skin type (P>0.05); however, patients with the TI genotype were more likely to have higher BMI than the other genotypes (P<0.05) (Table 3).

Table 3

Table 3

Similarily, family history, precipitating factors, and different clinical findings were indifferent in TT, TI, and II genotypes (P>0.05) (Table 3).

TI genotype had higher VNN1 expression compared with the II and TI genotypes but the difference was nonsignificant (P<0.05) (Fig. 2).

Figure 2

Figure 2

Although statistically nonsignificant, the TI group patients had higher levels of triglycerides and cholesterol (P>0.05), but they did have significantly lower levels of HDL (P<0.05) (Table 3).

Besides, PASI, disease duration, and patient age did not show significant differences among the three VNN1 genotypes (P>0.05 in all) (Table 3).

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Discussion

The present study showed higher expression of VNN1 mRNA (almost triple) in psoriatic lesions in comparison to the control group. These results are consistent with the others who reported significant expression of VNN1 in psoriatic epidermis 7,14.

The increased VNN1 expression might lead to increased levels of cysteamine and pantothenic acid in psoriatic epidermis. Both metabolities are major players in the inflammatory and oxidative response 4.

The increased expression of VNN1 in psoriatic epidermis might reflect the increased requirements for precursors in order to keep up with the increased demand for lipid synthesis.

Cysteamine is a strong transglutaminase (TGase) inhibitor 15. TGase is known to play an important role in epidermal differentiation. In psoriatic skin, TGase-3 expression is slightly downregulated 16.

In vitro, TGase inhibition caused hyperproliferation, parakeratosis, and cytokeratin expression, which are hallmarks of psoriasis 17. The high expression of VNN1 in psoriatic epidermis may induce large amounts of cysteamine that inhibits TGase, leading to the pathological changes in psoriasis.

VNN1-deficient mice lack free cysteamine in tissues and exhibit elevated stores of the reduced form of glutathione in multiple tissues instead 18.

Therefore, increased cysteamine levels in tissue means increased oxidative stress, rendering the tissues susceptible to damage by the reactive oxygen species generated during inflammation.

The aetiopathogenesis of psoriasis involves complex interaction of environmental and genetic factors that contribute to pathological changes in psoriasis.

In this study, we tried to investigate whether the elevated VNN1 expression is related to VNN1 polymorphism; we chose to test the T26I polymorphism as it is located in the coding region particularly in a splicing regulation site, thus regulating the mRNA expression levels.

The results of the current study revealed that the VNN1 (T26I) polymorphism of the T/T genotype was more frequent in controls, while the T/I genotype and the I/I genotypes were more frequent in psoriasis patients. However, these differences were statistically nonsignificant and this can be explained by the small sample size of our study.

The T/I genotype which had non-significantly higher VNN1 expression compared with the other two genotype groups showed a significantly higher BMI and lower HDL levels which means a higher risk for the development of metabolic syndrome that was previously linked to psoriasis in multiple studies 19.

Various studies were conducted in an attempt to link VNN1 polymorphisms and the risk of diseases. For example, Gensollen et al.20 who studied the genetic association between VNN1 and IBD and revealed that three SNPs in the regulatory region of the gene were associated with IBD (VN-1339T>C, VN-681C>T at 5′UTR and VNi88C>T in intron 3) on the other hand. Fava et al.21 failed to detect the association between T26I and the risk of major cardiovascular events and hypertension phenotypes in two large urban-based studies conducted in Sweden.

To the best of our knowledge, this is the first study to investigate the association between VNN1 gene (T26I) polymorphism and the VNN1 levels in psoriasis.

While investigating the association between the VNN1 gene polymorphism and psoriasis, we did not observe any statistically significant differences in genotype distribution or allele frequency between the patients and the controls.

Despite the fact that VNN1 seems to play an important role in the pathogenesis of psoriasis; the present evidence indicates that it is unlikely that this overexpression is a consequence of the studied polymorphism.

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Financial disclosure

No financial support.

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Conflicts of interest

There are no conflicts of interest.

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Keywords:

pantetheinase; psoriasis; vanin-1

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