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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 34  |  Issue : 3  |  Page : 960-965

Granzyme B gene polymorphism in vitiligo: does it have an association?


1 Department of Dermatology, Andrology and STDs, Menoufia University, Menoufia, Egypt
2 Department of Molecular Diagnostics and Therapeutics, Genetic Engineering and Biotechnology Research Institute, Sadat City University, Menoufia, Egypt
3 Medical Biochemistry and Molecular Biology, Menoufia University, Menoufia, Egypt

Date of Submission08-Aug-2020
Date of Decision31-Aug-2020
Date of Acceptance02-Sep-2020
Date of Web Publication18-Oct-2021

Correspondence Address:
Wafaa A Shehata
Department of Dermatology, Andrology and STDs, Faculty of Medicine, Menoufia University, Shebin El-Kom 32716
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_229_20

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  Abstract 


Objectives
To study the role of granzyme B (GZMB) gene polymorphism (rs8192917) in patients with nonsegmental vitiligo and to correlate the results with the available clinical information.
Background
Vitiligo is considered the most common acquired disease of depigmented skin characterized by depigmented patches of the skin, hair, and mucous membranes owing to destruction of melanocytes. Vitiligo is a multifactorial polygenic condition with a multifaceted pathogenesis, linked to both genetic and nongenetic aspects, such as autoimmune, cytotoxic, oxidant–antioxidant, and neural therories working in performance.
Patients and methods
GZMB gene polymorphism (rs8192917) was identified using real-time PCR technique in 40 patients having nonsegmental vitiligo and 40 age-matched and sex-matched healthy controls. Assessment of disease severity was done using Vitiligo Area Severity Index score, and disease activity was assessed using Vitiligo Disease Activity score.
Results
CC and TC genotypes were significantly higher in the patients' group [P = 0.005; odds ratio (OR) = 7.2, 95% confidence interval (CI) = 1.61–32.2 for CC and OR = 1.86, 95% CI = 0.42–8.15 for TC], with significant statistical preponderance of C allele in cases (P = 0.001; OR = 3.05, 95% CI = 1.55–6.01).
Conclusion
GZMB gene polymorphism (rs8192917) is associated with the susceptibility to nonsegmental vitiligo in a sample of Egyptian population.

Keywords: granzyme B, nonsegmental, polymerase chain reaction, polymorphism, vitiligo


How to cite this article:
Shehata WA, Maraee A, El-Kheir MA, Tayel N, El-Hefnawy SM. Granzyme B gene polymorphism in vitiligo: does it have an association?. Menoufia Med J 2021;34:960-5

How to cite this URL:
Shehata WA, Maraee A, El-Kheir MA, Tayel N, El-Hefnawy SM. Granzyme B gene polymorphism in vitiligo: does it have an association?. Menoufia Med J [serial online] 2021 [cited 2024 Mar 28];34:960-5. Available from: http://www.mmj.eg.net/text.asp?2021/34/3/960/328320




  Introduction Top


Vitiligo is an acquired skin disease with the presence of white and nonpigmented patches owing to a loss of functioning melanocytes with subsequent loss of melanin in the epidermis. The condition can be cosmetically harming, and the lesional skin is thus highly sensitive to sunburns. It affects 0.1–2% of the world's population, irrespective of sex and race [1].

Etiology of vitiligo is still unknown, but some evidence-based theories have been suggested to clarify the damage of melanocytes in epidermis; however, both environmental and genetic factors contribute to the risk of vitiligo [2].

Actually, family clustering of cases is frequent, as - 20% of patients have at least one affected first-degree relative, with a non-Mendelian form suggestive of multiple factors, such as polygenic inheritance [3]. Multiple analyses have proposed the involvement of several relating genes in diverse populations. Numerous genes and chromosomal regions have been associated with the predisposition to vitiligo, but none has been confirmed so far [4].

Granzyme B (GZMB) is a serine protease found in the cytoplasmic granules of cytotoxic T lymphocytes (CTL) and natural killer cells and plays an important role in the induction of apoptotic changes in target cells during granule exocytosis-induced cytotoxicity [5]. GZMB enters target cells via the cation-independent mannose 6-phosphate/insulin-like growth factor receptor in a perforin-independent manner [6]. However, in the existence of perforin, GZMB escapes from the endolysosomal compartment and achieves access to a number of chief proteins involved in the execution of apoptotic program [7],[8].

GZMB mediates direct and caspase-mediated apoptosis of target cells [9], as well as proteolytic cleavage of autoantigens, creating or exposing autoimmune epitopes that may initiate or propagate the autoimmune process [10].

GZMB gene is a protein-coding gene that is located at the long arm of chromosome number 14 at locus 12 and has 5 exons, with a length of 3320 base pairs. Genome-wide association studies of vitiligo have demonstrated genetic association with GZMB, encoding granzyme B, a marker of activated CTLs that mediates target-cell apoptosis, as well as autoantigen activation and consequent initiation and propagation of autoimmunity [11].

The aim of the current study was to study the role of GZMB gene polymorphism (rs8192917) in nonsegmental vitiligo and to correlate the results with the available clinical data.


  Patients and methods Top


This case–control study included two main groups:

  1. Patients' group (cases): this group included 40 patients with different clinical varieties of nonsegmental vitiligo who were randomly selected during the period from June 2019 to January 2020.
  2. Control group: it comprised of 40 age-matched and sex-matched healthy participants, who voluntarily consented to participate in the study.


According to the Declaration of Helsinki 1975 (revised in 2000), ethical approval was obtained from the Local Ethical Research Committee before conducting this study, and written informed consent was obtained from every participant before enrollment.

Inclusion criteria were all cases with different clinical varieties of nonsegmental vitiligo, irrespective of age and sex.

Exclusion criteria: patients who met any of the following criteria were excluded and considered ineligible for participation:

  1. Any participant with associated inflammatory disease (such as infections and autoimmune disorders).
  2. Immune-compromised patients.
  3. Patients who had recent major surgical procedures.
  4. Patients who were taking any treatment (including vitamins and anti-inflammatory drugs) within the previous 3 months.
  5. Patients who were experiencing spontaneous repigmentation of depigmented patches at the time of presentation.


Full history of the patients was taken. They underwent complete general examination and detailed dermatological examination with identification of type and distribution of vitiliginous lesions.

Patients were classified according to Taïeb and Picardo [12], and assessment of disease severity was conducted using Vitiligo Area Severity Index score where Vitiligo Area Severity Index = all body sites (hand units)×residual depigmentation [13].

Vitiligo Disease Activity score was used to assess disease activity, and a low Vitiligo Disease Activity score indicated less activity [14].

Grading was as follows: +4, activity of 6 weeks or less duration; +3, activity of 6 weeks to 3 months; +2, activity of 3–6 months; +1, activity of 6–12 months; 0, stable for 1 year or more; and − 1, stable with spontaneous repigmentation for 1 year or more.

Every participant included in the study underwent determination of GZMB gene polymorphism (rs8192917) genotypes utilizing real-time PCR technique.

Blood sampling

Under complete aseptic conditions, 2 ml of venous blood was drawn under complete aseptic conditions from every participant and transferred to EDTA-containing tube and stored at –20°C or used directly for DNA extraction.

DNA extractions were done from whole blood samples by a GeneJET Whole Blood Genomic DNA Purification Mini extraction kit, by Thermo Fisher Scientific, Applied Biosystems: town and state: USA, CANADA, Jasper. Genotyping of the rs8192917 polymorphism within GZMB gene was performed by real-time PCR and allelic discrimination assay using a TaqMan probe (Applied Biosystems, USA). The primers/probes and Master Mix (×40) were provided by Thermo Scientific as well. The context probe sequences (VIC/FAM) were AGCAGCTGTCAGCACGAAGTCGTCT [T/C] GTATCAGGAAGCCACCGCACCTCTT. Overall, 1.25 μl of the primer/probe mixture was applied to a mix of 10 μl of Master Mix and 3.25 μl of nuclease-free water. Then, 5 μl of our extracted DNA was added to every one reaction. The cycling conditions were set as follows: 10 min of denaturation at 95°C, then 45 cycles as 0.5 min for denaturation at 95°C, 1 min for primer annealing at 55°C, and 1.5 min for extension at 74°C, and eventually, 1 min at 72°C as a final extension. Data were analyzed by the software supplemental to the ABI7500 real-time PCR instrument, version 2.0.1 [Figure 1]a and [Figure 1]b.
Figure 1: (a) Allelic discrimination plot showing different genotypes of granzyme B gene. (b) Amplification plot displaying fluorescence versus cycle number

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Statistical analysis

Statistical analysis was done using an IBM personal computer with Statistical Package of Social Science (SPSS) version 22 (SPSS, Inc, Chicago, Illinois, USA), version 20 and Epi Info 2000 programs. Two types of statistics were used: (a) descriptive statistics, where quantitative data were presented in the form of mean, SD, and range and qualitative data were presented in the form of numbers and percentages, and (b) analytical statistics, including χ2 test and Kruskal–Wallis test (K).

P value less than or equal to 0.05 was considered to be statistically significant. Odds ratio (OR) was used to measure the strength of association between two categorical variables, and the statistical significance of the measured OR is assessed by a special formula.


  Results Top


Patients comprised 17 (42.5%) males and 23 (57.5%) females, with a male:female ratio of 1:1.3. Their age ranged from 7 to 64 years, with mean ± SD value of 33.0 ± 17.5 years. Controls represented 15 (37.5%) males and 25 (62.5%) females, with a male:female ratio of 1:1.9. Their age ranged from 7 to 65 years, with a mean ± SD value of 32.8 ± 17.5 years. There were no significant differences between cases and controls regarding their age and sex (P > 0.05 for both).

Clinical data of patients are shown in [Table 1].
Table 1: Clinical data of the studied cases (n=40)

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Regarding GZMB genotyping in studied groups, the CC genotype was present in 24 (60%) cases, TC genotype was present in 13 (32.5%) cases, and TT genotype was present in three (7.5%). In the control group, CC genotype was present in 10 (25%) controls, TC genotype was present in 21 (52.5%) cases, and TT genotype was present in nine (22.5%). CC genotype was significantly higher in patients compared with controls, with OR of 7.2 and 95% CI: 1.61–32.2, and for TC genotype, OR was 1.86 and 95% CI: 0.42–8.15 [Table 2].
Table 2: Comparison between cases and controls regarding granzyme B genotype and alleles (rs8192917) (n=80)

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Regarding GZMB allele distribution, in the studied cases, C allele was present in 61 (76.2%) cases and T allele in 19 (23.8%) cases, whereas in the control group, C allele was present in 41 (51.2%) controls and T allele in 39 (48.8%) controls. C allele was statistically higher in the patients' group compared with controls (P = 0.001), with OR = 3.05 and 95% CI: 1.55–6.01 [Table 2].

There was a nonsignificant relationship between GZMB genotypes and clinical data of the studied cases (P > 0.05) [Table 3].
Table 3: Relationship between granzyme B gene polymorphism (rs8192917) genotype and clinical data of studied cases (n=40)

Click here to view



  Discussion Top


Vitiligo is an autoimmune disorder in which depigmented patches of skin and overlying hair result from autoimmune damage of melanocytes within the lesions. Vitiligo results from multifactorial causation, involving multiple underlying susceptibility genes and environmental triggers. Over the past several years, there has been considerable progress in defining the genetic epidemiology and genetic pathogenesis of vitiligo, and its relationships to other autoimmune diseases and almost all genetic studies have been of generalized or 'nonsegmental' vitiligo [15].

GZMB encodes the enzyme granzyme B, which plays an important role in cytotoxic T cell-induced apoptosis, and it has been considered one of the candidate genes for vitiligo because of its connections with human immune system [2].

The aim of the current study was to assess if there is an association between GZMB gene polymorphism (rs8192917) in nonsegmental vitiligo and to correlate the results with the available clinical data.

In the current study, upon studying GZMB gene polymorphism (rs8192917) in cases with nonsegmental vitiligo, there was a significant difference between cases and controls regarding GZMB genotype (P = 0.005), with predominance of CT and TC genotypes in cases and the risk for vitiligo development by ~7.2 folds. This was nearly similar to Jin et al. [16], who found a significant association between vitiligo and SNPs rs8192917 (P = 5.54 × 10−6; OR = 1.28) and concluded that GZMB mediates two processes: immune-induced target-cell apoptosis mediated by cytotoxic T cells and natural killer cells and activation-induced cell death of effector type 2 helper T cells, which terminates the immune response.

In the current study, regarding the frequency of GZMB allele, there was a significant difference between cases and controls regarding GZMB allele (χ2 = 10.8, P = 0.001), and the C allele was significantly higher in the patients' group, with an increased risk for vitiligo development by about 3.05 folds. This was nearly similar to Xu et al. [2], who found that SNP rs8192917 was significantly associated with vitiligo and C allele of this SNP increased the risk of vitiligo by - 40% (P = 1.92 × 10−8).

GZMB has been genetically associated with other autoimmune diseases, specifically including juvenile idiopathic arthritis[17] and Behçet's disease [18]. This suggests the possibility that GZMB may be relatively specific for melanocyte-directed autoimmune susceptibility. In addition to the effector function of GZMB in target cell killing by CTLs, perhaps particularly relevant to GV may be the role played by GZMB cleavage in activating autoantigens in autoimmune disease [10].

Therefore, GZMB (CC) polymorphism can be considered as a risk factor of vitiligo in Egyptian population. However, as vitiligo is the result of multiple genes and environment, single gene studies cannot fully clarify the interactions between genes and their effects on phenotype. So, additional analysis of multiple genetic loci is required.


  Conclusion Top


GZMB gene polymorphism (rs8192917) is associated with the susceptibility to nonsegmental vitiligo in a sample Egyptian population.

The present study had a number of limitations. First, sample size was very small to come into any conclusion, which makes it necessary to confirm our findings in large-scale studies. Small sample size will contribute to low statistical power. Second, there was a lack of knowledge about the functional influence of the identified polymorphisms with vitiligo, which remains to be determined.

Unresolved questions: the cause of vitiligo remains unknown, and it is still unclear whether the abnormalities described in the single pathogenetic theories are a cause or rather an effect of the disease. This fact is reflected over the lack of a definitive effective therapy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Bergqvist C, Ezzedine K. Vitiligo. Dermatology 2020; 2020:1–22.  Back to cited text no. 1
    
2.
Xu M, Liu Y, Liu Y, Li X, Chen G, Dong W, et al. Genetic polymorphisms of GZMB and vitiligo: a genetic association study based on Chinese Han population. Sci Rep 2018; 8:13001.  Back to cited text no. 2
    
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Spritz RA, Santorico SA. The Genetic Basis of Vitiligo. J Invest Dermatol. 2021;141:265-73.  Back to cited text no. 3
    
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Turner CT, Lim D, Granville DJ. Granzyme B in skin inflammation and disease. Matrix Biol 2019; 75-76:126–140.  Back to cited text no. 5
    
6.
Motyka B, Korbutt G, Pinkoski MJ, Heibein JA, Caputo A, Hobman M, et al. Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis. Cell 2000; 103:491–500.  Back to cited text no. 6
    
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Voskoboinik I, Whisstock JC, Trapani JA. Perforin and granzymes: function, dysfunction and human pathology. Nat Rev Immunol 2015; 15:388–400.  Back to cited text no. 7
    
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Veugelers K, Motyka B, Goping IS, Shostak I, Sawchuk T, Bleackley RC. Granule-mediated killing by granzyme B and perforin requires a mannose 6-phosphate receptor and is augmented by cell surface heparan sulfate. Mol Biol Cell 2006; 17:623–633.  Back to cited text no. 8
    
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Hiebert PR, Granville DJ. Granzyme B in injury, inflammation, and repair. Trends Mol Med 2012; 18:732–741.  Back to cited text no. 9
    
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Ferrara TM, Jin Y, Gowan K, Fain PR, Spritz RA. Risk of generalized vitiligo is associated with the common 55R-94A-247H variant haplotype of GZMB (encoding granzyme B). J Invest Dermatol 2013; 133:1677–1679.  Back to cited text no. 11
    
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Taïeb A, Picardo M. Clinical practice. Vitiligo. N Engl J Med 2009; 360:160–169.  Back to cited text no. 12
    
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Kawakami T, Hashimoto T. Disease severity indexes and treatment evaluation criteria in vitiligo. Dermatol Res Pract 2011; 2011:750342.  Back to cited text no. 13
    
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Bhor U, Pande S. Scoring systems in dermatology. IJDVL 2006; 72:315–321.  Back to cited text no. 14
    
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Spritz RA. Modern vitiligo genetics sheds new light on an ancient disease. J Dermatol 2013; 40:310–318.  Back to cited text no. 15
    
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Jin Y, Birlea SA, Fain PR, Gowan K, Riccardi SL, Holland PJ, et al. Variant of TYR and autoimmunity susceptibility loci in generalized vitiligo. N Engl J Med 2010; 362:1686–1697.  Back to cited text no. 16
    
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Donn R, Ellison S, Lamb R, Day T, Baildam E, Ramanan AV. Genetic loci contributing to hemophagocytic lymphohistiocytosis do not confer susceptibility to systemic-onset juvenile idiopathic arthritis. Arthritis Rheum 2008; 58:869–874.  Back to cited text no. 17
    
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Kucuksezer UC, Duymaz-Tozkir J, Gül A, Saruhan-Direskeneli G. No association of granzyme B gene polymorphism with Behçet's disease. Clin Exp Rheumatol 2009; 27 (2 Suppl 53):S102.  Back to cited text no. 18
    


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