What’s Known

Vitiligo may be associated with disruption of apoptosis regulatory molecules and altered expression of BCL2 and BAX.

What’s New

Identification of molecular markers that can be investigated by rapid methods, such as Polymerase chain reaction (PCR), can help provide warning information about the conditions of people at risk. Investigation of BAX 248G>A and Bcl-2 938C>A polymorphisms shows whether they increase the risk of vitiligo.

Introduction

Vitiligo, an acquired and progressive disorder, is characterized by the emergence of circumscribed white macules on the skin. This depigmenting condition results from the chronic and progressive loss of functional melanocytes in the epidermis. ¹ It seems that vitiligo is affected by various factors, including environmental and genetic factors, that can cause melanocyte dysfunction. ²

In vitiligo disease, melanocytes seem to be destroyed through apoptosis. ³ Apoptosis in melanocytes is induced through active caspases and BAX/BCL2 systems. ⁴ Anti-apoptotic B-cell lymphoma-2 (BCL2) protein prevents cell apoptosis, contrary to pro-apoptotic Bcl-2-associated X (BAX) protein, which activates the apoptosis pathway. ^{5 , 6} It was found that the expression level of BCL2 in the melanocytes around the lesion is lower than in control melanocytes, and the expression level of BAX protein is high, showing that the melanocytes of vitiligo patients are involved in the process of apoptosis. ⁷

BCL2-938C>A and BAX-248G>A polymorphisms are in the promoter region and can bind to various transcription factors and regulatory proteins. ⁸ Therefore, polymorphisms located in the promoter of BCL2 and BAX genes can alter gene expression or protein function, which may affect apoptosis regulatory mechanisms. ⁹ Polymorphisms are considered to be one of the most influential factors in people’s sensitivity to diseases and drugs. ¹⁰ The study of polymorphisms and gene expression is necessary to discover the diseases’ molecular mechanisms, determine the disease’s genetic risk, and use it in personalized medicine in the future. ¹¹ In this regard, the pathogenesis of vitiligo and the association of polymorphisms with this disease are not fully understood. ¹² Considering the role of apoptosis in vitiligo disease and the effectiveness of the BAX/BCL2 expression ratio in the process of apoptosis, the study of polymorphisms of BCL2 and BAX genes and their association with vitiligo is particularly important. Apoptosis plays an exceptionally vital part in causing immune system reactions and the devastation of melanocytes in vitiligo. Apoptosis is mediated by BCL2 and BAX proteins in the intrinsic apoptosis pathway. Vitiligo may be associated with disruption of apoptosis regulatory molecules and altered expression of BCL2 and BAX. ^{13 , 14} BCL2-938C>A and BAX-248G>A polymorphisms are in the promoter region of BAX and BCL2 genes. ¹⁵ These polymorphisms can cause changes in the binding sites of transcription factors and have significant functional effects in regulating gene expression, ultimately affecting disease susceptibility. Therefore, single-nucleotide polymorphisms (SNPs) can act as biomarkers for the diagnosis and prognosis of diseases. ¹⁶

Therefore, this cross-sectional study aimed to investigate the association of BAX-248G>A (rs4645878) and BCL2-938C>A (rs2279115) polymorphisms with vitiligo disease in Shirazi patients in the Iranian population. The studied polymorphisms had not previously been investigated in vitiligo patients.

Patients and Methods

This cross-sectional study was approved by the Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (ID number IR.SUMS.REC.1400.095). The inclusion criteria of this case-control study were age (18 years old or older) and clinical diagnosis of vitiligo. The case group consisted of vitiligo patients, while the control group included healthy individuals who were matched for age and sex across both groups. The sample size was confirmed by the post hoc power analysis that uses the observed sample size and estimated effect, along with the assumed type I error, to calculate the observed power. ¹⁷ The vitiligo evaluations were clinically performed by dermatologists, with the skin lesions examined under a Wood’s lamp. We designed the sample size based on the average number of vitiligo patients visiting the dermatology clinic.

For all volunteers referred to dermatology clinics at Shiraz University of Medical Sciences, a clinical history form and general information (age, sex, smoking, parental consanguinity, age of onset, duration of vitiligo) were completed by the researcher. Next, whole blood samples were collected in Ethylenediaminetetraacetic acid (EDTA) tubes (Easy, Iran) for genetic analysis. In this study, all ethical principles were reviewed and approved by the ethics committee of Shiraz University of Medical Sciences.

The genotypes of BAX-248G>A (rs4645878) and BCL2-938C>A (rs2279115) gene polymorphisms were investigated in all subjects enrolled in this study using the tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) technique. ¹⁸

DNA Extraction and Genotyping of Samples

Sampling Procedure

Whole blood samples collected from the case and control groups were transferred into EDTA tubes and stored. Then, the genomic Deoxyribonucleic acid (DNA) was extracted from the blood samples using a FavorPrep^TM Blood/Cultured Cell Genomic DNA Extraction Mini Kit (Favorgen Biotech Corp., Taiwan), following the manufacturer’s instructions.

The Quality Control of the Extracted DNA

The DNA optical absorbance was determined using a nano-drop device, and the samples with Optical Density (OD) values between 1.8 and 2 were confirmed. In addition, the electrophoresis technique was used to validate the quality of DNA extraction. DNA samples were loaded and run on an agarose gel (Sigma-Aldrich, US). The DNA bands were visualized using the gel documentation imaging system, and molecular markers confirmed the band’s validity on the gel.

Tetra-primer ARMS PCR

The tetra-primer ARMS PCR reaction was utilized to amplify the polymorphisms of the target gene. In this technique, four primers are needed, including an outer primer pair and an internal primer pair that contain mismatches at the 3′-end so that two bands for a homozygous individual and three bands for a heterozygous individual will appear in the agarose gel.

Table 1 presents the primers used in this study. The genotype determination was investigated in a single tube with a total volume of 20 μL: master mix (10 μL), 100 ng of template DNA, and one μL each primer (10 pmol).

The PCR condition was set as follows: 5 min of denaturation at 95 °C, then 35 cycles of 30 sec for denaturation at 95 °C, 30 sec for primer annealing at 59 °C, and 20 sec for the extension at 72 °C, and 5 min at 72 °C as a final extension.

Finally, the PCR products of different samples were run on a 2% electrophoresis gel, and the bands related to alleles were identified according to figure 1.

Figure 1.BAX-248G>A (rs4645878) and BCL2-938 C>A (rs2279115) polymorphisms are shown on 1.5% agarose gel electrophoresis; A) BAX-248G>A; the individuals homozygous for the G allele (GG genotype) were identified by the presence of products of 323 bp and 209 bp. The individuals homozygous for the A allele (AA genotype) were identified by the presence of products of 323 bp and 160 bp. The heterozygous individuals (AG genotype) were identified by the presence of all products of 323 bp, 209 bp, and 160 bp. B) BCL2-938 C>A; the individuals homozygous for the A allele (AA genotype) were identified by the presence of products of 300 bp and 220 bp. The individuals homozygous for the C allele (CC genotype) were identified by the presence of products of 300 bp and 121 bp. The heterozygous individuals (AC genotype) were identified by the presence of 300 bp, 220 bp, and 121 bp products. DNA ladder; 100 bp DNA size marker.

Statistical Analysis

Statistical analysis was done using an IBM personal computer with Statistical Package of Social Sciences (SPSS) version 22 (SPSS, Inc., Chicago, Illinois, USA). 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 the Hardy-Weinberg equilibrium (HWE) for each single nucleotide polymorphism (SNP) were used, which was calculated among controls. The Chi square test assessed the distribution of genotypes between groups. A t test was also used to analyze data using quantitative variables and continuous values. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to investigate the association of polymorphisms with vitiligo risk in two genetic models.

Results

Profile of the Participants in the Study

This study had 219 participants, 112 of whom were in the control group and 107 were in the patient group. Based on post hoc power analysis, this sample volume obtained a power equal to 94.4% in detecting differences, which is acceptable. In terms of gender, the two groups were homogenous (The sex distribution was compared between the two groups using Pearson’s Chi square test). The mean age was 41.39 years in the patient group and 41.08 years in the control group. There was no significant difference between the means (age was compared between the two groups using the Independent Samples t test, depending on the result of Levene’s test of the homogeneity of the variances) (table 2).

The results are presented as frequency and percentages or mean±SD. The Chi square test and the independent samples t test was used; P<0.05 was considered statistically significant.

Investigation of Hardy-Weinberg Equilibrium in the Population

To check for balance in the population, the allelic and genotypic frequencies in the healthy population are given below for BAX-248G>A (rs4645878) and BCL2-938C>A (rs2279115) polymorphisms.

The results of our investigation showed that the genotypic frequencies of the BAX-248G>A gene polymorphism (rs4645878) in the healthy group (χ²=0.035, P=0.987) followed the Hardy-Weinberg equilibrium. Additionally, as to the BCL2 gene, the research results showed that the genotypic frequencies of polymorphism 938C>A (rs2279115) in the healthy group (χ²=0.98, P=0.611) followed the Hardy-Weinberg equilibrium.

Assessment of Genotypic and Allelic Frequencies between the Control and Patient Groups

Data analysis showed that the GG genotype of the BAX-248G>A polymorphism significantly increased the probability of vitiligo. Compared to the A allele of this polymorphism, the G allele increased the risk of vitiligo disease. In other words, the results indicated that the A allele in genotypes could decrease the probability of vitiligo (table 3).

Regarding the BCL2 gene, data analysis showed a significant relationship between the CC genotype of the BCL2-938C>A polymorphism and the risk of vitiligo disease. The C allele was also associated with the vitiligo risk, and it increased the disease risk. In other words, the results revealed that the presence of the A allele in genotypes could reduce the probability of vitiligo (table 3).

Association of Vitiligo with the Concurrent Effect of BAX and BCL2 Gene Polymorphisms

Another investigation showed the effect of BAX and BCL2 gene polymorphisms simultaneously with the risk of vitiligo. The results of this study are given in table 4.

The results in table 4 show that the combination of AA+AG genotypes of BAX-248G>A polymorphism, along with AA+AC genotypes of BCL2-938C>A polymorphism, has a significant association with reducing the risk of vitiligo. The combination of GG genotypes of BAX-248G>A polymorphism and CC genotypes of BCL2-938C>A polymorphism has a significant association with an increased risk of vitiligo.

Association between Parental Consanguinity, Smoking, and Vitiligo

The data analysis of this study showed a significant association between parental consanguinity, which indicates the genetic background of vitiligo patients. Consequently, parental consanguinity is a risk factor for vitiligo. Moreover, other results showed that there was no significant association between smoking and vitiligo (table 5).

Discussion

In the present study, data analysis showed that the CC genotype of the BCL2-938C>A polymorphism significantly increased the probability of vitiligo. Considering the role of the C allele as mentioned, it can be concluded that the CC genotype is associated with a decrease in the BCL2 expression level. The increase in the frequency of the CC genotype in vitiligo compared to the control group shows that the increase in apoptosis may destroy more melanocytes and, as a result, the appearance of vitiligo symptoms. Until now, no study has been conducted on the association between BCL2-938C>A polymorphism and vitiligo disease. However, since the process of apoptosis in melanocytes causes the appearance of vitiligo symptoms, studies were conducted that showed the association between BCL2-938C>A polymorphism and the expression level of this gene.

Considering the role of the C allele, it can be concluded that the CC genotype is associated with a decrease in the BCL2 expression level. ¹⁵ Hirata and others observed that kidney cancer tissues carrying the CC genotype had a significantly lower expression of BCL2 than those carrying the CA+AA genotype. ¹⁹ Our study shows similarities with previous reports on the association of the CC genotype with increased apoptosis. Considering the role of BAX in apoptosis, G (-248) A polymorphism can cause changes in BAX expression and disrupt the apoptosis process. ^{8 , 20}

Previous studies have shown that the GG genotype is associated with decreased BAX expression, which is inconsistent with our study. ²¹ Ma and others found that the GG genotype might lead to overexpression of BAX and cause abnormal cell apoptosis. ²² These results are consistent with our study. Additionally, the results of our research showed that the GG genotype of the BAX-248G>A polymorphism significantly increased the probability of vitiligo, and considering the role of apoptosis in vitiligo, it can be concluded that the GG genotype is associated with a higher expression of BAX, which leads to clinical manifestations of vitiligo.

Wang and others demonstrated that the 938AA genotype of the BCL2 gene might be a susceptible genotype for non-Hodgkin lymphoma (NHL), and this genotype is associated with a larger tumor size in NHL. ²³ Yao and others showed a statistically significant association between rs2279115 and cancer susceptibility. Subgroup analysis indicated that rs2279115 was associated with a significantly higher risk of cancer susceptibility in Asia but not in Caucasians. Furthermore, rs2279115 was associated with a considerably higher risk of digestive system cancer and endocrine system cancer, but not breast, respiratory, and hematopoietic cancers. ²⁴

Cingeetham and others found that the BCL2-938CA and BAX-248GG genotypes are significantly associated with an increased risk of acute myeloid leukemia (AML). The BAX-248A allele showed a lower risk of AML. They concluded that the gene polymorphisms BCL2-938C>A and BAX-248G>A might contribute to the development of AML. In Addition, the GG genotype and G allele frequencies were significantly higher. Conversely, the A allele was linked to a reduced risk of AML. ²⁵

Moreover, in previous chronic lymphocytic leukemia (CLL) studies, the GG genotype and G allele of the polymorphic locus 248 G>A were associated with an elevated risk of CLL. ²⁶ However, the rs4645878 variant (G125A) of the BAX promoter showed an association with decreased CLL risk. ²⁷ Fernandes and others have provided evidence that G allele carriers in the promoter regions of BAX-248G>A are related to the risk of squamous intraepithelial neoplasia development but not the disease severity. ²⁸ In contrast, Al-Zubaidy and others concluded that there was no significant difference in the BCL2-938C>A polymorphism of all genotype models compared to healthy models in Iraqi women with breast cancer. ²⁹

Results from previous studies and our research show that polymorphisms in the promoter of BCL2 and BAX genes can significantly alter protein function or expression, thereby disrupting the regulation of apoptosis. This disruption can lead to a disturbance in cellular homeostasis and potentially contribute to malignant transformation.

In general, the results of different studies related to the association between BAX-248G>A and BCL2-938C>A polymorphisms and BAX and BCL2 expression levels are dissimilar. However, further confirmation in more extensive studies is needed to clarify the role of these polymorphisms in vitiligo.

One limitation of this study was not using the matching process, which is recommended to be used in future studies. Moreover, contrary to previous studies, we found no significant association between gender and smoking and the risk of vitiligo in men and women. ^{30 , 31} One of the key findings of the present study was a significant association between parental kinship and the risk of vitiligo, suggesting that inherited risk factors from parents may contribute to the development of the condition. These results are in line with previous studies that have shown that individuals with consanguineous parents have an increased risk of developing vitiligo. ^{32 , 33}

Conclusion

Our data support the role of apoptosis-related BAX-248G>A (rs4645878) and BCL2-938C>A (rs2279115) polymorphisms in vitiligo susceptibility. Therefore, these polymorphisms can be utilized as biomarkers for the early diagnosis of vitiligo.

Acknowledgment

The authors would like to thank the Molecular Dermatology Research Center staff, Shiraz University of Medical Sciences, and all those who helped in this study.

Authors’ Contribution

A.D: Data gathering, data analysis, drafting, and critical reviewing; N.H: Data gathering, data analysis, and critical reviewing; F.H: Data gathering and critical reviewing; I.J: Study design, data gathering, data analysis, and critical reviewing. All authors have read and approved the final manuscript and agree to be accountable for all aspects of the work to ensure that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflicts of Interest

None declared.

References

1. Elbuluk N, Ezzedine K. Quality of Life, Burden of Disease, Co-morbidities, and Systemic Effects in Vitiligo Patients. Dermatol Clin. 2017; 35:117-28. DOI | PubMed
2. Spritz RA, Andersen GH. Genetics of Vitiligo. Dermatol Clin. 2017; 35:245-55. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
3. Chen J, Li S, Li C. Mechanisms of melanocyte death in vitiligo. Med Res Rev. 2021; 41:1138-66. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
4. Bialczyk A, Welniak A, Kaminska B, Czajkowski R. Oxidative Stress and Potential Antioxidant Therapies in Vitiligo: A Narrative Review. Mol Diagn Ther. 2023; 27:723-39. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
5. Qian S, Wei Z, Yang W, Huang J, Yang Y, Wang J. The role of BCL-2 family proteins in regulating apoptosis and cancer therapy. Front Oncol. 2022; 12:985363. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
6. Raisova M, Hossini AM, Eberle J, Riebeling C, Wieder T, Sturm I, et al. The Bax/Bcl-2 ratio determines the susceptibility of human melanoma cells to CD95/Fas-mediated apoptosis. J Invest Dermatol. 2001; 117:333-40. DOI | PubMed
7. Xuan Y, Yang Y, Xiang L, Zhang C. The Role of Oxidative Stress in the Pathogenesis of Vitiligo: A Culprit for Melanocyte Death. Oxid Med Cell Longev. 2022; 2022:8498472. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
8. Mirmajidi SH, Najafi M, Mirmajidi ST, Nasri Nasrabadi N. Study of regulatory promoter polymorphism (-248 G&gt;A) of Bax gene in patients with gastric cancer in the northern provinces of Iran. Gastroenterol Hepatol Bed Bench. 2016; 9:36-44. Publisher Full Text | PubMed [ PMC Free Article ]
9. Moawadh MS, Mir R, Tayeb FJ, Asim O, Ullah MF. Molecular Evaluation of the Impact of Polymorphic Variants in Apoptotic (Bcl-2/Bax) and Proinflammatory Cytokine (TNF-alpha/IL-8) Genes on the Susceptibility and Progression of Myeloproliferative Neoplasms: A Case-Control Biomarker Study. Curr Issues Mol Biol. 2023; 45:3933-52. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
10. Chiarella P, Capone P, Sisto R. Contribution of Genetic Polymorphisms in Human Health. Int J Environ Res Public Health. 2023; 20Publisher Full Text | DOI | PubMed [ PMC Free Article ]
11. Gummadi AC, Guddati AK. Genetic Polymorphisms in Pharmaceuticals and Chemotherapy. World J Oncol. 2021; 12:149-54. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
12. Marchioro HZ, Silva de Castro CC, Fava VM, Sakiyama PH, Dellatorre G, Miot HA. Update on the pathogenesis of vitiligo. An Bras Dermatol. 2022; 97:478-90. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
13. Su Q, Wang F, Dong Z, Chen M, Cao R. IFNgamma induces apoptosis in human melanocytes by activating the JAK1/STAT1 signaling pathway. Mol Med Rep. 2020; 22:3111-6. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
14. Wu X, Yang Y, Xiang L, Zhang C. The fate of melanocyte: Mechanisms of cell death in vitiligo. Pigment Cell Melanoma Res. 2021; 34:256-67. DOI | PubMed
15. Olbromski PJ, Bogacz A, Bukowska M, Kaminski A, Moszynski R, Pawlik P, et al. Analysis of the Polymorphisms and Expression Levels of the BCL2, BAX and c-MYC Genes in Patients with Ovarian Cancer. Int J Mol Sci. 2023; 24Publisher Full Text | DOI | PubMed [ PMC Free Article ]
16. Degtyareva AO, Antontseva EV, Merkulova TI. Regulatory SNPs: Altered Transcription Factor Binding Sites Implicated in Complex Traits and Diseases. Int J Mol Sci. 2021; 22Publisher Full Text | DOI | PubMed [ PMC Free Article ]
17. Quach NE, Yang K, Chen R, Tu J, Xu M, Tu XM, et al. Post-hoc power analysis: a conceptually valid approach for power based on observed study data. Gen Psychiatr. 2022; 35:e100764. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
18. Munoz C, Gomez Talquenca S, Volpe ML. Tetra primer ARMS-PCR for identification of SNP in beta-tubulin of Botrytis cinerea, responsible of resistance to benzimidazole. J Microbiol Methods. 2009; 78:245-6. DOI | PubMed
19. Hirata H, Hinoda Y, Nakajima K, Kikuno N, Suehiro Y, Tabatabai ZL, et al. The bcl2 -938CC genotype has poor prognosis and lower survival in renal cancer. J Urol. 2009; 182:721-7. DOI | PubMed
20. Volkmann N, Marassi FM, Newmeyer DD, Hanein D. The rheostat in the membrane: BCL-2 family proteins and apoptosis. Cell Death Differ. 2014; 21:206-15. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
21. Moawad MS, Tayeb FJ, Mir R. Evaluation of polymorphic variants in apoptotic genes (Bcl-2/Bax) and their roles in the development of myeloproliferative disorders (hematological malignancies) a case-controlled study. Research Square. 2022. DOI
22. Ma G, Jiang D, Huang J. Genetic association of the polymorphisms in apoptosis-related genes with osteoarthritis susceptibility in Chinese Han population. Int J Clin Exp Pathol. 2018; 11:2221-6. Publisher Full Text | PubMed [ PMC Free Article ]
23. Wang WL, Tao YP, Han XL, Li X, Zi YM, Yang C, et al. Role of polymorphisms in BCL-2 and BAX genes in modulating the risk of developing non-Hodgkin lymphoma. Leuk Lymphoma. 2014; 55:1602-8. DOI | PubMed
24. Yao Z, Yang B, Liu Z, Li W, He Q, Peng X. Genetic polymorphisms of Bcl-2 promoter in cancer susceptibility and prognosis: a meta-analysis. Oncotarget. 2017; 8:25270-8. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
25. Cingeetham A, Vuree S, Dunna NR, Gorre M, Nanchari SR, Edathara PM, et al. Influence of BCL2-938C&gt;A and BAX-248G&gt;A promoter polymorphisms in the development of AML: case-control study from South India. Tumour Biol. 2015; 36:7967-76. DOI | PubMed
26. Bakirov BA, Karimov D, Viktorova T. Polymorphism of apoptosis regulatory and growth factor genes in patients with chronic lymphocytic leukemia. Russian Open Medical Journal. 2012; 1:0101.
27. Enjuanes A, Benavente Y, Bosch F, Martin-Guerrero I, Colomer D, Perez-Alvarez S, et al. Genetic variants in apoptosis and immunoregulation-related genes are associated with risk of chronic lymphocytic leukemia. Cancer Res. 2008; 68:10178-86. DOI | PubMed
28. Fernandes AT, Rocha NP, Vendrame E, Russomano F, Grinsztejn BJ, Friedman RK, et al. Polymorphism in apoptotic BAX (-248G&gt;A) gene but not in anti-apoptotic BCL2 (-938C&gt;A) gene and its protein and mRNA expression are associated with cervical intraepithelial neoplasia. Apoptosis. 2015; 20:1347-57. DOI | PubMed
29. Al-Zubaidy HFS, Majeed SR, Al-Koofee DAF. Evaluation of Bax and BCL 2 Genes Polymorphisms in Iraqi Women with Breast Cancer. Arch Razi Inst. 2022; 77:799-808. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
30. Patil S, Gautam M, Nadkarni N, Saboo N, Godse K, Setia MS. Gender differences in clinicoepidemiological features of vitiligo: a cross-sectional analysis. ISRN Dermatol. 2014; 2014:186197. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
31. Lee YB, Lee JH, Lee SY, Yu DS, Han KD, Park YG. Association between vitiligo and smoking: A nationwide population-based study in Korea. Sci Rep. 2020; 10:6231. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
32. Alenizi DA. Consanguinity pattern and heritability of Vitiligo in Arar, Saudi Arabia. J Family Community Med. 2014; 21:13-6. Publisher Full Text | DOI | PubMed [ PMC Free Article ]
33. Molla A, Alayoubi AM, Jannadi R. First Cousin Marriages and the Risk of Childhood-Onset Vitiligo: Exploring the Genetic Background: A Cross-Sectional Study. Clin Cosmet Investig Dermatol. 2024; 17:1471-9. Publisher Full Text | DOI | PubMed [ PMC Free Article ]