European Review for Medical and Pharmacological Sciences 2019; 23: 471-478 High incidence of MTHFR, CBS, and MTRR polymorphisms in vitiligo patients. Preliminary report in a retrospective study G. BENINCASA1, G. DI SPIGNA2, C. CAPPELLI1, R. DI FRANCIA3, M. OTTAIANO1, M. SANSONE4, L. IODICE5, E. DE MARINIS2, L. POSTIGLIONE2 1Pathological Anatomy, Pineta Grande Hospital, Castel Volturno, Caserta, Italy 2Department of Translational Medical Sciences, University of Naples “Federico II”, Naples, Italy 3Gruppo Oncologi Ricercatori Italiani (GORI), Pordenone, Italy 4Dia-Chem, Molecular Biology, Pineta Grande Hospital, Naples, Italy 5Healthcare Management, ASL Salerno, “S. Luca” Hospital, Vallo della Lucania, Italy Abstract. – OBJECTIVE: Vitiligo is a multifac- to get hypothesis about the putative pathogen- torial polygenic disorder with a complex patho- esis of vitiligo, and the precise cause remains genesis. It is related to both genetic and no ge- unclear. netic factors. The role of genetics is currently studied with several analytical approaches, Key Words: such as genetic linkage, candidate gene asso- MTHFR C677T, CBS I278T, MTRR A66G, Vitiligo ge- ciation studies, genome-wide association stud- netic markers. ies (GWAS), deep DNA re-sequencing and gene expression studies. To date, there are no genet- ic traits directly related to vitiligo pathogenesis. PATIENTS AND METHODS: 43 cases of vitiligo Introduction patients and 30 healthy donors recruited as con- trol, were screened by assaying the biochemical Vitiligo is a common pigmentary disorder of molecules involved in the self-cells cytotoxicity the skin with etiopathogenesis not yet well defi- (haptoglobin and homocysteine) and candidate ned. Clinically vitiligo is characterized by cir- genes involved in the regulatory process of the re-methylation cycles and transsulfuration. Can- cumscribed, depigmented macules and patches didate genes and their polymorphisms screened caused by melanocytes apoptosis that is suppo- are methylene-tetrahydrofolate-reductase (MTH- sed to be in the underlying epidermal layers. Ini- FR) C677T and A1298C; cystathionine-beta-syn- tial lesions occur most frequently on the hands, thase enzyme (CBS) I278T and Ins68bp; and me- forearms, feet, and face, favoring a perioral and thionine-synthase-reductase (MTRR) A66G. RESULTS: periocular distribution. The macules are chalk or A peculiar genetic profile in vitil- milk-white in color and are well demarcated. Le- igo patients are defined: 11.6% of vitiligo pa- 1 tients shown polymorphic variant MTHFR 677TT sions can be round, oval, or linear in shape . vs. 3.3% of healthy donor MTHFR 677CC profile The lack of an international consensus concer- (p=0.0017); 14.0% of vitiligo patients shown CBS ning the definition and classification of vitiligo polymorphic variant 278TT vs. 3.3% of healthy hampers international level research and com- donor 278II profile (p=0.0012); and 11.6% of vit- munication. The Vitiligo European Task Force iligo patients shown MTRR 66GG vs. 3.3% of (VETF) has proposed consensus definitions for healthy donor MTRR 677AA profile (p>0.0001). CONCLUSIONS: This is the first study report- the disease, but a need for broader international 2 ing the correlation between the polymorphic sta- consensus was felt . tus of MTHFR C677T, CBS I278T, and MTRR A66G Vitiligo is known to affect around 0.1-2% of the and vitiligo. The genetic screening of these poly- world population and could seem at any time from morphisms could be useful for early detection birth to senility3-5. The average age of beginning of the inheritance risk factor in a subject car- for vitiligo is most commonly observed approxi- rying relatives with vitiligo. Although these da- 6 ta could suggest a kind of dysregulation, genet- mately 20 years . The age of onset is unlikely to ically based, of thiols production mechanisms. vary between the sexes. The vitiligo incidence Based on these results, we have not been able has been reported female preponderance, but it is Corresponding Author: Raffaele Di Francia, Ph.D; e-mail: [email protected] 471 G. Benincasa, G. Di Spigna, C. Cappelli, R. Di Francia, M. Ottaiano, M. Sansone, et al. still not statistically confirmed. This discrepancy include genes associated with the biosynthesis of has been attributed to an increase in reporting of melanin, a response to oxidative stress, and regu- cosmetic concerns by female patients7-9. Etiopa- lation of autoimmunity28. thogenesis is almost completely unknown and the Substantial development has been made to un- clinical dates are conflicting. derstand the role of genetics in vitiligo, with se- Approximately 30% of vitiligo cases occur veral susceptibility genes being identified29. To with familial clustering of cases. The biopsy is appreciate the role of genetics in the pathogene- occasionally needed for differentiating vitiligo sis of vitiligo, several analytical approaches are from other hypopigmented or depigmenting di- currently used, such as genetic linkage, candidate sorders. Microscopic examination of involved gene association studies, genome-wide associa- skin shows a complete absence of melanocytes in tion studies (GWAS), DNA sequencing studies association with a total loss of epidermal pigmen- and gene expression studies29,30. To date, there are tation. Superficial perivascular and perifollicular no genetic traits directly related to vitiligo patho- lymphocytic infiltrates may be observed at the genesis. Currently, there are no effective therapies margin of vitiliginous lesions, consistent with a for vitiligo. The goal of our research is focused cell-mediated process destroying melanocytes10. on the genetic and biochemical assessment of me- Vitiligo is related to both genetic and no gene- chanisms involved in the self-cells cytotoxicity in tic factors. Although several theories have been patients with vitiligo and their family relatives. proposed about the etiopathogenesis of vitiligo, This study is performed by assaying the following the precise cause remains unknown11-13. Com- candidate biochemical and genetic issues: monly agreed upon principles are an absence Analysis of the binding proteins level (hapto- of functional melanocytes in vitiligo skin and a globin, ceruloplasmin) in order to evaluate the loss of histochemically recognized melanocytes, binding capability vs. molecules potentially har- owing to their obliteration. Though, the mela- mful to the cell membrane31. nocytes damage is most likely a slow process re- Analysis of the free plasmatic thiols (homocy- sulting in a progressive decrease of melanocyte steine, glutathione) as markers for the hyper-oxi- depletion activities. Theories regarding the de- dation and as a mediator of cytotoxicity. In addi- struction of melanocytes include many factors: tion, analysis of serum vitamin A and E levels, as autoimmune mechanisms, cytotoxic mechanisms, part of antioxidant potential, and folates, vitamins intrinsic melanocyte defects, oxidant-antioxidant B6 and B12 as co-enzyme of the enzymatic acti- mechanisms, and neural mechanisms14, 15. The vities regulating the methyls’ exchange and he- autoimmune theory proposes an alteration in hu- moglobin synthesis32. moral and cellular immunity in the destruction Genetic analysis of the regulatory enzymes of melanocytes of vitiligo. This is an actual re- genes of the re-methylation cycles and trans- levance model, given that no segmental vitiligo sulfuration. Candidate genes for polymorphism is more frequently associated with autoimmune screening are methylene-tetrahydrofolate-re- conditions than segmental vitiligo16-19. For these ductase (MTHFR) C677T and A1298C33; cysta- reasons, certain disorders have been linked to vi- thionine-beta-synthase enzyme (CBS) I278T and tiligo, such as Hashimoto thyroiditis, Graves dise- 844Ins68bp34; and methionine-synthase-reducta- ase, Addison disease, diabetes mellitus, alopecia se (MTRR) A66G35. aerates, pernicious anemia, inflammatory bowel In this issue, we report the genetic profile of the disease, psoriasis, and autoimmune polyglandu- vitiligo patients. lar syndrome20,21. The most substantial sign of autoimmune pathogenesis is the presence of cir- culating Immunoglobulin antibodies (Ab) against Patients and Methods melanocyte proteins in patients with vitiligo22-25. In addition, strong evidence indicates the invol- Patients vement of humoral cellular immune mechanisms. In this study, 43 patients showing vitiligo have As a consequence, melanocytes damaging could been retrospectively enrolled. In addition, 30 he- be directly mediated by T cells autoreactive CD8 althy donors were used as a control cohort. positive26,27. This retrospective study was performed in Vitiligo is characterized by incomplete pene- agreement with the Ethical values laid down by trance, multiple susceptibility loci, and genetic according to the Declaration of Helsinki, and in- heterogeneity. The inheritance of vitiligo may formed consent documentation of each patient was 472 High incidence of MTHFR, CBS and MTRR polymorphisms in vitiligo patients reviewed and agreed by the independent Ethics tion conditions were carried out for 5 minutes at Committee of AOU “Federico II”, Naples, Italy. 95°C followed by 35 cycles of 30 s at 94°C, 30 s at 58°C and 1,5 min at 72°C (extension) and a final Biochemical Assessment of Serum Levels extension of 7 min at 72°C. PCR product (ampli- of Circulating Binding Proteins con) was incubated at 37°C for 4 h for digestion and Vitamins (excluding CBS 844ins68). All products were vi- Analysis of haptoglobin