ASSESSMENT OF MC1R AND α-MSH GENE SEQUENCES IN IRANIAN VITILIGO PATIENTS.

BACKGROUND: Vitiligo is an acquired pigmentary disorder of the skin that is caused by unknown factors and is characterized by white and depigmented patches that enlarge and become more numerous with time. Genetic factors, oxidative stress, autoimmunity, and neurochemical agents, such as catecholamines might also contribute to vitiligo. Cutaneous pigmentation is determined by the amounts of eumelanin and pheomelanin synthesized by the epidermal melanocytes and interference of melanocortin-1 receptor (MC1R), a G-protein coupled receptor, its normal agonist, alpha-melanocyte stimulating hormone (α-MSH), and key enzymes, such as tyrosinase, to protect against sun-induced DNA damage. The MC1R, a 7 pass trans-membrane G-protein coupled receptor, is a key control point in melanogenesis. Loss-of-function mutations at the MC1R are associated with a switch from eumelanin to pheomelanin production, resulting in a red or yellow coat color. AIM: In this research, we aim to examine the genetic variety of MC1R and α-MSH gene in 20 Iranian vitiligo patients and 20 healthy controls.
MATERIALS AND METHODS: Analysis of the MC1R coding gene was performed with direct sequencing.
RESULTS: We found the following 9 MC1R coding region variants: Arg163Gl (G488A), Arg227Leu (G680A), Val 97Phe (G289T), Asp184Asn (G550A), Arg227Lys (G680A), Arg142His (G425A), Val60Leu (G178T), Val247Met (C739A), and Val174Ile (G520A). We also found 2 frameshift changes: one of them was the Insertion of C (frameshift in Pro136, stop at Trp148) and the other, Insertion of G (frameshift in Pro256, stop at Trp 333). Of all the changes, the most common was Val60Leu at 5% in patients vs 20% in controls, Val247Met at 15% in patients vs 0% in controls and Val174Ile at 15% in controls and 0% in patients. The other variants showed a frequency <5% in both patients and controls. Also in this study, we have examined the frequency of single nucleotide polymorphisms within the α-MSH genes with direct sequencing in 20 patients and 20 healthy subjects but found no changes along this gene.
CONCLUSION: We could not find any relationship between MC1R and α-MSH genes and their effect on the disease in Iranian vitiligo patients.

Introduction

Vitiligo is an acquired, idiopathic, hypomelanotic disease characterized by circumscribed depigmented macules.[1] Lack of melanocytes from the lesional skin due to their destruction has been suggested to be the key factor in the pathogenesis of vitiligo.[2] The significant role of cutaneous pigmentation in humans is protection against DNA destruction that is caused by harmful sunrays hitting the skin and fighting against carcinogenesis.[3] Vitiligo has been observed in 1% of the world population and is known to occur most commonly in pigmented races.[4] There are many theories in the etiology of vitiligo, including neurochemical, autoimmune, oxidative stress aspects, and genetic defects, but none of these hypotheses can explain the whole spectrum of vitiligo disorder. The importance of genetic factors in vitiligo has been suggested in reports of significant familial aggregation.[5] The inheritance pattern of vitiligo does not follow the simple Mendelian pattern and its mode of heredity suggests that it is a polygenic disease. Vitiligo seems to be a complex hereditary disease governed by a set of recessive alleles situated at several unlinked autosomal loci, which may be involved in the generation of oxidative stress, melanin synthesis, and autoimmunity, and could collectively confer the vitiligo phenotype.[6] The melanocortin-1 receptor (MC1R) gene, which codes for the melanocyte-stimulating hormone receptor (MSHR), is a key control point in melanogenesis. In humans, a number of loss-of-function mutations in the MC1R have been described.[7] The α-melanocyte-stimulating hormone (α-MSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. The binding of α-MSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments.[8] Melanogenesis is regulated, in part, by the binding of α-MSH and of agouti signaling protein (ASIP) to the MSHR. The binding of ASIP to MSHR precludes α-MSH-initiated signaling and thus blocks the production of c-AMP, leading to a downregulation of eumelanogenesis.[9] MC1R's mRNA expression is downregulated by ASIP.[10] It is also probable that MC1R, ASIP and α-MSH genes in relation with each other and together can have a role in the etiology of vitiligo, but in 2003 Gun Yoen Na et al .[11] stated that there was no significant relationship between MC1R and ASIP genes in Korean vitiligo patients, so for this probability there may be a connection between MC1R and α-MSH. Ohshiro et al . examined the whole of pro-opiomelanocortin (POMC) gene in the Japanese population[12] by direct sequencing, and only 2 silent mutations were detected. Thus, this gene is very conservative and preserved and the probability of any changes in α-MSH gene is very low. However, investigation into these genes can be useful for future studies and any probability of genetic hypothesis can be useful in the treatment with vitiligo patients in future. For this reason, in studying α-MSH and MC1R gene sequences, we put them under direct sequencing test until any changes were found in the vitiligo patients. Hence, by the assessment of these 2 genes, we intended to determine whether the MC1R and α-MSH changes were concerned with vitiligo.

Materials and Methods

Subjects for determination of single nucleotide polymorphisms in MC1R and α-MSH genes

Twenty Iranian vitiligo patients and 20 healthy controls [Table 1], from Guilan province were examined.

Table 1

Characteristics of Vitiligo patients and controls

DNA extraction

The total DNA of the peripheral leukocytes was extracted with QIAamp DNA extraction reagents (Qiagen). The DNA was treated with RNase A, extracted with phenolchloroform (1:1 by volume), precipitated with ethanol, resolubilized in 20 μL of distilled water, and quantified based on the absorbance at 260 nm.

Screening of single nucleotide polymorphisms screening

One pair of primers were used to amplify the region of the coding sequence of MC1R: MC1R-F (5′ -ATCCTTCCTGGACAGGACT-3′ ) and MC1R-R (5′ -CATTTAGTCCATCCTCTTTG-3′) and one pair of primers were used to amplify the part of coding region of α-MSH: α-MSH-F (5′ -TACGTCATGGGCCACTTC-3′) and α-MSH-R (5′ -AGTGCTCCATCCTGTAGG-3′) [Table 2]. The reaction mixture consisted of 50.0 ng of genomic DNA, 10× buffer, 2 mM MgCl2 200 μM of each dNTP, 1 μM of each primer, and 1 unit of Smart Taq DNA polymerase (Cinnagen, Iran). DNA fragments were amplified in an applied biosystem thermal cycler for both the genes using the following program: 94ºC for 6 min, then 30 cycles of the following: denaturation at 92ºC for 50 s, annealing temperature for 50 s at 60ºC, extension at 72ºC for 50 s, and one final step of primer extension at 72ºC for 7 min for both the genes. The amplification products were resolved by gel electrophoresis on 1% agarose gels stained with ethidium bromide in Tris-borate EDTA (TBE) buffer for 50 min at 80 volts. Subsequently, the gels were visualized on ultraviolet light and photodocumentation was performed. The DNA fragment sizes were estimated by comparison with the standard marker 1000-bp DNA ladder (fermentas).

Table 2

Primers used for MC1R and α-MSH amplification

The amplification products were purified with a kit (Qiagen) and sequenced on both forward and reverse directions and analyzed on a DNA sequencer (model 3100 sequencer; ABI) according to the manufacturer's specifications, with the intermediatory of Neday e Fan Company in Iran.

Direct sequencing of the MC1R and α-MSH coding sequences

Direct sequencing of the MC1R and α-MSH coding sequences was performed in all the subjects. The result of the direct sequencing of MC1R is summarized in Table 3; however, no changes were observed in α-MSH gene.

Table 3

Direct sequences result in MC1R gene

Results

Direct sequencing results of the MC1R and α-MSH coding sequences

The direct sequencing of the MC1R coding region was performed in the whole study group. This study showed some variants in both healthy and patients groups, but the frequency of some variants was high in both the groups. We could only find 9 variants in the coding region of MC1R, in all the subjects, whereas 2 frameshift changes were observed only in the patients group. Of all these changes, the most common was Val60Leu at 5% in the patients vs 20% in the controls, so it is possible that this change could be a natural variant in the Iranian population, because not only its frequency in the control subjects was high but also this change was reported as a natural variant in other researches that were carried out before this study [Table 3]. This residue is in the first transmembrane in the MC1R protein. A three-dimensional model of ligand-receptor complexes[13] showed a highly charged region, containing Glu94 (TM2), Asp117 and Asp121 (TM3), and a part of TM1 that has an interaction with an Arg residue in His-Phe-Arg-Trp (HFRW) pharmacophore core shared by the natural MCs. Figure 1 was made using the program MOLSCRIPT by Prusis et al.14 A network of aromatic residues located near the extracellular side of TM4, 5, and 6 could also contribute to the agonist binding by interacting with the aromatic residues of the pharmacophore, so any changes in these TM fragments can affect the agonist tendency to MCRs and in some way affect the c-AMP signaling pathway. The next change was Val247Met that was observed 15% in the patients vs 0% in the controls. This variant is in the third extracellular loops and this region of the MCRs is unusually conserved compared with other GPCRs (G-protein Coupled receptor). It is rich in Cys residues, suggesting a highly specialized function and its important role in MCR function.[15] However, as it is described above, agonist binding is mostly accounted for by a network of charged and aromatic residues located in several TM fragments, including TM6. Since TM6 is connected to the third extracellular loop, any changes in this fragment can affect the MCR function. The other variant that we found was Val174Ile that was 15% in the controls and 0% in the patients. This variant was observed in TM fragments too and can affect the MCR functions. The other variants were less than 5% in both the groups. However, these findings cannot reveal their critical role in the deficiency of vitiligo patients, since some of these changes are only variants and reported before as natural variants. Also in this study, the α-MSH coding sequence was under direct sequencing and we found no changes in all the subjects, since we could not find any relationship between these 2 critical genes and their effect on the disease in Iranian vitiligo patients.

Figure 1

Wall-eyed stereoplot showing positions of the 15 most conserved amino acids found in GPCRs (G-protein Coupled receptor) as viewed from the extracellular side.

Discussion

Several studies have suggested the crucial role of MC1R and α-MSH in regulating eumelanin synthesis and the pigmentary response of human skin to the ultraviolet rays. Recently, in relation to oxidative stress theory of vitiligo development, COMT(catechol O-methyltransferase), and catalase gene single nucleotide polymorphisms (SNPs) were studied for a possible association.[16] The MC1R in the human melanocytes has been reported by Cone et al.[17] and it is a single exon gene without introns; hence, very convenient for sequencing analysis. But this gene has many variants in healthy subjects, so it is not conservative and we cannot call any change a functional mutation. Recently,Széll et al. showed that some SNPs, such as Arg160Trp that caused by C478T, can be important factors against vitiligo disease. They examined C478T SNPs in 108 fair-skinned vitiligo patients and in 70 fair-skinned healthy control individuals, there was a significant difference in the allele frequency between the 2 groups. They revealed that this SNP in healthy subjects was higher than fair-skinned vitiligo patients.[18] This gene sequence directly affects the hair and skin color, because its sequence determines the agonist and antagonist tendencies toward MC1R protein and indirectly affects the c-AMP signaling pathway and induction of eumelanin synthesis. The search for vitiligo genes has been complicated by the fact that depigmentation can be a result of different clinical conditions and environmental factors. Because of the multifactorial features of vitiligo, some variants in MC1R gene or other genes that interfere in pigmentation process cannot illustrate the etiology of vitiligo. Also, in spite of the validity of MC1R gene or other genes, vitiligo appears in some people. Reasons for this ambiguity can be due to any other conditions, but probably due to the presence of some biophysical features of bilayer cell membrane, for example, the percentage of cholesterol or bilayer lipids can affect MC1R structural and functional features. Because biophysical and biochemical features of the bilayer cell membrane determine the fluidity of the cell membrane, and probably this fluidity has influence on resting of such membrane protein and the structure and exact function as an MC1R. Therefore, studies of cell membrane composition and fluidity effects of cell membrane on resting, structure, and function of this membrane protein will be a new hypothesis that probably makes clear the mystery of vitiligo.