Macrophage migration inhibitory factor gene polymorphisms (SNP ‐173 G>C and STR‐794 CATT5‐8) confer risk of plaque psoriasis: A case–control study

Abstract Background Macrophage inhibitory factor (MIF) is a pro‐inflammatory cytokine secreted by several cells, including those in the immune system and the skin. The MIF gene contains the SNP ‐173 G> C and STR ‐794 CATT5‐8 polymorphisms in the promoter region capable of affecting its activity. Our objective was to investigate the MIF polymorphisms as a risk factor for plaque psoriasis (PP) in the Mexican population. Methods We genotyped both MIF polymorphism (rs5844572 and rs755622) in 224 PP patients with a clinical and histopathological diagnosis and 232 control subjects (CS) by the PCR‐RFLP method. MIF serum levels were determined by an ELISA kit. Results We found significant differences in the genotypic and allelic frequencies for the MIF ‐173 G>C polymorphism; carriers of the GC genotype (OR 1.51, 95% CI 1.026–2.228, p = 0.03) and the C allele (OR 1.34, 95% CI 1.005–1.807, p = 0.04) had higher odds to present with PP. Moreover, the 6C haplotype was associated with PP risk (OR 2.10, 95% CI 1.22–3.69, p < 0.01). Also, the ‐173 CC genotype was associated with high MIF serum levels (p < 0.05). Conclusions The ‐173 GC genotype and the 6C haplotype of the MIF polymorphisms are associated with susceptibility to PP in the Mexican population.


| INTRODUC TI ON
Psoriasis is a highly prevalent skin disease that affects 2%-3% of the population worldwide. 1 It is a chronic dermatosis characterized by well-defined erythematous-squamous plaques with excessive keratinocyte proliferation and bilateral symmetric distribution, located mainly on the elbows, knees, sacral region, and scalp. 2,3 The severity of psoriasis is determined according to the PASI index (Psoriasis Area Severity Index), which evaluates the extent, erythema, and thickness of the scales present in the affected area. 4 The plaque-type lesions are a consequence of the inflammation of the skin, epidermal hyperplasia, and angiogenesis due to dysregulation of the skin immune responses. 5 Some authors have suggested that the cytokines expressed in psoriatic skin could explain most of the clinical features of this disease. 6,7 MIF is a cytokine synthesized by leukocytes and cells of tissues exposed to the environment, such as the epithelial lining of the skin. 8 The skin and serum of psoriatic patients express increased levels of MIF, but its pathogenic function in psoriasis is still not clear. 9 High MIF levels have been associated with two polymorphisms of the MIF gene (SNP -173 G>C and STR -794 CATT [5][6][7][8] ), 10,11 as well as with enhanced risk to develop autoimmune, infectious, and chronic inflammatory, [12][13][14][15] including psoriasis, 16,17 psoriatic arthritis, and systemic sclerosis in the Mexican population. 13,18,19 However, its role as a marker of genetic susceptibility to psoriasis has been poorly explored.
This study aimed to determine the association between the SNP -173 G>C and STR -794 CATT [5][6][7][8] polymorphisms of the MIF gene with MIF serum levels and the risk of presenting plaque psoriasis in a Mexican mestizo population.

| Study subjects
This case and controls study included 224 patients with a clinical and histopathological diagnosis of plaque psoriasis. The sample size was calculated using the OpenEpi v. 3.01 software considering 23.6% as the frequency of the minor allele at -173 G>C polymorphism according to previous reports in the Mexican population. 20 The patients were recruited from the Jalisco Dermatological Institute, "Dr.
José Barba Rubio," of the Mexican Ministry of Health, Guadalajara, Jalisco, Mexico. In addition, 232 clinically healthy subjects were also included as controls, with no family relationship to the patients or a psoriasis history. The inclusion criteria for the subjects of both study groups were Mexican mestizo individuals over 18 years of age without any other chronic disease.

| Ethical considerations
The research was carried out following the ethical aspects of the Declaration of Helsinki. Ethical approval (38/IDJ-JAL/2017) was obtained from the Dermatological Institute of Jalisco, "Dr. José Barba Rubio." The participation of the subjects was voluntary, and their consent was formally written and signed by each individual.

| Genomic DNA extraction
Genomic DNA (gDNA) was obtained from peripheral venous blood samples using Miller's modified salting-out technique. 21

| Genotyping
The target sequence of the MIF gene was amplified by the polymerase chain reaction (PCR) technique, using the following conditions: an initial denaturation of 95°C for 4 min, followed by 30 cycles of denaturation at 95°C for 30 s, alignment at 60°C for 30 s, and extension at 72°C for 30 s; and a final extension at 72°C for 2 min. The products amplified by PCR were visualized in polyacrylamide gels The amplified products were digested with the restriction enzyme AluI (New England BioLabs) for 16 h at 37°C. The enzyme generated restriction fragments according to the genotype; in all the amplified products, an additional fragment was obtained 98 bp, due to the presence of another restriction site: CC wild-type homozygous generates three bands (206, 98, and 62 bp); heterozygous GC generates four bands (268, 206, 98, and 62 bp) and homozygous polymorphic GG generates two bands (268 and 98 bp). The obtained products were visualized on 6% polyacrylamide gels ( Figure 1A). The results were confirmed by sequencing some randomly selected samples.
The STR -794 CATT 5-8 polymorphism was genotyped as reported by a previously described protocol. 18 Fragments were generated according to genotype: Allele 5 generated a 208 bp band, allele 6 a 212 bp, 7 a 216 bp, and 8 a 220 bp band. The PCR amplified products were visualized by electrophoresis on 10% polyacrylamide gels ( Figure 1B).

| MIF serum levels
MIF serum levels were quantified using the commercial ELISA kit, Human MIF Immunoassay (R & R&D). The procedure was carried out following the fabricant's instructions. The sensitivity of the assay was 6 pg/ml.

| Statistical analysis
The determination of the genotypic and allelic frequencies of the polymorphisms was carried out by direct counting. The Hardy-Weinberg equilibrium was calculated in the control group. Genotypic and allelic frequencies of the SNP -173 G>C and STR-794 CATT [5][6][7][8] polymorphisms in patients and CS were analyzed by Chi-square using 2 × 2 contingency tables. The association analysis between genotypes and psoriasis was estimated by odds ratio (OR) with a 95% confidence interval, considering a significance level of p < 0.05.
Linkage disequilibrium was evaluated with the SHEsis software.
The statistical analyses were done with the Stata 9.0 software and GraphPad Prism version 8.0.2.

| Clinical characteristics of study subjects
Clinical and demographic characteristics of PP patients and CS are described in Table 1. PP patients had a median age of 50  years, and 62% were male. The average time of evolution of the Linkage disequilibrium was identified between both MIF polymorphisms (D′ value = 0.62, r 2 = 0.17, p < 0.001, Figure 2

| MIF serum levels in PP patients and CS
We did not find significant differences of MIF serum levels between Also, the MIF serum levels were not associated with the clinical variables of the PP patients (data not shown). However, the -173 G>C polymorphism genotypes were associated with differences in MIF serum levels in PP patients ( Figure 4A). In general, the CC genotype carriers had higher MIF serum levels than those carrying the GC (p = 0.02) or GG genotypes (p = 0.01).
On the other hand, the MIF -794 CATT5-8 STR genotypes did not show an association with MIF levels in the study groups ( Figure 4C,D).
MIF serum levels were also analyzed according to MIF haplotypes, but we did not find significant differences (data not shown).

| DISCUSS ION
The main pathological manifestations of psoriasis are inflammation, keratinocyte hyper-proliferation, altered maturation of the  psoriasis (PASI score ≥20) in contrast with us, as 71% of our PP patients were under pharmacological treatment and with mild psoriasis (PASI = 6 ± 0.46). Although we did not find statistical differences in MIF levels by treatment, a trend of higher MIF levels is evident in those patients without treatment (median of MIF serum levels: 3.5 ng/ml vs. 4.9 ng/ml, respectively). On the other hand, if we assume that MIF has a key role in the severity of psoriasis, the lack of correlation in our study could be explained by the low PASI score of our patients.
It could be argued that MIF may play a role in the evolution of All of these would support the suggestions of MIF as a target for future therapies in psoriasis or other skin diseases.
In inflammatory skin diseases, as systemic sclerosis, the fibroblast and mononuclear infiltrating cells produce MIF in skin tissue. This action could be performed through an autocrine/paracrine mechanism, suggesting that MIF participates in the local pro-inflammatory loop that leads to tissue remodeling. 26 Genetic studies have uncovered several loci associated with susceptibility to psoriasis, highlighting the pathogenic involvement of genes related to inflammation; however, not all the underlying genes have been conclusively identified. 27 We observed a significant asso- To the best of our knowledge, this is the first study of the association between MIF polymorphisms and the susceptibility of PP in the

Mexican population. Studies involving ethnically diverse populations
and more detailed clinical manifestations should be conducted to verify our findings.
Some limitations of our study should be considered, such as the heterogeneity of comorbidities and treatments of our study subjects, which could influence the MIF levels. Moreover, the relation between the SNPs, MIF expression, and serum levels should be analyzed to further explore the effects of SNPs on serum levels and hence as a risk for PP.
In conclusion, we have provided evidence that the MIF -173C and MIF 6C haplotype (CATT 6 /-173C) are associated with PP disease risk.
These data also support the association of the MIF -173C allele with higher MIF serum levels, suggesting that overproduction of MIF may be important in mediating pathogenic effects in psoriasis.

ACK N OWLED G EM ENTS
We want to thank the Dermatological Institute of Jalisco, "Dr. José Barba Rubio," for their support; we recognize the work and help provided by Francisco Arturo Miranda-Gomez, Teresa Valencia Ponce, and Maria Isabel Medina-Ortega, who collected the samples for our study. We want to acknowledge the students in our laboratory that collaborated in the process, especially Néstor Oswaldo Alvarado López, Marco Ernesto Ramirez Messina, and we are grateful to the participants of this study.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest in this work.

E TH I C A L A PPROVA L
All experiments involving human subjects were conducted as per the ethical standards of the institutional and/or national research committee as well as the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

I N FO R M ED CO N S ENT
Informed consent was obtained from all individuals.