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Introduction

Psoriasis is a multisystem chronic inflammatory disease, which affects ~2% of the worldwide population (1). It is a multifactorial disease that is characterized by genetic heterogeneity, high heritability, varying age of onset and immunological risk factors (2). In addition, its prevalence has been shown to vary among different populations (2), and numerous clinical variants have previously been identified (3).

Due to the relatively high heritability of psoriasis, genetic analyses, including linkage and/or association studies, have been conducted worldwide in order to identify genes that may affect the risk of developing psoriasis (2). Genome-wide association studies have implicated 36 susceptibility loci in the pathogenesis of psoriasis (4), of which the most relevant is located within the major histocompatibility complex (MHC) on chromosome 6p21.3, adjacent to the MHC class I-C (HLA-C) gene, which is known as psoriasis susceptibility gene 1 (PSORS1) (5).

Various single nucleotide polymorphisms (SNPs) have previously been associated with an increased risk of developing psoriasis. These SNPs are involved in various processes, including: Skin barrier functions [late cornified envelope (LCE) 3E and 3C], interleukin (IL)-23 signaling (IL-23A, IL-23R, and IL-12B), nuclear factor-κB and interferon signaling [nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (NFKBIA), LINC01185, tyrosine kinase 2 (TYK2), potassium voltage-gated channel subfamily H member 7 (KCNH7), IL-28RA, tumor necrosis factor (TNF)-α-induced protein 3 (TNFAIP3), annexin A6 (ANXA6), and the IL-17 cell response [REV3L, TYK2, and IL-23R] (2). In contrast to their risk association, a number of these SNPs have also been shown to protect against psoriasis (6), whereas other studies have not detected an association between these SNPs and psoriasis (7,8). These discrepancies may be due to genetic variations in the populations studied.

Previous studies investigating the association between SNPs and psoriasis have predominantly analyzed European, American, and Asian populations (2,4,6,7); however, it is currently unclear whether these SNPs may also be associated with psoriasis in the Mexican population. Therefore, the present study aimed to investigate the genotypic and allelic frequencies of 32 SNPs at 24 loci, and their association with psoriasis, in a Mestizo population from northeast Mexico.

Materials and methods

Ethics

The present study (register no. DE11-004) was conducted at the Department of Genetics, University Hospital ‘Dr. José Eleuterio González’ (Monterrey, México), with approval from the Committee for Ethics, Research and Biosecurity of the School of Medicine, University Hospital ‘Dr. José Eleuterio González’, Autonomous University of Nuevo León.

Subjects

Written informed consent was obtained from all the patients enrolled, following an explanation of the nature of the study. A total of 46 patients (32 males and 14 females; age, 26–72 years), who had been diagnosed with chronic plaque psoriasis, were recruited from the Dermatology Outpatient Clinic at the University Hospital ‘Dr. José Eleuterio González’ between November 2011 and May 2012. Patients diagnosed with other clinical subtypes of psoriasis or other genetic diseases, as well as affected relatives of the patients in the study, were excluded.

Controls

A total of 103 peripheral blood samples (from 56 males and 47 females; age, 26–72 years) from control subjects without psoriasis (age, 26–72 years) were obtained from the DNA Bank of the Department of Genetics at the University Hospital ‘Dr. José Eleuterio González’.

SNP selection

A total of 32 SNPs from 24 genes and 2 intergenic regions, which were previously associated with psoriasis in various populations, were selected. The SNPs included in the study were associated with the following genes: HLA-C, IL-12B, IL-23R, IL-23A, IL-28RA, TNF-α, ring finger protein-114 (RNF114), cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (CDKAL1), LCE3D, signal transducer and activator of transcription 4 (STAT4), LINC01185, KCNH7, IL-13, ANXA6, endoplasmic reticulum aminopeptidase 1 (ERAP1), TNFAIP3, REV3L, Leptin, NFKBIA, F-box and leucine-rich repeat protein 19 (FBXL19), nitric oxide synthase 2 (NOS2), cluster of differentiation 40 (CD40), nuclear receptor coactivator 5 (NCOA5), and ADAM metallopeptidase domain 33 (ADAM33). These genes are primarily associated with immune and inflammatory responses. The included genes were selected on the basis of previously reported odds ratios (OR), which suggested that they were closely associated with psoriasis (2).

Genotyping

Molecular analyses were conducted using the TaqMan® assay (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and were analyzed using the OpenArray® NT Genotyping system (Applied Biosystems; Thermo Fisher Scientific, Inc.). DNA was extracted from 200 µL EDTA-treated whole blood samples, using the QIAamp Blood Mini kit on the automated QIAcube system (Qiagen, Hilden, Germany). Purified DNA was collected in a final volume of 150 µL and stored at −20°C until use. DNA for OpenArray (OA) analysis was diluted at a concentration of 50 ng/µl and a ratio of A260/280 and A260/230 of 1.6–1.9. A non-template control, which consisted of DNase-free double-distilled water, was used in each plate assay. The DNA sample (50 ng/µl) and TaqMan OpenArray Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) were mixed in a 384-well plate, and transferred to the OA plate using an autoloader, after which the OA plate was filled with immersion fluid and sealed with glue. Multiplex TaqMan® assay reactions were carried out in a Dual Flat Block GeneAmp PCR system 9700 (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the PCR cycling conditions were as follows: An initial step at 93°C for 10 min; followed by 50 cycles at 95°C for 45 sec, 94°C for 13 sec and 53°C for 2 min, 14 sec; followed by a final step at 25°C for 2 min. The samples were maintained at 4°C until further use. The plate was designed to analyze 32 TaqMan® assays for each sample. The allele analysis was conducted using the TaqMan® Genotyper Software 1.0, with the default parameters, according to the manufacturer's protocol (Applied Biosystems; Thermo Fisher Scientific, Inc.). The accuracy of the genotyping was assessed by comparisons with 15 samples, which had been genotyped three times, resulting in 45 comparison per SNP.

Statistical analysis

Genetic analyses were conducted using the Golden Helix SNP & Variation Suite 7.6 program (Golden Helix, Inc., Bozeman, MT, USA). The 32 SNPs were analyzed for deviation from the Hardy-Weinberg equilibrium using the Fisher's exact test (P<0.01 was considered to indicate a statistically significant difference). An association study was performed using the Basic Allelic test and Fisher's exact test in order to assess ORs, 95% confidence intervals (CIs) and P-values. In addition, Bonferroni P-values and false discovery rates (FDR) were calculated in order to exclude spurious associations. P≤0.05/32 SNPs (1.56×10−3) was considered to indicate a statistically significant difference.

Results

Clinical characteristics of the study population

The genetic analysis included samples from 46 patients and 103 controls (age range, 26–72 years). Of the patients, 32 were male (69.56%) and 14 were female (30.43%), of which 6 patients (13.04%) had a family history of psoriasis. A total of 17 patients (36.95%) had mild, 25 patients (54.34%) had moderate, and 2 patients (4.34%) presented severe psoriasis. Articular and nail involvement were detected in 4 (8.69%) and 18 (39.13%) patients respectively.

Genotype frequencies and OR

Genotype frequencies of the 32 SNPs in both groups are presented in Table I. For the control group, two of the SNPs (the rs1265181 allele of the HGC27 gene and the rs20541 allele of the IL-13 gene) were not in Hardy-Weinberg equilibrium (P<0.01).

Table I. SNPs analyzed in patients with psoriasis and controls.

Table I.

SNPs analyzed in patients with psoriasis and controls.

			Fisher's Exact				Allele Freq.		Fisher's HWE P
Gene/SNP	SNP	Chr	P	Bonf. P	FDR	OR (95% CI)	Cases	Controls	Cases	Controls
HLA-C	rs10484554	6	0.003	0.100	0.100	T 3.51 (1.54–7.99)	0.167	0.054	0.582	1.000
						C 0.28 (0.13–0.65)	0.833	0.946
HCG27	rs1265181	6	0.010	0.326	0.163	C 2.83 (1.29–6.23)	0.163	0.064	0.009	0.003
						G 0.35 (0.16–0.78)	0.837	0.936
RNF114	rs6125829	20	0.014	0.444	0.148	T 0.51 (0.30–0.86)	0.318	0.480	0.489	0.070
						G 1.98 (1.17–3.35)	0.682	0.520
IL-12B	rs3213094	5	0.018	0.569	0.142	T 0.51 (0.30–0.89)	0.267	0.414	0.701	0.100
						C 1.94 (1.13–3.36)	0.733	0.586
	rs3212227	5	0.025	0.805	0.161	G 0.53 (0.31–0.92)	0.267	0.406	0.701	0.408
						T 1.88 (1.09–3.24)	0.733	0.594
	rs6887695	5	0.269	1.000	0.614	C 0.72 (0.41–1.27)	0.239	0.304	0.698	0.484
						G 1.39 (0.79–2.44)	0.761	0.696
ANXA6	rs17728338	5	0.055	1.000	0.293	A 2.41 (1.00–5.77)	0.120	0.053	0.103	0.245
						G 0.42 (0.17 −0.99)	0.880	0.947
ADAM33	rs512625	20	0.078	1.000	0.356	A 1.75 (0.96–3.19)	0.250	0.160	1.000	0.284
						G 0.57 (0.31–1.04)	0.750	0.840
	rs597980	20	0.609	1.000	0.974	G 1.18 (0.71–1.94)	0.489	0.448	1.000	0.067
						A 0.85 (0.52–1.40)	0.511	0.552
	rs677044	20	0.524	1.000	0.931	G 1.27 (0.69–2.33)	0.217	0.180	0.661	0.736
						A 0.79 (0.43–1.45)	0.783	0.820
LTA; TNF; LTB	rs361525	6	0.084	1.000	0.337	A 2.08 (0.95–4.58)	0.144	0.075	0.206	0.433
						G 0.48 (0.22–1.06)	0.856	0.925
	rs1800629	6	0.097	1.000	0.346	A 2.20 (0.93–5.18)	0.120	0.058	0.103	1.000
						G 0.46 (0.19–1.07)	0.880	0.942
	rs1799964	6	0.642	1.000	0.978	C 0.84 (0.45–1.57)	0.185	0.213	1.000	0.039
						T 1.19 (0.64–2.23)	0.815	0.787
IL-13	rs20541	5	0.103	1.000	0.329	G 1.55 (0.94–2.54)	0.565	0.456	0.227	0.003
						A 0.65 (0.39–1.06)	0.435	0.544
NFKBIA	rs8016947	14	0.155	1.000	0.451	T 0.64 (0.36–1.15)	0.207	0.289	0.659	0.095
						G 1.56 (0.87–2.82)	0.793	0.711
NCOA5	rs2903908	20	0.222	1.000	0.592	C 0.62 (0.29–1.31)	0.109	0.165	1.000	1.000
						T 1.62 (0.76–3.44)	0.891	0.835
NOS2	rs4795067	17	0.267	1.000	0.657	G 1.39 (0.82–2.37)	0.337	0.267	1.000	0.799
						A 0.72 (0.42–1.22)	0.663	0.733
CDKAL1	rs6908425	6	0.286	1.000	0.610	T 0.68 (0.36–1.28)	0.174	0.235	0.601	0.788
						C 1.46 (0.78–2.74)	0.826	0.765
IL-28RA	rs4649203	1	0.287	1.000	0.574	G 0.56 (0.22–1.44)	0.065	0.110	1.000	1.000
						A 1.77 (0.69–4.53)	0.935	0.890
LCE3D	rs4112788	1	0.523	1.000	0.984	A 1.21 (0.74–1.99)	0.435	0.388	0.382	0.305
						G 0.83 (0.50–1.36)	0.565	0.612
KCNH7	rs17716942	2	0.597	1.000	1.000	C 0.67 (0.21–2.11)	0.043	0.064	1.000	1.000
						T 1.50 (0.47–4.72)	0.957	0.936
IL-23A;STAT2	rs2066808	12	0.651	1.000	0.948	G 1.29 (0.55–3.03)	0.098	0.078	0.351	1.000
						A 0.78 (0.33–1.83)	0.902	0.922
LEP	rs7799039	7	0.702	1.000	0.977	A 1.11 (0.67–1.83)	0.413	0.388	0.225	0.678
						G 0.90 (0.55–1.49)	0.587	0.612
TNFAIP3	rs610604	6	0.705	1.000	0.940	G 0.89 (0.54–1.46)	0.413	0.442	0.225	1.000
						T 1.12 (0.68–1.85)	0.587	0.558
LOC101927805	rs6809854	3	0.779	1.000	0.997	G 0.91 (0.52–1.60)	0.267	0.285	0.245	0.806
-LOC10537697						A 1.10 (0.63–1.92)	0.733	0.715
STAT4	rs7574865	2	0.800	1.000	0.984	T 1.10 (0.67–1.80)	0.435	0.413	0.227	0.546
						G 0.91 (0.56–1.50)	0.565	0.587
REV3L	rs240993	6	0.800	1.000	0.948	C 0.91 (0.55–1.49)	0.413	0.437	0.545	0.228
						T 1.10 (0.67–1.81)	0.587	0.563
IL-23R	rs7530511	1	0.858	1.000	0.980	T 0.92 (0.44–1.89)	0.130	0.141	0.561	0.414
						C 1.09 (0.53–2.25)	0.870	0.859
LINC01185	rs702873	2	0.887	1.000	0.978	T 0.92 (0.52–1.61)	0.250	0.267	0.242	0.449
						C 1.09 (0.62–1.92)	0.750	0.733
ERAP1;CAST	rs27524	5	0.887	1.000	0.946	A 0.94 (0.53–1.65)	0.250	0.262	0.430	0.071
						G 1.07 (0.61–1.88)	0.750	0.738
FBXL19	rs12924903	16	1.000	1.000	1.000	A 0.98 (0.59–1.63)	0.380	0.385	0.032	0.529
						G 1.02 (0.61–1.69)	0.620	0.615
CD40	rs4810485	20	1.000	1.000	1.000	T 1.03 (0.56–1.90)	0.207	0.201	1.000	1.000
						G 0.97 (0.53–1.78)	0.793	0.799

[i] P<0.05 indicates a statistically significant difference. CI, confidence interval; OR, odds ratio; SNP, single nucleotide polymorphism; P, P-value; Bonf. Bonferroni correction; FDR, false discovery rate; HWE, Hardy-Weinberg Equilibrium; Chr, chromosome; HLA-C, major histocompatibility complex class I-C; HCG27, HLA complex group 27; RNF114, ring finger protein-114; IL, interleukin; ANXA6, annexin A6; ADAM33, ADAM metallopeptidase domain 33; LTA, lymphotoxin alpha; TNF, tumor necrosis factor; LTB, lymphotoxin beta; NFKBIA, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha; NCOA5, nuclear receptor coactivator 5; NOS2, nitric oxide synthase 2; CDKAL1, cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1; LCE, late cornified envelope; KCNH7, potassium voltage-gated channel subfamily H member 7; STAT, signal transducer and activator of transcription; LEP, leptin; TNFAIP3, TNF-α-induced protein 3; REV3L, REV3-like polymerase (DNA directed) zeta catalytic subunit; LINC01185, long intergenic non-protein coding RNA 1185; ERAP1, endoplasmic reticulum aminopeptidase 1; CAST, calpastatin; FBXL19, F-box and leucine-rich repeat protein 19; CD40, cluster of differentiation 40.

Of the 32 SNPs, six were shown to confer risk for psoriasis, including: HLA-C rs10484554 (T allele; OR 3.51; 95% CI 1.54–7.99), HCG27 rs1265181 (C allele; OR 2.83; 95% CI 1.29–6.23), ANXA6 rs17728338 (A allele; OR 2.41; 95% CI 1.00–5.77), RNF114 rs6125829 (G allele; OR 1.98; 95% CI 1.17–3.35), IL-12B rs3213094 (C allele; OR 1.94; 95% CI 1.13–3.36) and IL-12B rs3212227 (T allele; OR 1.88; 95% CI 1.09–3.24) (Table I) (Fisher's exact test P<0.05). However, following FDR and Bonferroni correction (threshold for genome-wide significance, P<1.56×10−3), statistical significance was no longer detected.

Discussion

In the present study, six SNPs corresponding to different genes were associated with an increased risk of developing psoriasis, of which the HLA-C rs10484554 SNP exhibited the highest (T allele; OR 3.51) and the IL-12B rs3213094 SNP exhibited the lowest OR (C allele; OR 1.94).

The HLA-C gene has most frequently been associated with psoriasis in previous studies investigating other populations (9–11). This locus is known as PSORS1 and is located in the 6p21.3 genomic region. The SNP rs10484554 (12) is located 34.7 kb upstream of the HLA-C start site, and has previously been associated with immune responses, including antigen presentation and natural killer cell regulation (13).

The rs1265181 SNP of the HCG27 gene has previously been shown to have a markedly positive correlation with psoriasis in the Chinese population (2,13,14).

The rs6125829 SNP of the RNF114 gene was previously associated with psoriasis in British patients of north European descent (13), and in the present study. The RNF114 gene localizes to the 20q13 locus and encodes a cytosolic protein that has an important role in the ubiquitin pathway. A previous study has detected abundant RNF114 expression in disease-associated tissues, including CD4+ T-lymphocytes, dendritic cells and the skin (15).

The rs3212227 and rs3213094 SNPs of the IL-12B gene have previously been associated with psoriasis in European, North American, and Japanese populations (14–16). These SNPs are located in the 5q31.1–q33.1 chromosomal region, which is known as PSORS11. In addition, the rs3212227 SNP has previously been associated with various other autoimmune diseases, including type 1 diabetes mellitus, due to its effects on T-helper (Th)1 cells (16). The IL-12B gene encodes the p40 subunit of IL-12, which is also found in IL-23. This is consistent with previous studies that have associated IL-12 and IL-23 with psoriasis (16–18). IL-12 induces T-helper cells, whereas IL-23 expands and maintains Th17 cells. In previous studies, biological therapies against the p40 subunit were effective in patients with psoriasis (18–21). The results of the present study suggested that the IL-12B rs3212227 T and rs3213094 C SNPs confer an increased risk for psoriasis; however, the two IL-23 SNPs that were analyzed, including IL-23R rs7530511 and IL-23A rs2066808, did not. It would be interesting to determine whether the IL-12B polymorphisms are associated with the response to drugs targeting the p40 subunit in the Mexican population.

The ANXA6 gene belongs to the conserved annexin protein family, which is a group of Ca2+-dependent membrane binding proteins. It is an important candidate gene for developing psoriasis, since it is involved in plasma membrane repair. ANXA6 may have a role in receptor signaling at the cell surface, growth factor and lipoprotein receptor trafficking, Ca2+-channel activity and T cell activation (22). In the present study, ANXA6 was associated with psoriasis in the Mexican Mestizo population, and has also previously been associated with psoriasis in the Han Chinese population (23).

The present study demonstrated that the allele frequencies of rs20541 and rs1265181 were similar to reported frequencies for Mexican Ancestry in Los Angeles (CA, USA); therefore, the Hardy-Weinberg disequilibrium detected here may be due to a missense mutation in rs20541 (445 Gln>Arg). For rs1265181, the disequilibrium may be due to population substructure in this intergenic region between loci associated to multiple inmunes diseases and the HLA complex group 27, which is non-protein coding; however, further studies are necessary to fully elucidate this. The other SNPs analyzed in the present study did not exhibit an association with psoriasis in the Mexican Mestizo population. It is important to mention that the alternative non-risk associated alleles from the various genes may confer protection against psoriasis (protection indicated by OR<1).

To the best of our knowledge, the present study is the first to investigate the association between genetic regions that have previously been associated with psoriasis in other populations, including Asian and European populations, and psoriasis in the Mexican Mestizo population. The results of the present study suggested that SNPs in the HLA-C, IL-12B, HCG27, ANXA6 and RNF114 genes were associated with psoriasis in a Mestizo population from northeastern Mexico. Following FDR and Bonferroni correction (threshold for genome-wide significance P<1.56×10−3), a statistically significant difference between the patient and control groups was no longer evident; however, as the case control relationship was 1:2.3, it is necessary to assess a larger group of patients in the future in order to increase the statistical power and validate these preliminary results. Furthermore, future studies should include subjects from diverse Mexican populations, since there is significant genetic heterogeneity among the Mexican population. The Mexican population is a good model for genetic studies, due to the vast ethnic diversity among native and Mestizo populations. The results of the present study may be considered a reference in genetic studies of characterized populations investigating gene frequencies and associations.

In conclusion, the present study observed that in Mexican patients with plaque psoriasis, SNPs from different genes associated with immune response or membrane cell repair confer an increased risk for disease development.

References