• Authors:
  • Alejandra Villarreal‑Martínez
  • Hugo Gallardo‑Blanco
  • Ricardo Cerda‑Flores
  • Iris Torres‑Muñoz
  • Minerva Gómez‑Flores
  • Julio Salas‑Alanís
  • Jorge Ocampo‑Candiani
  Laura Martínez‑Garza

View Affiliations / Copyright

Affiliations:Department of Dermatology, University Hospital ‘Dr. José Eleuterio González’, Monterrey, Nuevo León 64460, México, Department of Genetics, University Hospital ‘Dr. José Eleuterio González’, Monterrey, Nuevo León 64460, México, Population Genetics Department, School of Nursing, Autonomous University of Nuevo León, Monterrey, Nuevo León 64460, México

Copyright: ©Villarreal‑Martínezet al.This is an open access article distributed under theterms ofCreative Commons Attribution License.
• Pages:1217-1222
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  Published online on:February 9, 2016
    https://doi.org/10.3892/etm.2016.3066
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Introduction

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

Due to the relatively high heritability ofpsoriasis, genetic analyses, including linkage and/or associationstudies, have been conducted worldwide in order to identify genesthat may affect the risk of developing psoriasis (2). Genome-wide association studies haveimplicated 36 susceptibility loci in the pathogenesis of psoriasis(4), of which the most relevant islocated within the major histocompatibility complex (MHC) onchromosome 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) havepreviously been associated with an increased risk of developingpsoriasis. These SNPs are involved in various processes, including:Skin barrier functions [late cornified envelope (LCE) 3E and3C], interleukin (IL)-23 signaling (IL-23A,IL-23R, andIL-12B), nuclear factor-κB and interferonsignaling [nuclear factor of kappa light polypeptide gene enhancerin B-cells inhibitor-alpha (NFKBIA), LINC01185, tyrosinekinase 2 (TYK2), potassium voltage-gated channel subfamily Hmember 7 (KCNH7),IL-28RA, tumor necrosis factor(TNF)-α-induced protein 3 (TNFAIP3), annexin A6(ANXA6), and the IL-17 cell response [REV3L,TYK2, andIL-23R] (2).In contrast to their risk association, a number of these SNPs havealso been shown to protect against psoriasis (6), whereas other studies have not detectedan association between these SNPs and psoriasis (7,8). Thesediscrepancies may be due to genetic variations in the populationsstudied.

Previous studies investigating the associationbetween 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 beassociated with psoriasis in the Mexican population. Therefore, thepresent study aimed to investigate the genotypic and allelicfrequencies of 32 SNPs at 24 loci, and their association withpsoriasis, in a Mestizo population from northeast Mexico.

Materials and methods

Ethics

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

Subjects

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

Controls

A total of 103 peripheral blood samples (from 56males and 47 females; age, 26–72 years) from control subjectswithout psoriasis (age, 26–72 years) were obtained from the DNABank 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 intergenicregions, which were previously associated with psoriasis in variouspopulations, were selected. The SNPs included in the study wereassociated with the following genes:HLA-C,IL-12B,IL-23R,IL-23A,IL-28RA,TNF-α, ringfinger protein-114 (RNF114), cyclin-dependent kinase 5regulatory 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 ofdifferentiation 40 (CD40), nuclear receptor coactivator 5(NCOA5), andADAM metallopeptidase domain 33(ADAM33). These genes are primarily associated with immuneand inflammatory responses. The included genes were selected on thebasis of previously reported odds ratios (OR), which suggested thatthey 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 Genotypingsystem (Applied Biosystems; Thermo Fisher Scientific, Inc.). DNAwas extracted from 200 µL EDTA-treated whole blood samples, usingthe QIAamp Blood Mini kit on the automated QIAcube system (Qiagen,Hilden, Germany). Purified DNA was collected in a final volume of150 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 ofA260/280 and A260/230 of 1.6–1.9. A non-template control, whichconsisted of DNase-free double-distilled water, was used in eachplate assay. The DNA sample (50 ng/µl) and TaqMan OpenArray MasterMix (Applied Biosystems; Thermo Fisher Scientific, Inc.) were mixedin a 384-well plate, and transferred to the OA plate using anautoloader, after which the OA plate was filled with immersionfluid and sealed with glue. Multiplex TaqMan® assay reactions werecarried out in a Dual Flat Block GeneAmp PCR system 9700 (AppliedBiosystems; Thermo Fisher Scientific, Inc.), and the PCR cyclingconditions 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°Cfor 2 min, 14 sec; followed by a final step at 25°C for 2 min. Thesamples were maintained at 4°C until further use. The plate wasdesigned to analyze 32 TaqMan® assays for each sample. The alleleanalysis was conducted using the TaqMan® Genotyper Software 1.0,with the default parameters, according to the manufacturer'sprotocol (Applied Biosystems; Thermo Fisher Scientific, Inc.). Theaccuracy of the genotyping was assessed by comparisons with 15samples, which had been genotyped three times, resulting in 45comparison per SNP.

Statistical analysis

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

Results

Clinical characteristics of the studypopulation

The genetic analysis included samples from 46patients and 103 controls (age range, 26–72 years). Of thepatients, 32 were male (69.56%) and 14 were female (30.43%), ofwhich 6 patients (13.04%) had a family history of psoriasis. Atotal of 17 patients (36.95%) had mild, 25 patients (54.34%) hadmoderate, 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 groupsare presented inTable I. For thecontrol group, two of the SNPs (the rs1265181 allele of theHGC27 gene and the rs20541 allele of theIL-13 gene)were not in Hardy-Weinberg equilibrium (P<0.01).

Table I. SNPs analyzed in patients withpsoriasis and controls.

Table I.

SNPs analyzed in patients withpsoriasis 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<0.05indicates a statistically significant difference. CI, confidenceinterval; 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 fingerprotein-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 lightpolypeptide gene enhancer in B-cells inhibitor-alpha;NCOA5,nuclear receptor coactivator 5;NOS2, nitric oxide synthase2;CDKAL1, cyclin-dependent kinase 5 regulatorysubunit-associated protein 1-like 1;LCE, late cornifiedenvelope;KCNH7, potassium voltage-gated channel subfamily Hmember 7;STAT, signal transducer and activator oftranscription;LEP, leptin;TNFAIP3, TNF-α-inducedprotein 3;REV3L,REV3-like polymerase (DNA directed)zeta catalytic subunit;LINC01185, long intergenicnon-protein coding RNA 1185;ERAP1, endoplasmic reticulumaminopeptidase 1;CAST, calpastatin;FBXL19, F-boxand leucine-rich repeat protein 19;CD40, cluster ofdifferentiation 40.

Of the 32 SNPs, six were shown to confer risk forpsoriasis, 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% CI1.00–5.77),RNF114 rs6125829 (G allele; OR 1.98; 95% CI1.17–3.35),IL-12B rs3213094 (C allele; OR 1.94; 95% CI1.13–3.36) andIL-12B rs3212227 (T allele; OR 1.88; 95% CI1.09–3.24) (Table I) (Fisher's exacttest P<0.05). However, following FDR and Bonferroni correction(threshold for genome-wide significance,P<1.56×10−3), statistical significance was no longerdetected.

Discussion

In the present study, six SNPs corresponding todifferent genes were associated with an increased risk ofdeveloping psoriasis, of which theHLA-C rs10484554 SNPexhibited the highest (T allele; OR 3.51) and theIL-12Brs3213094 SNP exhibited the lowest OR (C allele; OR 1.94).

TheHLA-C gene has most frequently beenassociated with psoriasis in previous studies investigating otherpopulations (9–11). This locus is known asPSORS1and is located in the 6p21.3 genomic region. The SNP rs10484554(12) is located 34.7 kb upstream oftheHLA-C start site, and has previously been associatedwith immune responses, including antigen presentation and naturalkiller cell regulation (13).

The rs1265181 SNP of theHCG27 gene haspreviously been shown to have a markedly positive correlation withpsoriasis in the Chinese population (2,13,14).

The rs6125829 SNP of theRNF114 gene waspreviously associated with psoriasis in British patients of northEuropean descent (13), and in thepresent study. TheRNF114 gene localizes to the 20q13 locusand encodes a cytosolic protein that has an important role in theubiquitin pathway. A previous study has detected abundantRNF114 expression in disease-associated tissues, includingCD4+ T-lymphocytes, dendritic cells and the skin(15).

The rs3212227 and rs3213094 SNPs of theIL-12B gene have previously been associated with psoriasisin European, North American, and Japanese populations (14–16).These SNPs are located in the 5q31.1–q33.1 chromosomal region,which is known asPSORS11. In addition, the rs3212227 SNPhas previously been associated with various other autoimmunediseases, including type 1 diabetes mellitus, due to its effects onT-helper (Th)1 cells (16). TheIL-12B gene encodes the p40subunit of IL-12, which is also found in IL-23. This is consistentwith previous studies that have associated IL-12 and IL-23 withpsoriasis (16–18). IL-12 induces T-helper cells, whereasIL-23 expands and maintains Th17 cells. In previousstudies, biological therapies against the p40 subunit wereeffective in patients with psoriasis (18–21). Theresults of the present study suggested that theIL-12Brs3212227 T and rs3213094 C SNPs confer an increased risk forpsoriasis; however, the twoIL-23 SNPs that were analyzed,includingIL-23R rs7530511 andIL-23A rs2066808, didnot. It would be interesting to determine whether theIL-12Bpolymorphisms are associated with the response to drugs targetingthe p40 subunit in the Mexican population.

TheANXA6 gene belongs to the conservedannexin protein family, which is a group ofCa2+-dependent membrane binding proteins. It is animportant candidate gene for developing psoriasis, since it isinvolved in plasma membrane repair.ANXA6 may have a role inreceptor signaling at the cell surface, growth factor andlipoprotein receptor trafficking, Ca2+-channel activityand T cell activation (22). In thepresent study,ANXA6 was associated with psoriasis in theMexican Mestizo population, and has also previously been associatedwith psoriasis in the Han Chinese population (23).

The present study demonstrated that the allelefrequencies ofrs20541 andrs1265181 were similar toreported frequencies for Mexican Ancestry in Los Angeles (CA, USA);therefore, the Hardy-Weinberg disequilibrium detected here may bedue to a missense mutation in rs20541 (445 Gln>Arg). Forrs1265181, the disequilibrium may be due to population substructurein this intergenic region between loci associated to multipleinmunes diseases and the HLA complex group 27, which is non-proteincoding; however, further studies are necessary to fully elucidatethis. The other SNPs analyzed in the present study did not exhibitan association with psoriasis in the Mexican Mestizo population. Itis important to mention that the alternative non-risk associatedalleles from the various genes may confer protection againstpsoriasis (protection indicated by OR<1).

To the best of our knowledge, the present study isthe first to investigate the association between genetic regionsthat have previously been associated with psoriasis in otherpopulations, including Asian and European populations, andpsoriasis in the Mexican Mestizo population. The results of thepresent study suggested that SNPs in theHLA-C, IL-12B, HCG27,ANXA6 andRNF114 genes were associated with psoriasis ina Mestizo population from northeastern Mexico. Following FDR andBonferroni correction (threshold for genome-wide significanceP<1.56×10−3), a statistically significant differencebetween the patient and control groups was no longer evident;however, as the case control relationship was 1:2.3, it isnecessary to assess a larger group of patients in the future inorder to increase the statistical power and validate thesepreliminary results. Furthermore, future studies should includesubjects from diverse Mexican populations, since there issignificant genetic heterogeneity among the Mexican population. TheMexican population is a good model for genetic studies, due to thevast ethnic diversity among native and Mestizo populations. Theresults of the present study may be considered a reference ingenetic studies of characterized populations investigating genefrequencies and associations.

In conclusion, the present study observed that inMexican patients with plaque psoriasis, SNPs from different genesassociated with immune response or membrane cell repair confer anincreased risk for disease development.

References