PSORS1 Locus Genotyping Profile in Psoriasis: A Pilot Case-Control Study

(1) Background: The psoriasis susceptibility 1 (PSORS1) locus, located within the major histocompatibility complex, is one of the main genetic determinants for psoriasis, the genotyping profile for three single-nucleotide polymorphisms (SNPs) comprising the PSORS1 locus: rs1062470 within PSORS1C1/CDSN genes, rs887466 within PSORS1C3 gene, rs10484554 within LOC105375015 gene, were investigated and correlated with psoriasis risk and severity. (2) Methods: This pilot case-controlled study involved 100 psoriatic patients and 100 healthy individuals. We investigated three SNPs and assessed the relative gene expression profile for the PSORS1C1 gene. We then correlated the results with both disease risk and severity. (3) Results: The most significantly associated SNP in PSORS1 locus with psoriasis was rs10484554 with its C/T genotype 5.63 times more likely to develop psoriasis under codominant comparison. Furthermore, C/T and T/T genotypes were 5 times more likely to develop psoriasis. The T allele was 3 times more likely to develop psoriasis under allelic comparison. The relative gene expression of PSORS1C1 for psoriatic patients showed to be under-expressed compared to normal controls. (4) Conclusions: Our study revealed the association of the three studied SNPs with psoriasis risk and severity in an Egyptian cohort, indicating that rs10484554 could be the major key player in the PSORS1 locus.


Introduction
Psoriasis is a common inflammatory skin disease of multifactorial origin that causes significant stress and morbidity [1]. It most often presents with well-demarcated, scaling and erythematous plaques, often at the extensor surfaces of knees and elbows [2]. Until now, the definite etiopathogenesis of psoriasis is not fully understood, however, it is widely regarded as a multifactorial disorder caused by the interaction between inherited susceptibility alleles and environmental triggers (e.g., stress, mechanical trauma and streptococcal infections) in combination with skin barrier disruption and immune dysfunction [3,4]. Recent advancements for expanding our understanding of psoriasis pathophysiology and targeted therapies are currently a hot topic in research [5,6].
To date, and to the best of our knowledge, no data has been reported about PSORS1 locus SNPs among any Egyptian cohort, specifically for rs1062470, rs887466 and rs10484554 genetic variants as possible risk factors for psoriasis. Therefore, this study is the first to provide data about the association between these SNPs and psoriasis predisposition in the Egyptian population. This might help in anticipating the disease and early prophylactic measures could be taken.  [15] and g:profiler tools https://biit.cs.ut.ee/gprofiler/gost (accessed on 1 March 2022) [16].
To date, and to the best of our knowledge, no data has been reported about PSORS1 locus SNPs among any Egyptian cohort, specifically for rs1062470, rs887466 and rs10484554 genetic variants as possible risk factors for psoriasis. Therefore, this study is the first to provide data about the association between these SNPs and psoriasis predisposition in the Egyptian population. This might help in anticipating the disease and early prophylactic measures could be taken.

Materials and Methods
A case-control study was conducted on two hundred participants. Written informed consent was taken from each patient before enrollment in the study. The study participants were divided into two groups: 100 Egyptian patients diagnosed with chronic plaque psoriasis of both genders with ages above 16 years old were recruited from the Dermatology Outpatient Clinics, and we excluded patients with psoriatic arthritis (PsA) or autoimmune diseases; and 100 healthy non-related participants of Egyptian descent, matched by age and gender to the patients with no family history of psoriasis or autoimmune diseases. All patients were subjected to full history taking and detailed dermatological examination. The severity of psoriasis was assessed using a PASI score that included an assessment of four body areas: head and neck (H), upper limbs (UL), trunk (T) and lower limbs (LL). Within each area, the severity of three signs, erythema (E), thickness/induration (I) and desquamation/scaling (D), is each assessed on a five-point scale: 0, none; 1, mild; 2, moderate; 3, severe; 4, very severe. According to the European consensus, interpretation of PASI is mild if the PASI score is <10, moderate if the PASI score is 10-20 and severe if PASI is >20 [17]. This study was performed in compliance with the guidelines of the Helsinki Declaration, 2013. Approval was taken from the Research Ethics Committee and the Institutional Review Board.

SNP Selection
The three studies' SNPs were selected based on the level of evidence demonstrated by the number of publications studied, with each SNP adopted from https://opensnp.org/ (accessed on 1 March 2022) [18]. The rs10484554 exhibited a high level of evidence equivalent to 37 publications, while rs1062470 and rs887466 values were 4 and 7 publications, respectively. Our selection was also based on the latest findings of Wiśniewski et al. [19], who studied the same SNPs with a proven significance in psoriatic Poland patients, supported by the fact that these SNPs were not investigated among Egyptians in any published research.

Molecular Analysis
Lab work was performed in the Center of Excellence in Molecular and Cellular Medicine & Genetics Unit using three milliliters of venous blood in an EDTA anticoagulant vacutainer. They were kept at −20 • C till DNA extraction was performed.

DNA Extraction
Genomic DNA was extracted using the Invitrogen Gene Catcher purification system (Thermofisher, Waltham, MA, USA) from the frozen venous blood according to the manufacturer's instructions. DNA concentration and purity were determined using a NanoDrop 2000 1C spectrophotometer (NanoDrop Tech., Inc. Wilmington, DE, USA).

Allelic Discrimination Analysis
The chosen genetic variants, rs1062470 (C__2438414_20), rs887466 (C___8941351_1) and rs10484554 (C__29612773_30) were genotyped using the TaqMan SNP Genotyping Assays (Thermofisher, Foster City, CA, USA) according to manufacturer's instructions. The Applied Biosystems StepOnePlus Real-Time PCR detection system was used to conduct reactions and allelic discrimination, respectively.

PSORS1C1 Relative Gene Expression Analysis
Total RNA was extracted from the plasma of psoriatic patients and controls using the Qiagen miRNeasy mini kit (Qiagen, Hilden, Germany, Cat. no. 217004) following the protocol supplied by the manufacturer. RNA purity and concentration were assessed by a NanoDrop 2000 1C spectrophotometer (NanoDrop Tech., Inc. Wilmington, DE, USA). Complementary DNA (cDNA) was generated from total RNA with the miScript II RT Kit (Qiagen, Cat. no. 218161) in which PSORS1C1 was polyadenylated by poly (A) polymerase and converted into cDNA by reverse transcriptase with oligo-dT priming. RT was carried Diagnostics 2022, 12, 1035 4 of 16 out in a Veriti™ 96-Well Thermal Cycler (Applied Biosystems, Bedford, MA, USA) at 37 • C for 1 h, followed by inactivation of the reaction by briefly incubating at 95 • C. GAPDH was used as the endogenous control where it exhibited a uniform and stable expression in plasma samples with no significant difference between psoriatic patients and controls. Triplicate PCR reactions were carried out in the StepOne Real-Time PCR system (Applied Biosystems) using the miScript SYBR Green PCR Kit (Qiagen, cat. no 218076) and specific PSORS1C1 primers: forward primer 5 -CTGACCGACTTTGCCACATGGA-3 , reverse primer 5 -GTGGGAAGAGGGAACCAGGATA-3 and GAPDH primers: forward primer: 5 -GGAGCGAGATCCCTCCAAAAT-3 , reverse primer 5 -GGCTGTTGTCATACTTCTCATGG-3 with negative controls in each run to exclude amplicon contamination.
The expression levels were done according to the quantitative real-time PCR experiments with minimal information required for publication (MIQE) guidelines. The relative PSORS1C1 expression levels were calculated using the LIVAK method 2 (−∆∆Cq) [20], where Delta-Delta quantitative cycle (C q ) = (C q PSORS1C1 − C q GAPDH) Psoriasis − (C q PSORS1C1 − C q GAPDH) controls . The PCR ran initially at 95 • C for 5 min, followed by 40 cycles at 95 • C (15 s), then at 55 • C (1 min), and finally at 72 • C (1 min) for denaturation, annealing and elongation, respectively.

Statistical Analysis
Data were analyzed using the Statistical Package for the Social Sciences (SPSS), version 20.0 software, and GraphPad Prism version 7.0. Quantitative data were expressed as means ± standard deviation, while qualitative data were expressed as numbers and percentages. Two-sided Chi-square, Student-t, and ANOVA tests were used for parametric data. A p-value of <0.05 was considered statistically significant. Analyses of allele frequencies (number of copies of a specific allele divided by the total number of alleles in the group) and carriage rates (number of individuals with at least one copy of the A allele divided by the total number of individuals within the group) was carried out. Genotype frequencies were assessed for deviation from the Hardy-Weinberg equation by the online program (https://www.snpstats.net) (accessed on 1 March 2022) [21]. The relationship between allele frequencies and the presence of psoriasis was determined under different genetic association models using odds ratio with multiple logistic regression analysis after adjustment for psoriasis risk factors was investigated using the same program.

Baseline Characteristics of the Study Population
The age of the patients and controls ranged from 18.0 to 60 years and 20.0 to 62.0 years, respectively, with no statistically significant difference between both groups. Regarding special habits, 77% of patients and 72% of controls were non-smokers. Concerning body mass index (BMI), the mean BMI was 26.80 ± 3.99 kg/m 2 for patients, while it was 27.70 ± 3.93 kg/m 2 in controls Table 1.

Clinical Assessment of Psoriasis Patients
The mean age of disease onset was 35.07 ± 13.43 and the mean duration was 6.75 ± 6.22 years. According to the age of disease onset, the patients were divided into three subgroups: (I) very early-onset psoriasis (vEOP): up to 20 years (21 patients); (II) middle early-onset psoriasis (mEOP): between 21 and 40 years (42 patients); late-onset psoriasis (LOP): above 40 years (37 patients). Forty-five percent of patients showed mild severity, 31% showed moderate severity, and 24% were severe (Table 2). There was a statistically significant difference between the age of onset of psoriasis in subgroups and gender; the vEOP group showed a higher percentage of females, while the median EOP and late EOP groups showed a higher percentage of males. There was no statistically significant difference between the age of onset of psoriasis in groups and the PASI score (Table 3).

Allelic Discrimination Analysis
The three studied polymorphisms were in accordance with Hardy-Weinberg equilibrium (rs887466: p = 0.53, rs1062470: p = 0.31, rs10484554: p = 0.19). On comparing the genotype frequency among the two study groups for rs887466 and rs1062470 genotypes, there was no statistically significant difference between patients and controls. On the contrary, the rs10484554 genotype showed a statistically significant difference between both groups ( Figure 2A). Regarding variants comparison between the two study groups, the rs887466 A variant was more frequent among patients (62% in patients versus 52% in the control group), and also for the rs10484554 variant T, which was more frequent among patients (35% in patients versus 15% in the control group). Meanwhile, rs1062470 variants did not show a statistical difference between patients and controls ( Figure 2B).

Association of PSORS1 Locus Gene Variants with Psoriasis Risk
The rs887466 genotype G/G was 0.4 times more likely to protect against psoriasis under a codominant comparison (OR = 0.4, 95% CI = 0.17 to 0.95) and recessive model (OR = 0.5, 95% CI = 0.23 to 1.05). Moreover, allele G, for this polymorphism, was 0.66 times more likely to protect against psoriasis under allelic comparison (OR = 0.66, 95% CI = 0.44 to 0.99).
For the rs1062470 genotype, only A/G was 1.84 times more likely to develop psoriasis under over-dominant comparison (OR = 1.84, 95% CI = 1.09 to 3.13); other genotypes did not show a significant effect on disease risk (Table 4).

Association of PSORS1 Locus Gene Variants with Psoriasis Risk
The rs887466 genotype G/G was 0.4 times more likely to protect against psoriasis under a codominant comparison (OR = 0.4, 95% CI = 0.17 to 0.95) and recessive model (OR = 0.5, 95% CI = 0.23 to 1.05). Moreover, allele G, for this polymorphism, was 0.66 times more likely to protect against psoriasis under allelic comparison (OR = 0.66, 95% CI = 0.44 to 0.99).

Association of PSORS1 Locus Gene Variants with Psoriasis Risk
The rs887466 genotype G/G was 0.4 times more likely to protect against psoriasis under a codominant comparison (OR = 0.4, 95% CI = 0.17 to 0.95) and recessive model (OR = 0.5, 95% CI = 0.23 to 1.05). Moreover, allele G, for this polymorphism, was 0.66 times more likely to protect against psoriasis under allelic comparison (OR = 0.66, 95% CI = 0.44 to 0.99).   The rs10484554 genotype C/T was 5.63 times more likely to develop psoriasis under codominant comparison (OR = 5.63, 95% CI = 2.9 to 10.9). Moreover, the TT genotype was 2.92 times more likely to develop psoriasis under codominant comparison (OR = 2.92, 95% CI = 0.97 to 8.8). Considering the dominant comparison model, C/T and T/T genotypes were 5 times more likely to develop psoriasis (OR = 5, 95% CI = 2.7 to 9.1). For the C/T genotype, it was 5 times more likely to develop psoriasis under over-dominant comparison (OR = 5, 95% CI = 2.59 to 9.4). Finally, allele T for this polymorphism was 3 times more likely to develop psoriasis under allelic comparison (OR = 3, 95% CI = 1.88 to 4.95) See Table 4.

Association of PSORS1 Locus Haplotypes with Psoriasis Severity
Gene-gene interaction analysis revealed that carriers for GGC genotype combinations had −0.73 times lower disease severity. On the contrary, carriers for GGT genotype combinations had −0.92 times more severe disease form (Table 5). These findings were based on SNPStats [19] web-based platform results, where descriptive statistics were used to estimate the relative frequency for each haplotype. Cumulative frequencies were also Diagnostics 2022, 12, 1035 9 of 16 calculated to help in the selection of the threshold cut point to group rare haplotypes. The association analysis of haplotypes was either presented using logistic regression results with OR and 95% CI or linear regression results with differences in means and 95% CI. The most frequent haplotype was automatically selected as the reference category and rare haplotypes were pooled together in a group.

Relative Expression Analysis of Plasma PSORS1C1 in Psoriasis
The relative gene expression of plasma PSORS1C1 for psoriatic patients showed to be under-expressed compared to normal controls with log-transformed values for median and quartile levels equivalent to −7.24 (−10.35-−2.07) ( Figure 4A). There were also significant differential expression levels among PSORS1C1 SNP rs1062470 genotypes (p < 0.001) ( Figure 4B).
Gene-gene interaction analysis revealed that carriers for GGC genotype combinations had −0.73 times lower disease severity. On the contrary, carriers for GGT genotype combinations had −0.92 times more severe disease form (Table 5). These findings were based on SNPStats [19] web-based platform results, where descriptive statistics were used to estimate the relative frequency for each haplotype. Cumulative frequencies were also calculated to help in the selection of the threshold cut point to group rare haplotypes. The association analysis of haplotypes was either presented using logistic regression results with OR and 95% CI or linear regression results with differences in means and 95% CI. The most frequent haplotype was automatically selected as the reference category and rare haplotypes were pooled together in a group.

Relative Expression Analysis of Plasma PSORS1C1 in Psoriasis
The relative gene expression of plasma PSORS1C1 for psoriatic patients showed to be under-expressed compared to normal controls with log-transformed values for median and quartile levels equivalent to −7.24 (−10.35-−2.07) ( Figure 4A). There were also significant differential expression levels among PSORS1C1 SNP rs1062470 genotypes (p < 0.001) ( Figure 4B).

Discussion
To date, there was only one comprehensive genome-wide association study (GWAS) done on the Egyptian population [22], identifying an association between MHC SNPs and psoriasis in a large Egyptian cohort, however, no data was reported from this study on the PSORS1 locus SNPs. The summary of the estimated genotype and allele frequency for each studied SNP based on worldwide previous publications was presented in Table 7. Table 7. Summary of the estimated genotype and allele frequency for each studied SNP based on worldwide previous publications (Data adopted from https://opensnp.org/ (accessed on 1 March 2022) [18]).  In conclusion, our results demonstrated that rs10484554, rs887466 and rs106247 netic variants within the PSORS1 locus encompassing the multiple genes LOC105375 PSORS1C3 and PSORS1C1/CDSN, respectively, are significantly associated with ps sis. This association is strongly dependent on genotype and less frequently the pati gender. Because of the complicated and extended LD pattern present in the MHC reg it is not clear whether the markers tested in this study confer the risk of psoriasi pendently or independently of other variants in this region. Our allelic discrimina analysis and association with disease or with the PASI score indicated the possibility rs10484554 has a higher effect than rs887466 and rs1062470 on the risk and severi psoriasis, and it is the major key player genetic variant in the PSORS1 locus. Finally results suggest that PSORS1C1 gene under-expression might be in psoriatic patients from arthritis. However, confirmation of this requires additional studies among Egyp and other populations. Moreover, the functional consequence of the polymorphism n to be investigated. Correlation analysis between the single peptide variant and expres levels in patients will add value to future studies outcomes.   In conclusion, our results demonstrated that rs10484554, rs887466 and rs1062470 ge netic variants within the PSORS1 locus encompassing the multiple genes LOC105375015 PSORS1C3 and PSORS1C1/CDSN, respectively, are significantly associated with psoria sis. This association is strongly dependent on genotype and less frequently the patient's gender. Because of the complicated and extended LD pattern present in the MHC region it is not clear whether the markers tested in this study confer the risk of psoriasis de pendently or independently of other variants in this region. Our allelic discrimination analysis and association with disease or with the PASI score indicated the possibility tha rs10484554 has a higher effect than rs887466 and rs1062470 on the risk and severity o psoriasis, and it is the major key player genetic variant in the PSORS1 locus. Finally, our results suggest that PSORS1C1 gene under-expression might be in psoriatic patients free from arthritis. However, confirmation of this requires additional studies among Egyptians and other populations. Moreover, the functional consequence of the polymorphism needs to be investigated. Correlation analysis between the single peptide variant and expression levels in patients will add value to future studies outcomes.   In conclusion, our results demonstrated that rs10484554, rs887466 and rs106247 netic variants within the PSORS1 locus encompassing the multiple genes LOC10537 PSORS1C3 and PSORS1C1/CDSN, respectively, are significantly associated with ps sis. This association is strongly dependent on genotype and less frequently the pati gender. Because of the complicated and extended LD pattern present in the MHC re it is not clear whether the markers tested in this study confer the risk of psoriasi pendently or independently of other variants in this region. Our allelic discrimin analysis and association with disease or with the PASI score indicated the possibility rs10484554 has a higher effect than rs887466 and rs1062470 on the risk and severi psoriasis, and it is the major key player genetic variant in the PSORS1 locus. Finally results suggest that PSORS1C1 gene under-expression might be in psoriatic patients from arthritis. However, confirmation of this requires additional studies among Egyp and other populations. Moreover, the functional consequence of the polymorphism n to be investigated. Correlation analysis between the single peptide variant and expre levels in patients will add value to future studies outcomes.   In conclusion, our results demonstrated that rs10484554, rs887466 and rs1062470 ge netic variants within the PSORS1 locus encompassing the multiple genes LOC105375015 PSORS1C3 and PSORS1C1/CDSN, respectively, are significantly associated with psoria sis. This association is strongly dependent on genotype and less frequently the patient' gender. Because of the complicated and extended LD pattern present in the MHC region it is not clear whether the markers tested in this study confer the risk of psoriasis de pendently or independently of other variants in this region. Our allelic discrimination analysis and association with disease or with the PASI score indicated the possibility tha rs10484554 has a higher effect than rs887466 and rs1062470 on the risk and severity o psoriasis, and it is the major key player genetic variant in the PSORS1 locus. Finally, ou results suggest that PSORS1C1 gene under-expression might be in psoriatic patients free from arthritis. However, confirmation of this requires additional studies among Egyptian and other populations. Moreover, the functional consequence of the polymorphism need to be investigated. Correlation analysis between the single peptide variant and expression levels in patients will add value to future studies outcomes.   In conclusion, our results demonstrated that rs10484554, rs887466 and rs106247 netic variants within the PSORS1 locus encompassing the multiple genes LOC105375 PSORS1C3 and PSORS1C1/CDSN, respectively, are significantly associated with ps sis. This association is strongly dependent on genotype and less frequently the pati gender. Because of the complicated and extended LD pattern present in the MHC re it is not clear whether the markers tested in this study confer the risk of psoriasi pendently or independently of other variants in this region. Our allelic discrimina analysis and association with disease or with the PASI score indicated the possibility rs10484554 has a higher effect than rs887466 and rs1062470 on the risk and severi psoriasis, and it is the major key player genetic variant in the PSORS1 locus. Finally results suggest that PSORS1C1 gene under-expression might be in psoriatic patients from arthritis. However, confirmation of this requires additional studies among Egyp and other populations. Moreover, the functional consequence of the polymorphism n to be investigated. Correlation analysis between the single peptide variant and expres levels in patients will add value to future studies outcomes.  In conclusion, our results demonstrated that rs10484554, rs887466 and rs1062470 ge netic variants within the PSORS1 locus encompassing the multiple genes LOC105375015 PSORS1C3 and PSORS1C1/CDSN, respectively, are significantly associated with psoria sis. This association is strongly dependent on genotype and less frequently the patient's gender. Because of the complicated and extended LD pattern present in the MHC region it is not clear whether the markers tested in this study confer the risk of psoriasis de pendently or independently of other variants in this region. Our allelic discrimination analysis and association with disease or with the PASI score indicated the possibility tha rs10484554 has a higher effect than rs887466 and rs1062470 on the risk and severity o psoriasis, and it is the major key player genetic variant in the PSORS1 locus. Finally, ou results suggest that PSORS1C1 gene under-expression might be in psoriatic patients free from arthritis. However, confirmation of this requires additional studies among Egyptians and other populations. Moreover, the functional consequence of the polymorphism needs to be investigated. Correlation analysis between the single peptide variant and expression levels in patients will add value to future studies outcomes. Institutional Review Board Statement: The study was conducted in accordance with the Declara 39 publications In this study, we found significant associations for the three selected SNPs located within the PSORS1 locus with psoriasis risk and severity, both in single SNP and gene-gene interactions haplotype approaches. Table 5 describes the average genotype and allele frequency for each studied SNP based on the level of evidence demonstrated by a number of publications that included each SNP.

SNP Genotype Frequency Allele Frequency Level of Evidence
The rs10484554 genetic variant within LOC105375015 was the most significantly associated with the disease, similar to that reported by Liu et al. [23], Képíró et al. [24], Kisiel et al. [25], Villarreal-Martínez et al. [26], and Strange et al. [27]. Our study odds ratio for the rs10484554 (T) minor allele (OR = 3, 95% CI = 1.88-4.95) was nearly the same as previously reported by Wiśniewski et al. [19] (OR = 2.68) and Villarreal-Martínez et al. [26] (OR=3). In comparing the minor allele frequency (MAF) for genetic variant (T) with other populations, the psoriatic patients in our study reported a MAF = 0.35, which is significantly higher than that reported worldwide (0.11); among Africans (0.08), Americans (0.08), East Asians (0.05), Europeans (0.14), and South Asians (0.2) [15]. In a comparison of different genotypes for this polymorphism with previous studies, the C/T genotype in our study was 5.63 times more likely to develop psoriasis under codominant comparison (OR = 1.84, 95% CI = 2.9-10.9); this is a much higher risk in comparison to Wiśniewski et al. [19] (OR = 3.38, 95% CI = 2.53-4.52). In contrast, the T/T genotype was 2.92 times more likely to develop psoriasis under codominant comparison (OR = 2.92, 95% CI = 0.97-8.8), and this is much lower than that reported by Wiśniewski et al. [19] (OR = 6.82, 95% CI = 4.11-11.30). In line with earlier publications [23,24,[28][29][30][31], rs10484554 SNP in our study showed a significant correlation with early disease onset. This repetitive finding suggests that rs10484554 could play a role in early-onset psoriasis. However, Hébert et al. revealed that HLA-Cw*06 is associated with late-onset psoriasis using dense genotyping [32]. From a gene perspective, there are no published data about the possible role of LOC105375015 in the pathogenesis of psoriasis. As rs10484554 belongs to a gene encoding lncRNA-LOC105375015-we assume that it might predispose to psoriasis via interacting with mRNA, DNA, protein and miRNA and consequently regulate gene expression at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels in a variety of ways [33]. The function of this SNP is also unknown, but being in close proximity to the exon/intron junction, this may suggest a role in the splicing process. However, an explanation of this point requires further studies.
The genetic variant rs887466 from PSORS1C3 was the only SNP associated with a protective effect in our study. We observed a protective effect only for the G/G genotype, which was 0.4 times more likely to protect against psoriasis under codominant comparison (OR = 0.4, 95% CI = 0.17 to 0.95) and recessive comparison (OR = 0.5, 95% CI = 0.23 to 1.05). In contrast, Wiśniewski et al. [19] reported that the A/A genotype was the protective one. Moreover, allele (G) for this polymorphism was 0.66 times more likely to protect against psoriasis under allelic comparison (OR = 0.66, 95% CI = 0.44 to 0.99), while Wiśniewski et al. [19] reported that the minor allele (A) was 0.77 times more protective against psoriasis, and De Bakker et al. [34] reported that the (G) allele was 5 times more likely to predispose to psoriasis and is considered a risk allele. Thus, the question is whether the (A) or (G) allele is the true marker of protection involving the PSORS1C3 gene and is still to be investigated in future studies. In comparing the MAF for this genetic variant (A) with other populations, the psoriatic patients in our study reported a MAF = 0.62. This is higher than that reported worldwide (0.43); among Africans (0.37), Americans (0.51), East Asians (0.48), Europeans (0.41), and South Asians (0.41) [15]. The function of the relatively novel PSORS1C3 gene is still under investigation. Specifically, nothing is known about the role of intronic rs887466. Previously, several SNPs in this gene have been tested in psoriasis in Swedish and Chinese populations [35,36], but rs887466 was just examined once in psoriasis. In our study, we found no significant association between rs887466 SNP and gender of patients or family history, contrasting Wiśniewski et al. [19]. In addition, when we stratified rs887466 SNP genotypes by both the age and gender of patients, we did not find any significant association between them, except with females aged from 41 to 50 years old. In consistence with Wiśniewski et al. [19], we found no statistically significant association between genotype frequencies of the rs887466 polymorphism and PASI score in both genders.
For the rs1062470 genetic variant, only the A/G genotype was 1.84 times more likely to develop psoriasis under over-dominant comparison (OR = 1.84, 95% CI = 1.09 to 3.13); other genotypes did not show a significant effect on disease risk. These results are contradicting those reported by Lesueur et al. [37] and Wiśniewski et al. [19] who stated that the AA genotype increased the risk of psoriasis over fivefold and was significantly associated with higher PASI score in males and explained it by the double effect of the (A) allele in the AA genotype, that may potentially elevate the expression of corneodesmosin in the skin and may result in increased severity of psoriasis. However, they were not able to explain why this effect was observed in males only. In comparing the MAF for this genetic variant (A) with other populations, the psoriatic patients in our study reported a MAF = 0.53, which is considered close to the reported range worldwide (0.45); among Africans (0.62), Americans (0.4), East Asians (0.51) and higher than that of the Europeans (0.35), and South Asians (0.31) [15].
We further assessed the association between the rs1062470 genetic variant and disease severity, and we observed a statistically significant association between rs1062470, and gender stratified by PASI. Interestingly, Wiśniewski et al. [19], Sakai et al. [38], and Hägg et al. [39] also observed that male patients, independently of rs1062470 genotype, had significantly higher PASI scores than female patients except for the earliest onset of disease.
Our study is among few available studies describing a possible gender-dependent association of the rs1062470 genotype with psoriasis severity worldwide and the first to report this finding in Egypt; therefore, further larger-scale studies are needed, including also other polymorphisms in the CDSN gene, to confirm our findings. In addition, corneodesmosin expression levels in the psoriatic skin in both genders should be compared and its correlation with the rs1062470 genotype is mandatory to evaluate the gender-dependent effect of this SNP on disease severity.
In haplotype analysis, we observed that carriers for GGC genotype combinations had lower disease severity and carriers for GGT genotype combinations had a more severe disease form. Unfortunately, we were unable to determine whether these haplotypes correspond to other HLA-C alleles.
Although the PSORS1C1 gene is located within the PSORS1 locus and is considered one of the potential psoriasis susceptibility genes, the function of its gene product remains unclear in this disease. The relative gene expression level of the PSORS1C1 gene in our cohort was significantly under-expressed, as shown in Figure 4A, opposing the expected assumption to be over-expressed. This can be justified by our cohort exclusion criteria, where we excluded psoriatic arthritis patients so as to decrease the study confounders. Our justification was based on Sun et al.'s findings which reported over-expression of the PSORS1C1 gene in blood and synovial tissues [40], supporting the hypothesis that PSORS1C1 plays a role in rheumatoid arthritis (RA) and is not in close association with the known HLA alleles.
In conclusion, our results demonstrated that rs10484554, rs887466 and rs1062470 genetic variants within the PSORS1 locus encompassing the multiple genes LOC105375015, PSORS1C3 and PSORS1C1/CDSN, respectively, are significantly associated with psoriasis. This association is strongly dependent on genotype and less frequently the patient's gender. Because of the complicated and extended LD pattern present in the MHC region, it is not clear whether the markers tested in this study confer the risk of psoriasis dependently or independently of other variants in this region. Our allelic discrimination analysis and association with disease or with the PASI score indicated the possibility that rs10484554 has a higher effect than rs887466 and rs1062470 on the risk and severity of psoriasis, and it is the major key player genetic variant in the PSORS1 locus. Finally, our results suggest that PSORS1C1 gene under-expression might be in psoriatic patients free from arthritis. However, confirmation of this requires additional studies among Egyptians and other populations. Moreover, the functional consequence of the polymorphism needs to be investigated. Correlation analysis between the single peptide variant and expression levels in patients will add value to future studies outcomes.  Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board and the Research Ethics Committee (protocol code #3974) (20 October 2020).

Informed Consent Statement:
A written informed consent was taken from each patient before enrollment in the study. Written informed consent has been obtained from the patients to publish this paper.

Data Availability Statement:
The data that supports the findings of this study are available from the corresponding author upon reasonable request.