Variants in PRKCE and KLC1, Potential Negative Regulators of Type I Psoriasis

Background: Psoriasis is a multifactorial disease with a complex genetic predisposition. The pathophysiology of psoriasis is associated with genetic variants, especially in negative regulatory genes. To better characterize gene variants in psoriasis and identify the relationship between clinical characteristics and variant genes in its pathogenesis. DNA was extracted from 282 type (cid:0) psoriasis patients and puried, and 13 variable genes were amplied and sequenced using the Sanger method. Results: Among the 13 investigated genes, the variants frequencies of protein kinase C epsilon (PRKCE) (c.240T>C, 35.9% vs 47.7%, P< .05) and kinesin light chain 1 (KLC1) (c.216A>G, 2.9% vs 98.1%, P< .01) were signicantly lower in patients than in normal Asian individuals. Additionally, we found considerable differences in the relationship between variants in genes CADM2, JPH2, SPTLC3 and clinical characteristics stratied by medical history and family history. Moreover, MEGF6 gene variants (39.52% vs. 22.50%, χ2=3.83, p < .05) showed a stronger association with the group as mild (PASI ≤ 10) than with the group as heavy. Conclusions: Our results provide a comprehensive correlation analysis of negative regulatory genes that are regulated in psoriasis. This integrated analysis offers novel insight into the pathogenic mechanisms involved in psoriasis.

Background Psoriasis is a common, genetic, chronic in ammatory skin disease that causes red, aky patches on the skin [1]. The worldwide prevalence of psoriasis is approximately 3.2%, or more than 7.4 million people, as estimated by Rachakonda et al [2]. Psoriasis has a high recurrence rate and is considered a complex disease due to environmental and genetic factors [3].
The genetic component is partly explained by its association with certain susceptibility loci [4]. At present, more than 70 susceptibility genes are associated with psoriasis [5][6]. The strongest susceptibility locus lies within the major histocompatibility complex (MHC) [7]. Although most studies have focused on psoriasis susceptibility genes, others have shown that certain genetic variants play a negative regulatory role in the pathogenesis of psoriasis. There was also article found the association of TRAF3IP2-rs33980500 variant with the susceptibility to psoriasis [8].
Because monozygotic twins carry the theoretically same genetic information, they are more conducive to the study of susceptibility genes than sporadic populations. Recently, we obtained the whole genome sequences of 9 pairs of monozygotic twins with psoriasis discordance. In addition to the C3 de novo mutation [9], we reported 13 loci that were signi cantly different between normal individuals and patients with psoriasis in this paper. Interestingly, the 12 of 13 loci found in the normal individuals whose twin's medical history are longer than 10 years, indicating the 12 loci play a negative regulatory role in the pathogenesis of psoriasis, especially in long medical history. According to the onset age, vulgaris psoriasis were classi ed as with psoriasis vulgaris (early onset, onset age < 40) and type (late onset, onset age ≥ 40) [10]. Many scholars suggested a genetic in uence upon its age at onset, especially in the type psoriasis [11][12]. To further investigate the contribution of these variants, we additionally examined these gene variants in 282 type psoriatic patients and analyzed the relation between variants and clinical manifestation based on the monozygotic twins investigation.

variant loci were ltered in the monozygotic twins
In this study, blood sample from 9 pairs of monozygotic twins with psoriasis discordance was analyzed by whole genome sequencing. We obtained 13 variants loci, such as kinesin light chain 1 (KLC1, c.216A > G), protein kinase C epsilon (PRKCE,c.  (Table 1). Interestingly, all of the 13 above mentioned variant loci were found only in the normal homozygote, but none of them in psoriatic homozygote. Variant exist in normal populations, indicating their potential prevention to psoriasis. ) and DENND5B (0.53% vs 0.6%) were lower in patients with psoriasis than in normal individuals, though all of P value was higher than 0.05. However, the variation frequencies of JPH2 (86.1% vs 85.12%) and RPAP3 (28.0% vs 26.9%) were higher in patients with psoriasis than in normal individuals ( Fig. 1).
Occurrence of psoriasis and its relationship with the sociodemographic characteristics of participants Medical history, the PASI score and family history were used to clarify the different clinical subtypes. Therefore, the relationship between medical history, the PASI score, family history and variant genes was investigated. CADM2(χ 2 = 9.29, P < 0.05), JPH2(χ 2 = 8.47, P < 0.05), SPTLC3 (χ 2 = 20.65, P < 0.01) was signi cantly different between patients with different medical histories (Fig. 2).
Analysis of the PASI score revealed a signi cant association with variation in MEGF6 (χ 2 = 6.06, P < 0.05). Next, we further analyzed the relationship between family history and variant genes and found that CADM2(χ 2 = 6.08, P < 0.05), JPH2(χ 2 = 10.98, P < 0.01), SPTLC3 (χ 2 = 8.51, P < 0.01) was signi cantly associated with family history, suggesting that the variation in CADM2,JPH2, SPTLC3 may be the risk factor for psoriasis in individuals who have a family member with psoriasis.
Univariate and multivariate analysis of family history on disease characteristics For research purposes, we rearranged the population data and divided individuals with psoriasis into two groups, as shown in Table 2. Research has shown that with the extension of time, new mutations will emerge and old ones will be repaired. Therefore, to eliminate the impact of time on mutation gene, we divided into two groups based on medical history and then compare different PSAI and family history of patients. In the under 20 years medical history group and over 20 year medical histroy group, we analyzed the relationship between gender, age, PASI, family history and gene mutation. For the comparison of family histroy of two group, we found that CADM2(χ 2 = 5.89,94.87% vs 76.56%, p<0.05) was a risk factor for having family histroy with type psoriasis. Similarly when compared to patients with under 20 years medical history group and over 20 year medical histroy group, there were differences in MEGF6(χ 2 = 3.86,39.52% vs 22.5%, p<0.05) according to PASI of type psoriasis.

Discussion
Based on screening studies and genome-wide association studies (GWASs), a large number of susceptibility genes that are involved in the pathogenesis of complex diseases, such as cancer, psoriasis, and other major human diseases, have been identi ed [17][18]. Concerning psoriasis, more than 70 susceptible genes have been identi ed in the last 30 years [19][20]. In our study, we use nextgeneration sequencing technique to assess the status of 13 genes, and we nally found that PRKCE and KLC1 had mutated in psoriasis patients. The results of the study indicated that genetic variation has a very close correlation with clinical features.
A pathogenic role for PRKCE has been found in several types of cancers, as it is able to promote proliferation and inhibit apoptosis [21]. Constitutively active PRKCE has been found in small cell lung cancer [22] and epithelial cells of the colon [23] associated with tumor formation [24]. The PRKCE acts as a regulator of PLD activity, and this inhibition is mediated by its regulatory domain [25]. Moreover, the novel partial loss-of-function defect in PRKCE impairs AKT activation via compromised mTORC2 complex function [26]. According to studies of patients with psoriasis, the PI3K/AKT pathway is an important signaling pathway that regulates the hyperproliferation of keratinocytes [27]. It has also been reported that a variation at the phosphorylation site of KLC1, Ser517/Ser520, affects AMP-activated protein kinase (AMPK) to suppress low glucose concentrations and block granule movement [28]. In this study, we demonstrated that the variation rates of PRKCE and KLC1 were signi cantly lower in psoriasis patients than in normal Asian patients, consistent with the results reported by Salo-Mullen EE [29], indicating that the described PRKCE and KLC1 variants may be a negative regulatory mechanism for the onset of psoriasis.
Medical history and the PASI score are commonly used in clinical trials on psoriasis, and this scoring algorithm greatly expands options for quantifying treatment outcomes in cost-effectiveness analyses of psoriasis therapies [30]. To further evaluate the relationship between variant genes and clinical characteristics, we grouped patients with psoriasis according to sex, age, medical history, PASI score and family history. The variation rate was distinctly different according to medical history, PASI score and family history. We found that the CADM2, JPH2 and SPTLC3 variation have correlation with more than 20 years of medical history in individuals with type 1 psoriasis. Moreover, moderate-to-severe (PASI score ≤ 10) variants were more commonly observed in individuals with a MEGF6 variation. Indeed, several studies have shown the signi cant impact of the onset of psoriasis not only in patients with a medical history and a high PASI score but also in their family and close relatives [31][32]. The literature supports some differences between familial and sporadic cases of psoriasis [33]. Differences in the strength of the association between the variant gene and family history have been reported for the MHC, including a strong association of HLA-C*06 and HLA-B*27 with psoriasis [34]. Similarly, in our study, we found that the variation frequency of CADM2, JPH2 and SPTLC3 was higher in the familial group than in the sporadic group. Our ndings are important for the promotion of large panels in patient populations with psoriasis and for clinical genetic testing for patient management. However, the speci c regulatory mechanisms involved need to be explored in the skin cells of psoriasis patients.

Conclusion
We demonstrated that the variation rates of KLC1 and PRKCE were signi cantly lower in psoriasis than normal, indicating these two genes could serve as potential novel negative regulatory genes for type psoriasis. We showed considerable differences in the relationship between clinical characteristics and variant genes.

Samples
MZ Twins Samples from 8 pairs of Chinese MZ twins who were discordant in psoriasis and samples from the twins' parents were collected with consent at Taiyuan Central Hospital.
282 patients diagnosed with vulgaris psoriasis were recruited from the outpatient center of the Taiyuan Central Hospital. All patients included in this study were of Han Chinese descent. Individuals with psoriasis were diagnosed by at least two dermatologists based on clinical and histopathological manifestations, and their clinical information was collected through a comprehensive clinical check-up by professional investigators. Self-reported information from a standard questionnaire was used to collect demographic and other characteristics (severity, medical history, and family history) from the patients and to exclude any other systemic, infectious, autoimmune, atopic, or malignant disease and to determine whether they received systemic treatment in the six months prior to collection. None of the patients had hypertension, gout, asthma or multiple coffee spots.

Characteristics of the subjects
The clinical features of MZ Twins Samples (4 pairs of women and 4 pairs of men, range from 11 to 51 years with the mean age 30.0 years) were shown in Table S1.
The clinical characteristics of the study subjects are shown in Table S2. In this study, we investigated 282 Chinese Han individuals with type psoriasis. Their age of onset is less than 40 and pertain to early onset, which all belong to the type 1 patients with psoriasis.
Regarding of medical history, PASI and family history, 282 patients were grouped, respectively. The medical history group was divided into two ranks: less than 20 years (58.2%) and more than 20 years (41.88%). Patients were selected based on disease severity, which was assessed using PASI score [13]. The number of patients in the mild group (PASI score ≤ 10) was 209(74.1%), and the number of patients in the moderate-to-severe group (PASI score > 10) was 73(25.9%). Patients were also grouped according to family history: positive (27.3%) and negative (49.3%).

Whole Genome Sequencing And Variant Identi cation
Each quali ed sample of genomic DNA from whole blood of 9 twins was sequenced on the Illumina HiSeq platform using paired-end reads according to the manufacturer's instructions. The following types of reads were removed: (1) containing a sequencing adapter; (2) low quality base > 50%; and (3) 'N' base > 10%. All clean data from each sample were mapped to the human reference genome HG19 by Burrows Wheeler Aligner software (BWA, v0.75) [14]. Duplicate reads were marked by Picard tools. We used 3 different methods to call SNPs and InDels and get overlap of them to ensure the accuracy of variant calling: SAMTOOLS mpileup [15], recommended best practices for variant analysis with the Genome Analysis Toolkit (GATK) [16] and Freebayes. We identi ed the DNM discordance between co-twins by checking the concordance genotype between them. We genotyped our DNMs with Sanger sequencing to con rm our DNM detection accuracy.

Dna Extraction
Genomic DNA was extracted from the peripheral whole blood of the 282 patients using a Blood Genomic DNA Midi Kit (Cwbio Biotech, Beijing, China) according to the manufacturer's instructions. All DNA samples were dissolved in water and stored at -20℃ until use.

Sanger Sequencing
Sequencing primers (Table S3)  All participants provided written informed consent. The study protocol was approved by the ethics committee of Taiyuan Central Hospital.

Consent to publish
All participants provided written informed consent. Availability of data and materials