Correlation Between Conventional Treatment-Insensitive Psoriasis Vulgaris And Gene Mutations In Exonic Regions

Objective:Psoriasis is a chronic inflammatory relapsing skin disease with a genetic basis.There is currently no clinical cure for psoriasis because the pathogenesis is unknown.The goal of treatment is to alleviate the patient’s discomfort and to remove the skin lesions.Clinicians use the evidence-based medicine model to develop a treatment plan for a patient by combining the patient’s condition with their personal clinical experience.The first-line treatment for patients with psoriasis vulgaris is narrow-spectrum ultraviolet radiation,systematic use of Acitretin capsules,and topical glucocorticoid ointment,i.e.,conventional psoriasis treatment.However,clinical studies have revealed that there is a subset of patients with psoriasis vulgaris who are resistant to conventional treatment,with no visible changes in skin lesions after treatment.The patients’ resistance is not related to the severity of the psoriasis lesions.Genetic factors,psoriasis co-morbidities,obesity,smoking,and mental status can affect the treatment outcome of patients with psoriasis,however,most of the existing studies have focused on the relationship between genetic variants of genes and psoriasis treatment outcome using candidate gene approaches,focusing on genes known to be associated with psoriasis susceptibility or the immune system,and are predominantly foreign,and the existing findings are not applicable to patients with psoriasis in China.Patients with psoriasis vulgaris who received conventional treatment were studied in this study,and psoriasis patients were divided into conventional treatment-insensitive and conventional treatment-sensitive groups based on disease severity(as measured by the PASI score),disease duration,recurrence frequency,and treatment effect.Exon sequencing technology and bioinformatics analysis were used to screen out specific genetic loci in psoriasis vulgaris that were not sensitive to conventional treatment,as well as to evaluate the effect of genes on the treatment effect of psoriasis patients.Methods:1.The subjects of this study were 345 patients with psoriasis vulgaris treated conventionally who attended the outpatient clinic of the Department of Dermatology of Taiyuan Central Hospital from January 2020 to January 2022 and were confirmed by clinical and pathological diagnosis.According to disease severity,disease duration,recurrence frequency,and treatment effect,the patients with psoriasis were divided into 166 cases in the conventional treatment-insensitive group and 179 cases in the conventional treatmentsensitive group.2.10 m L of peripheral blood was collected from the patients based on their general demographic characteristics and pertinent clinical information.3.After sequencing the coding regions of 345 study participants’ genomes with wholeexome sequencing technology,the candidate genes and mutation loci were screened out.To validate candidate gene mutation loci using Sanger sequencing technology,239 patients with psoriasis vulgaris who were not sensitive to conventional treatment were chosen for the study of Sanger sequencing among patients with psoriasis vulgaris who attended the outpatient clinic of Taiyuan Central Hospital’s Department of Dermatology from February 2022 to January 2023 and were confirmed by clinical and pathological diagnosis according to strict inclusion and exclusion criteria.Real-time fluorescence quantitative PCR was used to confirm the mRNA expression levels of mutant individuals identified through whole exome and Sanger sequencing.4.Two independent samples t-test,chi-square test,multi-factor logistic regression analysis and consistency test were performed using SPSS 26.0 statistical software.After filtering the downstream data(reads)from whole exome sequencing with the SOAPnuke filtering software,the clean reads were compared with the human genome reference sequence GRCh37 using BWA and GATK software,and the mutant sites were annotated using ANNOVAR software,and the mutant sites were functionally predicted using SIFT,Poly Phen2,LRT,and Mutation Taster bioinformatics analysis software.Species diversity analysis was performed using UGENE software.Sanger sequencing plots for all mutant sites in the sequences were viewed and compared with chromas software,and sequenced sequences were compared with normal sequences in the db SNP database.Graph Pad 8.0 was used to create statistical plots.Results:1.Gender,smoking,physical activity,age at onset,and duration of disease were statistically different between the two groups(P<0.05),according to a statistical analysis of the basic demographic characteristics and relevant clinical information of 166 patients in the conventional treatment-insensitive group and 179 patients in the conventional treatmentsensitive group.A multi-factor logistic regression model analysis revealed that smoking,age of onset,and disease duration were the most important predictors of the effect of conventional treatment(P<0.05).2.Whole-exome sequencing of 345 psoriasis patients yielded 15828 variant genes,of which 11391 were included in the study after being filtered through the ESP6500 database,gnom AD database,Ex AC03 database,and Anhui psoriasis database(filtered according to mutation frequency).In two independent samples,58 missense SNVs with low frequency(Minimum Allele Frequency,MAF<1%)were found to be statistically significant(P<0.05)using a chi-square test on 166 patients who were not sensitive to conventional treatment and179 patients who were sensitive to conventional treatment.The 58 mutated genes were interpreted by consulting the literature,functionally annotated by ANNOVAR software,and genes with non-synonymous mutation types and SNVs predicted to be "deleterious" by at least three of the four functional prediction software,SIFT,Poly Phen2,LRT,and Mutation Taster,were chosen.Finally,the genes TGFBI,BICD2,and VWDE were chosen.The mutation sites of the three genes were screened using four functional prediction software,namely SIFT,Poly Phen2,LRT,and Mutation Taster,to screen for SNVs with "deleterious" mutation sites that might affect protein function and sequence,and finally,12 different mutation sites were screened,namely TGFBI c.740T>C,TGFBI c.805C>T,BICD2 c.2366A>G,BICD2 c.2347C>T,BICD2 c.1741C>T,BICD2 c.752G>A,BICD2 c.295C>T,VWDE c.3907G>A,VWDE c.3181G>A,VWDE c.2878 T VWDE c.2878T>C,VWDE c.2631T>G and VWDE c.270＿271ins CC.3.Sanger sequencing was used to validate 12 mutant loci in 239 patients with psoriasis who were resistant to conventional treatment.A chi-square test revealed the presence of two low-frequency nonsynonymous coding variants with statistically significant differences in two independent samples(P<0.001),mutation at one locus in the mutant BICD2 gene:NM＿001003800: exon5: c.C1741T: p.R581 C and mutation at one locus in the mutant VWDE gene: NM＿001135924: exon12: c.T2631G: p.Y877 X,which is consistent with the results of whole exome sequencing.However,an interspecies conservativeness study found that BICD2-Arg581 Cys was the only altered amino acid in a highly conserved area between species.4.TGFBI mRNA expression levels were significantly lower in mutant individuals in the presence of the conventional treatment-insensitive group compared to non-mutant individuals in the conventional treatment-sensitive group(4.12±0.37 vs.5.27±0.26,P<0.001).In contrast,the BICD2 and VWDE genes were significantly up-regulated in mutant individuals compared to non-mutant individuals(3.97±1.19 vs.3.39±1.36,6.71±0.95 vs.5.16±2.40,P<0.05).When combined with the results of Sanger sequencing and species diversity analysis,as well as the fact that the BICD2 mutation was a missense mutation,the combined analysis allowed us to speculate that the BICD2 gene mutation(c.C1741T:p.R581C)could be associated with psoriasis insensitivity to conventional treatment.Conclusion:1.In this study,smoking,age of onset,and duration of disease are the main factors associated with the effectiveness of conventional treatment.2.The BICD2 gene NM 001003800: exon5: c.C1741T: p.R581 C and VWDE gene NM001135924: exon12: c.T2631G: p.Y877 X were statistically different in two independent samples,as confirmed by whole-exome sequencing technology,bioinformatics software analysis,and Sanger sequencing.By real-time fluorescent quantitative PCR,the expression of BICD2 mRNA and VWDE mRNA was found to be significantly higher in patients with mutations in the conventional treatment-insensitive group than in non-mutated patients in the conventional treatment-sensitive group.But the only region with amino acid residues highly conserved between species was BICD2-Arg581 Cys,and the BICD2 mutation was a missense mutation.The combined analysis suggested that the BICD2 gene c.1741C>T may be a specific mutation site associated with psoriasis vulgaris insensitivity to conventional treatment.