Expression And Significance Of ISX And HEATR1 Mutations In Patients With Psoriasis Vulgaris Who Are Not Sensitive To Conventional Treatment

Research BackgroundPsoriasis is a chronic,inflammatory,and systemic disease caused by both genetic and environmental factors and mediated by immunity.It is usually manifested as localized or widespread squamous erythema or plaques.It is prone to recurrence,often lifelong and incurable.Of these,psoriasis vulgaris accounts for more than 90% and is the most common manifestation.In the course of clinical treatment,there exists a special group--patients with psoriasis vulgaris who are not sensitive to conventional treatment.Studies have shown that genetic factors are closely related to the risk of psoriasis,the severity of the disease,and the differences in individualized medication.Currently,it has been found that gene polymorphism analysis can explain some phenomena of individual drug use differences,that is,the DNA sequence of patients will have a certain influence on drug response.In our previous study,170 patients with psoriasis vulgaris in the obvious group(effective to conventional treatment)and 180 patients in the refractory group(insensitive to conventional treatment)were collected.Through DNA exon sequencing analysis of peripheral blood,we compared the occurrence frequency of susceptible genes in the two groups,and screened out 26 gene sites with high frequency in the refractory group.Including genes ISX and HEART,suggesting that patients with these genes may need new and effective treatment options.Foreign studies have confirmed that ISX is involved in the occurrence and development of liver cancer,pancreatic cancer and gastric cancer,and in regulating the metabolism of vitamin A in the body.HEATR1 was up-regulated in gastric cancer,oral squamous cell carcinoma,non-small cell lung cancer,hepatocellular carcinoma,and glioblastoma.Therefore,it is speculated that the polymorphisms of genes ISX and HEATR1 may affect the development of proliferative cell diseases.Psoriasis is a skin disease characterized by excessive proliferation of keratinocytes.There are not many studies on ISX and HEATR1 in psoriasis,so it is necessary to further study the role of their polymorphisms in the treatment of psoriasis.In this study,patients with psoriasis vulgaris who are not sensitive to conventional treatment were collected to verify ISX and HEATR1 as stubborn and refractory gene sites indicating the disease,and the expression levels of ISX and HEATR1 m RNA in plasma were compared.It has been preliminarily proved that both mutations may reduce the sensitivity of drug therapy in the treatment of psoriasis.Methods1.Patients with psoriasis vulgaris who were not sensitive to conventional treatment were included as research objects,and basic clinical information of patients was collected and analyzed by means of questionnaire.2.Peripheral venous blood samples of the study subjects were collected,genomic DNA was extracted,ISX and HEATR1 gene mutation sites were amplified by PCR and Sanger sequencing was conducted,and gene sequences were compared with those of normal population and patients with effective conventional treatment,and mutations were screened.3.qPCR technology was used to measure the expression levels of ISX and HEATR1 m RNA in plasma of all mutation samples in patients who were not sensitive to conventional treatment and those who were effective in conventional treatment,and to compare the expression differences caused by target gene mutations.4.Alphafold2 tool and Schrodinger software were used to predict the tertiary structure of ISX and HEATR1 wild type and mutant proteins.After that,Imutant2.0 software was used to analyze the influence of protein mutation on structural stability and calculate the free energy(ΔΔG)of proteins.5.STRING database was used to construct the protein interaction network(PPI)with ISX and HEATR1 as the central proteins,to explore the relationships with key proteins,and to speculate the possible influence mechanism of gene mutations in patients with psoriasis vulgare who are not sensitive to conventional treatment.Results1.Among the 239 patients with psoriasis vulgaris who were not sensitive to conventional treatment,the male to female ratio was 1.25 to 1,the mean age was(41.32±13.076)years,the mean course of disease was(7.98±10.143)years,and the mean age of onset was(29.58±11.358)years.38.50% of patients had family genetic history;175patients were light in summer and heavy in spring and winter.85 patients had BMI of 24kg/m2 or greater.According to the severity of skin lesions,the same patient can be treated with local topical therapy,physical therapy,systemic therapy,and Chinese medicine.89 patients had one or more different types of psoriasis comorbidities,and 36 patients had two or more types of psoriasis comorbidities.2.According to Sanger sequencing,12 patients in the refractory group had missense mutation at a site on exon 3 of ISX gene(NM 001303508): C.895 T >C,p.Al85ala;15HEATR1 genes(NM 018072)A missense mutation occurred at a site on exon 22:c.3207C>T,p.Gly1027 Asp,which was consistent with the results of whole exon sequencing,confirming the existence of two mutation sites.239 patients with psoriasis vulgaris in refractory group and 170 patients with psoriasis vulgaris in obvious group were statistically analyzed,and the above two low-frequency variations were statistically significant(P<0.01).3.The relative expression level of ISX in the refractory group and the demonstrative group was 2.100±0.4339 and 1.006±0.1161,respectively,and the relative expression level of HEATR1 was 1.696±0.3708 and 1.070±0.3624,respectively.m RNA expression level in the refractory group was higher than that in the demonstrative group.The difference was statistically significant(P < 0.0001).4.After mutation,the free energy of ISX protein was changed,ΔΔG was-2.13kcal/mol,and the protein stability was decreased.The free energy of the HEATR1 protein changes(ΔΔG is-0.11kcal/mol),and the stability of the protein decreases slightly.5.PPI showed that the 10 proteins interacting with ISX were BCO2,SCARB1,BCO1,RASD2,CYGB,SPHK1,TOM1,POLDIP3,MCM5 and NPAT.The 10 proteins that interact with HEATR1 are UTP20,CIRH1 A,DCAF13,NOL6,WDR75,RRP9,UTP18,NOP58,UTP15,and UTP6.Conclusion1.In this study,missense mutations c.895T>C in ISX gene exon 3 and c.3207C>T in HEATR1 gene exon 22 were detected in 239 patients with psoriasis vulgaris who were not sensitive to conventional treatment.The mutant spectrum of psoriasis was further expanded.2.Genes ISX and HEATR1 may participate in the occurrence and development of psoriasis to a certain extent.Both mutations may affect related biological functions by reducing the expression level and altering the stability of protein molecular structure,leading to insensitivity of some patients with psoriasis vulgaris to conventional treatment.