Preliminary Study On The Mutations Of OSMR Gene And Gene Expression In Primary Cutaneous Amyloidosis

Background and objective : Primary cutaneous amyloidosis(PCA)is a chronic pruritic dermatosis in which amyloid material is deposited in skin tissue without involvement of other tissues and organs.It is common in southeast Asia,China,the Middle East and South America.Histopathology showed that a large amount of amyloid deposits in dermal papilla.The pathogenesis of PCA is not clear,which brings great trouble to the treatment of PCA.With the development of gene research technology,many researchers believe that genetic factors play an important role in the pathogenesis of PCA patients,especially in the family.Studies on patients of different races and regions have found that there are OSMR gene mutations in patients with PCA family,indicating that genetic susceptibility exists in patients with PCA.Previous studies have shown that the pathogenic genes of familial PCA are related to OSMR and IL31 RA genes.The OSMR gene belongs to the IL-6 cytokine receptor gene family.The OSMR?,a transmembrane protein encoded by the OSMR gene,is a component of the OSM type II receptor and the IL-31 receptor.The OSMR? protein and the gp130 protein constitute a heterodimer,which is the OSM type II receptor.OSMR? protein binds to IL-31 receptor ? protein to form IL-31 receptor.At present,the OSMR mutations in 12 different sites of family PCA patients reported in the literature are mostly concentrated in the FNIII region adjacent to the transmembrane region.Changes in the amino acid sequence of this region may result in an obstacle to the dimerization process of the OSMR? molecules with the gp130 and IL-31 RA receptor subunits,affecting the activation of downstream signaling pathways after ligands binding to receptors.OSMR? protein cytoplasmic region contains multiple modules related to the recruitment function of Jak/STAT,MAPK and PI3K/Akt signals.Jak/STAT,MAPK and PI3K/Akt pathway are the main pathways of IL-6,and play an anti-apoptotic role in some tumor cell lines.Currently domestic and foreign researchers study PCA mainly focused on OSMR gene detection in individual families.There are few research report on OSMR gene detection in multiple families.Chinese researchers in Taiwan alone have reported studies on multiple families.Gene testing of sporadic PCA has also been reported twice by the Taiwan researcher.The expression of m RNA and protein levels and their effects on downstream signaling pathways in the dermal tissue of PCA patients with OSMR gene mutation are not clear.The objective of this study is to verify the clinical phenomenon that: to detect the OSMR gene mutations in 25 familial patients and 68 sporadic patients with PCA,to analyze the clinical manifestations of PCA,and to explore the relationship between OSMR gene mutations and clinical manifestations of PCA.Finally,In PCA patients with OSMR gene mututaion,OSMR gene expression at m RNA and protein levels and effects on downstream signaling pathways.Materials and Methods: 1.OSMR gene mutation and its correlation with clinical manifestations in patients with PCA(1)The peripheral blood samples were collected from 48 patients with PCA,and the genomic DNA was extracted using the N96 blood genomic DNA.Polymerase chain reaction(PCR)was used to amplify the 2-18 exons of OSMR gene,and its amplified products were sequenced.Four reported mutation sites were found at exons11 and 15,and the new single nucleotide polymorphism sites are found in exons 13,15 and 18.The results showed that four reported mutation sites in exons 11 and 15 and three new single nucleotide polymorphism sites(SNPs)were located in exons 13,15 and 18.Further,we collected 56 cases of PCA patients and psoriasis and normal people in total of 100 cases to verify the newly discovered SNP in exons 13,15 and 18,detection of the above four reported mutations in the distribution of the 56 patients.(2)The relationship between OSMR gene mutations and clinical manifestations were analyzed.2.The expression of OSMR gene in m RNA and protein levels and the effects on AKT and STAT3 signaling pathway in PCA patients with OSMR gene mutations 10 PCA patients were selected as the experimental group,10 normal skin tissues were selected as the control group.The distribution of OSMR? protein were detected by immunohistochemical staining.The m RNA levels of OSMR in the patients and normal controls were detected by real-time fluorescence quantitative PCR(q RT-PCR).Western Blot was applied to detect the expression of OSMR? protein and signaling pathway expression of AKT,P-AKT,STAT3 and P-STAT3.Results: 1.OSMR gene mutation sites were found in 45 of 104 patients with primary cutaneous amyloidosis(43.27%).The mutation sites were located on exons 11 and 15,and the corresponding amino acid changes were: p.G513 D,p.K697 T,p.P694 L,p.I691 T.36 A total of 36 patients were collected from 25 families,23 of whom had OSMR mutation sites(63.89%).OSMR gene mutation sites were found in 22 of 68 sporadic patients(32.35%).The mutation rate of OSMR gene in 25 families was significantly higher than that of sporadic patients(P<0.05).At the same time,four new single nucleotide polymorphism sites were detected: p.T617 A,p.I714 M,p.P936 S and p.N846 K,respectively.The above mutation sites were all nucleotide polymorphisms predicted and analyzed by the software,without pathogenicity.2.Among the 104 patients with PCA,there were 53 males and 51 females,and the ratio was 1:1.The disease occurs in young adults,and the median age of onset is 29.50 years.The main symptom of patients with PCA is pruritus(74.04% of patients with pruritus).The most common lesion is the lower leg,followed by the forearm and back. The onset age(median age of 25)of OSMR gene mutation patients was younger than that of non-mutated patients(median age of 33 years)(P<0.05).However,there was no significant difference between the OSMR gene mutation and gender ?pruritus and clinical type(P>0.05).3.the expression of OSMR?,P-AKT and P-STAS3 protein in the skin lesions of PCA were significantly down-regulated while the expressions of AKT and STAT3 did not change significantly.In PCA group,OSMR? protein distributed in the basal layer and the low half of stratum spinosum.While in control group,OSMR? protein expressed the whole stratum spinosum and basal layer.4.The m RNA expression level of OSMR in PCA group was not significantly different from that in the control group(P>0.05).Conclusions: 1.This study found that there was a high OSMR mutation rate in PCA family and sporadic patients,and the onset age of patients with OSMR gene mutations was younger than the onset age of non-mutant patients,Confirmed that the pathogenesis of primary cutaneous amyloidosis may be related to the OSMR gene mutations.The OSMR gene mutations found in this study are all reported mutation sites,and no new OSMR gene mutation sites were found.2.The expression of OSMR? protein in patients with PCA gene mutations was significantly lower than the control group,and the downstream signaling pathways activity of AKT and STAT3 was obviously reduced.This suggests that the pathogenesis of patients with primary cutaneous amyloid mutations may be after OSMR gene mutations affects the expression of the encoded protein OSMR?,resulting in decreased AKT and STAT3 activity of the downstream signaling pathway.There was no significant change in m RNA expression level of OSMR in lesions of PCA patients,which may be due to mutation of OSMR gene resulting in post-translational protein differences and no effect on transcription level.