Identification Of A Novel Gene Associated With Sporadic Acne Inversa And Its Pathogenic Study

Background:Acne inversa（AI,OMIM:142690）,also known as hidradenitis suppurativa（HS）,is a kind of recurrent,chronic and inflammatory skin disease.It often occurs in the wrinkled parts of the skin and the parts with dense distribution of sweat glands,including armpit,neck and buttocks.After healing,it will leave a lot of scars.The early clinical manifestations are mainly red papules and nodules,accompanied by pain.The infundibulum of expanded hair follicles and the contents released by the rupture of epidermal cysts cause a large number of local immune reactions,which can be followed by severe inflammatory reactions,leading to painful inflammation,abscess formation,and the formation of sinus and scar in the later stage.The main defect of AI in pathophysiology is hair follicle.The occlusion of hair follicle,rupture of hair follicle duct and foreign body type immune response are the necessary conditions for the occurrence and development of AI in clinic.Specific genetic characteristics and environmental factors,such as smoking,obesity and microbial colonization,contribute to the phenotype of AI.The accumulation of cell debris leads to the final formation of the cyst,but it is also possible that hypoxia in the hair follicle duct destroys the normal terminal differentiation of hair follicle keratinocytes,leading to hair follicle blockage.The etiology and pathogenesis of hair follicle obstruction have not been studied very clearly.At present,the most popular theory is due to y-secretase complex（GSC）results in the failure of notch to cut normally.According to the survey,about 30%-40%of AI patients have definite diagnosis GSC mutations are usually inherited by autosomal dominant inheritance.We speculate that the remaining 60-70%of AI patients may have new mutations or recessive mutations in other genes.Through a series of verification and functional studies,we will further explore the relationship between new gene mutations and the occurrence and development of AI,especially on the basis of previous work,as well as the relationship among new gene,GSC,Notch and EGFR signaling pathways This work will further enrich the understanding of the pathogenesis of AI and other related diseases.Objectives:In order to establish and improve the genetic database of AI patients,we collected the peripheral blood and clinical data of AI patients with and without family history.The whole exome sequencing/whole genome sequencing data of peripheral blood DNA of AI patients with family history were analyzed,and the mutation database of GSC was improved.The clinical characteristics of AI patients with family history and without family history were compared and summarized.The mutation genes of sporadic AI patients with different clinical manifestations were screened by bioinformatics analysis of the whole exome sequencing data and results of peripheral blood DNA of AI patients without family history.According to the bioinformatics results of AI patients without family history,we found and explored the possible pathogenic genes in sporadic AI patients,and explored their mechanism of action.Methods:1.Through the screening of outpatients in dermatology department,the clinical data,peripheral blood and skin samples of AI patients with family history and without family history were collected.On the basis of establishing the genetic resource database of AI patients,the pathogenesis and pathogenic genes of AI were studied.2.DNA was extracted from peripheral blood of patients with family history of AI.The mutation sites of GSC gene were determined by WES sequencing.The corresponding primers were designed and amplified by PCR.Sanger sequencing method was used to compare and analyze the sequences of the products to find out the pathogenic mutation of GSC gene.At the same time,the blood DNA of other members of the family was collected and identified again to draw the genetic family map.3.WES results of 30 sporadic AI patients were analyzed by bioinformatics,and the possible pathogenic genes were summarized and searched,and verified by Sanger sequencing.Then sporadic AI patients were divided into two groups according to the location of skin lesions,the only hip involvement group and without hip involvement group.To further explore and verify the possible pathogenic genes,combined with literature analysis,we focused on the pathogenic effect of one candidate gene-NCoR2.Based on the detection of mutations in the patients-parents’ DNA,we determined the specificity and genetic mode of the pathogenic genes,and detected the mRNA and protein levels of the target gene in the patient’s blood or skin lesions The mechanism of the pathogenic gene in the pathogenesis of AI was studied by cell experiment in vitro and gene knockout mice.Results:1.3 AI families with family history and 30 patients without family history were collected.Biological samples such as peripheral blood and skin laisons were collected.2.Through GSC sequencing analysis of AI patients with family history,new mutation sites were found（NCSTN c.1002₁003del/NCSTN c.1603C>G/NCSTN c.1637₁648del and PSENEN c.108G>A）,the genetic characteristics were autosomal dominant inheritance,and the mRNA expression level of NCSTN in the skin lesions of the proband was decreased.3.By exploring the differential genes between different phenotypes of SAI,we screened 10 genes that may be related to the clinical manifestations of hip group:PPP4R2,DZIP1L,GRM6,HBP1,SLC22A20,PRTG,FAM174B,IST1,TRIP10 and BAGE4.4.By Sanger sequencing,we did not detect NCoR2 mutations in the peripheral blood DNA of the patients-parents.NCoR2 may be a De novo mutation and its protein level is significantly reduced in the lesion area.In vitro NCoR2-knockdown HaCaT cellline,we found that cell migration would be accelerated,and cell adhesion,differentiation and apoptosis indexes were significantly decreased compared with the control group.The histopathological staining of mouse lesion showed that the stratum corneum was thickened,inflammatory cells were infiltrated,and the expression of terminal differentiation index of keratinocytes was decreased.Conclusions:1.We have collected more clinical data of AI patients,and further expanded and improved AI genetic resource database.2.AI is a genetic dermatosis with genetic heterogeneity.The new mutations of GSC were found（NCSTN c.1002₁003del/NCSTN c.1603C>G/NCSTN c.1637₁648del and PSENENc.108G>A）.Compared with SAI patients,patients with GSC mutation had more lesions,heavier phenotype,higher Hurley grade and earlier onset age.3.These 10 genes may be related to the pathogenesis of hip group,and may also indicate the outcome of AI disease and the risk of cancer.4.NCoR2 may be a new pathogenic gene of SAI.