| Background Acne inversa (AI; also known as hidradenitis suppurativa) is anautosomal dominantly inherited skin disorder characterized by recurrent abscessesand draining sinuses with subsequent scarring and chronic seepage. AI has originallybeen linked to the apocrine sweat glands. This link became merely historical in1990,when Yu and Cook showed follicular occlusion to be the primary event in AI. Atpresent, the disease was considered the result of the hyperplasia of epithelial cell ofthe infundibular portion of the hair follicle which led to the obstruction of hairfollicles mouth and sebaceous gland, the incomponent exhaust of cortical andsecondary bacterial infection. Genetic factors are the primary etiological factor, butother factors such as environment and endocrine also play an important role in theonset of the disease. AI usually occur in the apocrine gland-bearing areas of the skinsuch as the armpit, groin and buttocks, manifesting as infarctate, painful, inflamednodules that can result in abscesses, sinuses, and varying degrees of chronic drainingsinus tracts. Anemia, quickened speed of ESR and serum rheumatoid factor negativeare often found in the laboratory tests. AI is often associated with acne conglobata anddissecting cellulitis of the scalp, collectively known as follicular occlusion triad. Inaddition to skin lesions, the disease can be associated with arthritis or secondary tosquamous cell carcinoma, which not only has great influence on the health andappearance of the patients but also can cause serious physical and mental burden forthe patients. Its global prevalence has been estimated to be1–4%. AI usually occursafter puberty, most of the cases occurs in the age of20-30, and the mean age of onsetis23years old. The disease affects women more than men, and the incidence ratio offemale to male is3.3:1.14. AI can happen in family groups, can be manifested asautosomal dominant, and can also be sporadic. In2006, Gao et al mapped the pathogenic gene of AI to a locus at chromosome1p21.1-1q25.3, but no specific genewas defined. Moreover,6q25.1-25.5and9p12-13.11are also reported to be associatedwith AI. Werth and Williams pointed out that AI may be a heterogeneous disease, andits incidence may be associated with several genes. Currently, the search for thepathogenic gene of monogenic disorder is usually by positional cloning. However,this method is time-consuming and the positioning area is usually large, so thepathogenic genes of the diseases often can not be discovered directly. Recently,exome sequencing has been successfully applied to discover the pathogenic genes ofrare monogenic diseases, such as the MYH3gene of Freeman–Sheldon Syndrome, theDHODH gene of Miller Syndrome and the SETBP1gene of Schinzel-GiedionSyndrome. The approach has been demostrated to be a powerful, effective means ofreducing the candidate genes and even discovering the pathogenic genes of raremonogenic diseases.Objective (1) To identify the candidate pathogenic genes of AI by exome sequencing.(2) To identify the pathogenic gene of AI by validating the candidate pathogenicgenes.(3) To explore the role of the pathogenic gene in the pathogenesis of AI bycomparing the difference of expression level of the pathogenic gene between casesand controls.Methods (1) In order to identify the candidate genes more effectively, we select thesame family(familyâ… ) as the object of the study which was used by genome-widelinkage analysis for mapping the candidate pathogenic region on chromosome1p21.1-1q25.3. Two cases(â…¡4and â…¢11) and one family control(â…¢10) were subjected toexome sequencing.(2) By a. filtering out synonymous mutations that do not affect thefunction of the genes, only retaining the mutation that affect the function of the genessuch as non-synonymous mutations and splice site mutations; b. filtering out commonmutations carrying out by normal people in the public genetic mutationdatabase(dbSNP), the normal individual mutations that have been published, and themutations carrying out by the control who subject exome sequencing in theexperiment; then making intersection of the mutation set of the two patients, we reduce the number of candidate pathogenic mutations.(3) By the candidatepathogenic region on chromosome1p21.1-1q25.3, we further reduce the number ofthe candidate pathogenic genes. And then using the software that can predict theaffection the mutations can make to the function of the protein, we further filter toidentify the possible mutation/gene of AI.(4) Using PCR sequencing methods, wevalidate the mutation identified by exome sequencing in familyâ… to observe whetherthe disease phenotype is caused by the mutation.(5) By PCR sequencing methods, wetest whether functional mutation occurs in the exons of the candidate pathogenic genein other family(familyâ…¡). And then, we test all the functional mutations in thecandidate pathogenic gene identified in the previous two steps in a wide range ofnormal population.(6) Using real-time quantitative PCR method, we test theexpression difference of the pathogenic gene between AI patients and normal controlsin the peripheral blood mononuclear cell on the mRNA level.Results (1) After exome sequencing, the total number of SNPs measured in the twocases(â…¡4and â…¢11) and one family control(â…¢10) are41,177,44,660and42,196respectively.(2) After filtering out synonymous mutations that do not affect thefunction of the genes, common mutations carrying out by normal people in the publicgenetic mutation database(dbSNP), the normal individual mutations that have beenpublished, and the mutations carrying out by the control(â…¢10) who subject exomesequencing in the experiment, the remaining mutant loci of â…¡4and â…¢11are227and203respectively. Subsequently, make intersection of the mutation set of â…¡4andâ…¢11, the remaining mutant loci are85.(3) Further filtering the85mutant loci by thecandidate region of chromosome1p21.1-1q25.3, there were only8remaining, whichare in the8following genes respectively: MYBPHL, KCNC4, FLAD1, NES, NCSTN,ITLN2, SLC9A11and Clorf220. After using ANNOVAR and GERP software topredict effects the mutation will have to the protein, we found that c.1352+1G> Asplice site mutation in NCSTN is the most possible pathogenic mutation of familyâ… .(4) After checking the NCSTN c.1352+1G> A mutation in familyâ… , we observedthat the mutation lead to the onset of AI of patients of family I.(5) By PCR sequencing methods, we found a two nucleotide deletion of c.210211delAG inexon3of NCSTN when testing the candidate pathogenic gene NCSTN in otherfamily(familyâ…¡). The mutation is not exist in all the controls of the family. The twofunctional mutations found in the candidate pathogenic gene NCSTN are not exist in90normal subjects as well after PCR sequencing testing.(6) Compared with normalsubjects, the expression of NCSTN mRNA in the peripheral blood mononuclear cellsof the patients reduced.Conclusion The research succeeded in discovering the pathogenic gene NCSTN ofAcne inverse by the most advanced method of exome sequencing. On this basis, theexpression study of NCSTN mRNA show that haploinsufficiency of the gammasecretase subunit gene NCSTN may be the genetic basis of some subtype of familialAI. Moreover, the results implicated that the gamma secretase-Notch pathway may beinvolved in the molecular pathogenesis of familial AI. In addition, the study furtherconfirmed that exome sequencing is a powerful and effective approach fordiscovering rare monogenic disorder. |
PDF Full Text Request