Research On The Pathogenic Genes And Related Pathogenic Mechanisms Of Acrokeratoelastoidosis

Background In this study,the pedigree of acrokeratoelastoidosis(AKE)was studied.The proband presented with multiple,firm and keratotic papules on both hands,the papules overlying the metacarpophalangeal and proximal interphalangeal joints protuberance of the proband.And multiple papules fused with a "paving stone" appearance,and predominantly distributed along the dosal palmar junction of the proband.Other members of his family had similar papules on the hands.Objective1)To explore the possible pathogenic genes of AKE through genetic studies within family.2)To study the related pathogenesis associated with AKE and lay a foundation for the diagnosis and treatment of the disease.Methods1)The four members(II-2,III-7,IV-3,IV-4)of an autosomal dominant AKE family were sequenced by whole-exome sequencing(WES).2)Primer Premier 5.0 software was used to design primers for mutation sites of the candidate genes,and the candidate genes were verified by PCR amplification and sanger sequencing in 19 members of the family and 60 healthy control individuals unrelated to the family,analyzed whether the gene is co-segregating with the disease in the family.3)Screening and validating of suspected pathogenic genes in another AKE family.4)Sh RNAs were cloned into lentivirus vector,and sh RNA-Encoding Lentivirus were packaged in 293 T cells,then transfected into human skin fibroblasts to form a stably transfected cell line p LKO.1 sh-CCDC91.The subcellular localization and possible mechanism of CCDC91 gene were studied by immunocytochemistry(ICC)and transmission electron microscopy(TEM).Results1)8 possible candidate genes were screened by WES,namely OVCH2: P.G257 D,CELSR3: P.A157 P,TRAIP: P.R157 H,CD180: P.N207 S,OTP: P.A71 V,ERAP2:P.T407,RIOK2: P.D140 N and CCDC91: C.993 + 1G > A.2)Sanger sequencing results showed that the pathogenic gene of the AKE family was CCDC91,the gene was present in all the affected individuals in the family,but was absent in the unaffected individuals.3)Sanger sequencing of the CCDC91 gene in another AKE family showed a missense mutation of the CCDC91 gene(c.941A>G,p.K314R).4)ICC results showed that the subcellular localization of CCDC91 gene was on the Golgi apparatus.TEM results showed that compared with p LKO.1 Sh-Cnotrol group,the Golgi apparatus of the p LKO.1 sh-CCDC91B2 group was swollen and fragmented,the number of lysosomes and autophages increased,and the accumulation of vesicles could be seen in the cytoplasm.Conclusion The subcellular location of CCDC91 gene is located in Golgi apparatus.The structural changes of CCDC91 protein may affect the transport between vesicles,which reduces the synthesis and secretion of elastin,leading to the occurrence of AKE.