Identification And Molecular Genetics Reasearch Of LOR Gene C.323G Mutation In A Pedigree Of Palmoplantar Keratodermas

Objective: Identification of pathogenic sites in a palmoplantar keratodermas family,analysis of its clinicopathological characteristics,to explore the pathogenesis of this family,to study the subcellular localization changes of LOR gene c.323G>C mutant protein in the family and its possible effects on apoptosis,cell cycle,proliferation and differentiation of Ha Ca T cell lines..Methods: Peripheral blood of related members of a palmoplantar keratodermas family was collected,genealogical map was drawn and genetic pattern was determined.267 genes known to be related to human skin diseases were detected by second-generation capture sequencing,and the suspected mutation sites were verified by sanger sequencing in the family line.The mutation pathogenicity and amino acid conservatism of LOR gene c.323G>C were analyzed by bioinformatics.Immunohistochemical method was used to analyze the clinicopathological characteristics of this family.Three expression vectors pc DNA3.1/V5-His-wild-LOR,pc DNA3.1/V5-His-c.323G>C-LOR and pc DNA3.1/V5-His-730 ins G-LOR were constructed and transfected into Ha Ca T cell lines,respectively.The localization of loricrin in cells was observed by laser scanning confocal microscopy,and the effect of mutant loricrin on cell apoptosis and cell cycle was detected by flow cytometry.Quantitative real-time PCR was used to detect the effect of mutant loricrin on cell cycle.QPCR)to detect the changes of the transcriptional levels of profilaggrin N-terminal domain(profilaggrin N-terminal domain,PND)and caspase14 protein in Ha Ca T cells..Results: Both the proband and his father with the disease carried heterozygous missense mutation c.323G>C on the LOR gene,which resulted in the replacement of Gly at 108 conserved position of the loricrin protein by Ala,while the proband’s mother with normal phenotype did not carry the mutation.Bioinformatics analysis indicated that the mutation had potential pathogenicity.Immunohistochemical results showed that the patients in this family had excessive proliferation of basal layer cells,thickening of the basel layer,granual layer and stratum corneum,excessive keratosis of the skin,and some residual nuclei in the stratum corneum.After the mutation,a large amount of loricrin was collected in the cytoplasm,and the expression of loricrin in the nucleus was low or even not expressed.I-Tasser results showed that the ligand type and the active site of the enzyme were changed after the mutation.The 2-D structure of the mutant loricrin constructed by Pymol showed that the hydrogen bonds between the mutant site 108 and two adjacent amino acid residues were lengthened and changed from 6 alpha helix to 8 alpha helix,and the helix sites did not match.Laser confocal microscope showed that:In the Ha Ca T cell lines transfected with pc DNA3.1/v5-His-730 ins G-LOR plasmid,the mutant protein was mainly distributed in the nucleus,while in the Ha Ca T cell lines transfected with pc DNA3.1/ v5-His-c.323G>C-LOR plasmid,the mutant protein was mainly distributed in the cytoplasm.The wild-type loricrin protein was evenly distributed in the nucleus and cytoplasm.No significant changes in cell cycle and apoptosis of Ha Ca T cell line were detected in the loricrin of the LOR gene c.323G>C mutant.The q PCR results showed that the overexpression of c.323G>C and 730 ins G mutant protein of LOR gene decreased the transcription level of PND in Ha Ca T cell line.The relative amount of PND gene m RNA transfected with wild-type LOR plasmid was 1.490.The relative amount of PND gene m RNA in c.323G>C was 0.91,and the relative amount of PND gene m RNA in 730 ins G positive control was 0.613(P <0.05).The relative amounts of caspase14 m RNA in Ha Ca T cell lines were 0.649,0.449 and 0.253 after overexpression of wild type LOR protein and c.323G>C and 730 ins G mutants(P <0.05).Conclusion: In the Loricrin Keratoderma(LK)family,the LOR gene heterozygous c.323G>C missense mutation is the cause of Loricrin Keratoderma.This is the first report that this mutation leads to delayed dominance and progressive aggravation of Loricrin Keratoderma.The change of spatial structure of c.323G>C mutation may weaken its nucleation ability,leading to its high concentration in the cytoplasm.It is speculated that c.323G>C mutation may interfere with the differentiation process of human keratinocytes and lead to palmoplantar keratodermas.According to the results of immunohistochemistry of the patient’s skin tissue and the experimental results of Ha Ca T cell line.Our study enriched the mutant profile of LOR gene and the genetic characteristics of Loricrin Keratoderma,and it was found for the first time that the highly aggregated mutant loculin in cytoplasm and the highly aggregated mutant loricrin in nucleus also lead to the occurrence of LK disease.