Molecular Genetics Investigation Of Two Monogenic Inherited Skin Diseases:Palmoplantar Keratodermas And Waardenburg Syndrome

Hereditary skin diseases can be divided into monogenic dermatosis and polygenic dermatosis.Monogenic dermatosis refers to skin diseases controlled by only a pair of alleles inherited according to the classical Mendelian inheritance model.There are many kinds of monogenic dermatosis,which often have no effective treatments.The causing genes and pathogenesis are unclear,which is the main reason for the limited treatments of monogenic dermatosis.Recent advances in sequencing technology have promoted the development of hereditary skin diseases.High-throughput sequencing,also known as next-generation sequencing,includes whole-exon sequencing,whole-transcriptome sequencing,etc.,which can simultaneously sequence millions of nucleic acid molecules,and is of great significance for the study of diseases with unknown pathogenic genes.Genetic testing for patients with hereditary skin diseases is beneficial to the clinical diagnosis and genetic counseling of patients.The discovery of new genes and loci will further expand the gene profile of pathogenic genes,and have great significance for revealing the pathogenesis of diseases,searching for therapeutic targets,and developing effective targeted drugs.This paper studied the genetic detection and pathogenesis of single-gene skin diseases,including palmoplantar keratoderma and Waardenburg syndrome.1.Research on the determination of SERPINA12 mutation,founder effect and pathogenesis of SERPINA12-related PPKBackground:Nagashima-type palmoplantar keratodermas mainly presents with erythema with clear boundary,often accompanied by keratosis,hyperhidrosis and other symptoms,belonging to the autosomal recessive non-mutilating type PPK.It occurs frequently in East Asia,and the known pathogenic gene is SERPINB7.However,our research group did not find SERPINB7 mutation in some patients clinically diagnosed with NPPK by Sanger sequencing.Objective:（1）Genetic testing was performed on the above 52 patients with unknown pathogenic genes clinically diagnosed as NPPK to identify their pathogenic sites.（2）Haplotype analysis was performed on the high-frequency site c.970₉71 del to explore whether it was founder mutation.（3）The possible pathogenesis of NPPK caused by SERPINA12 mutation was explored by enzyme activity experiment,immunofluorescence and other methods.Methods:（1）Clinical data of 52 NPPK patients and their families were collected,and the pathogenic mutations of the 52 patients were identified by whole exon sequencing technology and verified by Sanger sequencing.（2）By screening SNP sites upstream and downstream of SERPINA12 c.970₉71del mutation with the highest carrying rate,and conducting SNP site detection in patients carrying this mutation to explore whether c.970₉71del was founder mutation.（3）The inhibitory effect of SERPINA12 on KLK7 and KLK14 proteases was verified by detecting their enzyme activity,which may be one of the pathogenesis of NPPK caused by SERPINA12.Results:（1）There are 6 patients carried SERPINA12 gene mutation,including 4 mutations:c.970₉71del.c.662delA,c.656A>G,and c.635-7 A>G.（2）The patients carrying c.970₉71 del mutation have the same haplotype,which suggests that c.970 971 del might be a founder mutation.（3）SERPINA12 can inhibit the protease activity of KLK7 and KLK14,which explains the pathogenesis of NPPK.Conclusions:In this study,the pathogenic gene of 6 NPPK patients was identified as SERPINA12 by whole-exon sequencing,and c.970₉71 del was verified as a founder mutation by haplotype analysis.The loss-of-function of SERPINA12 increases the protease activity of KLK7 and KLK14,which leads to the NPPK.2.Genetic investigation of a patient with Waardenburg syndromeBackground:The typical clinical manifestations of Waardenburg syndrome are pigment changes in skin,hair and iris and congenital sensorineural deafness.Pigment changes affect the appearance of patients and bring serious psychological burden to patients.And deafness symptoms significantly reduce the quality of life of WS patients.At present,the pathogenic genes of Waardenburg syndrome have been found to be MITF,PAX3,SOXI0,EDN3,EDNRB and SNAI2.Identifying the pathogenic genes of patients with Waardenburg syndrome can help physicians provide genetic counseling and prenatal diagnosis to ensure that patients have healthy offspring.Objective:To further determine the etiology and diagnosis of a patient clinically diagnosed with Waardenburg syndrome by genetic testing.Methods:The clinical data of the proband and their parents were collected,the genomic DNA was extracted from their peripheral blood,the suspected pathogenic genes were screened by the genetic dermatosis exome sequencing technology,and the suspected loci were verified by Sanger sequencing.Results:The sequencing results indicated that the proband carried MITF c.649₆51delAGA（p.R217del）heterozygous deletion mutation,while neither of his parents had this mutation,suggesting that this mutation was a de novo mutation.Conclusion:MITF c.649₆51delAGA mutation was the pathogenic cause of the patient.Identifying the mutation could provide a prenatal diagnosis for the patient’s future fertility and avoid the mutation from being delivered to the next generation.