Epidermolytic Palmoplantar Keratoderma:Genotype-Phenotype Correlation, Prenatal Genetics Diagnosis And Knock-in Mouse Model

PART1Genotype-phenotype correlation and prenatal genetic diagnosis in12epidermolytic palmoplantar keratoderma kindredsBackground:Epidermolytic palmoplantar keratoderma (EPPK, OMIM:144200) is a relative common autosomal dominant inherited defect that often includes disabling diffuse hyperkeratosis of the palms and soles for which no satisfactory treatment is currently available. EPPK is mainly caused by dominant-negative mutations in keratin9(KRT9). Penetrance is100%.Objectives:To identify the causative mutations in12Chinese EPPK pedigrees, and to explore the DNA-based prenatal diagnosis for the fetus at risk of EPPK.Methods:Peripheral blood samples were drawn from the affected and unaffected family members in twelve EPPK pedigrees. DHPLC, RFLP, AS-PCR and Sanger sequencing of PCR products were utilized to screen and verify the potential mutations. The amniotic fluid-DNA-based prenatal testing at4at-risk pregnant EPPK women was performed.Results:All the EPPK-affected individuals in12pedigrees suffered severe diffuse palmar and plantar hyperkeratosis including hyperhidrosis and cuticle splitting. Five heterozygous missense mutations c.C487T (p.Arg163Trp), c.T470C (p.Met157Trp)、 c.T1373C (p.Leu458Pro)、c.G488A (p.Arg163Gln) c.A482G (p.Asnl61Ser) were detected in the affected individuals of11EPPK pedigrees, with no disease-causing gene found in one pedigree. The most frequent mutation was p.Argl63Trp, with the penetrance of41.7%(5/12). Knuckle pad-like keratoses was presented in67%(8/12) EPPK pedigrees. The phenotype of camptodactyly was also observed in three pedigrees, which referred to the possible consequence of mechanical friction. Fetus DNAs derived from amniotic fluid were tested by Sanger sequencing to identify the known mutation existed in the family. Three of four fetuses predicted to be normal with the KRT9wild type, while one was found to be a mutant.Conclusions:The genotype-phenotype correlation was complex in EPPK, which suggested the roles of environmental factors, gene polymorphisms or modifier gene in the development of non-classic EPPK phenotypes such as camptodactyly. In addition, knuckle-pad might be a congenital characteristic to common EPPK, regardless of friction. Precise knowledge of the KRT9mutation has facilitated DNA-based prenatal diagnosis in EPPK, which has proved to be of great benefit for individuals or couples at risk of having children with this disorder.PART2Construction of the Krt9knock-in mouse model with epidermolytic palmoplantar keratodermaBackground:The animal model will be useful for studying the role of KRT9in EPPK. And it has important implications for the development of new strategies for somatic gene therapy of EPPK and other dominant genodermatoses.Objectives:To establish the KRT9/500delAinsGGCT homolog mouse model for EPPK by gene knock-in techniques.Methods:The targeting vector was constructed by introduction of the point mutation (Krt9/c.434delAinsGGCT) into the mouse K9gene. Mouse C57BL/6ES cells were electroporated with the linearized targeting vector, and the recombinant ES cells were injected into albinic C57BL/6blastocysts after it was identified by southern hybridization and DNA sequencing. Chimeric offspring were crossed with wild type mice and descendants carrying the mutant allele were identified by PCR analysis of genomic DNA obtained from tail biopsies. The phenotypes, histology, protein expression and immunohistochemistry analysis were performed in the Krt9/c.434delAinsGGCT knock-in mice. Results:The Krt9/c.434delAinsGGCT knock-in mice that developed hyperpigmented calluses began to develop on the stress-bearing fore-paw and hind-paw footpads between3and4weeks after birth, which persisted into adulthood. In addition, callus size and hyperpigmentation state grew and intensified, respectively, over a-3-week-period on the major footpads before sloughing off. Histological, immunohistochemical, and Western blot analysis of these regions revealed hyperproliferation, impaired terminal differentiation, and abnormal expression of keratins K5, K2and K10. Furthermore, the absence of K9induces the stress-activated keratins K6and K16.Conclusions:Krt9/c.434delAinsGGCT mutation resulted in EPPK-like phenotype of mouse, mimicing EPPK at both the genetic and phenotypic levels. K9is required for terminal differentiation and maintaining the mechanical integrity of palmoplantar epidermis.