Next Generation Sequencing Reveals A Novel Splice Donor Site Mutation In2B Domain Of KRT1Underlies Diffuse Palmoplantar Keratoderm In A Four Generation Chinese Family

The diffuse palmoplantar keratoderma (DPPK) is an autosomal dominant genodermatoses. It is characterized by restricted, uniform hyperkeratosis on the palm and sole epidermis. It is normally associated with dominant-negative mutations in the KRT1; however in several cases the causative mutations were identified in KRT9. In our study, we investigated a four generation Chinese family has been suffering from diffuse palmoplantar keratoderma. After, next generation sequencing, we identified two novel mutations i.e. c.1254G>C, p.Gln418His in KRT1and c.228delG in GJB6as well as a previously reported pathogenic mutation i.e. c.1216T>C, p.Cys406Arg in KRT9in several family members. We validated the result of next generation sequence by direct sequencing. After studying the segregation of disease phenotype in the family members and subsequent linkage analysis enable us to exclude the c.228delG in GJB6and c.1216T>C, p.Cys406Arg in KRT9as a candidate mutation. Therefore, we found that a heterozygous novel point mutation in exon6splice donor site of KRT1(c.1254G>C) that segregates with DPPK in this four generation Chinese family. Functional analysis of this mutation has been done by in vitro exon trapping. As a splice donor site mutation, it completely abolishes the normal splicing consecutively causes aberrant splicing. Functional analysis of this mutation showed that the substitution of nucleotide (c.1254G>C) leads to complete loss of exon6and simultaneously utilization of a novel in-frame splice site54bases downstream of the mutation with the subsequent loss of42amino acids and insertion of18amino acids into2B domain of KRT1respectively. Bioinformatic analyses also support our finding. Multiple sequence alignment showed that KRT1Q418is evolutionary well conserved among several species as well as in all type Ⅱ keratin protein. As keratin is a coiled coil heterodimers, our protein structural studies showed that Gln418of KRT1located at the ’g’ position in heptad unit is engaged in a hydrogen bond with Lys386of KRT10. Q418H for KRT1mutation loses the hydrogen bonding interaction with Lys386of KRT10. So, as a result of this mutation, the coiled coil heterodimers of KRT1-KRT10become weaker than normal. This mutation appears to have a milder effect than previously described mutations in the helix initiation and termination sequence on the function of the rod domain, with regard to filament assembly and stability. This is the first report of a novel splice donor site mutation causes aberrant splicing followed by formation of two alternative transcripts simultaneously causing DPPK. In conclusion, next generation sequencing and in vitro exon-trapping studies demonstrated that the Q418H mutation completely abolishes the normal splicing of the mutant allele so this heterozygous novel missense mutation is classified as a deleterious mutation. Next-generation sequencing revolutionizes clinical medicine research. The gene mutations predisposing many palmoplantar keratoderma need to be resolved to facilitate definitive molecular diagnosis and genetic counseling. This work supports that the next generation sequencing technologies are revolutionizing in clinical genetics and their effects are becoming increasingly widespread.This study also suggests that alternation of alternative transcript levels caused by missense mutations may be one mechanism predisposing to diseases. Such information is valuable for unaffected family members who would benefit from predictive testing for this deleterious mutation.