Research Of Gene-phenotype Correlation Of Inherited Keratoderma

Objectives : To investigate the genetic basis of an atypical Chinese palmoplantar keratoderma pedigree,we performed mutation screening in several associated genes of palmoplantar keratoderma.We further explored the genotype-phenotype correlation by integrating functionl assays with in silico predictions.We also evaluate the efficiency of in diagnosis ing keratoderma cases with high heterogeneity.The above research is intend to develop a new pattern for studying the genotype-phenotype correlations of genodermatosis.Methods:A comprehensive sequencing of the entire coding region of several genes associated with palmoplantar keratoderma in the atypical PPK predigree was conducted.We dissected the structural changes of the protein resulted from the identified causative mutation by molecular modeling.m CSM and DUET prediction software gave an estimation of the free energy change(??G),to correlate genotype with phenotype.To investigate the apoptosis induction effects of four different amino acid substitution on Ha Ca T cells,cell viability was evaluated by CCK8 after transfection with various mutations in TRPV3 or wild type TRPV3 gene for 12,24,48 and 72 h.An annexin-V fluorescein APC/7-AAD double stain assay and flow cytometry analysis were performed to confirm cell apoptosis and to explore the differences in the apoptosis induction resulting from these four mutations versus the wild type.We further developed targeted depth sequencing platform to obtain the genetic etiology of four highly heterogeneous keratoderma cases.Results:(1)We indentified an identical pathogenic mutation c.2016 G > A in the proband,his mother and his maternal grandfather.(2)The only positive value was due to the Gln580 Pro mutation,which resulted in a relatively stable protein structure and caused the mildest phenotypic appearance.??G value resulting from other eight point mutations in TRPV3 were all negative values that were associated with more sever phenotypes.Among the eight mutations,the absolute energy change resulting from the Met672 IIe mutation in our case was less than the other seven mutations,which in turn caused a less dramatic conformational change on the ion channel and a relatively moderate clinical manifestation.(3)A CCK8 assay revealed that the four mutations all had apoptosis induction impacts upon Ha Ca T cell compared with that of the wild type.Cell viability was reduced remarkably after transfection for 48 h.The cytotoxicity exerted by G573 C and W692 G were more obvious,while the cytotoxicity imposed by M672 I and Q580 P were milder.(4)An annexin-V fluorescein APC/7-AAD double stain assay and flow cytometry analysis further revealed that transfection with p.Gly573 Cys,p.Trp692 Gly,p.Met672 IIe and p.Gln580 Pro increased the number of apoptosis cells in varying degrees compared with the wild type.In contrast,p.Gly573 Cys and p.Trp692 Gly exerted more significant induction of apoptosis upon Ha Ca T cells.The degree of the induction of apoptosis was consistent with the phenotype resulting from the corresponding mutation,which might hint towards the underlying mechanism mediating the genotype-phenotype correlation.(5)By using the next-generation targeted sequencing platform,we obtained the definitive diagnosis of two Neu-Laxova syndrome cases and two Mal de Meleda cases,which were all the first report of these two orphan diseases in Asian.Conclusions:(1)The mutation we identified : c.2016 G > A in TRPV3 was the causative mutation of the atypical Chinese PPK pedigree.Meanwhile,the scleodactyly-like appearance and tapered fingers may represent unreported clinical signs,thus broading the phenotypic spectrum of OS.(2)The in silico estimation and functional data consistently corroborated the underlying genotype-phenotype relationship of OS.(3)We further confirmed that the atypical cases with mutations in TRPV3,including the absence of aforementioned hallmarks and the presence of some unusual clinical features,actually belonged to OS.Our research thus redefined the phenotypic spectrum of OS.(4)By using next-generation targeted sequencing platform,we can identify the genetic etiology of rare and atypical cases economically and effectively.(5)Our research opened new avenues for studying the geneotype-phenotype of genodermatosis.