Investigation Of The Pathogenicity And Potential Mechanisms Underling PRRT2 Variants

PRRT2-related disorders are autosomal dominant hereditary diseases caused by PRRT2 variants,including paroxysmal kinesigenic dyskinesia(PKD),benign familial infantile seizures(BFIS),infantile convulsions with choreoathetosis(ICCA),hemiplegia migraine(HM),episode ataxia(EA)and so on.Primary PKD is the most common phenotype.To date,more than 100 variants have been reported,including nonsense variants,missense variants,splicing variants,small deletion,small insetion and so on.At least 90%of these variants lead to stop codon in advance and become the most common deleterious type.So far,cases with missense variants are far less than those with truncated variants,and there have been few functional studies about missense proteins.Whether these missense variants cause PRRT2-related disorders remains unclear.It brings difficulties to the diagnosis and treatment of diseases.PRRT2 is a membrane protein specifically expressed in the central nervous system and controls neuronal excitability by modulating Na+ channel activity and synaptic transmission.Truncated variants lead to conspicuous reduced protein level and abnormal subcellular localization.Haploinsufficiency may be the pathomechanism of PRRT2-related disorders.Studies of 2 missense variants(p.A287T and p.R308C)have showed that the variants also have decreased protein level or diffuse into the cytoplasm.However,our team did not find the same phenomenon in the previous study of another 2 missense variants(p.R266W and p.G305R).In 2016,our group generated induced pluripotent stem(iPS)cell derived from urinary epithelial cells of patients with PKD.However,these iPS cells differentiated into neurons with a long period of time and low efficiency in the previous experiments.It has been reported that iPS cells inherite epigenetic memory of their original somatic cells,which may have effect on differentiation and other cell properties.Human skin fibroblasts are the most frequently used original cells to generate iPS cell model of PKD patients.In this study,we performed cell experiments on all the PRRT2 missense variants reported before December 2017,and analyzed their pathogenicity according to American College of Medical Genetics and Genomics(ACMG)guidelines.For PKD patients with negative results in coding region tests of PRRT2,we further screened variants in regulatory region of PRRT2 gene and conducted functional experiments to analyze their pathogenicity.Meanwhile,we explored the pathogenesis of PRRT2 in term of protein degradation.Human iPS cell model derived from skin fibroblasts carrying PRRT2 hotspot variant was generated,which laid a foundation for further exploration of the pathological mechanism of PRRT2.Section I:Pathogenicity analysis of PRRT2 missense variantsObjective:To evaluate the pathogenicity of PRRT2 missense variants by performing in vitro experiments.Methods:We systematically reviewed the PRRT2-related disorders and summarized the PRRT2 missense variants reported before December 2017.Expression vectors containing EGFP at the N-terminus were constructed and transfected into cells.Western blotting was used to observe the effects of mutant PRRT2 on protein level.The subcellular localization of proteins was observed under confocal microscopy.The pathogenicity of PRRT2 missense variants was analyzed according to the ACMG guidelines.Results:29 PRRT2 missense variants were identified in PRRT2-related disorders.10 variants were observed affecting both subcellular localization and protein level,3 variants only altered protein membrane localization,and 2 variants only lowered protein level.According to ACMG guidelines,15 variants(p.S275F,p.P279L,p.W281R,p.A287T,p.A291V,p.R295Q,P.G305W,p.A306T,p.A306D,p.R308C,p.S317N,p.G323R,p.G323E,p.G324R and p.G324E)were finally classified as likely pathogenic variants,3(p.P 138A,p.D147H and p.P216L)were classified as benign variants,3(p.S208Y,p.A214P and p.P215R)were classified as likely benign variants and 8(p.E177K,p.P216R,p.P216H,p.R266W,p.R266Q,p.G305R,p.R311W and p.I327M)were classified as uncertain significance.Conclusion:51.7%(15/29)of reported PRRT2 missense variants can cause PRRT2-related disorders.The likely pathogenic variants are mainly concentrated in the C-terminal of PRRT2,indicating crucial physiological functions of these region.Section II:Investigation of variants in the untranslated region(UTR)of PRRT2 in PKDObjective:To screen variants in the untranslated region(UTR)of PRRT2 for PKD patients with negative results in coding region tests of PRRT2 and analyze their pathogenicity.Methods:The UTR of PRRT2 in 85 PKD patients with negative results in coding region tests of PRRT2 was screened.Expression vectors containing UTR variant were constructed.Cell transfection studies were preformed.Real-time quantitative PCR was applied to measure the mRNA level.Western blotting was used to observe the effects of the variants on protein level.Results:Of the 85 PKD probands,2 had heterozygous variants in the 3’UTR(c.*933A>G and c.*978A>G),and no variants was detected in the 5’UTR.In vitro cell experiments revealed that 3’UTR can significantly down-regulate mRNA level of PRRT2,leading to suppressed protein expression.The 2 variants attenuated the down-regulation of the 3’UTR,and up-regulated the mRNA and protein level of PRRT2 to some extentConclusion:Gain of function of PRRT2 may also play a role in PRRT2-related disorders.It is important to screen variants in 3’UTR for PKD patients with negative results in coding region tests of PRRT2.Section III:Preliminary study on the pathogenic mechanism of mutant PRRT2 proteinObjective:To preliminarily explore the pathogenic mechanism of mutant PRRT2 protein and generate human iPS cell model derived from skin fibroblasts carrying PRRT2 hotspot variantMethods:The hot spot mutant and wild-type expression vector were transfected into cells.Lactacystin or 3-MA were applied to inhibit proteasome-or Iysosome-mediated protein degradation,respectively.PRRT2 protein was detected by immunoblotting.Liquid chromatography-mass spectrometry combined with immunoprecipitation was used to find proteins that may be involved in the degradation of PRRT2.Human skin fibroblasts carrying PRRT2 hot-spot variant were cultured.Then,iPS cell was generated by using sendai virus reprogramming kit and differentiated into neurons.The PRRT2 protein in neurons was detected by immunoblotting.Results:Compared to wild-type PRRT2,application of lactacystin or 3-MA could both increase mutant PRRT2 at higher levels.The mutant protein was degraded by proteasome-and lysosome-mediated protein degradation.PRRT2 interacts with the E3 ubiquitin ligase AMFR.The human iPS cell carrying the PRRT2 hotspot variant was successfully generated and differentiated into neurons.A significant decreased mutant PRRT2 protein level was found in the neuronsConclusion:Abnormal degradation of mutant PRRT2 resulted in a decrease of mutant protein level.The iPS cell derived from skin fibroblasts carrying PRRT2 hotspot variant is one of appropriate cell models for studying the pathological mechanism of PRRT2.