The Role Of Rare Variants In The CTSD Gene In Neuronal Ceroid Lipofuscinosis Type 10

Objective: The CTSD gene encodes cathepsin D,which mainly degrades abnormal proteins in cells in lysosomes.Mutations in the CTSD gene lead to neuronal ceroid lipofuscinosis type 10(CLN10 disease),whose pathogenesis is currently thought to be related to changes in the enzyme activity of CatD.Given that the CatD protein has the function not related to its enzyme activity,inconsistency between the severity of the disease and different mutations in CTSD gene,and the lack of in-depth studies on the molecular mechanism,we intend to study the effects of different reported CTSD gene variants on the protein expression,intracellular transport and maturation,protease activity and other aspects of CatD,in order to clarify the molecular mechanism of disease caused by different CTSD gene mutations.Methods:We use the cDNA of human cells to construct a wild-type expression vector for CTSD gene,and on the basis of the wild-type,6mutant expression vectors are constructed through site-directed mutagenesis.The wild-type and 6 mutant expression vectors are transfected into HEK293 T cells and the cells with CTSD gene knock out,respectively,the mRNA and protein expression levels of wild-type and 6 mutant expression vectors in the two cells were detected respectively.In addition,the cell transport pathway of the protein expressed by the wild-type and 6 mutant expression vectors was detected in the knockout cells,and the cathepsin D activity detection kit was used to detect the cathepsin D activity of the wild-type and 6 mutant expression vectors in the knockout cells.Finally,the protein conformation of the mutant CatD protein was analyzed.Results: Real-time quantitative PCR experiments found that the 6mutant plasmids did not reduce their mRNA expression in both HEK293 T cells and CTSD gene knockout cells.Western blotting experiment found that the expression of mature protein encoded by CTSD gene in the 6 mutant plasmids was reduced in both cells,but the immature protein was no different from the wild type.Through immunofluorescence experiments,it was found that the proteins expressed by the 6 mutant plasmids changed in the cell transportation pathway,showing that the immature protein was no different from the wild type,but the mature protein transportation was blocked.Finally,the results of cathepsin D activity also showed that the cathepsin D of the 6mutant plasmids were greatly reduced,and it was found that the enzyme activity of the remaining CatD in the mutants was negatively correlated with the severity of the neurodegenerative disease.At the same time,the three-dimensional dynamic analysis of the 4 mutant CatD proteins also further explained the mechanism of the mutant protein causing the protein structure and function changes.Conclusion: Through a unified study of six kinds of CTSD gene missense sites,it is found that mutations at different sites of CTSD gene have different effects on the enzyme activity of CatD,and the molecular mechanisms are also different.The age of onset of CLN10 disease and the severity of the disease are highly negatively correlated with the enzymatic activity of the remaining CatD in the mutant,and may not be related to the non-enzymatic activity of the CatD protein.Objective: The CTSD genes encode cathepsin D.Mutations in the CTSD gene lead to neuronal ceroid lipofuscinosis type 10(CLN10)disease.This study investigated whether the c.863.A>G mutation of a newly discovered child neurodegenerative disease-related gene CTSD is a pathogenic variant.Methods:Collect cubital venous blood from patients and their parents,and extract genomic DNA and RNA from the blood.The DNA is sequenced for medical exons,and the PCR amplified products are subjected to first-generation sequencing to verify the mutations suggested by the medical exome sequencing.Reverse transcription of patient RNA was used to obtain cDNA,which was used as a template to amplify the coding region sequence of CTSD gene.The homologous recombination method was used to construct the CTSD gene mutant expression vector.The first part has been successfully constructed a wild-type expression vector of CTSD gene.These expression vectors were transfected into human embryonic kidney cells and CTSD gene knockout cells respectively,and the m RNA and protein expression levels of wild-type and mutant-type expression vectors in the two cells were detected.In addition,the cell transport pathway of the protein expressed by the wild-type and mutant expression vectors was detected in the knockout cells,and finally the cathepsin D activity detection kit was used to detect the cathepsin D activity level of the wild-type and mutant expression vectors in the knockout cells.Results:Real-time quantitative PCR experiments found that the mutant plasmid has no change in mRNA expression in human embryonic kidney cells(HEK293T)and the cells with CTSD gene knockout.Western blot experiments showed that the mutant plasmid did not reduce the CTSD gene encoding in both cells.Through immunofluorescence experiments,it was found that the protein expressed by the mutant plasmid did not change in the cell transportation pathway.The results of the cathepsin D activity detection experiment also showed that there was no difference between the mutant cathepsin D and the wild type.Conclusion: The c.863.A>G mutation of the CTSD gene encoding cathepsin D did not cause cathepsin D function changes.This mutation is a benign missense mutation in the CTSD gene.