Genetic Analysis And Pathogenetic Study Of Parkinson's Disease Associated With CHCHD2

Background:Parkinson's disease(PD)is one of the most common neurodegenerative diseases.Its clinical manifestations include resting tremor,rigidity,bradykinesia,gait disturbance and postural instabilitys.Most PD patients are sporadic,but few are inherited Parkinson's disease caused by genetic mutations.With the continuous advancement of genetic methods,new disease-causing genes of hereditary Parkinson's disease have been discovered in recent years.And the series of functional studies about these PD-causing genes have important implications for understanding the pathogenesis of PD.Nevertheless,a definitive genetic cause for some patients remains elusive.Objectives:In our previous work,we collected a Chinese Han parkinsonism pedigree.Based on the previous work,we will explore the disease-causing gene of the family by exome sequencing,and observe imaging characters of this family by PET-CT.At the same time,we will analyze the association between its polymorphism and sporadic Parkinson's disease.In addition,we further explore the possible pathological mechanism of this mutation using cell and mouse model.Methods:1.We used whole-exome capture and high-thrO?ghput sequencing technology followed by bioinformatic analysis in the two patients and one normal control to investigate the candidate disease-causing mutation.We then used Sanger sequencing technology to repeat the result of exome sequencing and validated the candidate disease-causing gene in cohort of relevant patients and healthy controls.2.Two patients and one normal control in the family were perfonrmed with[11C]CFT-labeled brain dopamine transporter PET imaging and 18F-FDG PET brain glucose metabolism imaging.3.364 sporadic PD patients and 384 normal controls were included.Sanger sequencing of the CHCHD2 gene was performed to investigate the association between CHCHD2 gene polymorphism and sporadic Parkinson's disease in Chinese Han population.4.We constructed wild type and mutant eukaryotic form of CHCHD2 expression vectors.We established stable over-expression of wild-type/mutant CHCHD2 and stable inhibition of CHCHD2 expression SHSY5Y cell lines.Then we investigated the expression of CHCHD2 protein under cell stress conditions,the stability and subcellular localization of the T61I mutant protein.5.Using the constructed CHCHD2 wild-type and mutant over-expression and expression-inhibited monoclonal cell lines,we studied the changes in cell viability,apoptosis,expression of apoptotic proteins,membrane potential,ROS,ATP,and cellular aerobic respiration levels.Isolate and culture patient-derived skin fibroblasts to study changes in the expression levels of CHCHD2 and related proteins6.We detected changes in phosphorylation levels of mutant CHCHDD2.Investigate the interacltion of CHCHD2 protein with RBPJ,COX1,CHCHDIO and MIC Sl proteins.Detect expression of CHCHD1O-related proteins and mitochondrial morphological changes in dermal fibroblasts derived from patient.7.The patient's peripheral blood mononuclear cells were isolated to induce iPS cell line.Using mouse embryonic stem cell targeting technology,a CHCHD2 T61I point mutation mouse model was constructed.Results:1.The parkinsonism pedigree included in this study met the autosomal dominant inheritance rules.In addition to Parkinson's symptoms,the proband had clinical features of multiple system atrophy,and another family member showed essential tremor.Using whole exome sequencing,combined with subsequent bioinformatics analysis,we found that the CHCHD2 c.182C>T(p.T61I)mutation is the pathogenic loci of this family.2.The PET-CT results of patients with CHC.HD2 c.182C>T(p.T61I)mutations in this family showed that the dopamine transporter imaging signal was significantly reduced,negatively related to clinical severity,which is similar to the results of sporadic Parkinson's disease.The levels of glucose metabolism in the putamen and globus pallidus were significantly higher,which is consistent with the Parkinson's disease related model.3.In Han sporadic PD patients,we found five known polymorphic sites:c.-1lG>A,c.-11G>A,c.5C>T,c.51-127G>A,C.456+125G>A.After statistical analysis,there was a significant difference in c.5C>T between the two groups(OR = 4.79,95%Cl 1.03-22.26,p = 0.03).4.We successfully constructed CHCHD2 wild-type and mutant eukaryotic expression vectors,and overexpression and shRNA suppression monoclonal cell lines.We found high expression of CHCHD2 protein under cell stress conditions,no significant changes in protein stability and subcellular localization after T61I mutation.5.Compared with the control,CHCHD2 knockdown cell lines were affected in cell viability,apoptosis levels,apoptotic proteins,membrane potential,ROS,ATP,and cellular aerobic respiration levels.In the steadily over-expressing cell line,compared with the wild-type protein,T61I mutation CHCHD2 loss the protective effect to the cells under stress conditions.The protein and mRNA level of CHCHD2 did not change in patient-derived skin fibroblast.6.Phosphorylation levels of muant CHCHD2 did not change.The CHCHD2 protein interacts with the CHCHD10 protein,and the CHCHD2 mutation has a higher binding level to CHCHD10.The levels of CHCHD10 and DRPI protein in patient-derived dermal fibroblasts were reduced,and the binding levels of CHCHD2 and MICS1 were reduced.Mitochondrial morphology was severely impaired in skin fibroblasts inpatients with CHCHD2 mutations.7.A disease-specific CHCHD2 mutant iPS cell line was constructed,and relevant indicators were identified correctly.A CHCHD2 T61I point mutation mouse model was constructed and identified correctly.Conclusion:1.The c.182C>T(p.T61I)mutation in the CHCHD2 gene is the pathogenic loci of this family.That is,CHCHD2 mutation can cause dominant hereditary parkinsonism.2.In sporadic Parkinson's disease,the CHCHD2 polymorphism site c.5C>T was statistically significant compared with normal controls,s?ggesting that carrying c.5C>T may increase the risk of PD.3.The wild-type CHCHD2 protein has a protective effect on cells.Although the T61I mutant CHCHD2 protein is not cytotoxic,it loses this cytoprotective function.4.The interaction between CHCHD2 and CHCHD1O may play an important role in the pathogenesis.5 The disease-specific CHCHD2 mutant iPS cell line and the CHCHD2 T61I point mutation mouse model were successfully constructed.