X-linked Parkinsonism Caused By A Novel Mutation Of RAB39B: Genetic Analysis And Mechanism Study

BackgroundParkinson's disease(PD)is the second most popular neurodegenerative disease in the elderly.Juvenile Parkinsonism(JP)is very rare and is caused by various genetic defects.Most patients are with adolescent onset,generally earlier than 21 years old.The clinical manifestations include slowly progressive tremor,muscle rigidity,bradykinesia and postural instability,and treatment with levodopa brings good relief.To date,there are more than 20 monogenic mutation forms are associated with JP.Pakin,Pink1,and DJ-1 mutations can cause typical Parkinsonism.However,patients with PLA2G6,ATP13A2,and DNAJC6 mutations usually present with not only Parkinsonism but also speech disorders,pyramid sign,dysarthria and other atypical symptoms.RAB39 B gene is located in the X chromosome and encodes a kind of vesicle trafficking-associated protein.It is reported that RAB39 B mutaions can cause early-onset PD with cognitive impairment.At present,there is no report of JP caused by RAB39 B mutation in Chinese Han population.ObjectiveTo determine the pathogenic mutation by whole exome sequencing(WES)in the pedigree with JP;to investigate the role of RAB39 B mutation in our population with sporatic PD;to preliminarily explore the role of RAB39 B underlying the pathogenesis of PD.Methods 1.In this study,we collected one pedigree with JP and 505 sporadic PD patients rom the First Affiliated Hospital of Zhengzhou University in China.All the atients were subjected to the detailed assessment from two neurologists ccording to the United Kingdom Parkinson's Disease Society Brain Bank.We lso included 510 age and sex-matched healthy individuals.All the subjects were nformed consent,registered clinical data and extracted DNA from blood amples.2.Two male patients in this pedigree were subjected to WES,and the sequencing esults were analyzed later to identify the causative mutation.3.Using Sanger sequencing,the RAB39 B gene mutation site was verified in all embers of the family.Sporadic PD patients and controls were genotyped by anger sequencing.The entire RAB39 B exons and exon-intron boundaries were equenced from genomic DNA.4.We constructed pcDNA3.1-HA-RAB39B-536 vector containing the mutation we dentified.We used liposome transfection,Real-time PCR,Western-blot,mmunofluorescence and other methods to confirm the expression level of utant RAB39 B,and the effect of RAB39 B on autophagy and ?-synuclein ?-Syn).Results 1.The two male patients in the pedigree showed limb tremor,bradykinesia,and tiffness with different degrees of cognitive impairment in their adolescence.rain MRI and CT showed bilateral basal ganglia calcification in two patients.The other members of the family are normal.2.According to WES data,we identified 31 suspicious pathogenic variants shared y two patients in this pedigree.RAB39B(c.536dupA)mutation was most likely o be the pathogenic mutation.3.?-2 and ?-1 carried with RAB39 B c.536 dupA hemizygous mutation.The roband's mother ?-3 carried with RAB39 B c.536 dupA heterozygous mutation. he other members in this family were normal.The genotype was cosegregated ith the phenotype in this family.We did not find any other disease-causing AB39B mutations in sporadic PD patients and controls.4.We successfully construced the pcDNA3.1-HA-RAB39B-536 vector.In N2 a cell odel,the mutant RAB39 B showed the higher transcriptional level than ild-type RAB39 B,but the protein level of mutant RAB39 B was significantly ower than that of wild-type.After adding MG132,the protein level of mutant AB39B protein increased.5.The LC3 B ?/? value of mutant RAB39 B group was significantly lower than hat of wild group.6.RAB39 B protein may interact with ?-Syn in vivo.Overexpression of wild-type nd mutant RAB39 B did not affect the levels of endogenous ?-Syn,while verexpression of wild-type RAB39 B resulted in the elevated level of exogenous ild-type and mutant ?-Syn(p.A53T).Conclusions 1.We firstly identified RAB39 B gene mutation caused X-linked JP in Chinese Han opulation,and RAB39 B mutation was rare in our population.2.The patients with RAB39 B mutation(c.536dupA)showed obvious calcification in ilateral basal ganglia.No such imaging change was found in the previous tudies.3.RAB39 B mutation(c.536dupA)may lead to the instability of RAB39 B protein,and the mutant protein may be degraded by ubiquitin-proteasome pathway.4.RAB39 B mutation(c.536dupA)may lead to the defect of autophagy.5.RAB39 B may interact with ?-Syn in vivo,and may be involved in the aintenance of steady-state levels of ?-Syn.