Mutation Analyses Of Neurodegeneration With Brain Iron Accumulation And The Functional Study Of PLA2G6

PART ⅠMutation analyses of neurodegeneration with brain iron accumulationNeurodegeneration with brain iron accumulation(NBIA)comprises a group of neurodegenerative diseases caused by gene mutation,which is characterized by extrapyramidal symptoms and iron deposition in the brain.Common clinical features of NBIA include progressive dystonia,spasticity,Parkinson’s syndrome and other complex clinical symptoms.The common imaging feature is abnormal iron deposition with or without cerebellar atrophy.NBIA has genetic and clinical heterogeneity.To date ten forms of NBIA has been described,with autosomal recessive inheritance,autosomal dominant form,and with X-linked dominant inheritance.The most common forms are pantothenase kinase-associated neurodegeneration(PKAN),due to mutations in the PANK2 gene,followed by phospholipase A2 associated neurodegeneration(PLAN)due to PLA2G6 gene mutations,mitochondrial membrane protein-associated neurodegeneration(MPAN)due to c19orf12 mutations,and beta-propeller protein-associated neurodegeneration(BPAN)due to WDR45 mutations.Different mutations of the same pathogenic gene can lead to the same subtype,and different pathogenic genes can cause the same disease.The obvious clinical and genetic heterogeneity of NBIA makes it difficult to diagnose clinically,easily causing missed diagnosis and misdiagnosis.So the diagnose relies on genetic testing.There have been a lot of studies using the traditional Sanger sequencing to screen pathogenic genes for patients with NBIA,but still many patients were not diagnosed clearly.Therefore,a new gene detection technique is urgently needed to identify the diagnosis of patients with significant clinical and genetic heterogeneity.Targeted next-generation sequencing(NGS),a high-throughput gene diagnostic technique,enables simultaneous sequencing of numerous interested genes to achieve rapid screening of pathogenic genes.Based on the advantages of target sequencing,the study applies this technique to detect the disease-causing genes of NBIA patients,and evaluates their application in the gene diagnosis.Method1.18 patients with extrapyramidal symptoms and imaging abnormalities were enrolled.Firstly,extract DNA from the patients、peripheral blood.Then,a panel of genes relevant to NBIA,Parkinson’s disease(PD)and hereditary spastic paraplegia(HSP)was designed.Subsequently,screen pathogenic genes on patients using targeted NGS.Finally,the raw data of targeted NGS were obtained.2.The data of targeted NGS were analyzed,filtered and validated by Sanger sequencing.Then,the variants identified were screened in the families and 500 controls.Subsequently,the classification of novel variants is based on the American College of Medical Genetics and Genomics(ACMG)standards and guidelines.3.The clinical characteristics of each pedigree were analyzed combined with the results of gene detection.Results1.We identified 7 PLA2G6 variants including five known variants(c.668C>T,c.991G>T,c.1117G>A,c.1982C>T,and c.2218G>A)and two novel variants(c.1511C>T,and c.1915G>A)in four index cases.And 4 PANK2 variants including three known variants(c.863C>T,c.1133A>G,c.1502T>A)and one novel variants(c.1679T>C)were identified in 3 cases.After molecular analyses of 7 families with identified PLA2G6 or PANK2 mutations,18 asymptomatic cases and a pre-symptomatic family member who carried two compound heterozygosity(c.1117G>A and c.1511C>T)mutation were found.2.Among 4 cases of PLAN,three cases had initial symptoms of difficulty walking or gait disturbance,and one case and his sibling developed mental handicap.Two cases exhibited a phenotype of ‘early parkinsonism’ and the other two cases mimicked a phenotype of ‘hereditary spastic paraplegia(HSP)’.Iron deposition in globus pallidus and substantia nigra was seen in three cases.Cerebellar atrophy was present in all four cases.Conclusion1.Our study expands the mutation spectrum of the PLA2G6 and PANK2.2.PLAN is clinically heterogeneous and can be characterized by Parkinson’s syndrome or hereditary spastic paraplegia.3.The efficiency of molecular diagnosis for patients with extrapyramidal symptoms and imaging abnormalities can be improved by targeted NGS,which reduces missed diagnosis and misdiagnosis.PART ⅡThe functional study of PLA2G6PLA2G6(Phospholipase A2 Group VI)is a protein coding gene,containing 17 exons.The protein encoded by this gene is an A2 phospholipase,a class of enzyme that catalyzes the release of fatty acids from phospholipids.The encoded protein may play a role in phospholipid remodeling,arachidonic acid release and the synthesis of leukotriene and prostaglandin.There are multiple m RNA isoforms resulting from alternative splicing,and the encoded protein play an important role in maintaining membrane metabolism and membrane stability.Diseases associated with PLA2G6 include Parkinson Disease 14,Autosomal Recessive and Infantile Neuroaxonal Dystrophy.Previous studies have suggested that the loss of enzyme activity caused by PLA2G6 mutations in infantile neuroaxonal dystrophy(INAD).However,whether the decrease in enzyme activity leads to early-onset Parkinson’s disease remains controversial.In the previous study,we found that the clinical manifestations of the patients with the PLA2G6 gene mutation were heterogeneous.In this study,cells functional research was conducted to clear the pathogenicity of novel variants.Method1.Wild-type and three mutant eukaryotic expression plasmid were constructed by restrictive enzyme digestion technique and site-directed mutagenesis,and full length sequences of the target gene were detected.2.HEK293 T cells were transfected with wild-type and mutant eukaryotic expression plasmid by Lipofectamine 3000 kit.Immunofluorescence study was conducted to analyze the subcellular localization of wide-type and mutant proteins.3.The expression difference between wild-type and mutant PLA2G6 protein was analyzed by Western Blot.4.The modified c PLA2 Enzyme Activity Assay Kit was applied to detect enzyme activity in the wild and mutant proteins.Results1.Wild and three mutant(c.668C>T,c.1511C>A,c.1915G>A)eukaryotic expression plasmid was constructed successfully.2.Immunofluorescence confirmed that mutant PLA2G6 does not affect subcellular localization of proteins.3.Western Blot results showed that two mutant and wild-type proteins have no difference expression.4.The result of the c PLA2 Assay Kit finds that the enzyme activity of the mutant PLA2G6 protein decreases.Conclusion1.This study successfully constructed the cell model of PLA2G6,which provides an effective tool for the function study of PLA2G6.2.Mutant PLA2G6 does not affect protein expression and subcellular localization,but can lead to the decrease of phospholipase A2 enzyme activity,which is possibly related to the pathogenesis of PLAN.