Pathogenesis Of PFBC Caused By Newly SLC20A2 Gene Mutation

Background Primary Familial Brain Calcification(PFBC),as known as Idiopathic Basal Ganglia Calcification(IBGC),Fahr's disease,is a rare neurogenetic degenerative disease,and clinical performances are diverse,including dystonia,headache,dementia,depression and other symptoms.Imaging examinations are characterized by extensive symmetry calcification in the brain,mostly located in the bilateral caudate nucleus,lenticular nucleus,thalamus,brainstem and cerebellar dentate nucleus,but the serum calcium and phosphorus levels as well as the thyroid function and parathyroid function of the affected individuals are normal,which is the main distinguishing point of PFBC from other brain calcification diseases.It is known that multiple gene mutations can lead to the occurrence and development of PFBC,including: SLC20A2,PDGFB,PDGFRB and XPR1 and so on.SLC20A2 at chromosome 8 is the major causative gene of PFBC,and multiple site mutations in this gene have been found to cause PFBC.The exact pathogenesis of PFBC has not been fully elucidated,which is effected by metabolism imbalance of calcium and phosphorus.Previous studies have shown that under physiological conditions,The inorganic phosphate(Pi)in the brain parenchyma is taken up into brain cells by Pi transporter 2(Pi T-2),and Pi is excreted from the brain cells by xenotropic and polytropic retrovirus receptor 1(XPR1).Under pathological conditions,SLC20A2 mutation leads to damage of the construction and function of Pi T-2,which causes a decrease in Pi uptake,and then Pi accumulates in the brain parenchyma,and intracellular Pi is low,which in turn leads to calcium deposition.Mitochondria and Golgi are important organelles within the cell.Studies have shown that mitochondrial and Golgi structures and functional abnormalities are involved in Alzheimer's disease(AD),Parkinson's disease(PD),spinalcerebellar ataxia(SCA),amyotrophic lateral sclerosis(ALS)and other neurodegenerative diseases,abnormal mitochondria and Golgi may be early pathological manifestations of neurodegenerative diseases.Changes in mitochondria and Golgi during the onset of PFBC have not been reported.At present,the neurons derived from PFBC patients are hard to obtain,and the research on the disease lacks a reliable cell model.In recent years,the rapid development of somatic cell reprogramming into stem cell technology has provided a new direction for obtaining patient-derived disease cell models.Induced pluripotent stem cells(i PSCs)are a class of cells with similar morphological and biological functions to embryonic stem cells(ESCs).In 2006,Japanese scientists Shinya Yamanaka transfected somatic cells by retrovirus with reprogramming genes.And firstly it successfully induced somatic cell reprogramming into i PSCs.The morphology and function of the embryonic stem cells were observed and detected,and the i PSCs could be further differentiated into various somatic cells in vitro,providing ideal and reliable cell model for exploring the pathogenesis,cell transplantation and drug screening of diseases.This study collected a family of PFBC patients,used Sanger sequencing technology to determine the disease-causing gene,obtained patient-derived somatic cells,reprograms into induced pluripotent stem cells,and then differentiated them into neural precursor cells,dopaminergic neurons and astrocytes,examined and analyzed different kinds of cells apoptosis morphology of mitochondria and Golgi.A new site mutation of PFBC pathogenic gene was found out,and we successfully constructed the healthy volunteer and PFBC patient-derived induced pluripotent stem cell lines,neural progenitor cell lines,dopaminergic neurons and astrocytes,and the pathogenesis of PFBC was preliminarily explored,which laid a foundation for further investigation of the pathogenesis and potential therapeutic measures of PFBC.Objective Identify the disease-causing genes carried by the collected families,construct healthy volunteer and the patient-derived i PSCs,and preliminary investigate the pathogenesis of PFBC.Methods 1.Using Sanger sequencing technology to determine the causative gene of the primary familial brain calcification family.2.Using reprogramming techniques to construct induced pluripotent stem cell lines derived from healthy volunteer and PFBC patient.3.In vitro differentiating healthy volunteer and PFBC patient-derived i PSCs into neural precursor cells,dopaminergic neurons and astrocytes.4.Flow cytometry,immunofluorescence staining and other experimental techniques were used to explore the role of SLC20A2 gene mutation in the pathogenesis of PFBC.Results 1.Sanger sequencing results showed that the family carried a new mutation in the SLC20A2 gene associated with PFBC(c.613G>A,p.Val205Met).2.Successfully obtained healthy volunteer and PFBC patient-derived somatic cells and successfully constructed healthy volunteer and patient-specific induced pluripotent stem cell lines.3.healthy volunteer and PFBC patient-derived induced pluripotent stem cells(i PSCs)have embryonic stem cell-like morphology,high expression of stem cell markers OCT4,Nanog,SOX2 and TRA-1-60,which have a normal diploid karyotype,can be successfully differentiated into a teratoma with three germ layers in NOD-SCID mice.And patient-derived induced pluripotent stem cells(i PSCs)still carry the SLC20A2 gene mutation.4.Successfully induced differentiation of i PS cells into neural precursor cells,further differentiated into dopaminergic neurons and astrocytes,and immunofluorescence staining showed that they expressed dopaminergic neuron marker proteins(TH and Beta-Tubulin III)and astrocyte marker protein(GFAP)respectively.5.In the detection of mitochondria and Golgi morphology,morphology and structural damage of mitochondrial and Golgi were found in neural progenitor cells,dopaminergic neurons and astrocytes.6.Apoptosis levels of neuronal precursor cells,dopaminergic neurons and astrocytes derived from PFBC patients are higher than the healthy volunteer group.Conclusions 1.SLC20A2 gene new-on-site mutation c.613G>A mutation may lead to primary familial brain calcification.2.The nervous cells in PFBC group have abnormal morphological and structural mitochondria and Golgi,which may be involved in the pathogenesis of PFBC.3.The c.613G>A mutation of SLC20A2 gene may lead to increased apoptosis and death of nervous cells,which may be the cause of PFBC.