PDGFB Gene Mutation Screening Of Idiopathic Basal Ganglia Calcification

Objective:The etiology of Idiopathic basal ganglia calcification (IBGC) is undefined, and the molecular mechanism of disease genes lead to disease is also unclear, until recently, Idiopathic basal ganglia calcificationhas been dete rmined by five genes, SLC20A2, PDGFRB, PDGFB, XPR1 and ISG15. No studies have been carried out to analyze the gene mutation of PDGFB in C hinese population.our aim is to screen mutations of PDGFB gene in a large cohort of Chinese IBGC patients and to discover the mutation spectrum of the population in ChinaMethods:The study recruited 144 IBGC patients including 10 index cases a nd 134 sporadic cases from China between February,2013 and January,2 016, Cases of source in fujian, jiangxi, anhui, Inner Mongolia and zhejiang. The diagnosis of each patient was performed following complete clinical eva luations, biochemical tests and neuroimaging examinations. Patients who were diagnosed with hypoparathyroidism were excluded from the study. All patie nts were previously screened negative for mutations in SLC20A2 by the San ger sequencing.200 healthy controls of Chinese Han population were recruit ed from the First Affiliated Hospital of Fujian Medical University and they were eliminated Idiopathic basal ganglia calcification by Brain CT. Peripheral venous blood samples of all included participants were collected. Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Hilde n, Germany) from the peripheral blood samples of all patients as well as th e controls. Six sets of primers designed for the PDGFB gene were used to amplify the coding sequence of PDGFB by polymerase chain reaction (PC R), and the PCR amplification products are performed direct sequencing by t he Sanger sequencing method. Then the sequencing results are read with Ch romas software sequence, PDGFB protein sequences from different species w ere aligned using the HomoloGene to check the evolutionary conservation of the identified mutations, The potential effects of the identified missense mut ations on protein function were assessed using Polymorphism Phenotyping (P olyPhen-2), Sorting Intolerant From Tolerant (SIFT), and Mutation Taster. Th e novelty of the identified variants was assessed by comparison with the db SNP,1000 Genomes Project and ExAC databases.Results:Three novel missense variants (c.232C>T and c.610C>A) of the PD GFB were identified in three sporadic IBGC patients. All of these three vari ants were not detected in 200 healthy controls. The c.232C>T occurred at hi ghly conserved regions and were predicted as damaging by at least two com putational predictive programs, suggesting that these two mutations were high ly likely to be causative for IBGC and it means that the mutations are path ogenic. Although variant c.610C>A also occurred at a highly conserved regio n, two computational predictive programs predicted it to be most likely beni gn, suggesting a uncertain role of this variant on.Conclusions:In conclusion, the present study identified a pathogenic mutatio ns and a variant of uncertain significance in PDGFB in a large cohort of C hinese IBGC patients. However, It is lack of the direct proof of effect upon function of the protein at present, Therefore, it is still required further stud y of PDGFB functional expression to confirm the pathogenic effect of novel missense mutations identified in this study.Now the other members of this team has successfully built a PDGFB wild t ype and mutant genes expression plasmid, Transfection 293 t cells, and plan to investigate mutations at a cellular level effect on protein expression, intr acellular localization and calcium and phosphorus metabolism, We will shed ligt on different mutations PDGFB influence on protein function and its exac t pathogenesis.