Identification Of Causative Gene Mutation And Pathological Mechanism In A Bardet-biedl Syndrome Family

Bardet-Biedl syndrome（BBS）is a rare autosomal recessive disorder with high clinical heterogeneity and genetic heterogeneity,which may affect multiple organ systems.The main characteristics of BBS include obesity,retinitis pigmentosa,mental retardation,polydactyly,hypogenitalism and renal anomalies.Patients with BBS showed a very poor prognosis with high disability.Once patients were diagnosed with BBS,they cannot have normal offspring.Unfortunately,the pathogenesis of BBS is not completely known and there is still no effective treatment for it.Prenatal testing and avoiding consanguineous marriage are the two main measures for BBS prevention.Exploring the genetic mutations to construct a full mutation spectrum for BBS is the basis of developing effective prenatal screening and potential gene therapy,which is of great clinical significance.Aim: To identify the phenotype,causative gene mutation and the molecular pathological mechanism for BBS diagnosed in two brothers from a Chinese family,we first did comprehensive physical examinations and ophthalmological examination to identify the disease phenotype.High throughout targeted exome sequencing was then used to identify the causative gene mutation,and the candidate mutations were further validated by Sanger sequencing.Finally,we constructed plasmid containing the identified mutations and transfected in human retinal pigment epithelium–derived cell line（ARPE-19）and human embryonic kidney cell line（HEK293T）,to investigate the molecular pathological mechanisms at both protein and cell levels.Methods: 1.The subjects were recruited among patients at Xiamen University affiliated Xiamen Eye Center.The study participants consisted of two BBS affected brothers firstly diagnosed retinitis pigmentosa and their phenotypically normal parents.All subjects underwent comprehensive ophthalmologic and physical examination and their medical history information was collected;2.Peripheral venous blood samples were obtained from the affected brothers and their phenotypically normal parents.Genomic DNA was extracted from whole blood samples using a DNA Extraction kit;3.We designed a customized panel containing 145 genes associated with inherited retinal diseases,and high throughout targeted exome sequencing（TES）was performed on genomic DNA samples from the two affected brothers to reveal the genetic mutation information.The candidate variations were further conducted bioinformatics analysis compared with reference databasesto identify potential pathogenic mutation.4.Primers for gene location of the potential pathogenic mutation were designed,and Sanger sequencing was performed on genomic DNA samples from the two affected brothers and their phenotypically normal parents as well as normal control to identify the causative gene mutations.5.To understand the molecular pathological mechanisms of the identified gene mutation,we constructed eukaryotic expression plasmid containing the specific mutations.The plasmids were transfected into human retinal pigment epithelium–derived ARPE-19 cells and human embryonic kidney –derived HEK 293 T cells by lipofectamine-based transfection reagents;6.Co-immunoprecipitation was performed to investigate the interaction of mutative BBS7 and other BBSome associated proteins,including BBS1,BBS4,BBS5,BBS8 and BBS9;7.We then further studied the influence of mutative BBS7 on cell proliferation,cell cycle and apoptosis of ARPE-19 cells using CCK-8 kits and flow cytometry.The cell apoptosis mechanism of ARPE-19 cells induced by mutative BBS7 were investigated by Western-blot and transmission electron microscope（TEM）.Results 1.There are currently two-generations existed in this family,and we collected clinical information for four family members.The two affected members in this family exhibited five BBS main features,including obvious retinitis pigmentosa,obesity,polydactyly,hypogenitalism and mental retardation,as well as some other secondary features,such as strabismus,dental abnormalities and developmental delay.The patients meet the diagnostic criteria for BBS,and can be diagnosed as BBS patients.BBS showed typical autosomal recessive inheritance in this family,which is in agreement with inheritance pattern of BBS;2.High throughout targeted exome sequencing revealed that among 145 genes associated with inherited retinal diseases,a shared,homozygous c.389-390 del AC deletion mutation in exon 5 of the BBS7 gene in the two affected brothers was identified,which results in p.N130 sf substitution in the amino acid sequence of the BBS7 protein（frame shift mutation）and led to a premature stop codon.Bioinformatics analysis suggested that this mutation was the potential pathological mutation for BBS in this family;3.Sanger sequencing showed the phenotypically normal parents had a heterozygous c.389-390 del AC mutation in the same location of BBS7 gene,while no such mutation was detected in DNA samples from normal control.The results confirmed that c.389-390 del AC mutation in exon 5 of the BBS7 gene was the causative mutation for BBS in this family;4.Co-immunoprecipitation showed that compared to wide-type BBS7,the interactions of mutative BBS7 protein with other BBSsome associated proteins,including BBS1,BBS4,BBS5,BBS8 and BBS9,were abnormal,which may lead to failure to form BBSome with normal function,and further resulted in BBS;5.Studies on ARPE-19 cells showed that compared to wild-type BBS7,mutative BBS7 significantly inhibited cell proliferation.The cell proliferation was inhibited by 11% after 3 days.In agreement with cell proliferation studies,cell apoptosis studies showed that mutative BBS7 significantly enhanced ARPE-19 cell apoptosis.Compared to cells transfected with wide-type BBS7 plasmid,the apoptosis of ARPE-19 cells transfected with mutative BBS7 increased 88 folds.However,mutative BBS7 didn’t significantly affect the cell cycle of ARPE-19 cells.Western-blot analysis suggested that the levels of activated caspase 3 and caspase 9 proteins in ARPE-19 cells transfected with mutative BBS7 plasmid were significantly increased,while no significant difference for caspase 8 protein was observed.TEM imaging of ARPE-19 cells showed that obvious mitochondrial lysis was observed in transfected with mutative BBS7 plasmid,while mitochondrial morphology was normal in cells transfected with wide-type BBS7 plasmid.These results suggested that c.389₃90del AC mutation in the BBS7 gene induced ARPE-19 cell apoptosis via mitochondrial pathway instead of cell membrane pathway.Conclusion: A homozygous c.389₃90del AC mutation on the BBS7 gene was identified in DNA samples from the two BBS affected patients,and this mutation has not been previously reported.Our study suggested that the potential pathological mechanism for this mutation could be the binding ability of BBS7 protein to BBS1,BBS4,BBS5,BBS8 and BBS9 was changed by c.389₃90del AC mutation,resulting in failure to form BBSome complex,and thus led to ciliary dysfunction.The ciliary dysfunction then inhibited cell proliferation and induced cell apoptosis in target organs,and thus led to the onset of BBS.This study reported the BBS7 gene c.389₃90del AC mutation for the first time.This new finding expanded the gene mutation spectrum for BBS,which is the basis for development of effective prenatal screening and clinical gene therapeutic methods,is of great clinical significances.Our study identified the causative gene mutation for BBS in this family,which may provide reliable reproduction advice for mutations carriers in this family.Furthermore,this study also revealed the molecular pathological mechanism for the newly identified mutation,enhanced the understanding of the BBS7 gene,and could be helpful for study of BBS molecular pathological mechanism.