Identification Of A Novel Pathogenic Mutation In TBX5 Gene In A Family With Holt-Oram Syndrome

BackgroundHolt-Oram syndrome,also known as heart-hand syndrome,is a congenital heart disease with upper limb deformity.The disease is an autosomal dominant disease with complete dominance,its clinical manifestations include congenital absence of atrial septum and ventricular septum,cardiac conduction dysfunction,upper limb skeletal abnormality and thumb deformity.There is a 50%probability that the offspring of the patient will have a birth defect,even a stillbirth,causing great burden to the family.The mutation in TBX5 gene was found to be a major genetic etiology in Holt-Oram syndrome,and 70%of whom have the mutation of this gene.TBX5 is located on chromosome 12,and TBX5 protein is an important transcription factor that regulates the development of heart and upper limbs during embryonic development.Mutations have been found in the coding,splicing and regulatory regions in TBX5.In addition,30%of patients have unknown etiology,there may be regulatory elements that affect the development of heart and upper limbs.The heterogeneity of TBX5 gene mutation and its correlation with Holt-Oram syndrome suggest that TBX5 has an important regulatory role and research value in embryonic development.ObjectiveTo identify the pathogenic gene and genetic pattern of the family with Holt-Oram syndrome and provide genetic counseling for patients;The identification of the novel mutation in TBX5 gene will enrich the understanding of mutation heterogeneity of Holt-Oram syndrome and broaden the mutation spectrum;Function studies of the novel mutation in TBX5 gene can reveal the effects of the mutation on gene expression,protein distribution,and transcriptional regulation.MethodsPrepare the medical history and family pedigree,analyze the clinical manifestations and genetic pattern;Discover the pathogenic gene via whole exome sequencing and Sanger sequencing;Analyze the conservation of amino acid sequence and predict the pathogenicity of TBX5 gene mutation;Construct the TBX5 plasmid and complete site-directed mutagenesis;The effects of the mutation on mRNA level of TBX5 gene and target gene NPPA were detected by real-time fluorescence quantitative PCR;The effect of the mutation on length and expression of TBX5 protein was performed with western blot;The distribution of TBX5 protein was detected by immunofluorescence;Double luciferase reporter assay showed the effect of the mutation on transcriptional regulation and synergistic reaction with NKX2-5 and TAZ.Results1.Patients in the Holt-Oram syndrome family had absence of atrial septum,ventricular septum and thumb deformity with an autosomal dominant inheritance.Three cases died due to severe congenital heart abnormalities.2.A novel heterozygous frameshift mutation in TBX5 gene was identified in exon 9,shown as c.992de1G(p.C331F fs*63).The database and bioinformatics analysis showed that the mutation was pathogenic.The amino acid sequence of mutant TBX5 was conserved across species.3.Cellular functional study of TBX5 mutation showed that the mRNA level of the mutant TBX5 was significantly lower than that of the wildtype gene;The mutation produced a truncated protein,decreased the expression of mutant protein and resulted in the abnormal distribution of mutant protein.4.Overexpression of the mutant TBX5 reduced the mRNA level of target gene NPPA.NPPA promoter luciferase reporter assay confirmed that mutation of TBX5 gene decreased its transcription function.The mutation impaired the transcription promotion of TBX5 with TAZ and NKX2-5.ConclusionsWe identified a novel TBX5 mutation in a Holt-Oram syndrome family and broaden the mutation spectrum.The study revealed that the mutation resulted the insufficient gene expression dose and abnormal protein distribution,reduced transcriptional regulatory activities and synergistic effect of the TBX5 transcription factor.