Identification Of A Novel Mutation TBX5 C.755+1 G>A In A Family With Holt-oram Syndrome And Preliminary Study On Its Pathogenesis

BackgroundHolt-Oram Syndrome(HOS)is an autosomal dominant disorder characterized by congenital heart disease and upper limb defect.Mutations in the TBX5 gene play a crucial role in the pathogenesis of HOS.The mutations related to HOS are distributed within TBX5 gene and most of them are located in T-box DNA binding domain.Recently,it has been reported that the mutations in the non-coding regions of TBX5,such as promoter region,3'-untranslated region and enhancer region,are related to the occurrence of HOS.We found a family in southern China in which three affected members presented with different degrees of congenital heart disease and hand deformity.They were clinically diagnosed as Holt-Oram syndrome.The purpose of this study was to identify the pathogenic mutation in this HOS family by performing genetic tests on the family members,and to explore the molecular mechanism by which the mutation causes HOS using a series of molecular biology techniques.The expected results would enrich the contents of the TBX5 gene mutation database.Combining with preimplantation genetic diagnosis and assisted reproductive technology,the completion of this study would contribute to blocking the inheritance of mutated genes and giving birth to healthy infants.Materials and MethodsIn the current study,3 patients with HOS underwent cardiac ultrasound and hand Xray examinations.After the informed consent approval was obtained,the peripheral blood samples were collected from 3 patients and the phenotypically normal family members and their genomic DNA was extracted.Whole exome sequencing was performed on the proband,and then bioinformatics analysis was used to screen for the candidate pathogenic mutation of the disease.Sanger sequencing and high-precision and high-depth sequencing of the mutation site were employed on the aformentioned affected family members to investigate the mutation frequencies of the suspicious disease-causing mutation in the family.Minigene splicing assay was carried out to study the effect of the suspected pathogenic mutation(SPM)on TBX5 messenger RNA(mRNA)splicing.Quantitative reverse transcription polymerase chain reaction(q RTPCR)and Western Blot were performed to study the impact of the SPM on the expression of TBX5 mRNA and protein.Immunofluorescence assay was used to analyze the effect of the SPM on the nuclear localization of TBX5 protein.Cell proliferation assay was used to analyze the effect of the SPM on cell viability.ResultsCardiac ultrasound examination showed that the proband and her brother(III-1 and III-2)had severe cardiac structural defects,including atrial septal defect(ostium secundum type),muscular ventricular septal defect,tricuspid regurgitation,pulmonary hypertension,and hand X-ray examination showed hypoplasia of the thumb,I-II syndactyly,hypoplasia of the first metacarpal bone and limitation of thumb curvature.The mother of the proband(II-2)had no obvious abnormality in the heart and hand except for limited thumb bending.By using whole exome sequencing,a new mutation TBX5 c.755+1 G>A was found in the proband III-1.Sanger sequencing confirmed that all three affected members had this mutation,while the phenotypically normal family members didn't.The mutation frequencies of TBX5 c.755+1 G>A of II-2,III-1 and III-2 were 38.08%,50.01% and 48.28%,respectively.Minigene splicing anlysis showed that TBX5 c.755+1 G>A mutation affected the normal splicing of exon 7,resulting in the retention of 56 bp of intron 7 and the production of abnormal TBX5 transcript.Further bioinformatics analysis revealed that TBX5 c.755+1 G>A mutation theoretically resulted in the change of protein reading frame and the generation of premature termination codon(PTC)after exon7.The amino acid change caused by the mutation was p.s252rfs2,which maybe damage its NID domain,resulting in the premature termination of TBX5 protein at the amino acid 252 and the production of truncated TBX5 protein.The q RT-PCR results showed that the TBX5 c.755+1 G>A mutation increased the transcription of TBX5 mRNA.The Western Blot results demonstrated that the mutant TBX5 translated truncated protein with smaller molecular weight.The expression of truncated TBX5 protein was significantly higher than the wild type TBX5 protein.Immunofluorescence assay showed that wild type TBX5 protein was located in nucleus,while mutant TBX5 protein was located in nucleus and cytoplasm,suggesting that nuclear localization of mutant TBX5 protein was significantly reduced.Cell proliferation assay showed that the cell viability transfected with TBX5 mutant recombinant plasmid decreased by about 10% at 24 h and 48 h as compared to those transfected with empty control plasmid and wild type TBX5 recombinant plasmid.ConclusionsTBX5 c.755+1 G>A is a novel pathogenic mutation in this HOS family.It probably affects the normal splicing of exon 7,resulting in the retention of 56 bp of intron 7,and the production of abnormal TBX5 transcript.The mutation promotes the expression of the abnormally spliced TBX5 mRNA and the translation of the truncated TBX5 protein in vitro,significantly reduces the nuclear localization of the truncated TBX5 protein and inhibites the cell proliferation in vitro.