| Small patella syndrome(SPS)is a rare autosomal dominant genetic disease.SPS caused by the TBX4 mutation is typically characterized by staped bones or dysplasia,patients often have abnormalities in the pelvis and femur.In addition,TBX4 mutations can also cause childhood onset pulmonary arterial hypertension(PAH).The main clinical features are pulmonary capillary dysplasia and acinar dysplasia.However,the pathogenesis of rare diseases after TBX4 mutation is still unclear.We established cellular models and performed a series of experiments for the characterization of the pathogenesis of those three mutations in TBX4.First,we collected whole blood samples of all patients from a family with SPS symptoms.By using high-throughput next generation sequencing analysis and Sanger sequencing validation,we found a novel TBX4 missense mutations,c.1241C>T(p.P414L),were present in patients of this family,and this site is not reported previously.In order to comprehensively understand the pathogenesis of TBX4 missense mutations,we selected previously reported c.256G>C(p.E86Q)and c.743G>T(p.G248V)mutations for a joint study.The two mutation sites are both located T-box DNA binding domain,which have been reported from patients with typical clinical SPS presentations,thus we speculated those two mutations are pathogenic,but their pathogenicity have not been functionally characterized.TBX4 mutations can cause SPS and PAH,which are related to hindlimb bone development and pulmonary vascular development,respectively.TBX4 protein is highly expressed in the mesenchyme of the lung and trachea and the limbs.Therefore,the selection of MSCs and A549 cells is the most suitable cell types.Both MSCs and A549 cells were transduced by the pseudo-lentiviral particles containing either TBX4mt or TBX4wt for exploring the pathogenic mechanism of SPS caused by TBX4 mutation.Since TBX4 affects mesodermal differentiation and plays an important role in hindlimb branching morphogenesis,we performed osteogenic differentiation experiments of mesenchymal stem cells.The results showed that the three missense mutations in TBX4 affected the osteogenic differentiation activities of MSCs.In order to further explore the cellular phenotypes caused from TBX4 mutations,MSCs and A549 with stable expression of either TBX4wt or TBX4mt were used for β-Galactosidase staining to identify senescent cells state.The results showed that all the three missense mutations had stronger accelerated effects on cell senescence than TBX4wt in MSCs and A549 cell model,and the TBX4 pathogenic variants may cause faster cell senescence.TBX4 is a transcription factor and FGF10 is its definite downstream target gene.TBX4 can bind to the promoter region of FGF10 and regulate the transcriptional function of FGF10.Studies in related literatures have found that the TBX4-FGF10 pathway has important regulatory functions for the development of hind limbs.The effect of TBX4 mutation on this pathway was explored.To test whether TBX4 mutations affect the expression of FGF10 in the cell lines,qPCR was performed to quantify relative FGF10 mRNA expression in MSCs expressing either TBX4mt or TBX4wt.It was found that the expression of FGF10 is significantly decreased in the cell lines with over-expression of TBX4 mutants,indicating that TBX4 involves the initiation or maintenance of FGF10 expression,mutation in TBX4 leading to reduced transcriptional activities.In order to further explain the reason of FGF10 transcription inhibition,we carried out chromatin immunoprecipitation(ChIP)and double luciferase reporter assay.The results showed that the TBX4 mutant could still bind to the promoter of its target gene FGF10 when it entered the nucleus,but the binding ability was weakened.To sum up,our study identified a novel TBX4 mutation site.Together with the two reported sites,we found that the TBX4 mutation causes an abnormality in the TBX4-FGF10 pathway,and initially revealed the molecular mechanism of SPS.Provides important information for the clinical management of SPS. |
