FHL1 gene expression and idiopathic congenital clubfoot: Comparative analysis of 15 patients and 3 normal controls / 中国组织工程研究

ABSTRACT

BACKGROUND: Preliminary study shows that the FHL1 gene expression is down-regulated in muscle tissue of patients with clubfoot, and the gel retardation experiments have verified HOXD13 and FHL1 gene promoter region transcription factor predicted binding sites in vitro, but the experimental results are not enough to truly reflect in vivo transcriptional regulatory protein and DNA binding conditions.OBJECTIVE: Western-blot technique is utilized to further validate FHL1 and HOXD13 gene expression in protein levels in muscle tissue of patients with congenital clubfoot, the in vivo HOXD13 and predicted binding sites in embryonic foot development was verified using chromatin immunoprecipitation technology.METHODS: Muscle tissues were samples from 15 children with congenital clubfoot, in the Department of Pediatric Surgery, at the Second Affiliated Clinical Hospital of China Medical University, 3 copies of normal children foot muscle tissues at the same age were provided by the Forensic Medicine College of China Medical University, 1 case of aborted embryo at pregnancy 13 weeks were offered by Department of Obstetrics and Gynecology, at the Second Affiliated Clinical Hospital of China Medical University. All specimens used are given informed consents by the patients and their families. Western-blot method was applied to detect the expressions of HOXD13 and FHL1 in foot muscle tissue of 15 patients of congenital clubfoot and 3 normal children at the same age; The FHL1 gene upper stream HOXD13 binding sites was predicted using software, the interaction of HOXD13and FHL1 during embryonic development was verified with chromatin immunoprecipitation experiment, brain tissue without HOXD13 protein expression served as controls.RESULTS AND CONCLUSION: Compared with normal children foot muscle tissues, both HOXD13 gene (5/15) and FHL1 (7/15)were down-regulated in 15 patients of congenital clubfoot. An enrichment of the predicted HOXD13 binding site was observed in the precipitated human embryo foot tissues chromatin. No enrichment of the predicted site containing sequence was observed in the control brain chromatin; additionally, there was no enrichment of the control sequence. This study further verifies that, the FHL1 and HOXD13 gene expression are down-regulated in the foot muscle tissue of congenital clubfoot children patients; during development of human embryos, HOXD13 protein can bind with FHL1 promoter region binding sites to play its role in transcriptional regulation. It is indicated that in the human foot embryonic development, the down-regulation of HOXD13expression may result in reduced expression levels of FHL1, thereby affecting the foot muscle growth and differentiation, leading to clubfoot deformity occurring.