Association Study Between Gli3 Gene And Idiopathic Congenital Talipes Equinovarus

ObjectiveIdiopathic congenital talipes equinovarus ( ICTEV) is a complex foot deformity presence at birth with an estimated incidence of 0. 6 ~ 1/1,000 live births in Chinese . The cause of ICTEV remains controversial. Many factors as skeleton development anomaly, neuromuscular disease, developmental arrest in uterus and so on seem to be responsible. It is considered that the genetic factors play an important role in the pathogenesis of ICTEV, and the heritability of ICTEV is about 65%. Recently, it is believed that abnormal expression of some limb development - related genes leads to the ICTEV. The transcription factor Gli3 is an important signaling molecule. During the early period of the limb development , it defines boundaries of genes expression along the anterior — posterior ( AP) axis, which can provide positional information for establishing asymmetric patterns. Mutation of Gli3 gene is the underlying cause of multiple human dysmorphology syndromes which are all characterized by digit anomalies. But there is no report about association between Gli3 gene and ICTEV. So, we choosed two single nucleotide polymorphisms ( SNPs ) rs846266,rs929387 in Gli3 gene and analyzed genotype of 2 cSNPs in 84 ICTEV nuclear pedigrees. Association analysis was performed between single SNP Locus and ICTEV respectively. At the same time, mutation sites in exon 9,10,11,12 of Gli3 gene were detected in 103 patients with ICTEV by denaturing gradient gel electrophoresis technique.In addition, we constructed animal model of talipes equinovarus with Wister rats , and discovered that there was up - regulation of Gli3 gene expression in dif-ferent time of limb development in the animal model by RT - PCR. So, for the first time, Glii gene was confirmed to be associated with ICTEV.Material and Methods1. Samples271 members from 84 core pedigrees and 19 patients came from the Second Affiliated Hospital, China Medical University. All patients had typical manifestation and were confirmed by X - rays and surgical operation. Veinous blood of all members was kept at -20°C for use after anti -coagulation.86 Wister rats were bought from the animal department of the CMU.2. PCR-RFLP(1) Choice of SNPs2 cSNPs in Glii gene were chosen for ETDT. Primers were designed according genome sequence.(2) GenotypeDNA was extracted from veinous blood routinely and all SNPs were amplified by polymerase chain reaction. PCR products were digested with enzyme.(3) Association between Glii gene and ICTEVGenotype of the members from 84 core pedigrees was achieved by PCR-RFLP and confirmed by DNA sequencing. The data was analyzed by Hardy -Weinberg Equilibrium Assay, and association analysis at single locus with ETDT software.3.PCR-DGGEMutation sites in exon 9,10,11,12 of GH3 gene were detected in 103 patients with ICTEV by denaturing gradient gel electrophoresis technique.4. Construction of rat model and RT - PCR135mg/kg body weight of ATRA was used to construct rat model on day 10 of gestation, which has shown a good consistency with talipes equinovarus malformation as that in humans. On day 15^ 17^ 19 and 21 of gestation, the lower limbs were cut from model fetus and normal controls, then mRNA was extracted. We studied the expression of Glii gene in rat model to confirm the relationshipbetween the gene and congenital talipes equinovarus deformation with RT -PCR.Results1. PCR - RFLP of GH3 geneThe PCR product at rs846266 was 217 bp. If the allele was " A" , PCR product was cut into 147 bp and 70 bp with Bts I. However, PCR product could not be cut when the allele was " G" .The PCR product at rs929387 was 367 bp. When the allele was " T" , PCR product was cut into 214 bp and 153 bp with BseY I. However, PCR product could not be cut when the allele was " C" .2. DNA sequencing and genotype of GU3 geneDifferent genotype of 2 cSNPs in GH3 gene was confirmed by DNA sequencing. Genotype of 84 core pedigrees was consistent with Mendel's pattern.3. Association between GH3 gene and ICTEVThe results of association analysis at single locus showed that x values at rs846266 and rs929387 were 3. 3582 ( P > 0. 05 ) and 7. 2466 ( P < 0. 05 ) , respectively. There was remarkable association at rs929387 loci with ICTEV.4. Result of PCR - DGGEA synonymous mutation in exon 9 was detected in one patient and his mother.5. The expression of GH3 gene in rat modelsRT - PCR results found the expression of GU3 gene up - regulated in different time of limb development in rat models with the clubfoot like deformity.Conclusion1. It is confirmed by SNP association analysis that there is association between GU3 gene and ICTEV for the first time.2. We discovered a synonymous mutation in exon 9 in one patient and his mother, and considered exon 9,10,11,12 was not GU3 gene mutation hot spotsin ICTEV.3. RT - PCR results found the expression of GU3 gene up - regulated in different time of limb development, which confirmed the association between Glii gene and talipes equinovarus deformation.