Association Analysis Between 5' HOXD Gene And Idiopathic Congenital Talipes Equinovarus

ObjectiveIdiopathic congenital talipes equinovarus (ICTEV) is a kind of congenital malformation that does severe harm to inborn'health with an estimated incidence of 1 ~8/1 ,000 live births. It is considered that the genetic factors play an important role in the pathogenesis of ICTEV. The heritability of ICTEV is about 65% , but it is not clear about the hereditary patterns and penetrance, and little advances have been made in the researches on the susceptibility genes of. ICTEV. Therefore, it is of great significance that the molecular genetic mechanisms of ICTEV should be discussed, which can provide the theorical bases for the genetic interfere of ICTEV, enrich the theory and practical application of human genomics and offer an useful method from which researches on other congenital malformations can draw lessons.Limb development is a complicated progress that involves not only spatial and temporal expression of many genes but also the migration, differentiation, proliferation of cells and accurate interaction between cells. With the studies on the limb development of chick embryo and mouse, some limb development - related genes have been identified, such as FGF , SHH , Wnt , EN - 1, Lmx -1 , Tbx - 4 , Tbx - 5 , pitx1,Hox , BMP and so on. The highly conservative characteristics of these genes provide an useful clue for us to look for Limb development - related genes.The HOX genes encode a highly conserved family of transcription factors which play a fundamental role in embryonic morphogenesis. The most 5 'members of the HOXA and HOXD clusters ( HOXA9 ~ HOXA13 and HOXD9 ~ HOXD13) are particularly important in vertebrate limb development. So, we chose 5' HOXD clusters as susceptibility gene according their function. We chose four single nucleotide polymorphisms ( SNP ) rs2593778, rs847154, rs847151, rs13392701 in HOXD10,HOXD12 , HOXD13 gene and analyzed genotype of 4 SNPs in 84 idiopathic congenital talipes equinovarus nuclear pedigree. Association analysis is directed between single SNP Locus and idiopathic congenital talipes equinovarus, respectively. Genotyping and transmission disequilibrium test were done in 252 members from 84 core pedigrees of simple ICTEV. We studied the expression of Hoxd12, Hoxd13 in rat model and the assotiation between Hoxd12, Hoxd13 gene and talipes equinovarus deformation.MethodsSamples252 members from 84 core pedigrees 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 252 members was kept at -20 C for use after anti - coagulation.1. Choose of SNP4 SNP in 5' HOXD clusters were chosen for ETDT. Primers were designed in Sangon according genome sequence and whether SNP changed enzyme site.2. GenotypingDNA was extracted from veinous blood routinely and all SNP were amplified by polymerase chain reaction. PCR products were digested with enzyme.3. Association between HOXD gene and simple ICTEVGenotyping of the members from 84 core pedigrees were achieved by PCR - RFLP and confirmed by sequencing. The following statistic analysis includes Hardy - Weinberg Equilibrium Assay, association analysis at single locus with ETDT software, paired likage disequilibrium test with 2LD software, and haplo-type analysis with TRANSMIT software ( version 2.5).4. The expression of Hoxd12, Hoxd13 in rat modelOur laborary utilized 135mg/kg body weight of ATRA for induction of club-foot - like deformity, which has shown a good consistency with talipes equinova-rus malformation as seen in humans. On day 21 of gestation, the lower limbs were cut from model fetus and normal controls, then mRNA was refined. We studied the expression of Hoxd12, Hoxd13 in rat model and the assotiation between Hoxd12, Hoxd13 gene and congenital talipes equinovarus deformation.RESULT1. PCR-RFLP of HOXD geneThe length of PCR products at rs847154 was 487 bp. When the allele is C, PCR products are cut into 355 bp and 133 bp with Sac I. However, PCR products can not be cut when the allele is T.The length of PCR products at rs2593778 was 353 bp. When the allele is G, PCR products are cut into 197 bp and 156bp with Sac I. However, PCR products can not be cut when the allele is C.The length of PCR products at rsl3392701 was 197bp. When the allele is C, PCR products are cut into 170 bp and 27bp with Dde I. However, PCR products can not be cut when the allele is A.The length of PCR products at rs847151 was 197bp.2. DNA sequencing and genotyping of HOXD geneDifferent genotypes of 3 SNPs in HOXD gene were confirmed by DNA sequencing. Genotypes of 84 core pedigrees were consistent with Mendel's pattern.3. Association between HOXD gene and ICTEVThe results of association analysis at single locus showed that x2 values at rs847154,rs2593778,rs13392701 were 13.838 (P <0.05), 1.485 (P >0.05) and 16.311 (P<0. 05), respectively. There existed remarkable association "at rs847154 and rs13392701 loci. Linkage Disequilibrium were found between rs847154 and rsl3392701 loci (D'=0.876056). The following haplotype analysis was performed at rs847154 and rsl3392701 loci. Four haplotypes were observed, The globe P value of these two haplotypes was over 0.05, which had no statistic significance.4. The expression of Hoxd12, Hoxd13 in rat model...