Location And Identification Of Human Idiopathic Congenital Talipes Equinovarus Susceptibility Genes

Location and Identification of human idiopathic congenital talipes equinovarus susceptibility genesIntroductionIdiopathic 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. The deformity consists of equinus and varus of the hindfoot and adductus and cavus of the midfoot. The etiology of ICTEV remains unknown, 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 progress 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 theoretical 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 lesions.Limb development is a complicated progress that involves not only spatial and temporal expression of many genes but also the cellular migration, differentiation, proliferation and accurate interaction between the cells. With the studies on the limb development of chick embryo and mouse, some limb development-related genes have been identified, such as DTDST,COL9A1,FGF,SHH,Wnt,EN-1,Lmx-1,Tbx-4,Tbx-5,Pitxl,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.At present, researches on human ICTEV mainly focus on the environmental factors at early stage of pregnancy and many syndromes with foot malformations. Candidate regions and genes for ICTEV such as 5q15-31.1,10p11.2-15,DTDST are identified. But little has been known about the pathogenesis of human ICTEV and further researches are urgent.Previous studies in our group showed that D6S348 locus in the region of 6q12-13 was remarkably associated with ICTEV(P＜0.01), suggesting that there might be ICTEV susceptibility genes around D6S348.In order to conform it ,ten microsatellite DNAmarkers were chosen around D6S348,which is D6S965, D6S1681, D6S280, D6S1036, D6S493, D6S313, D6S239, D6S493, 509-8B2F/509-8B2R, D6S1031. Genotyping and transmission disequilibrium test(TDT) were done in 252 members from 84 core pedigrees of ICTEV. For the first time, the ICTEV susceptibility gene was located to 6.04cM in 6q12-13. At the Same time,COL9Al gene in the candidate region was firstly conformed associated with ICTEV using candidate cloning stragety.MethodsSamples252 members from 84 core pedigrees came from the Second Affiliated Hospital, China Medical University. All patients had typical manifestation and were conformed by X-rays and surgical operation. Veinous blood of 252 members was kept at -20℃for use after anti-coagulation. Specimens of muscle and muscular tendon came from 25 patients of ICTEV.Clubfoot rat model was obtained from our group.Selection of microsatellite DNA markersAccording to the number of alleles, heterozygosity and polymorphic information content, 10 microsatellite DNA markers in 6q12-13 were chosen for TDT. All these microsatellite DNA markers were from Genome DataBase(GDB).GenotypingDNA was extracted from the veinous blood routinely and all microsatellite DNA markers were amplified by PCR-STR for genotype analysis.Transmission Disequilibrium Test and analysis on genetic polymorphismTDT was applied in 84 core pedigrees. Analysis on genetic polymorphism in Chinese North Han was performed when the microsatellite DNA markers were significant in TDT. The allele frequency, genotype distribution and heterozygosity at each microsatellite DNA marker were calculated directly. According to the Hardy-Weinberg Equilibrium, expected values of genotype frequency and expected heterozygosity were obtained, andχ~2 test was used evaluate the accordance with Hardy-Weinberg Equilibrium.Association between COL9A1 gene and ICTEVTwo coding-region SNPs in COL9AI gene, rs592121 and rs1135056, were chosen for studies.After regular amplification, genotype 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 linkage disequilibrium test with 2LD software, and haplotype analysis with TRANSMIT software(version2.5).The expression of COL9A1 in human ICTEV and in rat modelRT-PCR and immunohistochemistry methods were used to detect the expression of COL9A1 in ICTEV patients and rat model.Regulation of human COL9A1 gene expressionMutation screening of COL9A1 gene promoter was performed by using PCR-SSCP method. The promotor and the first intron sequence of human COL9A1 were cloned, respectively. Then luciferase report vector pGL3-COL9A1 was constructed and transient-transfection analyses performed in Bel, 823 and fibroblast cells. To delineate which regions of the COL9A1 promoter region are responsible for promoter activity, a series of COL9A1 promoter luciferase 5'deletion constructs were employed(990Luc,704Luc,548Luc,162Luc,420Luc,336Luc,217Luc and 157Luc).And transient- transfection analyses performed in Bel, 823 and fibroblast cells. One C/EBP binding elements at -403～-275 and one SOX9 binding elements at -275～-185 were predicted by using P-Match software.We constructed M1,M2,M3,M4 constructs , and each of the mutant constructs (M1mutLuc, M2mutLuc, M3mutLuc and M4mutLuc) were transfected separately into Bel cells to assess the effect of the mutations on promoter activity.ResultsGenotypingOnly one allele at D6S493 locus was observed and thus removed from analysis. The genotypes of 84 core pedigrees at other 9 microsatellite DNA markers accorded with Mendel inheritance.ransmission Disequilibrium TestThere existed linkage disequilibrium at D6S1036,D6S239,D6S421,D6S1681,509-8B2F/R,D6S280,D6S313(Which overlapped 6.04cM in 6q12-13),P＜0.05, While no linkage disequilibrium at D6S965 and D6S1031(P＞0.05) was found.Results of genetic polymorphism analysisThe numbers of the alleles and genotypes in D6S1036,D6S239,D6S1681,D6S421,5098B2F/R,D6S280 and D6S313 were 5,6,6,6,8,6,5and 12,18,19,14,27,19,10,respectively by screening 100 non-relationship individuals. There were no remarkably significance between the observed and expected members of different genotypes at each microsatellite DNA marker (P＞0.05), suggesting that the population was under the Hardy-Weinberg Equilibrium. The highest heterozygosities at the above 7 microsatellite were 85%(at D6S1681 and 509-8B2F/R),while the lowest heterozygosities were 77%(at D6S1036 and D6S313),which displayed that the highly genetic polymorphism at these loci was observed in Chinese North Han Population.PCR-RFLP of COL9A1 geneThe length of PCR products at rs592121 was 151bp.When the allele is G, PCR products are cut into 101bp and 50bp with HaeⅢ.However, PCR products can not be cut when the allele is A.The length of PCR products at rs1135056 was 287bp.When the allele is G, PCR products are cut into 160bp and 127bp with SmaⅠ.However, PCR products can not be cut when the allele turns to be A.DNA sequencing and genotyping of COL9A1Different genotypes of 2 SNPs in COL9A1 gene were confirmed by DNA sequencing.Genotypes of 84 core pedigrees were in accordance with Mendel's pattern.Association between COL9A1 and ICTEVThe results of association analysis at single locus showed thatχ~2 values at rs592121, rs1135056 were 12.488(P＜0.05) and 15.662(P＜0.05), respectively. There existed remarkable association at these two loci. Linkage Diseqiulibrium were found between rs592121 and rs1135056 (D'=0.917065).The haplotype analysis at these two loci showed four haplotypes and the globe P value of these haplotypes was over 0.05, which had no statistic significance.The expression of COL9A1 in ICTEVRT-PCR results found the expression of col9a1 gene up-regulated in 72.4%(21/29) model rat fetus, but no obvious change was detected in normal control fetus.The COL9A1 expression was higher in the muscle and muscular tendon tissues of ICTEV than that of in normal tissue(t=4.7500, P＜0.05). Immunohistochemistry results consistent with the results of RT-PCR. These results showed that the overexpression of COL9A1 gene is associated with ICTEV.The study of COL9A1 gene regulationThe results of enzyme digesting and sequencing confirmed that luciferase reporter vector pGL3-COL9A1(990Luc) and 990LucInt were successfully constructed. We demonstrate here that the proximal-promoter region of the human COL9A1 gene has the ability to drive expression of a reporter gene in Bel,823 and human fibroblast cells. But inclusion of the first intron had no effect on promoter activity. Transient-transfection of various deletion vectors showed that there might have positive regulation factors in -559～-403, -275～-195 and a negative regulation factor in -403～-275. Deletion and mutation analysis revealed that SOX9 binding sites within -275 to -257 are responsible for most of the COL9A1 promoter activity in Bel cells, and C/EBP binding sites within -321 to -312 could inhibite the COL9A1 promoter activity.Conclusion1,The ICTEV susceptibility gene is narrowed to 6.04cM in 6q12-13 by transmission Diseqiulibrium Test for the first time.2,It is confirmed for the first time that there is association between COL9A1 gene and ICTEV by cSNP association analysis, RT-PCR and immunohistochemistry methods.3,The cell tranfection results clearly demonstrate that SOX9 and C/EBP can regulated COL9A1 gene expression.