| Developmental dysplasia of the hip(DDH) is a kind of congenital malformation that does severe harm to inborn health,with an estimated incidence of 25-50/1000 live births among Lapps and Native Americans and a very low rate among the southern Chinese population and persons of African descent.The etiology of DDH remains unknown,and many factors contribute to DDH.Genetic and ethnic factors play a key role.A positive family history for DDH may be found in 12%to 33%of patients who have DDH.However,it is not clear about the hereditary patterns and penetrance and little progress have been made in the researches on the susceptive genes of DDH.Previous studies by our group showed that D17S1820 locus in the region of 17q21 was remarkably associated with DDH,suggesting that there might be DDH susceptive genes around D17S1820.In order to confirm that,11 STR DNA markers were chosen around D17S1820,and genotyping and transmission disequilibrium test(TDT) were done in 309 family members from 103 trio families of DDH.For the first time,the susceptive gene for DDH was located to 11.7Mb in 17q21.At the same time,COL1A1 gene in the located region was selected as a candidate gene.Mutation in COL1A1 transcription regulation region was detected in 109 patients with DDH,and preliminary function study associated with the mutation site was performed by site directed mutagenesis method and genetic recombination strategy.All the trio families of DDH came from department of paediatric orthopaedics, Shengjing Hospital of China Medical University.All the patients had typical manifestation and were confirmed by clinical and radiographic examination.Venous blood of the family members was collected and stored at -80℃for use after anti-coagulation.Genomic DNA was extracted from the venous blood by using QlAamp DNA Blood Mini Kit.All the parents gave informed consent to paricipate in the study,and this research had been approved by the Medical Ethics Committee of Shengjing Hospital,China Medical University.According to the number of alleles(≥5),heterozygosity(≥0.70) and polymorphic information content(PIC≥0.5),11 STRs in 17q21 were chosen for TDT.STR markers were amplified by PCR and genotypes were analyzed by capillary electrophoresis.TDT was applied in the 103 trio families.Analysis of genetic polymorphism was performed in a sample of 100 unrelated individuals from Chinese North Han population when the STR markers were significant in TDT.The allele frequency,genotype distribution and heterozygosity at each STR marker were calculated directly.According to the Hardy-Weinberg Equilibrium,expected values of genotype frequency and expected heterozygosity were obtained.Mutation detection in COL1A1 gene promoterMutation in transcription regulation region(-1024bp~+36bp) of COL1A1 gene was detected in 109 patients with DDH by PCR and gene sequencing.At the same time, the detection was also performed in 100 normal individuals for distinguishing mutation from polymorphism.Expression study of COL1 Al gene promoter mutationFor evaluating the effect of promoter mutation(-106C→T) in COL1A1 gene,the wide-type promoter sequence(-1024bp~+36bp) of human COL1A1 gene was amplified and the mutant was obtained by using site directed mutagenesis method.The promoter fragment length was 1060bp.Then,the wide-type and mutant promoter were subcloned to luciferase report vector PGL3 -basic to construct recombinant eukaryotic expression vector PGL3-COL1A1.The recombinant plasmids were transfected into NIH 3T3 cells by lipofectamin method,and the activity of promoters was defined by assaying the relaive activity of luciferase.Allele frequency,genotype frequency and PIC for 10 STR loci(except for D17S810) were analyzed in 100 unrelated individuals.The genotype distributions conformed to Hardy-Weinberg Equilibrium for all the analyzed loci.High genetic polymorphism was observed in Chinese North Han Population at all the STRs loci except for D17S810 and D17S931.The highest genetic polymorphism was observed at D17S806 and D17S787.Because of a low genetic polymorphism,D17S810 and D17S931 locus were removed from the TDT.For the other loci,transmission disequilibrium was detected at D17S855,D17S858,D17S806,D17S1877,D17S941,D17S752 and D17S790,which overlapped 11.7Mb in 17q21.However,no transmission disequilibrium was found at D17S1787 and D17S787.Thus,the susceptive genes for DDH were located in the chromosome region between D17S855 and D17S790.Mutation detection in COL1A1 gene promoterA heterozygous mutation of-106C→T was detected in the transcription regulation region of COL1A1 gene in two pedigrees,and a novel C/T polymorphism was found at -35 base.Enzyme digestion and sequencing analysis showed that the target sequences were cloned into recombinant vectors successfully,and the expression activity of human wild-type and mutant promoter was identified in the transfected NIH 3T3 cell.The result indicated that the mutation of -106C→T in the transcription regulation region of COL1 Al gene decreased the promoter activity by 64%. 1.The susceptive gene for DDH is narrowed to 11.7Mb in the chromosome region of 17q21.31-17q22,between STR locus D17S855 and D17S790.2.We identify a novel heterozygous mutation(-106C→T) in the transcription regulation region of COL1AI gene in patients with DDH.This mutation might play a role in the pathogenesis of DDH.3.The heterozygous mutation of -106C→T in the transcription regulation region of COL1Al gene inhibits the transcription activity,which is one of the reasons that resulted in the down-regulation of COL1A1 in the hip articular capsule and ligamentum teres in DDH.Key wordsDevelopmental dysplasia of the hip(DDH);Gene location;transmission disequilibrium test(TDT);COL1A1;Mutation detection;Transcription regulation Supported by1.National Natural Science Foundation of China(30600654)2.Science and Technique Program of Liaoning Province(2005225013-1)... |
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