Association Study Between COL1A1 Gene And Idiopathic Congenital Talipes Equinovarus

ObjectiveIdiopathic congenital talipes equinovarus (ICTEV) is a complex footdeformity presented at birth with 0.6‰～1‰birth rate in Chinese.The cause ofICTEV remains controversial. Many factors as skeleton development anomaly,neuromuscular disease,developmental arrest in uterus and so on seem to beresponsible. It is considered that the genetic factors play an important role in thepathogenesis of ICTEV, and the heritability of ICTEV is about 65%, it is yetunknown about the hereditary patterns and penetrance of ICTEV.Recently, it isbelieved that abnormal expression of some limb development-related genes leadsto ICTEV. At present,researches on human ICTEV mainly focus on manysyndromes with foot malformations,and little advances have been made in theresearches on the susceptibility genes of ICTEV. The researches provid somecandidate regions and genes for ICTEV such as 5q15-31.1,10p11.2-15 and soon,which provide an useful clue for us to look for the candidate genes of ICTEV.Researches indicat that the content of typeⅠcollagen is remarkable increasedin ICTEV.TypeⅠcollagen is the most abundant and widly expressed collagen inhuman. It provides the structural support for the body in skeleton, skin, bloodvessele,nerves,intestines,and in the fibous capsules of organs. TypeⅠcollagen is aheterotrimer of two identicalαl(Ⅰ) chains and one chα2(Ⅰ) chain. Theαl(Ⅰ) andchα2(Ⅰ) chains of typeⅠcollagen are encoded by the unlinked loci COL1A1 andCOL1A2 respectively. COL1A1 and COL1A2 locate in 17q21.3-q22 and7q21.3-q22 respectively.Now, it has discovered hundred of mutations in COL1A1gene.Mutations at these loci result primarily in the connective tissue disorders: osteogenesis imperfecta,Ehler-Danlos syndrome typesⅦA andⅦB,Osteoporosis.But there is no report about association between COL1A1 andICTEV.In our study, we will probe into the association between COL1A1 andICTEV furtherly on molecular level.In our studies,we constructed animal model of talipes equinovarus withWistar rats, and discovered that there was up-regulation of Collal gene expressionin different stage of limb development in the animal model by RT-PCR. Then,wealso discovered that there was up-regulation of COL1A1 gene expression in themusclar and tenden tissues of ICTEV patients by RT-PCR. At the sametime,mutation sites in transcription regular sequences(-1031bp～+30bp) and intron1 of COL1A1 gene were detected in 84 patients with ICTEV by denaturinggradient gel electrophoresis technique.The result was that we found a -161C→Theterozygous mutation and a +274C→G homozygous mutation in COL1A1gene.So,we thought COL1A1 gene was confirmed to be associated with ICTEV.Material and Methods1. Samples86 Wistar rats were bought from the animal department of China MedicalUniversity.Musclar and tenden tissues of 20 patients with ICTEV and correspondingnormal tissues came from the Second Affiliated Hospital, China MedicalUniversity.84 patients with ICTEV came from the Second Affiliated Hospital, ChinaMedical University. All patients had typical manifestation and were confirmed byX-rays and surgical operation. 100 normal DNA come from normal crowd ofnortheastern china.2.Construction of rat model and Study Collal gene in thehindlimbs of rat models with RT-PCR135mg/kg body weight of ATRA was used to construct rat model on day 10 ofgestation, which has shown a good consistency with talipes equinovarusmalformation as that in humans. On day 15,17,19 and 21 of gestation,thehindlimbs were cut from model fetus and normal controls,and total RNA was extracted,then reverse transcribed to cDNA. RT-PCR was used to detecte theexpression of COL1A1.3.Study COL1A1 gene in musclar and tenden tissues of ICTEVwith RT-PCRThe total RNA was extracted from musclar and tenden tissues of patients withICTEV and normal controls,then reverse transcribed to cDNA. RT-PCR was usedto detecte the expression of COL1A1.4.PCR-DGGEMutation sites in transcription regular sequences(-1031bp～+30bp) andintron 1 of COL1A1 gene were detected in 84 patients with ICTEV by denaturinggradient gel electrophoresis technique.Results1.The expression of Collal gene in rat modelsRT-PCR results found the expression of Collal gene up-regulated in differentstage of limb development in rat models with the clubfoot like deformity.2. The expression of COL1A1 gene in muscles and tenden ofpatients with ICTEVRT-PCR results found the expression of COL1A1 gene in musclar and tendentissues of ICTEV is higher than normal tissues.3. Result of PCR-DGGEThe result was that we found a-161C→T heterozygous mutation and a+274C→G homozygous mutation in COL1A1 gene.Conclusions1.RT-PCR results found the expression of Collal gene up-regulated indifferent stage of limb development, and the expression of COL1A1 geneup-regulated in musclar and tenden tissues of patients with ICTEV,whichconfirmed the association between COL1A1 gene and talipes equinovarusdeformation.2. We discovered a-161T→C heterozygous mutation and a +274C→Ghomozygous mutation in COL1A1 gene of patients with ICTEV. It illustrated that the mutation in transcription regular sequences could caused ICTEV.