| Congenital clubfoot (CCF) is one of the most common congenital foot malformations in pediatric orthopedics with an incidence of 0. 01 to 0. 08 percent. Genetic epidemical investigations and the incidences of different races suggest that there be a genetic background. CCF may require a predisposing gene acting in a particular background of poly genes or environmental influences. The development limb is a very complicated process, which involves in a set of genes to express at different periods and in different regions. The studies on embryonic development suggest that homeobox ( Hox) genes are closely related to the development of organisms as an important regulation family. Hox genes are critical for limb development based on experimental manipulation of mice and limb embryos. At present, few previous reports can be found on the genetic research of association between Hox genes and CCF. Transmission disequilibrium test (TDT) set up in 1993 by Spilman in the study of susceptive gene of insulin -development diabetes mellitus (IDDM ) , which can avoid population stratum error. The study chose the polymorphic markers D7S516 and D7S1808 at segment where HoxA genes locates. The polymerases chain reaction ( PCR) products were run to isolate alleles. By the transmission pettern of the alleles, a TDT table was formed and TDT test was done. The purpose of this study is to investigate the correlation between the congenital clubfoot and HoxA genes.Methods1. Sample collectionPeripheral venous blood samples were collected in cases of CCF nuclear families with one affected child. The Pediatric Orthopedics Department of 2ndClinical Hospital of China Medical University supplied the families. Each patient with CCF showed typical clinical manifestation and was diagnosed by X -ray and operation. Furthermore, the secondary clubfoot with other deformations was excluded.2. Selection microsatellite markersInformation about D7S516 and D7S1808 (microsatellite markers) was obtained from://www. gdb. org.3. GenotypingBlood samples were stored in 20℃. DNA was extracted using saturation sodium chloride method.PCR amplificationDNA was amplified in 10ul PCR reaction. 0. 1 ul a32 p - dCTP was added in this reaction. D7S516; PCR was done with an initial denaturation at 94℃ for 10min; followed by 35 cycle of denaturation at 94℃ for 30sec,a unealing at 56℃ for 30sec,and extension at 72℃ for 45sec,with a final 7 min extension at72℃ D7S1808: PCR was done with an initial denaturation at 94℃ for 10min; followed by 35 cycle of denaturation at 94℃ for 45sec, a unealing at 56℃ for 45sec,and extension at 721 for lmin, with a final 7 min extension at 72℃.3ul of PCR pruduct were denatured with 10ul of denaturing solution at 96℃. for 7 minutes. It was cooled rapidly and then run on 6% denatured poly-amine gel. The gels was tranfered to dark slide by filter paper, then it developed to a image. The results were stored in Kodak Digital system.Statistical analysisThe transmission disequilibrium test (TDT) table were formed according to the genotypes of families with heterozygous parents. We calculated the X2 value, verdicted the P value, then we got the result of TDT.Results1. The result of the alleles of D7S516 microsatellite marker and the result of the alleles of D7S1808 microsatellite marker were obtained. There were 7 alleles in D7S516 microsatellite marker and in D7S1808 microsatellite marker.2. The result of TDT was obtained. A TDT table was formed by the transmission pattern of the alleles of D7S516 microsatellite marker. A TDT table was formed by the transmission pattern of the alleles of D7S1808 microsatellite marke. There was transmission disequilibrium at the alleles of D7S516 (X2 = 20.3846 P < 0. 01). There wasnt transmission disequilibrium at the alleles of D7S1808(X2 =11.3196 P>0.05).3. A frequency table about the alleles of D7S1808 was formed.Conclusions1. There were 7 alleles at D7S516 microsatellite marker in the Chinese population. There was tranmission d... |
PDF Full Text Request