Study Of Transmission Test For Linkage Disequilibrium Between HoxD Gene And Congenital Clubfoot

ObjectiveCongenital clubfoot ( CCF) is one of the most common congenital foot malformations in pediatric orthopedics with an incidence of 0. 1 percent. Genetic epidemical investigations suggest that there be a genetic background. CCF may require a predisposing gene acting in a particular background of polygenes or environmental influences. Hie development limb is a very complicated process, which involves in a set of genes to express at different poeriods and in different regions. Recently, the studies on embryonic development suggest that as an important regulation family, homeobox (Hox) genes are closely related to the development of organisms. Hox genes are critical for limb development based on experimental manipulations of mice and chick embryos. At present, no 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 Spielman in the study of susceptive gene of insulin - dependent diabetes mellitus (IDDM), which can avoid population stratum error. The study chose the polymorphic marker Hox4Ep at segment where HoxD genes locates. The polymerases chain reaction(PCR) products were run to isolate alleles. By the transmission pattern 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 HoxD genes.Methods1. DNA preparationsPeripheral venous blood samples were collected in 65 cases of CCF nuclear families with one affected child. The Pediatric Orthopedics Department of the 2nd Clinical 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 deformities was excluded. Blood samples were stored in -70 C. DNA was extracted using phenol/chloroform method.2. PCR amplificationInformation about Hox4Ep ( microsatellite marker) was obtained from: http://www. gdb. org. DNA was amplified in 25ul PCR reaction. PCR was done with an initial denaturation at 94 C for 10min; followed by 30 cycles of denaturation at 94 C for 40sec, a unealing at 55 C for 40sec, and extension at 72 C for 40 sec, with a final 7min extension at 72 C. PCR products were examined on 2% agrose gels.3. GenotypingFirst, 10ul of PCR product was denatured with 10ul of denaturing solution at 96 C for 10 minutes. Second, it was cooled rapidly and then run on 6% denatured polyamine gel. The gels were stained by AgNO3/Na2CO3 method. The results were stored in Kodak Digital system.4. Statistical analysisThe TDT table was formed according to the genotypes of families with heterozygous parents. And transmission disequilibrium test (TDT) was performed by SPSS10.0 software.Results1. Genotypes of 126 members in 42 CCF families were analyzed by amplifying the fragment using PCR.2. There were 12 alleles at this microsatellite marker with fragment length from 110bp to 130bp, each named allele 1-12.3. Transmission disequilibrium test was performed on 32 CCF families with heterozygotes parents.4. A TDT table was formed by the transmission pattern of the alleles.5. For allele 12, transmission number was 15, and no transmission number was 6. There was transmission disequilibrium at the twelfth allele( x2=4.614, P<0.05).Conclusions1. There was transmission disequilibrium between HoxD gene and human CCF. HoxD gene may be a susceptive gene of CCF.2. There were 12 alleles at this microsatellite marker Hox4Ep in the Chinese population.