Transmission Disequilibrium Test Between HoxC Genes And Congenital Dislocation Of The Hip And Analysis Of Allele Frequency In D12S1686

ObjectiveCongenital dislocation of the hip ( CDH) is the one of the most common malformations in orthopedics with incidence 0. 91-3.8%. The genetic epidemical investigations indicate that CDH is a polygen-etic disease.Recently studies in the knockout animals have discovered that ho-meobox (Hox) genes involve and regulate the skeleton morphogenesis, and the animal experiments also indicate Abd - B related HoxC genes expressing in the hindlimb. In 1993 , Spielman was first to succeed in localizing the susceptibility gene of diabetes mellitus type I with transmission diseoquilibrium test (TDT) . This approach is very a-vailable to localize the susceptibility genes of polygenetic diseases in our country.But now there are no studies reported on the relationship between HoxC genes and CDH. To investigate the correlation between HoxC genes and CDH, a microsatellite marker D12S1686 was selected in the chromosome region where HoxC genes reside and then TDT was performed on 65 families with heterozygotes parents. The allele frequency in D12S1686 was also analyzed.Methods1. Study population: A total 101 CDH patients and their parents composed of experimental group, while 3 normal individuals as controlled group.2. DNA preparations; Genomic DNA was extracted from 5 -10ml peripheral blood anticoagulated by EDTA with a standard phenol/chloroform method.3. PCR amplification: The microsatellite marker D12S1686 was selected in the chromosome region where HoxC genes reside. PCR reactions were carried out in a 25ul cocktail, and amplifications proceeded for 10 minutes at 94 C , followed by 30 cycles of 94 C , 40 seconds; 55 C , 40 seconds; 72 C ,40 seconds with the last elongation at 72 C for 7minutes.4. Genotyping: The PCR products were electrophoresed on a 6% sequencing polyacrylamide gel for 3. 5-4 hours subsequently stained with silver. The genotypes of each CDH family were identified according to the standard alleles of 3 normal individuals while the fragment length of each allele was identified according to the known ones.5. Transmission disequilibrium test; According to the genotypes of families with heterozygoes parents, TDT was performed by FREQ program of SAS software.6. The analysis of allele frequency: The allele frequency, Hardy - Weinberg test and comparison of frequency between two differentpopulations were calculated according to the reference.Results1. The result of TDT:x2 = 6. 171, P = 0. 965 , which indicates there is no transmission disequilibrium between D12S1686 and CDH.2. The analysis of allele frequency in D12S1686 of Han ethnic population of Liaoning, ChinaThe allele frequency in D12S1686 can be analyzed among 188 parents as no - relationship population due to no transmission disequilibrium found between D12S1686 and CDH.a. There are 16 alleles and 59 genotypes detected in D12S1686 among 188 individuals of Han ethnic population of Liaoning, China with size from 215bp to 249bp, allele frequency from 0. 003 to 0. 245 , Het of 0.855.b. The genotype frequency data is consistent with Hardy - Wein-berg rule (X2=50.245,P>0.10) , which indicates that D12S1686 can be used to gene linkage analysis as an efficient genetic marker.c. The allele frequency in D12S1686 of Han ethnic population of Liaoning, China is significantly different from that of Caucasoid population (X2=48.456,P<0.001).Conclusion1. There is no transmission disequilibrium between D12S1686 and CDH, indicating that HoxC genes are not apparently associated with CDH.2. There are 16 alleles detected in D12S1686 among Han ethnic population of Liaoning, China, with size from 215bp to 249bp, allelefrequency from 0. 003 to 0. 245, Met of 0. 855. The significant difference of allele frequency in D12S1686 between Han ethnic population of Liaoning, China and Caucasoid population may be related to ethnic difference.