Transmission Test For Linkage Disequilibrium In Shh Gene And Anorectal Malformations

ObjectiveAnorectal malformations(ARMs)are common surgical problems affecting 1 in 1,500 to 1 in 5,000 live births.The spectrum of these abnormalities is complicate and the complications are multiply which affect pediatric health seriously.Genetic epidemical investigations suggest that there be a genetic background. ARMs may require a predisposing gene acting in a particular background of polygenes or environmental influences.Althought much work has been done,the genetic basis remains unclear.The development of hindgut is a very complicated process,which involves in a set of genes to express at different periods and in different regions.Recently,the studies on embryonic development suggest that as an important signal,shh gene is closely related to the development of organisms.Shh gene is critical for hindgut 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 shh genes and human ARMs.The study chose the polymorphic marker D7s500 that high linkage with shh gene. 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 association between ARMs and shh gene.Methods1,DNA extractionPeripheral venous blood samples were collected in 46 cases of ARMs nuclear families with one affected child.The Pediatric Surgery Department of the ShengJing Clinical Hospital of China Medical University supplied the families.Each patient with ARMs showed typical clinical manifestation and was diagnosed by operation. Furthermore,the ARMs with severe complications was excluded.White blood cells of these samples were stored in -20℃.DNA was extracted using phenol-chloroform method.2,PCR amplificationInformation about d7s500(microsatellite marker)was obtained from http://www.gdb.org.DNA was amplified in 25μl PCR reaction.PCR was done with an initial denaturation at 94℃for 3min;followed by 35 cycles of denaturation at 94℃for 40sec,a unealing at 57℃for 60sec,and extension at 72℃for 60 sec,with a final 7min extension at 72℃.PCR products were examined on 2%agarose gels.3,GenotypingFirst,5μl of PCR product was denatured with 5μl of denaturing solution at 96℃for 10 minutes.Second,the sample was cooled rapidly and then run on 12% denatured PAGE(polyacrylamide gel electrophoresis).The gels were stained by AgNO₃/Na₂CO₃ method.4,Statistical analysisThe TDT table was formed according to the genotypes of families with heterozygous parents.And transmission disequilibrium test(TDT)was performed by SPSS12.0 software.Results1,Transmission disequilibrium test was performed on 35 ARMs families with heterozygote parents.2,There were 12 alleles at this microsatellite marker,each named allele 1-12.3,For allele 10,transmission number was 10,and no transmission number was 2.The transmission disequilibrium was found at the tenth allele(x²=5.333,P＜0.05). Conclusions1,There was transmission disequilibrium between shh gene and ARMs.Shh gene may be a susceptive gene of human ARMs.2,There were 12 alleles at the microsatellite marker d7s500 in the Chinese population.