A형 혈우병에 있어서 중합효소연쇄반응의 응용

Abstract

Background :The polymerase chain reaction(PCR) which is used for the analysis of restriction fragment length polymorphism(RFLP) in the factor VIII gene and prenatal sex determination, is thought to be a useful tool for the diagnosis of hemophilia A. And thus, for carrier detection and prenatal diagnosis in hemophilia. A, we attempted a trial of application of PCR to analysis of XbaI- and BcII-RFLP in the factor VIII gene and to amplify the sequence specific for Y-chromosome Method :The samples were composed of peripheral blood from 120 kindreds(46 males and 64 females including 41 hemophilic patients) of hemophilia A families. In the PCR of BcII-RFLP, oligonucleotide pairs for intron 18 in factor VILL gene were used as primers. After digestion of 142 bp amplified products with BcII, two alleles were identified by negative allele(142 bp) and positive allele(99 and 43 bp). In the PCR of Xbal-RFLP, oligonucleotide pairs for intron 22 in factor VIII gene were used as primers. After digestion of 96 bp amplified products with XbaI, two allels were identified negative allele(96 bp) and positive allele(68 and 28 bp). In the PCR of sequence specific for Y chromosome. the samples composed of 27 amniotic fluids and 9 abortus of which sex was determined by chromosome analysis, were amplified using Y1.0 and Y2.0 primers Result :Frequencies of BcII negative allele and BcII positive allele were 18.0% and 82.0% respectively, and female heterozygosity rate was 36.5%. Frequencies of XbaI negative allele and BcII positive allele were 63.5% and 36.5% respectively, and female heterozygosity rate was 51.1%. It was shown that among 72 females tested 54(75.0%) were heterozygote BcII- and/or XbaI-RFLP. In the PCR of sequence specific for Y chromosome for evaluation of prenatal sex determination, amplified products of 154 bp fragments were detected in all 18 amniotic fluids and fetal tissues of which karyotypes were confirmed as male by cytogenetic method Conclusion :These results suggested that the PCR-based analysis of the BcII- and the XbaI-RFLP, and sex determination provides very useful tools for carrier detection and prenatal diagnosis in hemophilia A.