Study On Prenantal Gene Diagnosis Of Hemophila A

IntroductionIn 1978 Kan et al carried out gene diangnosis for scikle anemia by DNA recombination technique. From then on,the prologue of gene diagnosis was pulled away. With the quick progress of the molecular biology technique and deepening accomplish of Mankind Genome Project,gene diagnosis is developing rapidly. The methods have been increasing continually . Gene dianosis is dividied into direct diagnosis and indirect diagnosis, orthodox linkage analysis is main method in indirect diagnosis in which the linkage relationships of the polymorphism sites such as restriction length porlymorphism, short tandem repeated sequence, variable number tandem repeats et al and morbific gene are used. While direct diagnosis means that we directly detedct the gene associated with certain diease and determine it is normal or abnormal and mutation character, so direct diagnosis can provide more reliable and pre'ciser information, it will posses wide development prospect and scientific researth value, but it is also restricted due to the a-vailability of technique and economy .Hemophilia A is a inherited hemorrhagic disease caused by deficity or defect of the blood coagulation facror VIII, these patients have frequent spontaneous bleeding in joints, muscles and internal organs, bleeding into joint is most common,frequent bleeding can cause disorder even death. The patients degree of bleeding is related with the level of FVIII: C in blood plasm. At present, to infuse FVIII praeparatum or fresh whole blood is main treatment and prophylaxis method. It is also expected that factor VIII protein produced by biotechnology is used in treating the disease, but has no cure method yet. FVIII gene is loacated on the long arm of X chromosome and its lenth is 186 kb containing 26 exon and 25 intron. It is discovered that intron 22 - inversion is most common mutation in FVIII gene and responsible for about 40% ~ 50% of severe hemophiliaA . So itis very valuable in medical science to carry out hemophilia A especially severe hemophilia As gene diagnosis, carriers detdection and prenatal diagnosis, it can prevent hemophilia A carrier to give birth to hemophiliac A or hemophilia A carrier and cut down the morbidity of hemophilia A , it has important value to aristo-genesis , good growth and improving populations procatarxis.Materials and MethodWe obtained maternal blood samples from 31 patients , 11families member and a pregnant woman who gave birth to a dead hemophiliac A, amniotic fluid from 11 pregnant women at 8 -26 weeks'gestation . 5 normal man and 5 mormal women were used as negative contron.We extracted DNA from blood with saturated sodium chloride solution method and from amniotic fluid with boiling method, intron 22 -inversion mutation was detected using LD -PCR and I -PCR, then point mutation in exon 14 was screened using PCR - DGGE, at lsat we carried out linkage analysis selecting Stl4 ( DXS52) site as polymorphic site to conduct gene diagnosis and prenatal gene dianosis.ResultsWe determined 7 hemophiliacA carrying intron 22 - inversion mutatio from 31 patients using LD - PCR and I - PCR, determined 3 intron 22 - inversion mutation carriers from 4 mothers of hemophiliacA carrying intron 22 - inversion mutation, identical conclusion was arrived at from these two methods. A prenatal gene diagnosis of intron 22 - inversion mutation was completed using I -PCR:her fetus was normal and female,but not a carrier. Gene diagnosis and prenatal gene dianosis were carried out by linkage analysis, and got information from 8 families at St14 ( DXS52 ) polymorphic site, we successfully provided if every family member was carrier and prenatal gene dianosis evidence for pregnant woman.Conclusion1. We are the first in our country to use I - PCR to carry out detection of intron 22 - inversion mutation and complete prenatal gene dianosis of this mutation.2. Optimize the reaction condition and parameter of LD — PCR, make them more stable and operatable.3. Establish direct gene diagnosis method system in our laboratory, the rate of gene diagnosis, carrier detection and prenatal gene dianosis was rised . we propose the better projece for gene diagnosis of hemophilia A.