| Hemophilia A (HA) is caused by factor â…§(Fâ…§) gene defects, which is a common inherited bleeding disorder and resulted from deficiency and functional abnormality of coagulation factor â…§ . It is a classic example of X-linked recessive inheritance . The incidence is about one in five to ten thousand males and there is no difference in the various ethnic groups. The severity and frequency of bleeding in HA patients correlate with the activity of factor â…§ in plasma.The clinical presentation of HA is hemorrhage in skin, muscle, soft tissue .joint ,and internal organs .At present , a lot of studies indicated that HA was caused by various FVDI gene defects. Factor Vffl gene located on the human X chromosome, is 186kb long and contains 26 exons and 25 introns . Gene mutation of factor â…§ is highly particular , the amount of mutation is more than 500 , the main type includes deletion , insertion, inversion, duplication and point mutation .To investigate the molecular changes of Fâ…§ gene has important clinical significance for carrier detection .prenatal diagnosis and genetic counseling. Gene diagnosis for HA has been performed at abroad and Peking,Shanghai , Suzhou in our country .But the research only limited to simple case reports and clinical treatment in Shanxi . No paper has been published in gene diagnosis. Our aim is to provide a reliable detection methods in lab for HA genetic counseling and gene diagnosis in the future. Severe HA patients were analyzed intron 22 inversion ,directly detected carriers in the two pedigrees using long distance - polymerase chain reaction (LD-PCR) ;and the pedigree linkage analysis were performed by use polymorphic genetic markers.24 HA patients and 14 related pedigree members were selected from the Second Hospital of Shanxi Medical University, Children's Hospital of Shanxi Province and People's Hospital of Shanxi Province . Among them , 15 cases were positive in family history , 9 sporadic.The concentration of factor VII were detected with one stage method , the antigen of von-Willebrand factor with enzyme linked immunosorbent assay ( ELISA )method , DNA extraction was performed by QIAamp DNA mini kit purchased from QIAGEN company. After PCR, the production was performed by electrophoresis analysis.The diagnosis standard :The concentration of factor â…§ below 1% was severe; 1% ~ 5% moderate;5% - 25% mild; 25% ~ 50% sub clinical .Among 24 patients, 14 cases were severe, 8 moderate and 2mild .The level of vWF :Ag were all normal.Intron 22 inversion was detected by application of LD-PCR, 7 patients were positive in 14 severe cases, the positive rate was 50%, which is similar to relevant reports .Three female members in the two pedigrees were defined as carrier by direct detection intron 22 inversion. Two HA pedigrees were analyzed by utilization of entragenic and intragenic markers including Stl4 VNTR locus, BclI RFLP locus and two STR loci in intron 13 and 22. All results showed that one female member was a carrier in pedigree C, and another two females defined carriers in pedigree D.LD-PCR is a new method for HA gene diagnosis especially to analyze intron 22 inversion ,this method was rapidly, simply and accurately. The detection rate of inversion was 47.6% in Chinese population, same as other reports. Through this study , 50% severe HA patients in our province were caused by intron 22 inversion, carrier detection and prenatal diagnosis can be made by detection inversion in half of HA pedigree . The greatest advantage of direct detection was its speciality. Any member can be diagnosed if one patient or carrier has been detected with intron 22 inversion in this pedigree.The constitution of gene mutation in sporadic HA pedigree was same as that in positive family history. The direct detection of inversion has more priority in sporadic pedigree when polymorphism can't be used .Thus , the search for inversion of factor â…§ should be considered as the first DNA diagnostic option in genetic counseling of HA.The gene mutation of factor â…§ has ma...
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