| Background Hemophilia A is one of the most common recessive,X chromosome-linked hereditary bleeding disorders,caused by the genetic defect of factorⅧ(FⅧ).Its incidence is about 1 in 5,000 to 10,000 males.The recurrent bleedings of joints and muscles are the common clinical manifestations.Currently,the replacement therapy with factorⅧconcentrates is the most effective method to control the bleeding but associated with the development of factorⅧinhibitors,one of the most serious complications of the therapy.The prevalence of factorⅧinhibitor development in the treated patients was 5-30%.Although the mechanisms of FⅧinhibitor formation remained to be further explored,it had been shown that the development of the inhibitors were related to many environmental,genetic and immune factors.In China,the prevalence of the inhibitors in patients with hemophilia A was unknown,and there were no studies to demonstrate the risk factors responsible for the development of the inhibitors,including the genetic factors.The positive inhibitors were highly misdiagnosed since the most of the physicians were lack of awareness of the inhibitors.Therefore,it is necessary to investigate the frequency of the development of inhibitors in Chinese hemophilia A patients and to explore the related risk factors,which have significant importance for the prevention and treatment of the inhibitors.Objective To screen the inhibitors in patients with hemophilia A and explore its environmental risk factors and the relation between genetic defects and inhibitors formation.Methods 265 Outpatients and inpatients with hemophilia A have been recruited from April 2003 to April 2007 in Peking Union College Hospital.FⅧ:C were measured using one-stage coagulation assay.FⅧinhibitors were determined using Bethesda method.Intron 22 inversion was detected by long-distance polymerase chain reaction and intron 1 inversion,by multiplex polymerase chain reaction.DNA sequencing was performed using dideoxy sequencing method.Results1.Among 265 patients,22 were inhibitor positive(8.3%),19 patients(86.4%) were the low responders(titers less than 5BU) and 3 patients(13.6%) were high responders.2.97 out of 265 patients(36.7%) had severe disease(FⅧ:C＜1%),113 patients (42.6%) were moderate(FⅧ:C 1-5%),and mild(FⅧ:C＞5%) disease accounted for 20.7%(55 patients).The frequency of positive inhibitors in severe patients was 11.3%, higher than moderate(7.2%) and mild patients(6.2%).3.The age of 22 patients with positive inhibitor ranged from 6 to 68 years with a median age of 28 years.The inhibitors were more frequent in the patients older than 50 years with 50%of the positive detection.4.Among 158 patients having the detail medical history,the frequency of the inhibitors was higher in the patients whose annual FⅧconcentrate infusions were more than 12 times(12.8%vs 5.8%).In the patients whose first exposure to FⅧwas at the age less than 6 months,one(8.3%) was inhibitor positive with a titer of 80BU. The frequencies of the inhibitors in the patients with first exposure age 6-12 months and more than 12months were 4.8%and 8.0%,respectively.The frequency of the inhibitors in the patients with a history of continuous infusion(CI) of FⅧconcentrates was 28.3%,higher than those without the history(1.6%).It seemed that the inhibitor development was also related to FⅧproducts exposed.Its frequency was higher in the patients who had ever received more than one brand of FⅧconcentrates(9.3%vs 3.9%).5.In the 158 patients,60 patients(37.9%) were found having intron 22 inversion,2 had intron 1 inversion.Among 93 patients with severe disease,44 patients(47.3%) were intron 22 inversion.6.18 patients with negative detection of intron 22 and intron 1(9 severe and 9 mild-moderate cases,5 of them with positive inhibitors) were randomized to receive DNA sequencing.18 different genetic defects were found,8 small deletion(44.4%),7 point mutations(38.9%),2 insertion mutation(11.1%) and 1 splice error(5.6%).Among those defects,4 were nonsense mutations and 14 were missense mutations.7.Among 18 defects,eight were novel.They were①insertion A at codon 183 and 189;②del 4(AACA) at codon 297-8;③del G at codon 710;④del 2(AG) at codon 1442;⑤dei A at codon 1590;⑥Y1837C;⑦del 9(CTACCTTCG) at codon 2304-7;⑧del 4(TTGT) at codon 51-2.8.FⅧinhibitors were detectable in 2 of 60(3.3%) patients with intron 22 inversion.None of 2 patients with intron 1 inversions was inhibitor positive.9.In 4 patients with nonsense mutations,three of them had severe disease and their mutations were located in the different activity domains(C1,C2 and A3).The patient whose mutation site was C1 domain had positive detection of the inhibitor.Another nonsense mutation patient had moderate disease and his mutation site was located non-activation domain.6 of 14 missense mutations were severe cases,and 50%were inhibitors positive.8 missense mutations were found in the patients with mild-moderate disease,only 1 patient with inhibitors.Conclusions FⅧinhibitors were found in 8.3%of the patients with hemophilia A,which was slightly lower than that in the Western countries.The development of inhibitors were probably related to the age,the severity of the disease,the frequency of FⅧconcentrates infusion,the age of the first exposure to FⅧconcentrates and the mode of FⅧinfusion.Intron 22 inversion was the most common genetic defects in the severe patients,the next was intron 1 inversion.The point mutations and small deletions were more frequent gene mutation types.It has been shown that the types and the sites of gene mutations were possibly related to the severity of the disease and played an important role in the development of FⅧinhibitors.
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