SummaryThe biosynthesis of coagulation factor VIII (FVIII) is hampered by successive controls that limit its production. To improve this production, a truncated intron I sequence of factor IX (TFIXI1) was inserted in FVIII cDNA in place of FVIII introns 1, 12 and 13 and also as a combination between introns 1 and 12, and introns 1 and 13. The intron 12 and 13 locations were targeted because this region was previously shown to contain a transcriptional silencer. The expression of FVIII in CHO and HepG2 cells revealed important variations in the properties of the minigenes depending on the TFIXI1 insertion sites. In FVIII intron 13 location the TFIXI1 seemed to diminish the transcriptional silencer activity, whereas it was poorly spliced in intron 12 position. Among the five constructs, FVIII I1+13 leaded to a significant improvement in FVIII secretion (13 times) that was associated with a dramatic intracellular accumulation in cells. Therefore, the FVIII I1+13 minigene could represent a particular interest to produce recombinant FVIII in vitro as well as in the aim of gene therapy of haemophilia A.
We investigated the mechanisms responsible for severe factor IX (FIX) deficiency in two cross-reacting material (CRM)-negative hemophilia B patients with a mutation in the first and second epidermal growth factor (EGF) domains of FIX (C71Y and C109Y, respectively). We have determined the kinetics of mutant FIX biosynthesis and secretion in comparison with wild-type FIX (FIXwt). In transfected cells, FIXwt was retrieved as two intracellular molecular forms, rapidly secreted into the culture medium. One appeared to be correctly N-glycosylated, and corresponded to a form trafficking between the endoplasmic reticulum (ER) and Golgi apparatus. The other corresponded to the mature form, ready to be secreted, exhibiting correct N-glycosylation and sialylation. In contrast, the two mutants, FIXC71Y and FIXC109Y, were not secreted from the cells and did not accumulate intracellularly. Relative to FIXwt, they were retained longer in the ER and were only N-glycosylated. In addition, the intracellular concentration of the FIX mutants increased when ALLN, an inhibitor of cysteine proteases and of the proteasome degradation pathway, was added to the culture medium. Both the FIX mutants and FIXwt were associated in the ER with the 78-kDa glucose-regulated protein (GRP78/BiP) and calreticulin (CRT), though the amount of CRT associated with the two mutants was twice as strong as with FIXwt. These results strongly suggest that chaperone and lectin molecules act in concert to ensure both proper folding of FIXwt and the retention of mutant molecules.
We report here five surgeries successfully performed with a continuous infusion of Mononine (Armour Pharmaceutical Company, Kankakee, IL) in three hemophilic B patients. Before surgery the patients received a bolus dose of 40 to 100 U/kg according to the type of surgery. This injection was followed by a continuous infusion of Mononine, with an infusion rate of 3.5-7 U/kg/hr in order to maintain a factor IX level between 50 and 100% during the whole surgery and the following 6 days. The infusion rate was further adjusted according to the type of surgery until hospital discharge. This method appears to be safe and efficient, since no abnormal bleeding occurred during surgery and none of the patients presented any thrombotic complication. However, this alternative to intermittent administration of factor IX should be standardized and precisely evaluated, regarding the level and the amount of factor IX required, and the cost of the infused material. In our hands, this cost was decreased by 30-40% compared to previous therapeutic schedules at our institution.
Summary. Glanzmann thrombasthenia is the most common inherited disorder of platelets that may induce severe bleeding complications. Molecular biology techniques have offered the possibility to assess the basis of this chronic haemorrhagic disease at the molecular level. However, the accessibility of mRNA in platelets is limited by the availability of the patient's blood samples and the relatively weak amount of this material in these cells. Taking advantage of the genetic phenomenon of illegitimate transcription, we have demonstrated that glycoprotein IIb and glycoprotein IIIa mRNA could be detected in lymphoblastoid cell lines issued from normal EBV-transformed lymphoblasts. We further analysed the sequences of the two glycoprotein transcripts in lymphoblastoid cell lines from two previously characterized patients presenting with Glanzmann thrombasthenia. The results showed that illegitimate transcripts presented similar molecular abnormalities to those found in platelets. These data demonstrated that the nucleotide sequences of illegitimate transcripts were identical to tissue-specific mRNA found in platelets. We applied this methodology to screen for the genetic defect in a new thrombasthenic patient, and found a homozygous nonsense mutation GCA →TGA converting Arg8 to stop in the glycoprotein IIIa gene. This immortalized source of genetic material is therefore particularly useful for molecular genetic studies in inherited platelet disorders, avoiding repetitive and large blood samplings in frequently anaemic patients.
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