Analysis of inversion in intron 22 of the factor F VIII:C gene in patients with hemophilia A in the Slovak population

Hemophilia A is the result of Factor F VIIIC (F8C) gene mutations. Predominating mutation is inversion, occurring in about 50% of patients with severe form of the disease. Inversion is the result of homologous recombination between gene A located on the 22. introne of the F8C gene and one of its telomeric copies located about 500 kb from 5'end of the factor F VIIIC gene. This study presents the results of this mutation screening in 84 nonrelated patients with hemophilia A. Inversion was identified in 22 (50%) of 44 patients with severe form and in 1 (from 13) with moderate form of the disease. Distal type of inversion was more frequent (82.6%) than proximal one. The identification of iversions enabled direct DNA diagnosis in 50% of patients with severe form of the disease and will be successfully used in the prenatal diagnosis and carrier testing, mainly in families with sporadic occurrence of the disease. (Tab. 1, Fig. 2, Ref. 18.)     

Analysis of the F8 gene in individuals with high plasma factor VIII: C levels and associated venous thrombosis

High FVIII:C levels have previously been shown to be an independent risk factor for thrombosis with 4.8 times higher potential risk of thrombosis in individuals with FVIII:C levels greater than 1.5 u/ml. Recently, we found that raised FVIII:C levels are largely attributable to elevated FVIII:Ag levels. The determinants of FVIII:Ag levels are unclear and might be partly genetic. The promoter of the F8 gene has recently been characterised we therefore investigated the promoter and the 3' terminus of the F8 gene for possible polymorphisms associated with raised FVIII:Ag levels in 62 selected individuals with a thrombotic tendency. We confirm previous reports that raised FVIII:C levels are largely attributable to an elevation in FVIII:Ag and this is also associated with elevation of vWF; non-O blood group: relatively short APTT and relatively low APC ratio. We screened 1140 bp of the proximal promoter including the protein binding sites identified by DNase I footprint analysis by SSCP, however no polymorphisms were identified. Direct DNA sequence analysis of the region -542 to +165 failed to identify any sequence polymorphisms. The recently described polymorphism in the polyadenylation cleavage site in the prothrombin gene associated with increased prothrombin activity prompted us to screen the region surrounding the 3' terminus of the F8 gene for polymorphisms but we found none.     

Cross-reactivity to porcine factor VIII of factor VIII inhibitors in patients with haemophilia in Australia and New Zealand

BACKGROUND: Inhibitory antibodies which neutralise factor VIII develop in 10-20% of individuals with inherited haemophilia A and rarely as autoantibodies in normal individuals to cause acquired haemophilia. The antibodies are directed against human factor VIII but cross-react to varying degrees with porcine factor VIII. Porcine factor VIII can be used for treatment in individuals with low cross-reactivity. AIMS: To determine the cross-reactivity of factor VIII inhibitors between human factor VIII and porcine factor VIII, in a population of patients with inherited and acquired haemophilia A. Also, to determine whether patients with inherited haemophilia and inhibitors have a higher incidence of factor VIII gene inversion in intron 22. METHODS: Samples and data sheets from 43 patients with inherited and ten with acquired haemophilia were submitted from hospitals in Australia and New Zealand. Inhibitor levels to human and porcine factor VIII were measured by the Bethesda method in 39 with inherited and nine with acquired haemophilia A. RESULTS: Of 39 patients with inherited haemophilia A, cross-reactivity was 0% in 17 patients, 1-19% in six, 20-39% in 11 and 40-80% in five. In six of nine patients with acquired haemophilia cross-reactivity was < or = 7%. In inherited severe haemophilia A, the frequency of the intron 22 inversion was not greater in 37 study patients than in 28 patients without an inhibitor. CONCLUSIONS: Many patients in Australia and New Zealand with inhibitors to human factor VIII presently show a low or absent level of cross-reactivity to porcine factor VIII. These may respond to treatment with this concentrate at least in the short term. There remains a group of patients with high cross-reactivity who will respond only to recombinant factor VIIa or prothrombin complex concentrates.     

Immunosuppressive treatment in haemophiliacs with inhibitors to factor VIII and factor IX

9 patients with severe haemophilia A and inhibitors (inhibitor levels between 0.1 to 5.8 U/ml) and 3 patients with severe haemophilia B and inhibitors (inhibitor levels between 0.1 to 11 U/ml) were treated on a total of 16 and 13 occasions, respectively, with a large dose of antigen (factor VIII or factor IX) and cyclophosphamide (10-15 mg/kg b.w. i.v. initially and then 2-3 mg/kg b.w. orally for 7-10 days) in connection with severe bleeding and surgery. All the patients had proved not to respond to treatment with factor VIII or factor IX concentrate alone, and all except one had shown strong secondary antibody increases. In 6 of the patients with haemophilia A the treatment (11 occasions) had a satisfactory haemostatic effect and even permitted neurosurgery without bleeding complications. The inhibitor level remained at zero for 5-10 days, after which it gradually began to return towards its original level. In these cases it was possible to give factor VIII in amounts which neutralised the inhibitor and afterwards raised the factor VIII initially to at least 50%. In the 3 patients with haemophilia B treatment (13 occasions) was successful except on one occasion, and surgery was performed without abnormal bleeding. The factor IX level was initially raised to at least 50% except in the one failure. The inhibitor level remained at zero for 12 days to 3 months, after which it gradually rose towards its original level. One patient was treated on 8 occasions.     

Continuous infusion of porcine factor VIII in the management of patients with factor VIII inhibitors

Increased detection thresholds for pure tones were observed in a large cohort of children exposed to lead environmentally while smaller studies in lead-exposed workers have reported conflicting results on assessments of auditory function. Pure tone detection thresholds were determined in a group of monkeys (Macaca fascicularis) dosed with 2 mg/kg/day of lead from birth through testing at 13 years of age. Blood lead concentrations were stable at about 30 micrograms/dl until monkeys were 10-11 years of age at which time they increased to between 50 and 70 micrograms/dl. Five age- and rearing-matched monkeys served as controls. Detection thresholds were determined at six frequencies between 0.125 and 31.5 kHz. Ear phones were fit over both ears, and thresholds were determined for each ear separately. The monkey signaled detection of the tone by breaking contact with a stainless steel bar. Three lead-exposed monkeys exhibited normal pure tone detection functions. Three monkeys had thresholds outside of the control range at some frequencies; there was a tendency for higher frequencies to be differentially more affected. These findings are consistent with reports of elevated pure tone thresholds in humans exposed to lead developmentally, although the effect is smaller than might have been predicted given the concurrent blood lead concentrations of these monkeys.     

Factor VIII inhibitors in patients with hemophilia A: epidemiology of inhibitor development and induction of immune tolerance for factor VIII

Factor (F) VIII inhibitor development remains one of the most serious complications in the treatment of hemophilia A. Former and recent studies on inhibitor development revealed that patients with severe hemophilia A and positive inhibitor family history are at highest risk of developing an inhibitor. Comparison of recent inhibitor incidence studies on previously untreated patients indicate that the risk of inhibitor development under treatment with recombinant FVIII concentrates is comparable to the inhibitor incidence under FVIII substitution by plasma-derived concentrates. However, longer observation periods are necessary to draw final conclusions. Since inhibitor development may result in inefficacy of FVIII concentrates in the treatment of severe bleedings, the induction of immune tolerance (IT) is still of main concern. Various regimens to induce IT by application of FVIII concentrates have been conducted up to now. Success rate appears to be influenced by low to high responder status, number of exposure days before onset of treatment, and dosage of therapeutic regimen. Especially, discontinuation of IT therapy seems to be associated with failure of therapy. Taking into account available data on IT therapy, we recommend early onset of a high dosage regimen in high responder patients as soon as possible after inhibitor detection, as this is associated with higher success rate and shorter elimination time.     

A human alloantibody interferes with binding of factor IXa to the factor VIII light chain

Inhibitory antibodies directed against factor VIII develop in a substantial number of patients with hemophilia A as a consequence of factor VIII replacement therapy. These antibodies usually recognize discrete epitopes within the A2 and/or the C2 domains of factor VIII. Here, we have characterized the antibodies present in the plasma of a patient affected by severe hemophilia A. The antibodies reacted readily with the metabolically labeled factor VIII light chain and fragments thereof when analyzed by immunoprecipitation. The inhibitory activity could be neutralized by the complete light chain, whereas only slight neutralization occurred with a fragment comprising the isolated C2 domain. Binding of the majority of antibodies to in vitro synthesized factor VIII fragments was dependent on the presence of amino acid residues Gln1778-Met1823, a region known to contain a factor IXa binding site. Functional characterization showed that purified IgG from the patient's serum inhibited binding of factor IXa to immobilized factor VIII light chain in a dose-dependent manner. These data indicate that human alloantibodies may inhibit factor VIII activity by interfering with factor IXa-factor VIIIa complex assembly.     

Combined hereditary deficiency of factors VII and VIII: a distinct coagulation disorder due to the 'lack' of an autosomal gene controlling factor VII and VIII activation?

A patient with a combined hereditary deficiency of factors VII and VIII is presented together with a family study. The main bleeding manifestations were easy bruising and bleeding after tooth extractions. No hemarthrosis was ever observed. The main laboratory features consisted in a mild prolongation of prothrombin time and of partial thromboplastin time. TG test was abnormal and was corrected by the addition of adsorbed normal plasma. Specific assays revealed a moderate defect of factors VII and VIII. All other clotting factors were within normal limits. The factor VII antigen in the propositus was normal or nearly normal. The factor-VIII-associated antigen was also normal. Five additional family members presented the same coagulation pattern and were variably symptomatic. The hereditary transmission pattern seems to be autosomal dominant. The defect appears to be due to a structural abnormality of a gene controlling factors VII and VIII activation.     

Variant of intron 22 inversions in the factor VIII gene in severe hemophilia A

Recurrent DNA inversions, which disrupt the factor VIII (FVIII) gene, generally occur between a region of intron 22 (int22h) and one of two homologous copies of this region, located 300 to 400 kb telomeric to the FVIII gene. This report describes a patient with severe hemophilia A and a high level inhibitor with atypical hybridization patterns. A Bcl I Southern blot assay was altered to 17.5, 16, and 14 kb. His mother and two out of four aunts tested had normal and abnormal restriction patterns which led to a total of five different fragments, suggesting that they were carriers. The Xba I plus Kpn I restriction fragment-length polymorphism in intron 22 by Southern blotting using the same probe (probe a) yielded the 6.2 kb polymorphic band, with a clearly separated 6.6 kb band from the non-factor VIII region; an alternative int22h hybridization probe (probe x) detected no additional fragment. These results suggest that probe a as well as probe x could recognize an intron-22-sized fragment. This report shows a variation in the number of int22h copies although we could not find the inversion junction.     

Transcriptional activation of the factor VIII gene in liver cell lines by interleukin-6

PURPOSE: The pathogenesis of thrombocytopenia in patients with thrombocytopenia with absent radii (TAR) syndrome has not been clarified yet. PATIENTS AND METHODS: This is the first report of a Japanese patient with TAR syndrome. We studied his megakaryopoiesis in vitro and serum levels of thrombopoietin (TPO). RESULTS: Serum levels of TPO in the patient with TAR syndrome were comparable with those of an age-matched control. The bone marrow cells from the patient with TAR syndrome actually generated megakaryocyte colonies in the presence of TPO and the numbers were significantly greater than those from the age-matched control marrow. However, megakaryocyte colonies from the marrow cells with TAR syndrome contained a much lower number of cells per colony and the size of the individual megakaryocytes appeared to be smaller. CONCLUSION: These data suggest that megakaryocyte progenitors from patients with TAR syndrome may have decreased proliferative and differentiative capacity to respond to TPO, leading to thrombocytopenia.     

Localised sequence regions possessing high melting temperatures prevent the amplification of a DNA mimic in competitive PCR

The polymerase chain reaction is an immensely powerful technique for identification and detection purposes. Increasingly, competitive PCR is being used as the basis for quantification. However, sequence length, melting temperature and primary sequence have all been shown to influence the efficiency of amplification in PCR systems and may therefore compromise the required equivalent co-amplification of target and mimic in competitive PCR. The work discussed here not only illustrates the need to balance length and melting temperature when designing a competitive PCR assay, but also emphasises the importance of careful examination of sequences for GC-rich domains and other sequences giving rise to stable secondary structures which could reduce the efficiency of amplification by serving as pause or termination sites. We present data confirming that under particular circumstances such localised sequence, high melting temperature regions can act as permanent termination sites, and offer an explanation for the severity of this effect which results in prevention of amplification of a DNA mimic in competitive PCR. It is also demonstrated that when Taq DNA polymerase is used in the presence of betaine or a proof reading enzyme, the effect may be reduced or eliminated.     

Efficacy of prothrombin-complex concentrates in hemophiliacs with antibodies to factor VIII: a multicenter therapeutic trial

The therapeutic efficacy of prothrombin-complex concentrates in patients with hemophilia and inhibitors (antibodies) to factor VIII has been increasingly debated. We therefore entered 51 hemophiliacs with factor VIII inhibitors into a double-blind randomized crossover study to compare two commercial prothrombin-complex concentrates (Konyne and Proplex) and an albumin placebo. Acute hemarthrosis of the elbow, knee, or ankle was treated with a single dose of a test preparation and assessed six hours later with objective and subjective criteria. In all measurements the concentrates were significantly more effective than the placebo. The data indicate that although prothrombin-complex concentrates, when used in a single dose, are only partially effective in the treatment of joint hemorrhage in hemophiliacs with inhibitors, their continued use for acute hemarthrosis is justified in the absence of any other effective and readily available therapy for this disorder.     

Apoptosis-prone phenotype of human colon carcinoma cells with a high level amplification of the c-myc gene

Although apoptosis can be induced by the enforced expression of exogenously introduced c-myc genes, it is not clear whether overexpression resulting from the amplification of the resident c-myc gene in tumor cells is sufficient to induce apoptosis. We have investigated the relationship between c-myc gene amplification and the propensity of tumor cells to undergo apoptosis, using the SW613-12A1 and SW613-B3 cell lines, which are representatives, respectively, of tumorigenic and non-tumorigenic clones isolated from the SW613-S human colon carcinoma cell line. Tumorigenic clones are characterized by a high level of amplification and expression of the c-myc gene, whereas cells of non-tumorigenic clones have a small number of copies and a lower level of expression of this gene. Analysis of c-myc mRNA level in cells cultured under low serum conditions indicated that the expression of the gene is tightly regulated by serum growth factors in non-tumorigenic B3 cells, whereas it is poorly regulated in tumorigenic 12A1 cells, the level of mRNAs remaining relatively high in serum-starved 12A1 cells. Under these conditions, 12A1 cells showed clear evidence of apoptosis, whereas B3 cells were completely refractory to the induction of apoptosis. Moreover, the study of cell lines derived from non-tumorigenic apoptosis-resistant clones following the introduction by transfection of exogenous c-myc gene copies showed that they have acquired an apoptosisprone phenotype. Altogether, our results strongly suggest that deregulated c-myc expression due to high-level amplification confers an apoptosis-prone phenotype to tumor cells. The possible consequences of these observations for cancer therapy are discussed.     

Analysis of factor VIII gene polymorphisms in Brazilian blacks reveals further differences in the black population

We report here the analysis of four intragenic (BclI, HindIII, XbaI, and BglI) and two extragenic (MspI and TaqI/St14) polymorphisms associated with the factor VIII gene in the Brazilian Black population. No difference was observed for the allelic frequencies of the BclI, HindIII, and BglI polymorphisms when we compared our results with those reported for North American Blacks. For the first time, the XbaI and MspI polymorphisms were investigated in a Black population. Interestingly, the XbaI polymorphism is almost monomorphic for the Brazilian Black population, differing in this respect from all other populations studied thus far. The MspI polymorphism presents inverse allelic frequencies for the Brazilian Blacks when compared with Caucasians, as observed for the BclI and HindIII polymorphisms. Analysis of the TaqI/St14 multiallelic system revealed five novel alleles (ranging from 5.8 to 11.0 kb) which may represent unique alleles for the Black population. The results show that analysis of factor VIII gene polymorphisms and haplotypes may be successfully used for investigating interrelationships between human populations. The results also allow definition of a strategy for carrier detection and prenatal diagnosis of hemophilia A in this population based on analysis of factor VIII gene polymorphisms, which is considered an alternative approach for those families where identification of gene mutation is not feasible.     

A multiplex RT-PCR assay for analysis of relative transcript levels of different members of multigene families: application to Arabidopsis calmodulin gene family

In systemic amyloidosis, widespread amyloid deposition interferes with organ function, frequently with fatal consequences. Diagnosis rests on demonstrating amyloid deposits in the tissues, traditionally with histology although scintigraphic imaging with radiolabeled serum amyloid P component (SAP) has lately been developed as a specific noninvasive alternative. We report a detailed analysis of the abnormal turnover of SAP in patients with systemic amyloidosis and an assessment of its clinical value. METHODS: Iodine-123-labeled human SAP (200 MBq) SAP was injected intravenously into 49 patients with histologically proven systemic AA- or AL- amyloidosis and in 7 control subjects. Plasma clearance and whole-body retention of labeled SAP were analyzed over 48 hr using plasma sampling, whole-body gamma camera imaging and measurement of radioactivity in the urine. The rate of SAP synthesis and interstitial exchange were determined, and the size of the amyloid compartment was compared with clinical estimates of whole-body amyloid load and patient survival. RESULTS: All plasma time-activity curves were biphasic. In comparison with control subjects, patients with amyloidosis showed significantly faster plasma disappearance [4-hr value: AA 48% +/- 18%, AL 45% +/- 15% versus 65% +/- 8% (p < 0.05)], higher total-body retention 48 hr p.i. [AA 74% +/- 14%, AL 73% +/- 17% versus 46% +/- 15% (p < 0.01)] and especially higher extravascular retention 48 hr p.i. [AA 59% +/- 16%, AL 58% +/- 19% versus 30% +/- 14% (p < 0.01)]. Extravascular retention correlated with clinical estimation of the amyloid load. If extravascular retention values in patients with AL amyloidosis were over 60%, survival was decreased (median 4 versus 23 mo, p < 0.001). Markedly increased interstitial exchange rates were present in amyloidosis (AA 64 +/- 61, AL 50 +/- 37 versus 18 +/- 8 mg/hr), whereas the SAP synthesis rate did not differ from the control values (AA 5.0 +/- 3.0, AL 5.5 +/- 3.2 versus 4.5 +/- 1.4 mg/hr). CONCLUSION: The presence of systemic amyloidosis is characterized by accelerated initial clearance of 123I-SAP from the plasma and increased interstitial exchange rate and extravascular retention. These findings reflect reversible binding of radiolabeled SAP to amyloid deposits and provide clinically useful information for diagnosis, monitoring of therapy and prognosis in patients with systemic amyloidosis.     

Genotype/phenotype correlations for coagulation factor XIII: specific normal polymorphisms are associated with high or low factor XIII specific activity

Factor XIII is a transglutaminase essential for normal hemostasis. We have studied the plasma FXIII levels and FXIII activity in 71 individuals and found these to be normally distributed. FXIII specific activity is also normally distributed. However, we show that FXIII activity is not directly dependent on FXIII levels, and individuals with low FXIII levels may have high FXIII activity and vice versa. We have determined the FXIIIA genotype in these individuals to assess whether the variation observed in FXIII specific activity is dependent on specific polymorphisms in the FXIIIA gene. Our data show that the Leu34 and Leu564 variants give rise to increased FXIII specific activity, while the Phe204 variant results in lower FXIII specific activity. We also report preliminary evidence that the Phe204 polymorphism may be associated with recurrent miscarriage. Overall, we have identified 23 unique FXIIIA genotypes. Certain specific FXIIIA genotypes consistently give rise to high, low, or median FXIII specific activity levels, while others appear to have little or no consistent influence on the FXIII phenotype. These genotype to phenotype relationships are discussed in light of the growing interest in the role of FXIII in clinical problems involving an increased thrombotic tendency.     

某高碳低硫石英脉型金矿石浮选试验研究

针对新疆某高碳低硫石英脉型金矿石性质,进行了浮选试验研究。通过考察磨矿细度及碳酸钠、硫酸铜、异戊基黄药、酯-205用量等因素对浮选指标的影响,确定了最佳药剂制度:磨矿细度-0.074 mm占70%,碳酸钠2 000 g/t、硫酸铜200 g/t、异戊基黄药230 g/t、酯-205 150 g/t。在此最佳条件下,浮选闭路试验获得了金精矿金品位130.14 g/t、金回收率90.93%的较好指标。     

Prostate carcinoma associated spontaneous factor VIII:C inhibitor hemophilia. Successful therapy of severe hemorrhagic complication with porcine factor VIII in a 75-year-old patient

We have employed fluorescence in situ hybridization (FISH) in combination with standard morphology (MGG/FISH) to identify the clonal involvement of different bone marrow cell lineages in 20 AML patients (14 MDS-AML, 6 de novo AML). Even though the number of cells belonging to the abnormal clone varied between individual cases, the percentage of clonal blasts was similar in MDS-AML and de novo AML patients. The erythropoietic cells appeared to be part of the abnormal clone in 13 of 14 patients with MDS-AML, but only in 1 of 6 with de novo AML. Similarly, clonal granulocytes were detected in 13 of 14 patients with MDS-AML, compared to 2 of 6 with de novo AML. Lymphocytes consistently displayed normal, diploid karyotype. The results suggest that it is possible to distinguish between MDS-AML and de novo AML by the use of MGG/FISH; in de novo AML the abnormal chromosomal clone is generally confined to the immature myeloid cells, while in MDS-AML mature granulocytes and erythroid cells are of clonal origin. It is, however, not possible to conclude that MDS-AML is a "multipotent" type of leukaemia, since it cannot be ruled out that the chromosomally aberrant erythroid cells and granulocytes represent surviving cells from the original MDS clone.     

Rapid methods for the analysis of immunoglobulin gene hypermutation: application to transgenic and gene targeted mice

Hypermutation of immunoglobulin genes is a key process in antibody diversification. Little is known about the mechanism, but the availability of rapid facile assays for monitoring immunoglobulin hypermutation would greatly aid the development of culture systems for hypermutating B cells as well as the screening for individuals deficient in the process. Here we describe two such assays. The first exploits the non-randomness of hypermutation. The existence of a mutational hotspot in the Ser31 codon of a transgenic immunoglobulin V gene allowed us to use PCR to detect transgene hypermutation and identify cell populations in which this mutation had occurred. For animals that do not carry immunoglobulin transgenes, we exploited the fact that hypermutation extends into the region flanking the 3'-side of the rearranged J segments. We show that PCR amplification of the 3'-flank of VDJH rearrangements that involve members of the abundantly-used VHJ558 family provides a large database of mutations where the germline counterpart is unequivocally known. This assay was particularly useful for analysing endogenous immunoglobulin gene hypermutation in several mouse strains. As a rapid assay for monitoring mutation in the JH flanking region, we show that one can exploit the fact that, following denaturation/renaturation, the PCR amplified JH flanking region DNA from germinal centre B cells yields mismatched heteroduplexes which can be quantified in a filter binding assay using the bacterial mismatch repair protein MutS -Wagner et al. (1995) Nucleic Acids Res. 23, 3944-3948-. Such assays enabled us, by example, to show that antibody hypermutation proceeds in the absence of the p53 tumour suppressor gene product.     

The estimation of the number of binding sites for antibody on antigen molecules with reference to factor VIII and its antibody

The theoretical basis for determining the number of antibody sites on antigen molecules is examined. The theoretical considerations are applied to factor VIII molecules. Examples based on data available at the Oxford Haemophilia Centre are calculated to illustrate the approach. It is concluded that there are few sites on each factor VIII molecule for human antibody. The three antibodies for which reasonable data were available suggest 1-3 sites for human antibody. The data for rabbit antibody suggest 5-6 sites per factor VIII molecule.