Synergy between transcription factors DBP and C/EBP compensates for a haemophilia B Leyden factor IX mutation.

Haemophilia B Leyden is characterized by low childhood levels of factor IX which gradually increase after puberty, eventually resulting in a return to health. The disease is the result of single nucleotide substitutions within a 40 bp region encompassing the major transcriptional start site. We have characterized transcription factor binding sites within the factor IX promoter. Five sites were identified and a Leyden mutation at nucleotide -5 was shown to interfere with the binding of proteins to one of three newly identified sites. The correlation between the post-pubertal recovery of these mutants and the induction of the transcription factor DBP led us to the discovery of a synergistic interaction between DBP and C/EBP responsible for the recovery of normal transcriptional activity of the -5 mutant promoter and may play a role in the resolution of other Leyden mutants.

Characterization of the original Christmas disease mutation (cysteine 206----serine): from clinical recognition to molecular pathogenesis.

Christmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952. The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals. This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

Molecular diagnosis of the fragile X [Fra (X)] syndrome: calculation of risks based on flanking DNA markers in small phase-unknown families.

We studied two small, two-generation families with the fragile X [Fra (X)] syndrome. The absolute phase of the DNA markers in relation to the disease in the mother was not known in either family. We present the derivation of risks for these families using flanking markers, taking into account the uncertainty regarding maternal phase. Since the use of flanking markers fails to yield useful counseling data in the 1/3 to 1/7 of all cases where a single recombination event occurs between the two flanking markers, we calculate the probability that this method is likely to be successful or unsuccessful when prenatal diagnosis is attempted using linked RFLPs.

Carrier detection and prenatal diagnosis of Pelizaeus-Merzbacher disease using a combination of anonymous DNA polymorphisms and the proteolipid protein (PLP) gene cDNA.

We report carrier identification and a prenatal diagnosis using DNA polymorphisms in 2 families with X-linked Pelizaeus-Merzbacher disease (PMD). In both families, the proteolipid protein (PLP) gene in the single affected male could be traced back to his unaffected maternal grandfather. Therefore, each family contains a new mutation. In the case of the prenatal diagnosis, the fetus was shown by cytogenetic analysis to be a female, who we predict will be a noncarrier of PMD based on her genotype with the PLP intragenic polymorphism.

Recombination between the factor VIII gene and the DXS52 locus gives the most probable genetic order as centromere-fra(X)-DXS15-DXS52-F8C-telomere.

The linkage relationship between the factor VIII gene (F8C) and the DXS52 locus was examined in 8 families. Two recombinations were identified in 35 informative meioses (Zmax = 5.67; theta = 0.05), one in a family with hemophilia A, the other in a family with the fra(X) syndrome. Based on the latter recombination, the most probable order of loci was determined to be centromere-fra(X)-DXS15-DXS52-F8C-telomere. When these data are added to those reported previously the most probable genetic distance between F8C and DXS52 is 3 cM (Z = 14.62). Identification of these and other recombinations suggests that the use of DXS52 as a genetic marker for carrier detection and prenatal diagnosis of hemophilia A has an error rate between 3-5%.

Heterogeneity of laboratory test results for antiphospholipid antibodies in patients treated with chlorpromazine and other phenothiazines.

Ninety-seven psychiatric patients who have been treated with the antipsychotic drug chlorpromazine or another phenothiazine have been investigated for the presence of antiphospholipid antibodies. A variety of coagulation studies and specific antiphospholipid immunoassays were performed to define the spectrum of antigen specificity of these antibodies. Coagulation studies showed an increasing sensitivity for the lupus anticoagulant with reagents of differing phospholipid content. Prolonged activated partial thromboplastin times (APTTs) were found in five patients with the use of an insensitive APTT reagent and in 14 patients with a lower phospholipid content reagent. In every case, attempted correction of the clotting time with normal plasma was unsuccessful. Twenty-one patients had abnormal kaolin clotting time profiles. In seven of these patients, test results with both APTT reagents had been normal. Antibody reactivity was tested against three negatively charged phospholipids, phosphatidyl-serine, cardiolipin, and phosphatidylinositol. Only five patients demonstrated reactivity against phosphatidylinositol, whereas high antibody titers were observed in 28 patients against one or both of phosphatidylserine and cardiolipin. Twenty-three of these patients were found to have elevated anticardiolipin-specific IgM antibodies. Overall, 41 of the patients had at least one laboratory abnormality suggestive of antiphospholipid antibody activity. Seven of the 26 patients, taking phenothiazines other than chlorpromazine, had positive test results for antiphospholipid antibodies. No clinical thromboembolic events were recorded in any patient. These findings demonstrate the heterogeneity of antiphospholipid antibody specificity induced in patients treated with various phenothiazine drugs and indicate that none of these patterns of reactivity marks a predisposition for thromboembolism in this population.

Absence of a bleeding tendency in severe acquired von Willebrand's disease. The role of platelet von Willebrand factor in maintaining normal hemostasis.

A 67-year-old male with a prolonged activated partial thromboplastin time (APTT) of 43 seconds (normal, 25-40 seconds) was found to have laboratory features of von Willebrand's disease and IgA myeloma but had a normal bleeding time and no bleeding tendency. Plasma Factor VIII coagulant activity (F.VIII:C) was 80 U/L (0.08 U/mL), Factor VIII antigen (F.VIII:Ag) 70 U/L (0.07 U/mL), and von Willebrand's factor antigen (vWF:Ag) 50 U/L (0.05 U/mL) and ristocetin cofactor (vWF:RiCoF) 10 U/L (0.10 U/mL). The platelet vWF:Ag level was normal, whereas both platelet lysate and plasma vWF high molecular weight multimers were decreased. Patient plasma had no inhibitory effect on either F.VIII:C or vWF:RiCoF. However, when patient plasma was incubated with normal plasma, crossed immunoelectrophoresis for vWF:Ag demonstrated the presence of immune complexes. Infusion of 1-desamino-8-D-arginine vasopressin led to a transient correction of the plasma vWF:Ag multimer pattern. The survival of all components of vWF/F.VIII was decreased, as also occurred after cryoprecipitate. The levels of plasma F.VIII/vWF increased as the IgA values decreased after chemotherapy, whereas the platelet high molecular weight multimers remained decreased. The data suggest that the plasma vWF/F.VIII deficiency results from complexing of the IgA myeloma protein with vWF, resulting in premature clearance of the vWF/F.VIII complex. The absence of clinical bleeding likely results from the combination of a normal platelet vWF:Ag level and persistence of intermediate molecular weight vWF multimers.

Sensitivity of PCR in detecting monoclonal B cell proliferations.

AIMS: To evaluate the rapid detection of various forms of monoclonal B cell proliferations by using the polymerase chain reaction (PCR) to identify clonal immunoglobulin heavy chain genomic rearrangements. METHODS: Thirty four B cell lymphomas defined by morphology, immunophenotyping, and positive immunoglobulin heavy chain gene rearrangements detected by Southern blot analysis were examined. An additional 22 cases representing miscellaneous lymphoproliferative and non-lymphoproliferative disorders were also studied. RESULTS: Monoclonal rearrangements were identified in 19 (56%) cases of B cell lymphoma. The method was less sensitive in the detection of follicular centre cell lymphomas (15 of 28, or 54%) than non-follicular centre cell lesions (four of six, or 67%). Monoclonal rearrangement was not identified in 19 control cases, including T cell lymphomas, Hodgkin's disease, reactive lymphadenopathy and metastatic carcinoma. Three cases showed positive immunoglobulin gene rearrangement by PCR but were negative on Southern blotting. Two of these cases had definite clinical, morphological, and immunophenotypic evidence of monoclonal B cell proliferation suggesting that PCR could, on occasion, pick up cases missed by Southern blotting and that the two methods are complementary in clonal lymphoproliferative disease diagnosis. The third case represented a "false positive" PCR reaction involving a colonic adenocarcinoma. CONCLUSIONS: PCR analysis, using the primer sequences outlined in this study, will detect about 55% of clonal lymphoproliferative proliferations with increased sensitivity for non-follicular centre cell lesions. With these levels of detection in mind, this testing strategy can still be especially useful in cases which prove diagnostically problematic with standard morphological and immunophenotypic analysis, and in instances where the quantity and type of diagnostic material is limiting (needle aspirates and cellular fluids).

Erythroblast multinuclearity in bone marrow and spleen. Congenital dyserythropoietic anemia-like abnormalities without functional evidence of dyserythropoiesis.

A dyserythropoietic syndrome with coincidental immune thrombocytopenia seen during pregnancy is discussed. The morphological and serological results in this case most resemble type II congenital dyserythropoietic anemia. However, functional evidence of dyserythropoiesis was absent and the patient was not anemic. Splenectomy was performed for the resistant thrombocytopenia and microscopic examination of the spleen showed evidence of extramedullary dyserythropoiesis. The significance of these findings is discussed with regard to the spectrum of recognized dyserythropoietic disorders and the unusual discrepancy between the abnormal morphology and the absence of functional dyserythropoiesis.

Multicentric warfarin-induced skin necrosis complicating heparin-induced thrombocytopenia.

Two patients developed catastrophic multicentric skin necrosis while receiving warfarin to treat venous thromboembolism complicated by immune-mediated heparin-induced thrombocytopenia (HIT). Patient 1 developed skin necrosis involving the breasts, thighs, and face, as well as venous limb gangrene and bilateral hemorrhagic necrosis of the adrenal glands, resulting in death. The second patient developed bilateral mammary necrosis necessitating mastectomies, as well as skin necrosis involving the thigh. Neither patient had an identifiable hypercoagulable syndrome, other than HIT. HIT may represent a risk factor for the development of multicentric warfarin-induced skin necrosis (WISN).

Prolonged thrombocytopenia in post-transfusion purpura (PTP) associated with changes in the crossed immunoelectrophoretic pattern of von Willebrand factor (vWF), circulating immune complexes and endothelial cell cytotoxicity.

A 39-year-old multigravida presented with a platelet count of 2 X 10(9)/112 d after a blood transfusion. The clinical presentation was typical of post-transfusional purpura (PTP). She was successfully managed with intensive plasma exchange with plasma replacement. Replacement with albumin saline was unsuccessful. Elevated levels of immune complexes were detected on presentation but were reduced to normal levels by plasma exchange but were elevated again during relapse. Raised levels of circulating platelet aggregates and platelet-aggregating activity were found during relapse but were absent during remission. Crossed immunoelectrophoresis of vWF showed a left shift suggesting the presence of abnormally high molecular weight multimeric forms. In contrast, multimeric assay of the same samples were within normal limits suggesting that the shift noted on crossed immunoelectrophoresis may have resulted from complexing of vWF with some other components. Immunological studies demonstrated the presence of endothelial cell cytotoxicity and lymphocytotoxicity in the patient's serum. It is concluded that the thrombocytopenia observed in PTP may not occur as a direct result of the immunological event initiating the disorder but rather indirectly via the release of vWF consequent upon endothelial cell damage which, either alone or in combination with another factor, possible immune complexes, induces intravascular platelet aggregation and sequestration. There were significant similarities between the case reported and recent observations in thrombotic thrombocytopenic purpura suggesting that these syndromes may have aetiological factors in common.

Somatic mosaicism and female-to-female transmission in a kindred with hemophilia B (factor IX deficiency).

Studies have shown that hemophilia B (Christmas disease; factor IX deficiency) results from many different mutations in the factor IX gene, of which greater than 95% are single nucleotide substitutions. This study has identified a previously unreported form of hemophilia B in a patient who was a somatic mosaic for a guanine-to-cytosine transversion at nucleotide 31,170 in the factor IX gene. This point mutation changes the codon for residue 350 in the catalytic domain of factor IX from a cysteine to a serine. We used differential termination of primer extension to confirm and measure the degree of mosaicism. Our study shows that a varying proportion of cells from hepatic, renal, smooth muscle, and hematopoietic populations possessed normal as well as mutant factor IX sequences. These results indicate that the mutation in this patient occurred either as an uncorrected half-chromatid mutation in the female gamete or as a replication or postreplication error in the initial mitotic divisions of the zygote preceding implantation. In addition, this kindred also contains two females in successive generations who have moderately severe factor IX deficiency. The molecular pathogenesis of this latter phenomenon has been studied and seems to relate to the unaccompanied expression of the mutant factor IX gene consequent upon a second, as yet undefined, genetic event that has prevented inactivation of sequences including the mutant factor IX gene on the X chromosome inherited from the affected male.

Association of lupus anticoagulant with severe valvular heart disease in systemic lupus erythematosus.

Two cases of systemic lupus erythematosus with hemodynamically significant mitral valve dysfunction and associated lupus anticoagulant are reported. Both patients underwent valve replacement and both had thrombus formation on the mitral valve, one pre- and the other postoperatively. Both patients suffered a number of extracardiac thromboses at different times in the course of their illness. The contribution of the lupus anticoagulant to the thrombotic problems, and its possible relationship to the pathogenesis of Libman-Sacks endocarditis are discussed.

Lymphoproliferative disease of "LAK cell" precursor large granular lymphocytes in association with celiac disease.

We have investigated a case of lymphoproliferative disease of large granular lymphocytes (LDGL) occurring in association with celiac disease, anemia, neutropenia, and carcinomas of the endometrium, breast, and skin. The large granular lymphocyte (LGL) proliferation was monoclonal, T cell in origin, with T cell receptor beta-chain gene rearrangement, and a CD3+, CD8+, CD16+/- phenotype. In spite of the high frequency of LGL, natural killer (NK) cell activity was absent. Stimulation with interleukin-2 in vitro, however, resulted in high lymphokine-activated killer (LAK) cell activity against NK-resistant targets. The T-cell nature of the LAK precursor cells is in contrast to the majority seen in normal peripheral blood. Therapeutic trials of cyclosporin A, low-dose cyclophosphamide, and levamisole were unsuccessful in reducing transfusion requirements. This case is unique in the association of LDGL with celiac disease. It is also unique in that the patient had been followed for several years prior to the onset of the LDGL. The case extends the list of lymphoproliferative disorders documented to be associated with celiac disease and, conversely, adds to our knowledge of lymphoproliferative disorder of LGL and its "dysimmune" manifestations.

What is a cure and how do we get there?

The absence of adequate treatment for most of the world's 400 000 individuals with haemophilia makes the development of a cure compelling. Advances in the basic molecular sciences over the past 20 years have resulted in the feasibility of curing haemophilia through the application of gene therapy. However, the reality of this therapeutic strategy is highly complex. In addition, challenges to achieving a cure exist beyond the basic scientific hurdles. Thoughtful attention must also be given to a number of interrelated issues, including ethical considerations in patient recruitment, informed consent and geographical variables of global clinical trials. The global inequalities in healthcare mean that the ethics of international medical research, especially when it includes countries where people usually do not receive quality care, become much more complicated. The majority of haemophiliacs lives in developing countries and is a valuable resource of human subjects who could be enrolled in clinical trials. When recruiting subjects globally, investigators must be ever mindful that the patient population is a precious resource, which must be treated with respect and care. This presents a major challenge for investigators engaged in trials of haemophilia gene therapy to ensure that the informed consent process is current and comprehensive, that therapeutic misconceptions are appropriately managed, and that the roles of the researcher and physician are clear. Global clinical gene-therapy trials are an important and appropriate component in the quest to achieve a cure for haemophilia. When trials follow identical internationally accepted standards, a successful outcome can be achieved for trials including developing countries, if country specific cultural and economic aspects are considered.

Inherited thrombocytopenia, elevated serum IgA and renal disease: identification as a variant of the Wiskott-Aldrich syndrome.

A kindred with X-linked hereditary thrombocytopenia in association with elevated serum IgA and a mild nephropathy is described. Thirteen males with thrombocytopenia were identified in three generations amongst 49 family members who were available for screening. Serious infective sequelae were absent but five patients had suffered from severe eczema since infancy. The platelet volume as measured by an automated counter and electron microscopy was reduced compared with normal and in vitro tests demonstrated minor abnormalities of immune function in three patients. The disorder is identified as a novel variant of the Wiskott-Aldrich syndrome and comparisons are made with previously described kindreds showing different patterns of expression.

Risk of venous thromboembolism associated with the common hereditary haemochromatosis Hfe gene (C282Y) mutation.

A high prevalence of a common mutation in the Hfe gene (C282Y) has recently been reported in patients with the factor V Leiden mutation and a history of thrombosis. The aim of this study was to estimate the relative risk of venous thromboembolism in a large case-control study. 56/481 patients (11.6%) and 57/497 controls (11.5%) were heterozygous for the C282Y allele giving an odds ratio of 1.02 (95%CI 0.69-1.51). 12/81 patients with the factor V Leiden mutation were heterozygous for the C282Y allele compared to 1/13 controls, odds ratio 2.09 (95%CI 0.25-17.6). An analysis of a further group of patients and controls selected for the factor V Leiden mutation did not indicate a higher prevalence of the C282Y allele in symptomatic patients, odds ratio 0.17 (95%CI 0.34-0.81). This study does not support the hypothesis that the C282Y allele is an additional risk factor for venous thrombosis in patients with the factor V Leiden mutation.

Carrier detection in the hemophilias.

The subject of carrier detection in the hemophilias has received new impetus in the past several years. Treatment complications arising from clotting factor concentrates have become more evident and earlier prenatal diagnosis and new genetic markers for the clotting factor genes have focused interest on this area. Until now, carrier diagnosis has relied upon standard pedigree analysis and clotting factor assays. The results obtained using these methods are probabilistic, and the coagulation tests are unavoidably influenced by the effects of random X chromosome inactivation and the inherent variability of the methods involved. With the cloning and characterization of both factor IX and factor VIII genes, has come the capability of using gene analysis to diagnose the carrier state. This usually involves the detection of restriction fragment length polymorphisms (RFLPs) and their use as linked markers for the defective clotting factor gene. In hemophilia A, the combined use of three intragenic RFLPs and two closely linked, highly polymorphic extragenic markers will make carrier information available to approximately 90% of kindred. In hemophilia B, phenotypic analysis has been complicated by the more heterogeneous expression of the gene defect. To date, five intragenic and one closely linked RFLP have been reported, as well as two protein polymorphisms detectable by monoclonal antibody immunoassays. With the combined use of these genetic markers it is likely that accurate carrier assignment will be available to more than 80% of hemophilia B families.

Premature stroke in a family with lupus anticoagulant and antiphospholipid antibodies.

Lupus anticoagulant and antiphospholipid antibodies are associated with thromboembolic phenomena in individuals both with and without systemic lupus erythematosus. A 32-year-old woman (the index case) with lupus anticoagulant, multiple cerebrovascular events, and a family history of premature stroke raised the possibility of a familial diathesis. Histories or interviews, examinations, and blood tests were obtained for 23 members of four generations of her family. Four individuals had suffered strokes and three more had suffered neurologic symptoms. Two living individuals who had suffered strokes, two individuals with neurologic symptoms, and five asymptomatic individuals had antiphospholipid activity in their blood. In addition, a cousin of the index case was found to have systemic lupus erythematosus and antiphospholipid activity. Elevated concentrations of von Willebrand factor antigen were found associated with some positive lupus anticoagulant assays, the highest concentrations in the two individuals with stroke. The characteristic presentation of the index case and her good response to treatment suggests that further studies of families in whom antiphospholipid antibodies may represent a risk factor for stroke is worthwhile.