Genetic diagnosis of haemophilia and other inherited bleeding disorders

Summary.  Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500.000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders.     

Recessively inherited coagulation disorders

Deficiencies of coagulation factors other than factor VIII and factor IX that cause bleeding disorders are inherited as autosomal recessive traits and are rare, with prevalences in the general population varying between 1 in 500 000 and 1 in 2 million for the homozygous forms. As a consequence of the rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects, and the actual management of bleeding episodes are not as well established as for hemophilia A and B. We investigated more than 1000 patients with recessively inherited coagulation disorders from Italy and Iran, a country with a high rate of recessive diseases due to the custom of consanguineous marriages. Based upon this experience, this article reviews the genetic basis, prevalent clinical manifestations, and management of these disorders. The steps and actions necessary to improve the condition of these often neglected patients are outlined.     

Extrahepatic portal vein thrombosis in children and adolescents: Influence of genetic thrombophilic disorders

AIM:To explore the prevalence of local and genetic thrombophilic disorders as risk factors for portal vein thrombosis (PVT) in our series,the largest ever published in pediatric literature. METHODS:We conducted a case-control study enrolling 31 children with PVT and 26 age-matched controls. All were screened for thrombophilia,including genetic disorders,protein C,protein S and homocysteine deficiencies. All coagulation parameters were studied at least 3 mo after the diagnosis of portal vein obstruction.RESULTS:In our study we showed that most pediatric patients with PVT have local prothrombotic risk factors,which are probably the most important factors leading to PVT. However,there is a clear association between the presence of prothrombotic disorders and PVT,suggesting that these increase the risk of thrombosis in patients with local factors such as perinatal umbilical vein catheterization or sepsis. CONCLUSION:Patients with PVT should be screened for inherited prothrombotic disorders regardless of a history of an obvious local risk factor.     

Rare coagulation factor deficiencies: a countrywide screening data from India

As compared to haemophilia, although the clinical features and the management strategies for rare coagulation factor deficiencies are discussed, little is known about them. This study was undertaken to assess the distribution, clinical presentation and treatment of patients with rare coagulation factor deficiency disorders in a cross‐sectional population of India. Blood samples and other clinical details from patients suspected of rare coagulation factor deficiencies were collected by the Haemophilia Treatment Centers across India and were diagnosed at National Institute of Immunohaematology, Mumbai. A total of 321 cases of rare clotting factor deficiencies were diagnosed, of which 88% were severe, 10% moderate and 2% mild. Commonest deficiency encountered was factor XIII (FXIII) (30%) followed by FX (15.6%), FVII (15%), fibrinogen (12.1%), FXI (9%), combined V and VIII deficiency (5.6%) and congenital multiple vitamin K‐dependent coagulation factor deficiency (MCFD, 2.1%). Major representation of these deficiencies was from Southern and Western India (82%). Mucocutaneous bleeding was the commonest clinical presentation (59%); intracranial (IC) haemorrhage was seen in 18% of the patients; menorrhagia was an important clinical pointer in women in the reproductive age group (78%); 8% of the severe cases had no history of bleeding and 73% of the FXIII deficiency cases had umbilical stump bleeding. The major therapeutic products used was fresh frozen plasma (64%), cryoprecipitate (15%), whole blood (15%), antifibrinolytics (5%) and recombinant FVIIa (1%). A distinct pattern in the distribution of rare clotting factor deficiencies was observed which was based on multiple factors that include ethnicity and the available diagnostic facilities in different regions of this vast country.     

Rare coagulation deficiencies

Deficiencies of coagulation factors (other than factor VIII and factor IX) that cause a bleeding disorder are inherited as autosomal recessive traits and are generally rare, with prevalences in the general population varying between 1 : 500 000 and 1 : 2 000 000. In the last few years, the number of patients with recessively transmitted coagulation deficiencies has increased in European countries with a high rate of immigration of Islamic populations, because in these populations, consanguineous marriages are frequent. Owing to the relative rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects and the actual management of bleeding episodes are not as well established as for haemophilia A and B. This article reviews these disorders in terms of their clinical manifestations and characterization of the molecular defects involved. The general principles of management are also discussed.Keywords: afibrinogenaemia, autosomal recessive disorders, factor VIII, factor XI, factor XIII.     

Rare bleeding disorders

Summary.  Deficiencies of coagulation factors other than factor VIII and factor IX (afibrinogenemia, FII, FV, FV+FVIII, FVII, FX, FXI, FXIII) that cause bleeding disorders (RBDs) are inherited as autosomal recessive traits and are rare, with prevalences in the general population varying between 1 in 500.000 and 1 in 2 million for the homozygous forms. As a consequence of the rarity of these deficiencies, the type and severity of bleeding symptoms, the underlying molecular defects, and the actual management of bleeding episodes are not as well established as for hemophilia A and B. The study of the genetic basis of these disorders could represent an important tool for prevention through prenatal diagnosis. Treatment of patients with RBDs during bleeding episodes or surgery is a challenge because of the lack of experience and the paucity of data. For some deficiency factor concentrates are still non available and severe complications can occur. These complications can be minimized by assessment of risks of bleeding and thrombosis, use of haemostatic means other than blood components or no therapy at all. The RBDs pose a problem for guideline writers because there are no suitable clinical trials to supply good evidence for how these people are best treated. The lack of adequate information on clinical manifestations, treatment and genetic basis of RBDs could be improved by the collection of data in an International Database ( http://www.rbdd.org ), linkable to others previously published. This could be a useful tool to fill the gap between clinical data and clinical practice. This article reviews the genetic basis of RBDs, problems and complications of treatment, problems in the preparation of suitable guidelines for treatment and the future perspectives of the International Registry on RBDs.     

Congenital combined deficiency of coagulation factors VII and X – different genetic mechanisms

Combined coagulation factor VII (FVII) and factor X (FX) deficiency (combined FVII/FX deficiency) belongs to the group of bleeding disorders in which both factors show reduced plasma activity. It may arise from coincidental inheritance of separate coagulation factor deficiencies or a common cause as large deletions comprising both gene loci. The F7 and F10 genes are located on the long arm of chromosome 13. Here, we describe 10 cases with combined FVII/FX deficiency representing both genetic mechanisms of occurrence. Genetic analyses included direct sequencing of the F7 and F10 genes and MLPA (multiplex ligation‐dependent probe amplification) for detection of heterozygous large deletions. In four patients, the combined deficiency was due to a large deletion within the terminal end of chromosome 13. In the remaining six cases the deficiency resulted from coincidental inheritance of different genetic alterations affecting both genes independently. In most cases, the genetic defects were heterozygous, presenting with prolonged PT, normal aPTT and mild or no bleeding symptoms. Only in one case compound heterozygous mutations were detected in the F10, resulting in prolonged aPTT and a more severe bleeding phenotype. To avoid a misdiagnosis of combined FVII/FX deficiency, analyses of single factor activities have to be performed in all cases with prolonged PT even if aPTT is normal. Genetic analyses are substantial for correct prediction of an inheritance pattern and a proper genetic counselling.     

Advances in the treatment of inherited coagulation disorders

Inherited coagulation disorders constitute a broad spectrum of coagulation factor deficiencies that include X‐linked factor (F)VIII or FIX deficiency that causes haemophilia, and autosomal recessive disorders producing heterogeneous deficiencies in fibrinogen (FI), prothrombin (FII), FV, FVII, FX, FXI, FXIII and combined FV+FVIII. Significant advances in treatments for patients with congenital haemophilia A (FVIII deficiency) and B (FIX deficiency) over the last two decades have resulted from improvements in the production, availability and patient access to factor replacement products. Translation of advances in biotechnology, namely recombinant protein technology, targeted protein modifications to improve function and potentially reduce immunogenicity, and advanced formulations to optimize bioavailability and sustain activity offer promisingly new treatments for haemophilia as well as recessively inherited bleeding disorders in patients who otherwise have few therapeutic options. Though a theoretical risk remains for blood‐borne viral infections with pooled plasma‐derived products, this concern has diminished with breakthroughs in purification and viral inactivation methods. Development of inhibitory antibodies is still the most daunting problem for patients with inherited bleeding disorders, complicating treatment approaches to control and prevent bleeding, and posing risks for allergic and anaphylactic reactions in susceptible patients. The objectives of this review are to (i) highlight emerging advances in hemostatic therapies that are bioengineered to improve pharmacokinetic properties and bioavailability, sustain functional activity, and possibly eliminate immunogenicity of recombinant factor proteins; and (ii) present an overview of key clinical trials of novel factor products currently in the development pipeline.     

Approaches to investigating common bleeding disorders: an evaluation of North American coagulation laboratory practices

Bleeding disorders commonly result from deficiencies or defects in von Willebrand factor (VWF), platelets, coagulation factors, or fibrinolytic proteins. The primary goal of our study was to assess current North American coagulation laboratory practices for diagnosing bleeding disorders, using an on-line patterns-of-practice survey of diagnostic laboratory members of the North American Specialized Coagulation Laboratory Association. The survey examined laboratory approaches to evaluating bleeding disorders, with specific questions about the tests and test panels offered and compliance to recent guideline recommendations on diagnosing von Willebrand disease (VWD) and platelet function disorders. All laboratories responding to the survey performed a prothrombin time/international normalized ratio, an activated partial thromboplastin time, and coagulation factor assays, and many tested for VWD and platelet disorders. However, few laboratories had test panels that evaluated the more common bleeding disorders and few performed some assays, including VWF multimer assessments and assays for fibrinolytic disorders. Additionally, the cutoffs used by laboratories to diagnose type 1 VWD varied considerably, with only a minority following the National Heart Lung Blood Institute recommendations. In contrast, laboratories that tested for platelet function disorders mostly complied with aggregation testing recommendations, as published in the recent North American guidelines. Our results indicate that there are some gaps in the strategies used by laboratories to diagnose bleeding disorders that might be addressed by development of further guidelines and test algorithms that emphasize evaluations for common bleeding disorders. Laboratories may also benefit from guidelines on test interpretation, and external evaluation of their bleeding disorder testing strategies.     

Thrombophilia and outcome in severe infection and sepsis

Thrombophilia is associated with a prohemostatic state and consequently with an increased tendency to develop thrombosis. In severe infection and sepsis, activation of coagulation frequently occurs, which contributes to the development of multiple organ dysfunction. Hypothetically, patients with thrombophilia may suffer from more severe coagulopathy in the presence of severe infection or sepsis, which may result in a more serious clinical course and an unfavorable outcome. This article reviews experimental and clinical evidence regarding such a relationship, with a particular focus on deficiencies of natural anticoagulant proteins (protein C and antithrombin), the factor V Leiden mutation, and genetic variation in the fibrinolytic system.     

Congenital thrombotic disorders

The investigation of kindreds with recurrent thrombotic disease has advanced the understanding of the mechanisms of coagulation and fibrinolysis. In those cases where an etiology has been established, congenital thrombotic disorders are associated either with deficiencies or qualitative abnormalities in inhibitors of activated coagulation factors, qualitative abnormalities of fibrinogen, fibrinolytic defects that impair clot lysis, or an inborn error of metabolism, homocystinuria. The etiologies of congenital thrombotic disorders, their clinical features, and an approach to their laboratory diagnosis are summarized in this review.     

Thrombosis in rare bleeding disorders

Inherited deficiencies of blood coagulation factors are usually associated with lifelong bleeding tendency. In addition to Haemophilias A and B and von Willebrand disease, congenital deficiencies of such factors as fibrinogen, prothrombin (FII)), FV, FVII, FX, FXI, FXIII, and combined deficiencies occur and can lead to a diversity of clinical conditions. Paradoxically, for some of these disorders associated with significant bleeding tendency there are reports of thrombotic events, both arterial and venous. Thrombosis in hemophilia patients has a multifactorial pathogenesis and the main conditions associated with this complication are the use of long-term central venous catheters, intensive replacement therapy usually in the setting of surgical procedures, the use of bypassing agents or the coexistence of acquired or inherited prothrombotic risk factors. Regarding other rare bleeding disorders, thrombotic phenomena has been described particularly in patients with afibrinogenemia, FXI and FVII deficiency and the events can occur even in young patients, in the presence of concomitant risk factors or spontaneously. Replacement therapy must be individualized and should take into account past history of haemostatic challenges, family history of bleeding and thrombosis, just like the level of factor. For mild deficiencies when patients are asymptomatic the use of antithrombotic prophylaxis must be considered with or without concomitant use of replacement therapy. In patients with history of thrombosis it may be helpful to perform a thrombophilia screening to exclude coexisting prothrombotic defects and for all patients it is recommended to control known cardiovascular disease risk factors.     

Is there a ‘Basque’ profile regarding autosomal recessive deficiencies of coagulation factors?*

When excluding haemophilia and von Willebrand disease, coagulation factors deficiencies constitute rare autosomal recessive disorders (<1 in 500,000) of less precisely defined epidemiology. We have reported herein the distribution of these entities in the French Basque Country, a genetic isolate of very old individualization with peculiar biological specificities. The prevalence of these disorders was markedly high, especially, as already shown, factor XI deficiency. This unusual profile needs to be discussed in the view of population genetics.     

Hemostatic complications associated with paraproteinemias

Hemorrhagic events are a common complication at diagnosis, during treatment, and with disease progression of lymphoproliferative disorders that produce monoclonal immunoglobulins or paraproteins. Typical causes of bleeding include vascular infiltration by malignant cells, hyperviscosity syndrome, thrombocytopenia secondary to bone marrow infiltration or treatment, uremia, and coagulopathies secondary to liver dysfunction or sepsis. However, there are several unique hemostatic derangements associated with lymphoproliferative disorders that express paraproteins and these are the subject of this article (eg, inhibition of fibrin polymerization, qualitative platelet dysfunction, acquired von Willebrand factor deficiency, heparin-like circulating anticoagulants, light chain amyloid associated hemostatic disorders, acquired coagulation factor deficiencies, and acquired hypercoagulable states). The exact role that paraproteins play in many of these conditions is unknown or incompletely investigated. Often, abnormal hemostasis test results are not accompanied by clinically apparent hemostatic complications. Management of paraprotein-related hemostatic complications varies in strategy and efficacy, depending on the underlying mechanism and type of lymphoproliferative disease.     

Homocysteine. The cardiovascular risk factor of the next millennium?

Several epidemiologic studies have demonstrated that hyperhomocysteinemia is a risk factor for arteriosclerosis in coronary, cerebral, peripheral and aortic arteries. This risk is independent of other cardiovascular risk factors, and it is dose related. However, prospective studies show contradictory findings. Hyperhomocysteinemia is also associated with a higher risk of venous thrombosis to which other coagulation disorders, such as factor V Leiden, could contribute. Hyperhomocysteinemia can be due to genetic defects in the enzymes that control homocysteine metabolism, and also to other factors, mainly nutritional (deficiencies in vitamin B6, vitamin B12, or folic acid). Dietary supplements of these vitamins reduce plasma homocysteine levels. Randomized clinical trials are still needed to demonstrate that reducing plasma homocysteine levels will reduce the risk for cardiovascular disease.     

Introduction

The recognition of severe coagulation deficiencies of congenital origin is relatively easy through family histories, and clinical bleeding which is usually apparent, although occasionally it is diagnosed even as battered child syndrome. The laboratory abnormalities are usually quite definitive. In contrast, however, adults with even mild congenital or acquired bleeding disorders are much more difficult to diagnose. Like other areas of haematology, like DIC for example, a variety of factors can combine: the setting which can be as diverse as the dentist's office, the gynaecologist's examination room, the surgical amphitheatre or the geriatric ward; an astute clinician and going beyond the routine testing to reveal the abnormality. This symposium focuses on exploring these settings both from a clinical and laboratory standpoint and an approach to both the diagnosis and treatment of these occult bleeding disorders.     

The Use of Factor IX Concentrates in Man: a 9-Year Experience of Scottish Concentrates in the South-East of Scotland

S ummary . During the last 9 years, two factor IX concentrates produced in Scotland, PPSB and DEFIX, have been used for the treatment of haemophilia B and acquired coagulation disorders, including those due to liver disease, coumarin therapy, neonatal immaturity and post-operative bleeding. During the period of study 112 batches of DEFIX and 40 batches of PPSB were used in the Edinburgh and South-East Scotland Region. Data were analysed from 575 non-haemophilic patients, receiving 968 treatment episodes, as well as 24 haemophiliacs. Serial coagulation studies and analysis of retrospective data showed that both concentrates corrected the coagulation deficiencies in all the above patient groups; in no case was there any evidence of intravascular coagulation resulting from concentrate infusion.     

Clinical and prognostic role of plasma coagulation factor XIII activity for bleeding disorders and 6-year survival in patients with chronic liver disease.

BACKGROUND/AIMS: Alterations of plasma coagulation factor XIII may contribute to bleeding disorders in patients with liver cirrhosis. As standard clotting tests such as prothrombin time or activated thromboplastin time (aPTT) cannot detect factor XIII deficiency, this may often be overlooked in clinical practice. We aimed to define factor XIII's clinical and prognostic role in chronic liver disease. PATIENTS AND METHODS: Factor XIII activities were assessed among various other parameters in 111 patients with chronic liver diseases during evaluation for liver transplantation in a prospective study. RESULTS: Unlike coagulation factors II, V or VII, factor XIII activity was maintained in the majority of patients with liver cirrhosis. However, although rarely, factor XIII deficiencies (<50%) occurred, especially in Child C cirrhosis. Factor XIII levels correlated with liver's biosynthetic capacity (cholinesterase activity, albumin, total protein) as well as with platelet count, global coagulation tests and other single coagulation factors. Patients reporting a current systemic bleeding tendency at study entry had significantly reduced factor XIII. In a 6-year follow-up, patients with factor XIII<50% had a significantly increased risk of severe upper gastrointestinal bleed, and reduced factor XIII (<50%, 50-75% vs. normal) was associated with increased mortality. CONCLUSIONS: Factor XIII deficiency is rare in patients with liver cirrhosis, but is associated with a clinical bleeding tendency and an unfavorable prognosis for future hemorrhages and survival.     

Basic mechanisms of hemostasis

Hemostasis governs two essential processes of human life in that it maintains the fluidity of blood under physiological conditions and prevents excessive blood loss after injury. Hemostasis is regulated by components of the vessel wall and blood cells and by humoral coagulation factors. Under normal conditions, these components are involved in an active equilibrium through activation, propagation, and termination of the hemostatic pathways. This equilibrium is disturbed upon vascular injury, leading to a procoagulant response when needed. Unfortunately, pathological disturbances can occur as a result of inherited or acquired coagulation factor deficiencies that may lead to bleeding or thrombotic disorders.     

Combined FV and FVIII deficiency

Summary. Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders. The severity of these disorders is generally inversely proportional to the degree of factor deficiency. Among all the autosomal recessive rare bleeding disorders, which include afibrinogenaemia, factor (F) II, FV, FV + VIII, FVII, FX, FXI, FXIII, the combined deficiency of coagulation FV and FVIII (F5F8D or FV + FVIII) is exceptional because it is due to mutations in genes encoding proteins involved in the FV and FVIII intracellular transport (LMAN1 and MCFD2) rather than DNA defects in the genes that encode the corresponding coagulation factors. F5F8D is estimated to be extremely rare (1:1.000.000) in the general population, but an increased frequency is observed in regions where consanguineous marriages is practiced. F5F8D is characterized by concomitantly low levels (usually between 5% and 20%) of both FV and FVIII, and is associated with a mild to moderate bleeding tendency. Treatment of bleeding episodes requires a source of both FV and FVIII; replacement of FV is achieved through the use of fresh frozen plasma, and that of FVIII by desmopressin or specific FVIII concentrates, plasma‐derived or recombinant FVIII products. We focus here on the clinical, molecular, treatment‐related and diagnostic features of F5F8D.