Long-term prophylaxis in severe factor VII deficiency.

INTRODUCTION: The spectrum of bleeding problems in FVII deficiency is highly variable and FVII levels and causative genetic mutations correlate poorly with the bleeding risk. Long-term prophylaxis is generally initiated in order to prevent subsequent CNS bleeding after a first event or in patients with other major/ life threatening/ frequent bleeding symptoms as gastrointestinal bleeding or hemarthrosis. However few data are available in the literature regarding FVII prophylaxis and clinical decisions cannot be based on evidence. AIMS: We report the data available in the literature on FVII prophylaxis and our personal experience regarding three patients affected by severe FVII deficiency. METHODS: Specific papers on long-term prophylaxis in severe FVII deficiency were identified using the database, PUBMED. RESULTS: The most frequent indications for long-term prophylaxis were CNS bleeding (58%), hemartrosis (15%) and GI bleeding (9%). Patients were treated with various dosages and frequency. Prophylactic treatment with 10-30U/kg (pdFVII) or 20-30mcg/kg (rFVIIa) twice or three times/weeks was described to be effective. CONCLUSIONS: In the literature and in our experience, prophylaxis can be considered in patients with severe FVII deficiency and severe bleeding phenotype. A dose of 10-30U/kg (pdFVII) or 20-30 microg/kg (rFVIIa) twice or three times/week is usually administrated, but dose and frequency can be tailored based on the clinical follow-up of the patients. Since hemarthrosis is a frequent manifestation, a suggestion to improve the outcomes of patients with severe FVII deficiency is to monitor joint condition in order to identify early arthropathy that could be another indication to start secondary prophylaxis.

Prenatal diagnosis of haemophilia B: the Italian experience.

This article describes prenatal diagnosis (PND) of haemophilia B (HB) within the framework of Italian haemophilia centres and genetics laboratories. The study details the experience from six haemophilia genetic centres (three in the North, one in the Centre and two in the South of Italy) and summarizes the different techniques used to perform PND of HB during the last 15 years. To date, the Italian HB database includes 373 characterized unrelated patients and their genetic information has permitted the identification of 274 carriers of childbearing age. This database represents the main instrument for timely and precise PND. Sixty-six prenatal diagnoses were performed on 52 HB carriers whose average age at the time was 34 (ranging from 24 to 44 years). In 44 cases, genetic counselling for carrier status determination was performed before pregnancy, while eight were not studied prior to pregnancy. Foetal samples were obtained by chorionic villus sampling in 52 cases, by amniocentesis in 12 while two were diagnosed by analysis of free foetal DNA obtained from maternal peripheral blood. In 35 (53%) pregnancies the foetus was female. For 31 men (47%), haemophilia status was determined by analysis of previously determined informative markers or familial mutations (12 affected and 19 unaffected). There may be more than one laboratory involved in the PND diagnostic pathway (providing DNA extraction, karyotype analysis, gender determination, maternal contamination detection, molecular diagnosis and sequencing). Good communication between all the parties, coordinated by the haemophilia centre, is essential for a successful and rapid process.

Pregnancy complications and obstetric care in women with inherited bleeding disorders.

Women with inherited bleeding disorders (IBD) require the input of a multidisciplinary team to improve outcomes of pregnancy. The role of the haemophilia nurse within the multidisciplinary team is to provide educational and emotional support to the women and to facilitate and co-ordinate patient-centred care. Prenatal diagnosis in cases of haemophilia is an integral part of the management of early pregnancy with a recent drive towards non-invasive prenatal diagnostic techniques. There is a current lack of data on the risk of miscarriage and bleeding complications during pregnancy. A clear association has only been established in women with fibrinogen and factor XIII deficiency. In the affected neonate with severe bleeding disorders such as haemophilia, the risk of head bleeding is significant, and appropriate management of labour and delivery has an important impact on reducing the risk. Women with IBD are at risk of both primary and secondary postpartum haemorrhage. Appropriate risk assessment and advance planning for haemostatic cover can reduce the bleeding risk.

Prenatal diagnosis and preimplantation genetic diagnosis: novel technologies and state of the art of PGD in different regions of the world.

Prenatal diagnosis (PND) aims to provide accurate, rapid results as early in pregnancy as possible. Conventional PND involves sampling cells of foetal origin by chorionic villus sampling at 11-14th weeks of pregnancy or amniocentesis after 15th week. These are invasive procedures and have a small but significant rate of 0.5% to 1% for loss of pregnancy. An alternative to existing methods for conventional PND for couples at risk of transmitting a genetic disease to their child is preimplantation genetic diagnosis (PGD). PGD is a newly emerging form of a very early prenatal diagnosis. The technique combines assisted reproductive technology with molecular genetics and cytogenetics to allow the identification of abnormality in embryos prior to implantation. The diagnosis of genetic disease in human preimplantation embryos was pioneered in the late 1980s for testing of aneuploidy, single gene and X-linked disease, such as cystic fibrosis, haemophilia and chromosomal abnormalities. The PGD-related legal and ethical issues have been debated at many levels both nationally and internationally. The attitude towards PGD varies substantially not only in different parts of the world but also within the Europe, owing to scientific, cultural and religious differences. PGD has become widely practised throughout the world for various indications and can substantially decrease the eventual risks of passing a genetic undesired condition of the offspring. Nevertheless, its extension to some new and non-medical indications has raised ethical concerns, in particular its potential eugenic dimension.

Non-invasive tool for foetal sex determination in early gestational age.

Free foetal DNA in maternal blood during early pregnancy is an ideal source of foetal genetic material for non-invasive prenatal diagnosis. The aim of this study was to evaluate the use of free foetal DNA analysis at early gestational age as pretest for the detection of specific Y-chromosome sequences in maternal plasma of women who are carriers of X-linked disorders, such as haemophilia. Real-time quantitative PCR analysis of maternal plasma was performed for the detection of the SRY or DYS14 sequence. A group of 208 pregnant women, at different gestational periods from 4 to 12 weeks, were tested to identify the optimal period to obtain an adequate amount of foetal DNA for prenatal diagnosis. Foetal gender was determined in 181 pregnant women sampled throughout pregnancy. Pregnancy outcome and foetal gender were confirmed using karyotyping, ultrasonography or after birth. The sensitivity, which was low between 4th and 7th week (mean 73%), increased significantly after 7+1th weeks of gestation (mean 94%). The latter sensitivity after 7+1th week of gestation is associated to a high specificity (100%), with an overall accuracy of 96% for foetal gender determination. This analysis demonstrates that foetal gender determination in maternal plasma is reliable after the 9th week of gestation and it can be used, in association with ultrasonography, for screening to determine the need for chorionic villus sampling for prenatal diagnosis of X-linked disorders, such as haemophilia.

Timing and severity of inhibitor development in recombinant versus plasma-derived factor VIII concentrates: a SIPPET analysis.

Essentials Recombinant factor VIII (rFVIII) was contrasted with plasma-derived FVIII (pdFVIII). In previously untreated patients with hemophilia A, rFVIII led to more inhibitors than pdFVIII. Inhibitors with rFVIII developed earlier, and the peak rate was higher than with pdFVIII. Inhibitors with rFVIII were more severe (higher titre) than with pdFVIII. SUMMARY: Background The development of neutralizing antibodies (inhibitors) against factor VIII (FVIII) is the most severe complication in the early phases of treatment of severe hemophilia A. Recently, a randomized trial, the Survey of Inhibitors in Plasma-Product Exposed Toddlers (SIPPET) demonstrated a 2-fold higher risk of inhibitor development in children treated with recombinant FVIII (rFVIII) products than with plasma-derived FVIII (pdFVIII) during the first 50 exposure days (EDs). Objective/Methods In this post-hoc SIPPET analysis we evaluated the rate of inhibitor incidence over time by every 5 EDs (from 0 to 50 EDs) in patients treated with different classes of FVIII product, made possible by a frequent testing regime. Results The highest rate of inhibitor development occurred in the first 10 EDs, with a large contrast between rFVIII and pdFVIII during the first 5 EDs: hazard ratio 3.14 (95% confidence interval [CI], 1.01-9.74) for all inhibitors and 4.19 (95% CI, 1.18-14.8) for high-titer inhibitors. For patients treated with pdFVIII, the peak of inhibitor development occurred later (6-10 EDs) and lasted for a shorter time. Conclusion These results emphasize the high immunologic vulnerability of patients during the earliest exposure to FVIII concentrates, with the strongest response to recombinant FVIII products.

Prediction of factor VIII inhibitor development in the SIPPET cohort by mutational analysis and factor VIII antigen measurement.

Essentials A residual factor VIII synthesis is likely to be protective towards inhibitor (INH) development. Mutation type-inhibitor risk association was explored in 231 patients with severe hemophilia A. A 2-fold increase in INH development for in silico null vs. non-null mutations was found. A 3.5-fold increase in INH risk for antigen negative vs. antigen positive mutations was found. SUMMARY: Background The type of F8 mutation is the main predictor of inhibitor development in patients with severe hemophilia A. Mutations expected to allow residual synthesis of factor VIII are likely to play a protective role against alloantibody development by inducing immune tolerance. According to the expected full or partial impairment of FVIII synthesis, F8 variants are commonly classified as null and non-null. Objectives To explore the mutation type-inhibitor risk association in a cohort of 231 patients with severe hemophilia A enrolled in the Survey of Inhibitors in Plasma-Product Exposed Toddlers (SIPPET) randomized trial. Methods The genetic defects in these patients, consisting of inversions of intron 22 (n = 110) and intron 1 (n = 6), large deletions (n = 16), and nonsense (n = 38), frameshift (n = 28), missense (n = 19) and splicing (n = 14) variants, of which 34 have been previously unreported, were reclassified according to two additional criteria: the functional effects of missense and splicing alterations as predicted by multiple in silico analyses, and the levels of FVIII antigen in patient plasma. Results A two-fold increase in inhibitor development for in silico null mutations as compared with in silico non-null mutations (hazard ratio [HR] 2.08, 95% confidence interval [CI] 0.84-5.17) and a 3.5-fold increase in inhibitor development for antigen-negative mutations as compared with antigen-positive mutations (HR 3.61, 95% CI 0.89-14.74] were found. Conclusions Our findings confirm an association between the synthesis of minute amounts of FVIII and inhibitor protection, and underline the importance of investigating the residual FVIII antigen levels associated with causative variants in order to understand their clinical relevance.

Advances in the treatment of bleeding disorders.

Historically, the bleeding episodes in subjects with coagulation disorders were treated with substitution therapy, initially with whole blood and fresh frozen plasma, and more recently with specific factor concentrate. Currently, patients with hemophilia have the possibility of choosing different effective and safe treatments, including novel extended half-life and alternative hemostatic drugs. The availability of novel extended half-life products could probably overcome current prophylaxis limitations, particularly in hemophilia B patients, by reducing the frequency of injections, achieving a higher trough level, and improving the quality of life of the patients. In addition, subcutaneous administration of alternative therapeutics would simplify prophylaxis in patients with hemophilia A and B with and without inhibitors. Regarding von Willebrand disease, a recombinant von Willebrand factor was recently developed to control bleeding episodes in patients with this disease, in addition to available von Willebrand factor/factor VIII concentrates. The management of patients affected by rare bleeding disorders (RBDs) is still a challenge, owing to the limited number of specific products, which are mainly available only in countries with high resources. Some improvements have recently been achieved by the production of new recombinant factor (F) XIII A subunit-derived and FX plasma-derived products for the treatment of patients affected by FXIII and FX deficiency. In addition, the development of novel alternative therapeutics, such as anti-tissue factor pathway inhibitor, ALN-AT3, and ACE910, for patients with hemophilia might also have a role in the treatment of patients affected by RBDs.

Role of the 2 adenine (g.11293_11294insAA) insertion polymorphism in the 3' untranslated region of the factor VII (FVII) gene: molecular characterization of a patient with severe FVII deficiency.

Polymorphic variants in the gene encoding factor VII (F7) affect the plasma levels of this coagulation protein and modify the clinical phenotype of FVII deficiency in some patients. In this study we report the in vitro functional analysis of a novel polymorphic variant located in the 3' untranslated region of F7: g.11293_11294insAA. To determine whether this variant regulates FVII expression, we initially compared an expression vector containing FVII cDNA with g.11293_11294insAA with the FVII wild-type (WT) construct. The kinetics of mRNA production showed that the insertion decreases the steady-state FVII mRNA levels. To assess whether the insertion influences the phenotype of FVII-deficient patients, we evaluated its effect on the expression of FVII in a patient with severe FVII deficiency (undetectable FVII activity and antigen) carrying two additional homozygous missense variations (p.Arg277Cys and p.Arg353Gln). The two substitutions alone reduced the expression of FVII activity and antigen in vitro, but with the insertion polymorphism in our expression vector the patient's phenotype of undetectable plasma FVII was recapitulated. The insertion polymorphism in the 3' untranslated region of F7 is another modifier of FVII expression that might explain the poor genotype-phenotype correlation in some FVII-deficient patients.

Cytogenetic abnormalities and microsatellite instability in endometrial adenocarcinoma.

Recently various authors described a new mechanism involved in the genesis of some tumors, which is characterized by a tendency for replication mistakes and by genomic instability of microsatellite repeats. This instability can be revealed through the shift in the electrophoretic mobility of the analyzed fragments, which is due to a different number of repeat units. This phenomenon is widely documented in colorectal tumors of patients affected by hereditary nonpolyposis colorectal carcinoma (HNPCC). We performed a cytogenetic and molecular study of 23 endometrial adenocarcinomas to investigate the presence of genomic instability and to evaluate the possibility of a positive correlation with specific chromosomal changes. The study of genomic instability was performed using 23 microsatellites localized over 8 chromosomes. Genomic instability of microsatellites was observed in 3 cases over all 8 analyzed chromosomes. The tumoral stage of cases with microsatellite instability does not differ significantly from the remaining tumors. As a matter of fact several cases showing no evidence of instability were more advanced (II B, III A) than tumors with instability. In ten cases we observed trisomy of chromosome 10, in some as a sole anomaly. The 3 cases with genomic instability revealed a near-diploid karyotype and all showed the presence of a supernumerary marker derived from chromosome 1 rearrangements. A derivative chromosome 1 was revealed in 4 cases without evidence of microsatellite instability. It should be noted that the presence of many unidentified markers and the small number of tumors with instability do not allow us to give a definitive significance to this observation. Our results indicate that there is not an apparent correlation between microsatellite instability and specific chromosomal abnormalities. Moreover, we did not find any correlation between pathological characteristics of the tumor and genomic instability. Microsatellite instability appears to be a relatively rare event in endometrial carcinoma.

Losses of heterozygosity in oral and oropharyngeal epithelial carcinomas.

We analyzed 25 oral and oropharyngeal epithelial carcinomas for loss of heterozygosity (LOH) and microsatellite instability by using 55 oligonucleotide repeat markers located in 45 chromosomal regions. The aim was to identify which chromosomal regions and tumor-suppressor genes (TSGs) are preferentially lost in these tumors and to relate LOH at specific loci to clinicopathologic data. The analysis was performed on tumor tissue and on a corresponding normal tissue (blood lymphocytes) with the use of the polymerase chain reaction technique followed by microsatellite allele separation with denaturing gel electrophoresis. Thirty-two of 45 chromosomal regions demonstrated a significant (>/=20%) incidence of LOH. An allelic loss of >/=50% was found in 9p21 (77.8%), 8p22-23 (70%), 3p12 (61.5%), 1p36.1 and 12q22 (60%), 3q28 (57.1%), 5q23.3 (54.5%), 3p25-26, 3p24, and 7q35 (50%). We did not find any microsatellite instability. Our results suggest that in addition to a group of TSGs, pleiotropic for several tumor types, other suppressor genes are specifically involved in oral and oropharyngeal carcinogenesis.

Losses of heterozygosity in endometrial adenocarcinomas: positive correlations with histopathological parameters.

We analyzed 37 samples of endometrial adenocarcinoma for loss of heterozygosity (LOH) by using a panel of 44 microsatellites located in 29 chromosomal regions. The aim of our study was to investigate the existence of a possible preferential involvement of some tumor suppressor genes in endometrial carcinogenesis. The analysis was performed on tumoral tissue and on a corresponding normal tissue by the use of polymerase chain reaction (PCR) and the comparison of the amplified alleles. We observed significative LOH (>20%) in the chromosomal regions of 2q14 (33.33%), 7q35 (24.00%), 10q22.1 (37. 50%), 11q13-q14 (44.12%), 15q26 (40.63%), 17p13 (25.71%), and 17q21. 3 (37.04%). We defined a 1-cM minimal common deletion in 11q13-q14 between D11S911 and D11S937 markers. A statistical analysis revealed a positive correlation between LOH of 11q13-q14 and clinicopathological data.

Future of coagulation factor replacement therapy.

Over a million patients worldwide currently suffer from hemophilia and other congenital clotting factor deficiencies. Patients affected with hemophilia A and B are treated by intravenous replacement therapy of factor VIII and factor IX, respectively. Current hemophilia treatments have favorably supported their efficacy, tolerability, and safety profiles. The onset of alloantibodies inactivating the infused coagulation factor is the main problem in hemophilia patients rendering replacement therapies ineffective; another disadvantage is the short half-life of the infused clotting factors with the need for multiple and frequent infusions to manage a bleeding episode. Now, the challenge in the management of hemophilia treatment is the prolongation of the half-life and reduction in the immunogenicity of recombinant clotting factors. The bioengineering strategies, previously applied successfully to other therapeutic proteins, encourage the current efforts to produce novel coagulation factors with more prolonged bioavailability, with increased potency and resistance to inactivation and potentially reduced immunogenicity.

Source and purity of factor VIII products as risk factors for inhibitor development in patients with hemophilia A.

BACKGROUND: Inhibitor development is influenced by several factors and the type of factor VIII (FVIII) products may play a role. OBJECTIVES: In order to explore such a role, we designed a cohort study whose novelty resides in the classification of products not only according to the source of FVIII (plasmatic, pd, or recombinant, r) but also to their degree of purity (expressed as specific activity). PATIENTS/METHODS: Treatment data up to inhibitor development or 150 exposure days were collected in 377 patients with hemophilia A. RESULTS: Inhibitors developed in 111 patients (29%; 96 high-responders, 25%). The cumulative incidence was progressively higher from patients treated with low/intermediate-purity pdFVIII compared with those treated with high-purity pd and rFVIII. The adjusted hazard ratio of inhibitor development was 4.9 with rFVIII and 2.0 with high-purity pdFVIII (95% CI, 2.9-8.3 and 1.1-4.0), taking as reference low/intermediate-purity pdFVIII. There was no difference in the frequency of inhibitor testing between treatment groups. Sensitivity analyses (in patients who never switched product type, previously untreated patients, those treated on-demand and those with high-risk F8 mutations) confirmed an increased inhibitor risk with rFVIII and high-purity pdFVIII. CONCLUSIONS: This study shows that the degree of purity of FVIII products influences inhibitor development independently from other risk factors, and emphasizes that differences exist within pdFVIII products.

Gynecological and obstetrical manifestations of inherited bleeding disorders in women.

Patients affected by bleeding disorders present a wide spectrum of clinical symptoms that vary from a mild or moderate bleeding tendency to significant episodes. Women with inherited bleeding disorders are particularly disadvantaged since, in addition to suffering from general bleeding symptoms, they are also at risk of bleeding complications from regular haemostatic challenges during menstruation, pregnancy and childbirth. Moreover, such disorders pose important problems for affected women due to their reduced quality of life caused by limitations in activities and work, and alteration of their reproductive life. These latter problems include excessive menstrual bleeding or menorrhagia, miscarriage, bleeding complications during pregnancy and after delivery and their related complications such as acute or chronic anaemia. The management of these women is difficult because of considerable inter-individual variation. Moreover, reliable information on clinical management is scarce, only a few available long term prospective studies of large cohorts provide evidence-based guideline about diagnosis and treatment.

Severe hemophilia with mild bleeding phenotype: molecular characterization and global coagulation profile.

BACKGROUND: Patients with severe hemophilia may show very varied bleeding tendencies, and the reasons for this heterogeneous clinical expression are unclear. The factor VIII/FIX genotype is the main determinant of the residual factor activity; however, different bleeding phenotypes have also been reported in patients sharing the same mutation. Such global coagulation tests as thrombin generation assays are tools with which to investigate different coagulation profiles among severe hemophiliacs. OBJECTIVES, PATIENTS AND METHODS: This case-control study was aimed at comprehensively evaluating the role of genotype and endogenous thrombin potential (ETP) as predictors of the clinical phenotype in severe hemophiliacs with an extremely mild bleeding tendency (cases, n = 22), in comparison with those showing a typical bleeding tendency (controls, n = 50). RESULTS: Cases were more frequently affected by hemophilia B than by hemophilia A, and showed a lower incidence of severe FVIII/FIX gene defects (referred to as null mutations), higher FVIII and FIX antigen levels and higher ETP values in platelet-rich plasma than controls (P < 0.05). By multivariate logistic regression, only non-null mutations were confirmed as an independent predictor of a mild clinical phenotype. CONCLUSIONS: These results indicate that non-null mutations represent the main determinant of the bleeding tendency, and that ETP measurement in platelet-rich plasma is able to identify severe hemophiliacs with a mild clinical phenotype.