Haemophilia (2008), 14, 1201–1208
DOI: 10.1111/j.1365-2516.2008.01845.x
ORIGINAL ARTICLE
Combined FV and FVIII deficiency M. SPREAFICO and F. PEYVANDI A. Bianchi Bonomi Hemophilia and Thrombosis Center, University of Milan and Department of Medicine and Medical Specialties, Luigi Villa Foundation, IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Milan, Italy
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
Introduction Combined deficiency of factor (F)V and FVIII (F5F8D, OMIM 227300) is an autosomal recessive bleeding disorder, meaning that both parents must carry the defective gene to transfer the disease. F5F8D, characterized by concomitantly low levels (usually between 5% and 20%) of the two coagulation factors FV and FVIII [1], is completely separate from FV deficiency and FVIII deficiency. The latter are transmitted with different patterns of inheritance (autosomal recessive for FV, X-linked for FVIII) and involve proteins encoded by two different genes. F5F8D was first described by Oeri et al. in 1954 [2]. In this report, Oeri postulated that F5F8D Correspondence: Marta Spreafico, A. Bianchi Bonomi Hemophilia and Thrombosis Center, University of Milan and Department of Medicine and Medical Specialties, IRCCS Maggiore Hospital, Mangiagalli and Regina Elena Foundation, Via Pace 9, 20122 Milan, Italy. Tel.: +39 02 55035414; fax: +39 02 54100125; e-mail:
[email protected] Accepted after revision 9 July 2008 2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd
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. Keywords: combined FV + FVIII deficiency, F5F8D, LMAN1, MCFD2
resulted from a defect in a gene that encodes a common precursor of both clotting factors [2]. The failure of cross transfusions of plasma from haemophilia patients performed in the 1960s was the first indication that the precursor hypothesis could be abandoned [3]. Subsequently, the two distinct genes encoding respectively for FV, on chromosome 1 [4], and FVIII, on chromosome X [5], were identified. The molecular mechanism of the association of the combined factor deficiency was not understood until 1998, when Nichols et al. [6,7], using the positional cloning method, discovered that the cause of the deficiency was associated with null mutations in the ERGIC-53 gene now called LMAN1 gene (Lectin Mannose Binding Protein), encoding the ER-Golgi intermediate compartment (ERGIC) marker protein. Mutations in LMAN1 were found in 70% of affected patients but 30% of this population had no detectable mutation in LMAN1. In 2003, Zhang et al. [8] identified a second locus associated with the deficiency in about 15% of affected families with no mutation in LMAN1. The MCFD2 (Multiple Coagulation Factor Deficiency 2) gene encodes for a
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cofactor for LMAN1 [9]. Even if a debate existed on the possible existence of other loci involved in the intracellular transport of FV and FVIII and associated with the disease, until now previous biochemical studies failed to identify additional components of the LMAN1–MCFD2 receptor complex [9], supporting the idea that F5F8D might be limited to the LMAN1 and MCFD2 genes [10].
Materials and methods All the information included in this review have been derived from the analysis of the available literature on F5F8D as well as on our long personal experience on clinical, phenotype and genotype analysis of patients affected by F5F8D coming from all over the world (http://www.rbdd.org). Data on the distribution of F5F8D around the world were also taken from the WFH global survey (WFH, http://www.wfh.org/2/7/ 7_0_Link7_GlobalSurvey2005.htm) as also from single national registries reported during International meetings and the Rare Bleeding Disorders database (RBDD, http://www.rbdd.org), and also from data by single National Registries reported in the frame of the SSC meeting during International Congress (WFH International Congress, II Rare Bleeding Disorders (RBDs) SSC ISTH working group RBDD Steering Committee meeting, Vancouver, Canada 2006; XXI ISTH Congress, SSC Working group on Factor VIII and Factor IX, RBDs, Geneva 2007, minute available at http://www.rbdd.org/news.htm). Incidence, racial/ethnic predilection Congenital F5F8D is estimated to be extremely rare (1:1.000.000) in the general population [11], affecting males and females in equal numbers. However, this disorder was reported to be particularly prevalent among Middle Eastern Jewish and non-Jewish Iranians, where the incidence was estimated at 1:100.000 [12]. This high frequency is probably attributable, at least in part, to the high incidence of consanguineous marriages in these populations [13]. Most reported cases are from Iran [14–18] and Italy [10,17–21], but also from Pakistan [16,17], Iraq [10,18], Lebanon [22], Algeria [16]. F5F8D cases were also reported from other European countries such as Turkey [8,13,18,19], Austria [10], Greece [10], Kosovo [10], Serbia [10], Poland [10], France [19], UK [17] and Belgium [10], from India [23–26] and Asian countries such as Japan [10,19,27], China [17,28] and Thailand [29], from South Africa [17], United States [19] and South America countries [18,30], such as Venezuela [19] and Argentina [10].
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Unfortunately, a reliable picture of the global distribution of RBDs could not be developed, because available data are not homogeneous and information from many countries is lacking, because of the limited number of reliable national registries especially in developing countries. However, an indication of the worldwide prevalence of F5F8D compared to other rare coagulation disorders can be derived from the WFH global survey, the RBDD (http://www.rbdd.org), and by data from single reported National Registries. According to these data, patients affected by F5F8D around the world seem to reach approximately the number of 200, representing 3% of the total number of patients affected by RBDs (7000) and representing, together with FII deficiency, the most rare coagulation disorder. The distribution of F5F8D patients is variable in different regions of the world, ranging from 1–2% in North America, Europe and Africa to 4–5% in South America and Asia and reaching the highest frequency in MiddleEastern countries and India (15–16%). Pathophysiology F5F8D is characterized by concomitantly low levels (usually between 5% and 20%) of the two coagulation factors, FV and FVIII, both as coagulant activity and antigen [1]. Within the coagulation cascade, FV and FVIII are two large plasma glycoproteins that function as essential cofactors for the proteolytic activation of prothrombin (FII) and FX respectively. Both these proteins have the same A1-A2-B-A3C1-C2 structure with 40% amino acid sequence homology in the A and C domains [31]. The F5F8D is an autosomal recessive disorder distinct from the coinheritance of both FV deficiency (chromosome 1) and FVIII deficiency (chromosome X). F5F8D is essentially because of defects in the secretion pathway of coagulation FV and FVIII, involving a qualitative or quantitative defect in the lectin mannose binding protein type 1 (LMAN1, previously referred as ERGIC-53) or in the MCFD2 [7,8]. LMAN1 is a 53-kDa type-1 transmembrane nonglycosylated protein with homology to leguminous lectin proteins [32]. LMAN1 has a signal sequence mediating translocation into the ER. It consists of a luminal, a transmembrane, and a short cytoplasmatic domain, in total 513 residues. The luminal domain can be divided into two subdomains, an N-terminal carbohydrate recognition domain (CRD) (residues 31–285) and a membrane-proximal a-helical coiled domain, the stalk domain (residues 290–460). The CRD is responsible for the calcium-ion dependent 2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd
F5F8D
binding of the protein to mannose-rich glycans. The cytoplasmatic tail binds to the COPII component of vesicle coats, allowing efficient ER export, and also has a prototype signal to drive ER-retrieval from the intermediate compartment and the Golgi complex. LMAN1 displays different oligomerization states, monomer, dimer and hexamer, which have been implicated in its exit/retention within the ER. The crystal structures of the CRD in apo- and Ca2+bound form were determined [33,34]. LMAN1 resides in the early secretory pathway, with highest concentration in the endoplasmic reticulum/Golgi intermediate compartment (ERGIC), where it facilitates the intracellular transport of both FV and FVIII through mannose-selective and calcium dependent binding/release cycles [32]. Efficient transport of coagulation FV and FVIII along the secretory pathway requires the integrity of their heavily glycosylated B-domains and a functional LMAN1 protein, and an interaction between FVIII and LMAN1 has been demonstrated [9,31]. LMAN1 has thus been implicated to act as a sorting receptor, mediating transport of certain glycoproteins from the ER to Golgi. In support of such an important function, homologues of LMAN1 have been identified in levels of the animal kingdom ranging from Caenorhabditis elegans to man and display a high degree of sequence identity [35]. Multiple Coagulation Factor Deficiency 2 is a small (146 residues) soluble protein of 16 kDa with a signal sequence mediating translocation into the ER and two EF-hand motifs that may bind Ca2+ ions. MCFD2 forms a Ca2+-dependent 1:1 stechiometric complex with LMAN1, that works as a cargo receptor for efficient ER-Golgi transfer of coagulation FV and FVIII during their secretion [8]. Recent studies reported that LMAN1 can bind cargo glycoproteins in an MCFD2-independent fashion and suggest that MCFD2 is an essential recruitment factor for blood coagulation FV and FVIII [30,35– 37]. Genetics/molecular basis of disorder LMAN1 is encoded by a gene of 29 kb located on chromosome 18q21 and containing 13 exons [16], while MCFD2 is encoded by a gene of 19 kb located on chromosome 2p21 and containing 4 exons [8]. Mutations in MCFD2 and LMAN1 are associated with very similar phenotypes [8,18]. However, a selective delay in secretion of the proteins procathepsin C has been observed in HeLa cells overexpressing a dominant-negative form of LMAN1 [38]. These results were confirmed by 2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd
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Nyfeler et al., showing that LMAN1 also interacts with the two lysosomal glycoproteins cathepsin Z and cathepsin C whereas MCFD2 is dispensable for the binding of them to LMAN1 [36]. To date, 47 mutations are described in LMAN1 (n = 32, 68%) and MCFD2 (n = 15, 32%) genes [http://www.med.unc.edu/isth/mutations-databases/ FVandVIII_2006numbers.htm, 18,26,28,30] (Figs 1 and 2). A vast majority of these mutations (nonsense, frame shifts, splicing defects or missense mutations) predict null alleles and are predominantly identified in LMAN1 (95%, n = 38/40). Among them, also recurrent mutations in both the LMAN1 and MCFD2 genes are described. LMAN1 mutations, such as p.M1T, c.86_89insG, p.R202X, c.822G>A, p.K302X, c.1140 + 2T>C, have been observed in more than four patients [7,13,17– 20,27]. Similarly, the c.149 + 5G>A and p.I136T mutations in MCFD2 have also been commonly reported [18,26,27]. The MCFD2 c.149 + 5G>A appears to be one of the most common mutation causing F5F8D and it has now been identified in at least 13 unrelated families from different geographic regions and particularly from India (n = 6) [25,26], followed by Italy (n = 4) [10,18], USA (n = 1) [8], Serbia (n = 1) [10] and Germany (n = 1) [8]. In case of recurrent mutations in a particular geographic area, haplotype analysis has to be considered as a tool to evaluate whether or not a founder effect exists. The founder effect is, indeed, a potential diagnostic tool of genetic disease, particularly in countries with a high prevalence of the disease and low economic facilities. In the LMAN1 gene, for example, the nt89– 90insG mutation leading to a premature stop at codon 102 was found to be particularly frequent in Iranians and Iraqi Jews, while the IVS9 + 2T>C mutation was frequent among Jews originating from the island of Djerba [13]. In vitro expression studies have proved to be an invaluable tool to understand the nature of the genetic defect and to unravel the underlying molecular mechanism of deficiencies. In vitro expression studies of mutations in LMAN1 and MCFD2 genes and characterization of the activity of the corresponding recombinant proteins in the secretion of coagulation FV and FVIII could thus significantly help to describe the mechanism of the deficiency. In the literature, all the five missense mutations identified in MCFD2 were expressed in COS-1 transfected cells [8,18]. All the other identified mutations were demonstrated to be associated with the deficiency through analysis performed on Epstein Barr Virus-transformed lymphoblasts of the patients that showed the presence/ absence of the mutated protein [8,10,17].
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Fig. 1. The mutation found in LMAN1 gene as reported in http://www.med.unc. edu/isth/mutations-databases/ FVandVIII_2006numbers.htm and Zhang B et al., [18], Jayandharan G et al., [26], Ma ES et al., [28] and Nyfeler B et al., [30].
Clinical manifestations The concomitant presence of two coagulation defects does not enhance the haemorrhagic tendency that was observed in each defect separately [14,15]. F5F8D is associated with a mild to moderate bleeding tendency [11–15,24]. Mild bleeding symptoms such as easy bruising, epistaxes and gum bleeding are not uncommon in affected individuals. Other common bleedings include bleeding after surgery; dental extraction and trauma; menorrhagia and postpartum haemorrhage are also commonly seen in affected women [12,15]. In F5F8D patients, circulating levels of FV and FVIII, varying between 5% and 20% [1], are usually sufficient to prevent more severe spontaneous bleeding episodes [12,15,39]. However, severer symptoms including haemarthroses, (occurring in about one fourth of the patients) and umbilical cord bleeding can be observed. Soft-tissue haematomas are unusual [14,40]. Other severe symptoms, reported in few patients include gastrointestinal and central nervous
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system bleedings [14,15]. Excessive bleeding after circumcision was reported in two-third (8/12) and approximately half (6/13) of the male patients [14,15] but surprisingly was not reported among non-Ashkenazi Jews [12]. Diagnosis F5F8D patients are characterized by normal platelet count and bleeding time and by prolongation of both prothrombin time (PT) and partial thromboplastin time tests (PTT) [11]. Specific assays of FV and FVIII coagulant activity are then necessary to evaluate the residual FV and FVIII coagulant activity. Factor antigen assays are not strictly necessary for diagnosis and treatment. Molecular analysis Direct mutational analysis to identify the genetic defect underlying the F5F8D could be performed on patientsÕ DNA by direct sequence analysis of all 2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd
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Fig. 2. The mutation found in MCFD2 gene as reported in http://www.med.unc.edu/isth/mutations-databases/FVandVIII_2006numbers. htm and Zhang B et al., [18], Jayandharan G et al., [26], Ma ES et al., [28] and Nyfeler B et al., [30].
coding regions, intron–exon junctions and 5¢- and 3¢-UTR regions of LMAN1 and MCFD2 genes. Any identified mutation has to be confirmed by repeat sequencing and restriction enzyme digestion if associated with the creation or loss of a specific restriction enzyme and has to be verified in at least 200 alleles of a control population coming from the same geographical area as the patient to discriminate between mutations and polymorphisms. As about 70% of the found mutations are located in LMAN1 gene, the molecular analysis should start at this gene, followed by the sequencing of the MCFD2 gene. This does not seem to be true for the Indian population, for whom a higher frequency of recurrent mutations in MCFD2 gene has been reported [26]. 2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd
Prenatal diagnosis in rare coagulation disorders As F5F8D has a mild to moderate bleeding tendency, prenatal diagnosis is not currently performed and not recommended. Prevention of F5F8D through prenatal diagnosis of the underlying mutations could be feasible, but only in couples that already have at least one severe affected child. Gene mutations found in both parents need to be verified via DNA extracted from chorionic villi at 10–12 gestational weeks. Management In F5F8D, treatment of bleeding episodes is dependant on the nature of the bleed and the FV and FVIII levels of affected individuals. F5F8D bleeding epi-
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sodes are usually treated on demand and do not require regular prophylaxis. However, prophylaxis could be chosen in cases of recurrent severe haematomas and haemarthroses. Sources for both FV and FVIII are needed and their plasma half-life (FV: 36 h; FVIII: 10–14 h) have to be taken into consideration for treatment of spontaneous bleeding episodes. Because FV concentrates are not available and FV is not present in cryoprecipitate or prothrombin complex concentrates [41], replacement of FV can be achieved only through the use of fresh frozen plasma (FFP), preferably with virus-inactivated plasma. For FVIII replacement, a large number of products are available, including FFP, plasma-derived concentrates or recombinant FVIII (rFVIII) (different generations), because of their use to treat haemophilia A. However, a debate exists in the literature on the type of product (plasma-derived vs. recombinant) associated with the higher risk of inhibitor development [42]. More detailed analysis and in-depth studies are required to address this issue. For minor bleeding episodes, FVIII levels should be raised to at least 30–50 IU dL)1; for more severe bleeds, they should be raised to at least 50–70 IU dL)1. Plasma-derived FVIII concentrate or rFVIII concentrate are treatments of choice [39]. The synthetic hormone (desmopressin, 260 lg intranasal or 0.3 lg kg)1 subcutaneous), can also be successfully used for minor bleeding episodes to further raise FVIII when the post-FFP trough levels of this factor are thought to be inadequate for haemostasis, but its efficacy on the patient need to be tested [11]. Replacement of FV is achieved by using FFP, preferably with virus inactivation (15–20 mL kg)1) [43], in order to increase the FV level to at least 25 IU dL)1 [39]. The initial dose should be 15–20 mL kg)1 followed by smaller amount, such as 5 mL kg)1 every 12 h, adjusting the dosage on the basis of FV levels, PT and partial thromboplastin time (PTT) [38]. Studies of FV recovery recommend maintaining a level of 20–25% of FV activity for surgery or in case of severe bleeding [39]. It has been suggested that in cases of severe bleeding not controlled with FFP replacement, platelet transfusions may be considered, although this treatment modality has not been proven to be efficacious in the literature [43]. Platelets provide a concentrated supply of FV. Therefore, following alpha granule release upon platelet activation, FV can presumably bind immediately to surface receptors optimizing prothrombinase complex activity [39]. Development of alloantibodies to FV in FFP is a potential complication of hereditary FV deficiency [39] and might occur in F5F8D patients usually
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treated with FFP. The occurrence of inhibitors, especially transient ones of low level, following FFP replacement therapy may not be uncommon [39]. In the event of a bleeding problem, it has been suggested that low-level inhibitors can be neutralized using large amounts of FFP [41]. Intravenous immunoglobulin may be also effective in eradicating the FV inhibitor [39]. The use of large amount of plasma leads to a fluid overload and sometimes needs to be corrected by diuretics [11]. Surgical procedures should be addressed by administering FVIII 30 min before surgery and then every 12 h to maintain FVIII levels above 50 IU dL)1 and with FFP every 12 h to achieve minimum levels of FV of 25 IU dL)1 until wound healing is established [39]. There are no published data on managing pregnant women with F5F8D [39]. FV levels in pregnancy do not consistently increase or decrease, whereas FVIII levels will increase throughout pregnancy. Any possible bleeding is therefore likely to be dependent on the FV level during labour and postdelivery. In keeping with general recommendations for the management of bleeding disorders in pregnancy, affected women should be managed by an obstetric unit in close liaison with a haemophilia centre. FV and FVIII levels should be confirmed in the third trimester so that the delivery can be planned with regard to haematological intervention. FV levels should be maintained above the haemostatic level of 15 IU dL)1 during labour, using virus-inactivated plasma as the source of the factor. FVIII levels should remain at greater than 50 IU dL)1 throughout this period. If a Caesarean section is carried out, it would be prudent to continue FV replacement to maintain PT and PTT in the normal range until wound healing is complete in women with FV levels of
