MCFD2 Research Articles

Decreased Factor V Ameliorates Bleeding Diathesis in Patients with Combined Deficiency of Factor V and Factor VIII

Combined factor V (FV) and factor VIII (FVIII) deficiency (F5F8D) is a rare autosomal recessive bleeding disorder caused by mutations in LMAN1 and MCFD2 gene. Though characterized with deficiencies of both FV and FVIII, it failed to lead to more severe bleeding than single defect of FVIII in similar degree. Our study on F5F8D patients revealed that mild decrease of FV and FVIII had counteractive effect on blood coagulation. Out of 5 subjects investigated, three showed potent thrombin generation as observed in thrombin generation assay (TGA), with peak heights ranging from 138 nM to 166 nM, compared with 81 nM in normal plasma when coagulation was initiated with 1pM TF. The same trend was seen when the TF concentration was increased to 5 pM.Supplement of FV and FVIII, however, demonstrated distinct impact in TGA, with FVIII addition inducing more thrombin generation whereas higher FV level failed to further boost thrombin generation despite the shortened lag time.FV is a Janus-faced protein and suppresses blood coagulation by protecting TFPI from degradation.Similar to patients with severe FV deficiency, both total and free TFPI levels also decreased slightly in F5F8D patients. Total TFPI levels decreased from healthy donors (65.95±8.35 ng/mL) to F5F8D patients (65.79±12.99 ng/mL), to severe FV-deficient patients (51.30±11.15 ng/mL, P=0.037). A more pronounced trend was observed for free TFPI levels, as free TFPI in healthy controls were 12.68±2.91 ng/mL, higher than those in F5F8D (4.73±1.31 ng/mL, P=0.004), and in severe FV-deficient patients (3.81±1.03 ng/mL, P=0.004).The lowered TFPI in patients with F5F8D may neutralize the impact of FV and FVIII deficiency on blood coagulation.Desmopressin (DDAVP) increases endogenous FVIII and is used in the management of mild hemophilia A patients. In current study, 4 patients with F5F8D were treated with DDAVP. The administration of DDAVP saw no elevation of FV yet the increase of FVIII. The mean basal FV and FVIII levels were 11.5±3.4% and 15.1±4.8% respectively. The peak value of FVIII levels rose to 66.1±20.5% 30 minutes after the administration, and was sustained at two-folds of the baseline four hours post administration.The distinctive changes brought by combined FV and FVIII deficiencies on blood coagulation functions indicates the lone FVIII replacement without FV supplement might be sufficient in bleeding management of patients with F5F8D. DDAVP can be considered as a potential substitute for FVIII concentrate in the treatment of F5F8D patients. DisclosuresNo relevant conflicts of interest to declare.

Successful percutaneous coronary intervention in a patient with combined deficiency of FV and FVIII due to novel compound heterozygous mutations in LMAN1

Percutaneous coronary intervention (PCI) in patients with congenital coagulation factor deficiencies presents a unique challenge. They are not only at increased risk of perioperative bleeding but can also suffer thrombosis of the stent as preventive anticoagulation and antiplatelet therapy is difficult. Several cases of successful PCI have been described in patients with haemophilia A and B, but there are no reports in patients with combined coagulation factor deficiencies. We used PCI to treat the coronary artery disease in a patient with the combined deficiency of factor V and factor VIII (F5F8D) and analysed the molecular basis of the disorder for this patient. A 68-year-old patient was admitted for urgent PCI with bare metal stent placement after the diagnosis of the F5F8D. Peripheral blood DNA was extracted for the sequence analysis of LMAN1 and MCFD2 genes. Mutations in LMAN1 was confirmed by molecular cloning of the PCR product and resequencing of the resulting clones. The patient underwent successful PCI with good long-term outcome. Our patient tolerated anticoagulation therapy well, with unfractionated heparin, and double antiplatelet therapy while he was initially supported with fresh frozen plasma and recombinant FVIII. Molecular analysis revealed that the patient carries unusual compound heterozygous frameshift mutations on the same microsatellite repeat region in exon 8 of LMAN1, one of which is a novel mutation (c.912delA). Our results suggest that patients with F5F8D can safely undergo PCI for coronary artery disease, with the treatment individualized to the specific patient.

Unveiling the Unfolding Pathway of F5F8D Disorder-Associated D81H/V100D Mutant of MCFD2 via Multiple Molecular Dynamics Simulations

Combined factor deficiency (F5F8D) is a rare autosomal recessive disorder caused by mutations in the LMAN1 or MCFD2 genes. It has been proposed that this pathogenic process occurs via a multi-step pathway involving metal loss, EF-hand-Ca2+ dissociation and assembly of misfolded MCFD2-LMAN1 complex. Here, we have investigated the solution conformations of the MCFD2(D81H,V100D) protein mutant through extensive molecular dynamics (MD) simulations. The V100D, one of the many MCFD2 mutations known to be associated to F5F8D, is difficult to be reconciled with the pathway model because it is located far from the metal sites and the MCFD2/LMAN1 interface. Consequently, an inspection of all the steps involved in D81H/V100D MCFD2 misfolding is expected to provide hints in the understanding of the molecular basis of the disease. A comparison with parallel studies carried out for the Wild-Type (WT) MCFD2 pointed out that the mutation decreases the affinity of the protein for the Ca2+ ion. Multiple explicit solvents MD simulations (50 ns) performed on the two proteins revealed that in the WT protein, stable H-bond network and compact hydrophobic core region are created thus confirming a pivotal role of this region in driving the biophysical properties of the entire protein. In fact it is shown that the V100D mutation, although located far away the EF-hand domain, may induce subtle modification in the structural core of MCFD2 leading to the loosening of metal binding and to the formation of metastable intermediate states along the unfolding pathway. The native-like hydrophobic cluster formed near the V100 residue in the wild-type protein is disrupted by the negatively charged Asparagine residue. Furthermore, the presence of the D81H mutation in the EF-1 hand domain may also increase the protein unfolding rate and consequently prevent the formation of the MCFD2-LMAN1 complex. The detailed structural insights obtained from our large-scale simulations complement the clinical features and offer useful insights into the mechanism behind MCFD2 protein misfolding.

Analysis of newly detected mutations in the MCFD2 gene giving rise to combined deficiency of coagulation factors V and VIII

Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is a rare autosomal recessive disorder characterized by mild-to-moderate bleeding and reduction in FV and FVIII levels in plasma. F5F8D is caused by mutations in one of two different genes, LMAN1 and MCFD2, which encode proteins that form a complex involved in the transport of FV and FVIII from the endoplasmic reticulum to the Golgi apparatus. Here, we report the identification of a novel mutation Asp89Asn in the MCFD2 gene in a Tunisian patient. In the encoded protein, this mutation causes substitution of a negatively charged aspartate, involved in several structurally important interactions, to an uncharged asparagine. To elucidate the structural effect of this mutation, we performed circular dichroism (CD) analysis of secondary structure and stability. In addition, CD analysis was performed on two missense mutations found in previously reported F5F8D patients. Our results show that all analysed mutant variants give rise to destabilized proteins and highlight the importance of a structurally intact and functional MCFD2 for the efficient secretion of coagulation factors V and VIII.

Inherited and acquired factor V deficiency

The clotting factor V, also known as proaccelerin or labile factor, is synthesized by the liver and possibly by the megakaryocytes. Factor V exerts a pivotal role in hemostasis, as it participates in both procoagulant and anticoagulant pathways, being an essential cofactor of the prothrombinase complex in the former case and participating in the inactivation of factor VIII (FVIII) in the latter. Isolated factor V deficiency due to mutations in the F5 gene is a rare inherited coagulopathy typically associated with a broad spectrum of bleeding symptoms, ranging from easy bruising, delayed bleeding after haemostatic challenges such as trauma or surgery to more severe joint bleeds. The combined deficiency of factor V and FVIII, commonly known as F5F8D, is a recessive disorder not attributable to the association of isolated factor V and FVIII deficiencies, but rather to defective intracellular processing of both proteins due to mutations involving the LMAN1 and MCFD2 genes, which encode two proteins forming an essential cargo receptor complex. Overall, patients affected by F5F8D do not bleed more in terms of both frequency and severity than those carrying specific deficiencies of both factors and the bleeding phenotype is generally mild. Although now increasingly rare, inhibitors directed against factor V may also develop in individuals of any age and are characterized by a very heterogeneous clinical phenotype. The aim of the current review is to provide an overview on the physiopathology, diagnostics, and clinical management of both inherited and acquired factor V deficiency.

Molecular analysis in two Tunisian families with combined factor V and factor VIII deficiency

Combined factor V (FV) and factor VIII (FVIII) deficiency (F5F8D) is a rare autosomal recessive disorder caused by mutations in LMAN1 or MCFD2 genes which encode proteins that form a complex involved in the transport of FV and FVIII from the endoplasmic reticulum to Golgi apparatus. We report two novel mutations in MCFD2 gene and one recurrent mutation in LMAN1 gene that caused combined FV and FVIII deficiency in two unrelated Tunisian Muslim families. For the first family two patients were homozygous for a new missense mutation Asp81His in exon 3 of MCFD2 and heterozygous for a second new missense mutation Val100Asp in the same exon. Replacement respectively of the hydrophilic Asp residue with hydrophobic positively charged His and of the hydrophobic neutral Val residue with the Asp residue most likely disrupts the MCFD2-LMAN1 interaction, thus leading to the disease phenotype. For the second family a reported Arg202X mutation in exon 5 in the LMAN1 gene was identified in the homozygous state.

Genetic diagnosis of four combined deficiency of factor V and VIII patients

Objective To investigate the genetic diagnosis and molecular pathogenesis of four patients with combined deficiency of coagulation factor Ⅴ and Ⅷ and their family members. Methods The APPT, FT, FⅤ: C, FⅧ: C were detected for phenotypic diagnosis. Thrombin generation assay was applied to determine the generation condition of thrombin in patients and healthy controls. Cenomic DNA was extracted from peripheral blood using the TianGen RelaxCene Blood DNA System;amniotic fluid DNA was extracted with phenol-ethyl ether method. The LMAN1 and MCFD2 genes were analyzed by PCR. Gene mutations were detected with nucleotid sequences by using end-labeling dideoxy method. Results The APTT of Proband 1 was significantly prolonged to 88. 2s and her PT was prolonged to 19. 6 s. The combined deficiency was identified with FⅧ (FⅧ: C 24. 2% ) and FV(FⅤ: C 9. 1% ). Proband 2 and 3 were sisters. The coagulation studies revealed that both of them had prolonged APTT (71.6 s and 74.6 s respectively) and PT (22. 1 s and 18. 3 s respectively). The combined deficiency of FⅤ (FⅤ: C 7. 6% and 14. 5% respectively) and FⅧ( FⅧ: C 25% and 19.6% respectively) were identified. Proband 4 was detected to have the prolonged APTT (70.3 s),PT (18.2 s) and the deficiency of FⅤ(FⅤ: C 9. 4% ) and FⅧ (15. 7% ). The remaining phenotype indicators test of the 4 probands were normal. The diagnosis for the 4 probands was combined deficiency of factor Ⅴ and Ⅷ. The proband 1 was detected to have compound heterozygous mutations in LMAN1 gene while having the LMAN1 and MCFD2 direct gene sequencing. One mutation was a small insertion located on exon 8 [ nt912insA (X71661. 1)] that resulted in p. 305frameshiftX20 and her mother was detected to have the same heterozygous mutation on the the locus. The other mutation was located on exon 11: nt1366C > CT ( X71661. 1 ) , p. 456Arg > Stop which was inherited from her father. Amniocyte DNA was detected to have only one heterozygous mutaion [nt1366C > CT (X71661. 1) , 456Arg > Stop] inherited from the father. No mutation in MCFD2 gene was found in proband 1 and her parents. The analysis of the MCFD2 gene in proband 2 and 3 revealed a novel homozygous single base substitution (nt411T>C) in exon 4, which results in the exchange of the amino acid isoleucine by the amino acid threonine at amino acid position 136 (p. Ile136Thr). Sequencing of the whole LMAN1 gene showed that the proband 4 had one homozygous nonsence mutation in the exon 5 of the LMAN1 ( nt615C >T,p. 202 Arg> Stop). All of the 4 probands with combined deficiency of FⅤ and FⅧ showed declined endogenous thrombin potential in the thrombin generation tests. Conclusion The combined deficiency of FⅤ and FⅧ in the proband 1 results from the compound heterozygous mutations ( nt1366C > CT and nt912insA) in LMAN1 gene, which are inherited from her parents respectively. The prenatal genetic investigation for the patient mother with preganency indicates that the fetus is a female carrier with one mutation (nt1366C > CT) inherited from the father. The homozygous missence mutation ( nt411T > C, p. Ile136Thr) in the MCFD2 gene accounts for the proband 2 and 3. The daughter of the proband 2 is a carrier with a heterozygous mutation inherited from her mother. The homozygous nonsence mutation in the LMAN1 gene of the proband 4 results in the deficency of F Ⅴ and FⅧ. Key words: Factor Ⅴ deficiency; Factor Ⅷ deficiency; Membroane proteins; Mannose-binding lectin; Vesicular transport proteins; Pedigree

Percutaneous Coronary Intervention (PCI) in Combined FV and FVIII Deficiency with a Novel LMAN1 Mutation.

Abstract 4448 INTRODUCTIONPatients with congenital coagulation factor deficiencies undergoing PCI are at increased risk of both perioperative bleeding and thrombosis of coronary stents. Several cases of successful PCI have been described in patients with factor VIII (FVIII) and factor IX deficiency. We report on the first PCI in combined factor V (FV) and FVIII deficiency (F5F8D). F5F8D is an autosomal recessive disorder associated with moderate bleeding diathesis. This genetically heterogeneous disorder involves the genes LMAN1 and MCDF2, which encode for a cargo receptor complex that plays a key role in the trafficking of FV and FVIII from the endoplasmic reticulum to the golgi complex. CASE DISCUSSIONA 68 year old white male presented with dyspnea on exertion and palpitations. A myocardial perfusion stress test showed anteroseptal ischemia with a LVEF of 64%. He was referred for cardiac catheterization. He had life-long history of episodes of self-limiting mucosal bleeds, easy bruising and a large hematoma on his upper extremity provoked by a minor injury. Coagulation work up revealed a FV level of 11% and FVIII level of 7%. Before cardiac catheterization, the patient was given a loading dose of 30ml/kg of FFP. The trough levels of FV and FVIII were 37% and 31% after the procedure. The patient was given 25U/kg of recombinant FVIII (helixate FS ©) at 12 and 24 hours post PCI with a resultant FVIII level of 71% at 36 hours post PCI. Coronary angiography revealed the patient had a 95% stenosis of the left anterior descending artery. A metal stent was used to minimize the duration of antiplatelet therapy needed postoperatively. During the procedure anticoagulation was achieved using intravenous unfractionated heparin. There were no signs of local bleeding at the femoral access site through the postoperative period after using an Angioseal closure device. The patient was treated with clodipogrel 75 mg and aspirin 81 mg by mouth daily for 30 days after the procedure followed by aspirin alone long term. He had two episodes of mild self-limiting nose bleeds while on double antiplatelet therapy, no other bleeding. He remains well with no symptoms of cardiac ischemia at nine months after the procedure. Subsequent genetic analysis of both LMAN1 and MCFD2 genes showed two mutations: a novel 912delA mutation and a previously described 912-913insA mutation, both at the same location in exon 8. The mutations were confirmed first by sequencing, followed by cloning the PCR product and sequencing individual clones. Normally the LMAN1 gene has a string of 9A's at this location from 904 to 912, while our patient had 10 A's on one chromosome and 8 A's on the other chromosome. This could be explained by the patient having inherited one mutation on one chromosome and gaining a de novo mutation on the other chromosome, since this microsatellite repeat is a mutation hot spot. Unfortunately this question cannot be answered since no parent DNA was available. Informed consent was obtained prior to publication of this manuscript. CONCLUSIONSThis patient's presentation was consistent with published findings in other patients with F5F8 deficiency caused by LMAN1 mutations, with reported levels of FV 13.7% (range 1.7% to 35%) and FVIII 16% (range 2.2% to 35%). Our experience suggests that patients with F5F8D and mild bleeding diathesis can tolerate PCI for coronary artery disease followed by double antiplatelet therapy of limited duration and low-dose aspirin long term. Disclosures:No relevant conflicts of interest to declare.

The first case of combined coagulation factor V and coagulation factor VIII deficiency in Poland due to a novel p.Tyr135Asn missense mutation in the MCFD2 gene

Congenital combined coagulation factor V and coagulation factor VIII deficiency (F5F8D) is a rare bleeding disorder due to mutations in the LMAN1 or MCFD2 genes. Here we report the first Polish family with F5F8 deficiency due to a mutation in the MCFD2 gene. The proposita suffered from mild bleeding including epistaxis, menorrhagia, bleeding after dental extraction, and bruising after minor traumas. The F5F8 deficiency was diagnosed due to an excessive postpartum bleeding at the age of 31. Analysis of further family members revealed a second affected individual. Sequencing of the MCFD2 gene and its flanking regions in both patients demonstrated a novel homozygous missense mutation within the second elongation factor hand domain resulting in a substitution of tyrosine by asparagine at amino acid position 135 (p.Tyr135Asn). This variant represents the third missense mutation found in the MCFD2 gene and most likely disrupts the MCFD2-LMAN1 interaction, thus leading to the disease phenotype.

The first case of combined coagulation factor V and coagulation factor VIII deficiency in Poland due to a novel p.Tyr135Asn missense mutation in the MCFD2 gene

The first case of combined coagulation factor V and coagulation factor VIII deficiency in Poland due to a novel p.Tyr135Asn missense mutation in the MCFD2 gene

Mutations in the MCFD2 gene are predominant among patients with hereditary combined FV and FVIII deficiency (F5F8D) in India

Combined FV and FVIII deficiency (F5F8D) is a rare (1:1.000.000) autosomal recessive disorder caused by a defect in the LMAN1 or MCFD2 genes, encoding for a FV and FVIII cargo receptor complex. We report the phenotype and genotype analyses in nine unrelated Indian patients with low FV and FVIII coagulant activity [FV:C, range: 5.6-22.4% and FVIII:C, range: 8.3-27.1%]. Four homozygous mutations, including two frame shift, one missense and one splice site, were identified in all the nine patients. Three of them, a 72-bp deletion in LMAN1 (c.813_822 + 62del72, p.K272fs), a 35-bp deletion in MCFD2 (c.210_244del35) and a missence mutation in MCFD2 (p.D122V), identified in four patients, were novel mutations. A previously reported c.149 + 5G > A transition in MCFD2 was identified in the remaining five patients. Haplotype analysis of MCFD2 gene in patients with p.E71fs and c.149 + 5G > A defects suggested an independent origin of both these mutations. The identification of two common mutations (p.E71fs, c.149 + 5G > A) in MCFD2 gene in seven of nine patients, particularly the c.149 + 5G > A (55,6% of patients), suggests that this gene could be the first to be analysed during the genetic diagnosis of F5F8D in this population. This is the first report describing the molecular analysis of a consistent number of F5F8D patients of South Indian origin, a population with a high frequency of such recessive bleeding disorders.

Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2

Mutations in LMAN1 (ERGIC-53) or MCFD2 cause combined deficiency of factor V and factor VIII (F5F8D). LMAN1 and MCFD2 form a protein complex that functions as a cargo receptor ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. In this study, we analyzed 10 previously reported and 10 new F5F8D families. Mutations in the LMAN1 or MCFD2 genes accounted for 15 of these families, including 3 alleles resulting in no LMAN1 mRNA accumulation. Combined with our previous reports, we have identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 71 of 76 families. Among the 5 families in which no mutations were identified, 3 were due to misdiagnosis, with the remaining 2 likely carrying LMAN1 or MCFD2 mutations that were missed by direct sequencing. Our results suggest that mutations in LMAN1 and MCFD2 may account for all cases of F5F8D. Immunoprecipitation and Western blot analysis detected a low level of LMAN1-MCFD2 complex in lymphoblasts derived from patients with missense mutations in LMAN1 (C475R) or MCFD2 (I136T), suggesting that complete loss of the complex may not be required for clinically significant reduction in FV and FVIII.

A mutation in LMAN1 (ERGIC-53) causing combined factor V and factor VIII deficiency is prevalent in Jews originating from the island of Djerba in Tunisia.

Combined deficiency of factor V and factor VIII is a rare autosomal recessive bleeding disorder that is caused by mutations in the LMAN1 or MCFD2 genes. These genes encode for proteins that form a complex that takes part in the transport of factor V and factor VIII from the endoplasmic reticulum to Golgi. Two mutations in LMAN1 have been observed in Jews: a guanine (G) insertion in exon 1 among Middle Eastern Jewish families, and a thymidine (T) to cytosine (C) transition in intron 9 at a donor splice site among Tunisian families. For each mutation, haplotype analysis revealed a founder effect. Because all affected Tunisian families belong to an ancient Jewish community in the island of Djerba off the coast of Tunisia, we screened members of this community for the intron 9 T --> C transition. Among 233 apparently unrelated individuals five heterozygotes were detected, predicting an allele frequency of 0.0107 (95% confidence interval, 0.0035-0.0248), while among 259 North African Jews none was found to carry the mutation. The prevalence of the mutation in Djerba Jews is consistent with the observation that all affected Tunisian Jewish families have origins in Djerba and with the finding of a common haplotype for the 9 + 2 T --> C mutation. The G insertion in exon 1 was found in one of 245 Iraqi Jews, predicting an allele frequency of 0.0022 (95% confidence interval, 0.0001-0.0123), but in none of 180 Iranian Jews examined. In view of the relatively low frequency of the mutations in the respective populations it seems reasonable to advocate carrier detection and prenatal diagnosis only in affected families.

Inherited Combined Factors V and VIII deficiency is caused by impaired association of two ERGIC proteins LMAN-1 and MCFD2

Inherited Combined Factors V and VIII deficiency is caused by impaired association of two ERGIC proteins LMAN-1 and MCFD2