Platelets from Calreticulin mutated essential thrombocythemia patients are less reactive than JAK2 V617F mutated platelets.

Both JAK2V617F and calreticulin (CALR) mutated essential thrombocythemia (ET) patients have different clinical characteristics, with lower thrombosis risk in patients with CALR mutations. To elucidate the mechanism for this lower risk we studied platelet function in ET patients with either JAK2V617F or a CALR mutation. Platelet activation state was similar in ET and healthy controls at baseline using P-selectin and PAC1 flow cytometry analysis. However, CALR mutated platelets were significantly less activated following ADP stimulation, compared to control or JAK2 mutated platelets (P < .001). In live-cell imaging of platelet attachment to immobilized fibrinogen by Interference Reflection Microscopy (IRM), the number of attached CALR mutated platelets was lower compared to control and JAK2 mutated platelets, with lower fractions of platelets achieving the fully spread state (90%, 78% and 54% of adherent cells for control, JAK2 and CALR mutated subjects, respectively). Compared to controls, ET patients, regardless of the mutation type, had increased numbers of immature platelets (IP) and leukocyte platelet aggregates (LPA), as well as plasma sP-selectin. These were all correlated with the platelet count and not to the state of platelet activation. We also found that intracellular free Ca2+ was increased in resting ET compared to control platelets. Note, CALR had a more dispersed localization in activated ET platelets compared to healthy controls, and mutated CALR interact physically with TpoR in CALR mutated platelets. We hypothesize that defects in platelet activation and spreading in CALR mutated patients can explain, at least in part, the lower thrombotic tendency in CALR mutated ET patients.

The reduced form of coagulation factor XI is associated with illness severity and coagulopathy in critically-ill septic patients.

Coagulation Factor XI (FXI) contributes to the pathobiology of sepsis-associated thrombosis and is a target for new therapeutics. Through cleavage of disulfide bonds, FXI becomes reduced (rFXI), accelerating intrinsic coagulation cascade activation. The role of rFXI in human sepsis has never been studied. We determined levels of total FXI and rFXI in critically-ill septic patients with and without overt disseminated intravascular coagulation (DIC, a dysregulated pro-thrombotic condition). Total FXI and rFXI plasma levels were measured on ICU admission in prospectively enrolled septic patients (n = 32) from three academic medical centers and matched, healthy controls (n = 15). In septic patients, hematologic and physiologic parameters and pathological thrombosis (presence or absence of overt DIC) were determined. rFXI was higher in septic patients than controls (p < 0.05). In septic patients, rFXI was significantly associated with platelet count (r = 0.3511, p < 0.05) and APACHE II score (r = - 0.359, p < 0.05), indices of illness severity. rFXI was lower in patients with overt DIC (p = 0.088), suggesting a consumptive coagulopathy. In contrast, while total FXI levels were reduced in sepsis, they failed to correlate with illness severity, thrombosis, or hematologic parameters. We establish, for the first time, that rFXI is increased in patients with sepsis and correlates with illness severity (APACHE II score and platelet count) and pathological coagulopathy (overt DIC). Total FXI levels, in contrast, are decreased in sepsis but fail to associate with any indices. These findings suggest that rFXI has unique activity in human sepsis.

Persistent neonatal thrombocytopenia can be caused by IgA antiplatelet antibodies in breast milk of immune thrombocytopenic mothers.

Immune thrombocytopenia (ITP) in pregnant women can cause neonatal thrombocytopenia by transport of antiplatelet autoantibodies across the placenta. Usually, an infant's platelet count normalizes within 2 months. We observed neonatal thrombocytopenia that persisted more than 4 months and disappeared following discontinuation of breastfeeding. The aim of our study was to discern whether breast milk of ITP mothers contained antiplatelet antibodies causing persistent thrombocytopenia. We collected milk samples from 3 groups of women: ITP group, 7 women who had ITP during pregnancy; R-ITP group, 6 women who recovered from ITP before pregnancy; and 9 healthy controls. We found increased levels of antiplatelet antibodies of the immunoglobulin A type in the milk of ITP patients compared with the other 2 groups. Similar increase was demonstrated for antibodies binding to αIIbß3 expressed in cultured cells. Thus, transfer of antiplatelet antibodies from ITP mothers by breastfeeding can be associated with persistent neonatal thrombocytopenia.

Thromboembolic events in patients with severe inherited fibrinogen deficiency.

Inherited afibrinogenemia and hypofibrinogenemia are rare bleeding disorders characterized by markedly reduced levels of fibrinogen in blood. Thrombotic complications in these disorders have been rarely described. We performed a multicenter retrospective study and reviewed the occurrence of thrombotic complications among patients with inherited fibrinogen deficiency. Cases were identified during a review of medical records of all patients with inherited fibrinogen deficiency followed at three different university hospitals in Israel. Nine patients were included in this study: five were afibrinogenemic and four hypofibrinogenemic. There were seven thrombotic events, mostly venous, that occurred in four out of nine patients (44 %). All thrombotic events occurred in afibrinogenemic patients. Mean age at the time of thrombosis was 45 (range 28-61) years. Thrombophilic evaluation performed was negative in all cases. At the time of thrombosis in five out of seven (71.4 %) events, fibrinogen replacement therapy was concurrently given. Therapeutic approach was different among patients ranging from supportive therapy alone, antiplatelet agents and anticoagulant therapy with the concurrent administration of fibrinogen replacement therapy. This study discloses a high rate of thrombosis in patients with afibrinogenemia. Events were both venous and arterial and may be recurrent. Management is highly problematic due to the precarious balance between bleeding and thrombotic risk in these patients. Fibrinogen replacement therapy should be cautiously used in these patients as most thrombotic events followed the administration of fibrinogen replacement therapy. Larger cohorts are warranted to better characterize the best management strategy in these paradoxical events.

Unique disulfide bonds in epidermal growth factor (EGF) domains of ß3 affect structure and function of αIIbß3 and αvß3 integrins in different manner.

The ß3 subunit of αIIbß3 and αvß3 integrins contains four epidermal growth factor (EGF)-like domains. Each domain harbors four disulfide bonds of which one is unique for integrins. We previously discerned a regulatory role of the EGF-4 Cys-560-Cys-583 unique bond for αIIbß3 activation. In this study we further investigated the role of all four integrin unique bonds in both αIIbß3 and αvß3. We created ß3 mutants harboring serine substitutions of each or both cysteines that disrupt the four unique bonds (Cys-437-Cys-457 in EGF-1, Cys-473-Cys-503 in EGF-2, Cys-523-Cys-544 in EGF-3, and Cys-560-Cys-583 in EGF-4) and transfected them into baby hamster kidney cells together with normal αv or αIIb. Flow cytometry was used to measure surface expression of αIIbß3 and αvß3 and their activity state by soluble fibrinogen binding. Most cysteine substitutions caused similarly reduced surface expression of both receptors. Disrupting all four unique disulfide bonds by single cysteine substitutions resulted in variable constitutive activation of αIIbß3 and αvß3. In contrast, whereas double C437S/C457S and C473S/C503S mutations yielded constitutively active αIIbß3 and αvß3, the C560S/C583S mutation did not, and the C523S/C544S mutation only yielded constitutively active αIIbß3. Activation of C523S/C544S αvß3 mutant by activating antibody and dithiothreitol was also impaired. Molecular dynamics of C523S/C544S ß3 in αIIbß3 but not in αvß3 displayed an altered stable conformation. Our findings indicate that unique disulfide bonds in ß3 differently affect the function of αIIbß3 and αvß3 and suggest a free sulfhydryl-dependent regulatory role for Cys-560-Cys-583 in both αIIbß3 and αvß3 and for Cys-523-Cys-544 only in αvß3.

Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex.

Mutations in LMAN1 (also called ERGIC-53) result in combined deficiency of factor V and factor VIII (F5F8D), an autosomal recessive bleeding disorder characterized by coordinate reduction of both clotting proteins. LMAN1 is a mannose-binding type 1 transmembrane protein localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC; refs. 2,3), suggesting that F5F8D could result from a defect in secretion of factor V and factor VIII (ref. 4). Correctly folded proteins destined for secretion are packaged in the ER into COPII-coated vesicles, which subsequently fuse to form the ERGIC. Secretion of certain abundant proteins suggests a default pathway requiring no export signals (bulk flow; refs. 6,7). An alternative mechanism involves selective packaging of secreted proteins with the help of specific cargo receptors. The latter model would be consistent with mutations in LMAN1 causing a selective block to export of factor V and factor VIII. But approximately 30% of individuals with F5F8D have normal levels of LMAN1, suggesting that mutations in another gene may also be associated with F5F8D. Here we show that inactivating mutations in MCFD2 cause F5F8D with a phenotype indistinguishable from that caused by mutations in LMAN1. MCFD2 is localized to the ERGIC through a direct, calcium-dependent interaction with LMAN1. These findings suggest that the MCFD2-LMAN1 complex forms a specific cargo receptor for the ER-to-Golgi transport of selected proteins.

A unique interaction between alphaIIb and beta3 in the head region is essential for outside-in signaling-related functions of alphaIIbbeta3 integrin.

The main interface of the 2 subunits of platelet integrin alphaIIbbeta3 comprises the beta-propeller domain of alphaIIb and the betaA domain of beta3. In the center of the beta-propeller, several aromatic residues interact by cation-pi and hydrophobic bonds with Arg261 of betaA. In this study, we substituted alphaIIb-Trp110 or beta3-Arg261 by residues abundant in other alpha or beta subunits at corresponding locations and expressed them in baby hamster kidney cells along with normal beta3 or alphaIIb, respectively. These mutant cells displayed normal surface expression and fibrinogen binding but grossly impaired outside-in signaling-related functions: adhesion to immobilized fibrinogen, cell spreading, focal adhesion kinase phosphorylation, clot retraction, and reduced alphaIIbbeta3 stability in EDTA (ethylenediaminetetraacetic acid). Expression of mutants with substitutions of Arg261 in beta3 by alanine or lysine with normal alphav yielded normal surface expression of alphavbeta3 and soluble fibrinogen binding as well as normal outside-in signaling-related functions, contrasting findings for alphaIIbbeta3. Structural analysis of alphaIIbbeta3 and alphavbeta3 revealed that alphavbeta3 has several strong interactions between alphav and beta3 subunits that are missing in alphaIIbbeta3. Together, these findings indicate that the interaction between Trp110 of alphaIIb and Arg261 of beta3 is critical for alphaIIbbeta3 integrity and outside-in signaling-related functions.

Discerning the ancestry of European Americans in genetic association studies.

European Americans are often treated as a homogeneous group, but in fact form a structured population due to historical immigration of diverse source populations. Discerning the ancestry of European Americans genotyped in association studies is important in order to prevent false-positive or false-negative associations due to population stratification and to identify genetic variants whose contribution to disease risk differs across European ancestries. Here, we investigate empirical patterns of population structure in European Americans, analyzing 4,198 samples from four genome-wide association studies to show that components roughly corresponding to northwest European, southeast European, and Ashkenazi Jewish ancestry are the main sources of European American population structure. Building on this insight, we constructed a panel of 300 validated markers that are highly informative for distinguishing these ancestries. We demonstrate that this panel of markers can be used to correct for stratification in association studies that do not generate dense genotype data.

The association of factor V leiden and prothrombin gene mutation and placenta-mediated pregnancy complications: a systematic review and meta-analysis of prospective cohort studies.

BACKGROUND: Factor V Leiden (FVL) and prothrombin gene mutation (PGM) are common inherited thrombophilias. Retrospective studies variably suggest a link between maternal FVL/PGM and placenta-mediated pregnancy complications including pregnancy loss, small for gestational age, pre-eclampsia and placental abruption. Prospective cohort studies provide a superior methodologic design but require larger sample sizes to detect important effects. We undertook a systematic review and a meta-analysis of prospective cohort studies to estimate the association of maternal FVL or PGM carrier status and placenta-mediated pregnancy complications. METHODS AND FINDINGS: A comprehensive search strategy was run in Medline and Embase. Inclusion criteria were: (1) prospective cohort design; (2) clearly defined outcomes including one of the following: pregnancy loss, small for gestational age, pre-eclampsia or placental abruption; (3) maternal FVL or PGM carrier status; (4) sufficient data for calculation of odds ratios (ORs). We identified 322 titles, reviewed 30 articles for inclusion and exclusion criteria, and included ten studies in the meta-analysis. The odds of pregnancy loss in women with FVL (absolute risk 4.2%) was 52% higher (OR = 1.52, 95% confidence interval [CI] 1.06-2.19) as compared with women without FVL (absolute risk 3.2%). There was no significant association between FVL and pre-eclampsia (OR = 1.23, 95% CI 0.89-1.70) or between FVL and SGA (OR = 1.0, 95% CI 0.80-1.25). PGM was not associated with pre-eclampsia (OR = 1.25, 95% CI 0.79-1.99) or SGA (OR 1.25, 95% CI 0.92-1.70). CONCLUSIONS: Women with FVL appear to be at a small absolute increased risk of late pregnancy loss. Women with FVL and PGM appear not to be at increased risk of pre-eclampsia or birth of SGA infants. Please see later in the article for the Editors' Summary.

Genotype-phenotype correlation in combined deficiency of factor V and factor VIII.

Combined deficiency of factor V and factor VIII (F5F8D) is caused by mutations in one of 2 genes, either LMAN1 or MCFD2. Here we report the identification of mutations for 11 additional F5F8D families, including 4 novel mutations, 2 in MCFD2 and 2 in LMAN1. We show that a novel MCFD2 missense mutation identified here (D81Y) and 2 previously reported mutations (D89A and D122V) abolish MCFD2 binding to LMAN1. Measurement of platelet factor V (FV) levels in 7 F5F8D patients (4 with LMAN1 and 3 with MCFD2 mutations) demonstrated similar reductions to those observed for plasma FV. Combining the current data together with all previous published reports, we performed a genotype-phenotype analysis comparing patients with MCFD2 mutations with those with LMAN1 mutations. A previously unappreciated difference is observed between these 2 classes of patients in the distribution of plasma levels for FV and factor VIII (FVIII). Although there is considerable overlap, the mean levels of plasma FV and FVIII in patients with MCFD2 mutations are significantly lower than the corresponding levels in patients with LMAN1 mutations. No differences in distribution of factor levels are observed by sex. These data suggest that MCFD2 may play a primary role in the export of FV and FVIII from the ER, with the impact of LMAN1 mediated indirectly through its interaction with MCFD2.

[Inherited bleeding disorders common in Jews].

Four inherited disorders of hemostasis have been identified in Jews with a relatively high frequency: Factor XI deficiency, factor VII deficiency, combined factor V and VIII deficiency and GLanzmann thrombasthenia. During the past decades, the bleeding manifestations of these disorders, the diagnosis, the molecular-genetic basis and therapy have been discerned. Furthermore, the prevalence of the respective mutant genes have been delineated in various Jewish Communities. Each one of the disorders can serve as a model enabling better understanding of the pathophysioLogy of the coagulation systems. On the basis of data obtained from the research of Glanzmann thrombasthenia, several widely used drugs have been developed as effective antithrombotic agents.

Recombinant activated factor VII and tranexamic acid are haemostatically effective during major surgery in factor XI-deficient patients with inhibitor antibodies.

One-third of patients with severe factor XI (FXI) deficiency caused by homozygosity for null alleles develop inhibitor antibodies following exposure to plasma. Haemostasis during surgery is achievable in such patients by recombinant activated factor VII (rFVIIa) at doses used in haemophilia A patients with an inhibitor to FVIII. However, thrombosis has occurred in three of 12 such patients. In this study we discerned whether low-dose rFVIIa would secure haemostasis and cause no thrombosis in patients with severe FXI deficiency and an inhibitor during surgery. In vitro, a very low concentration of rFVIIa (0.24 microg/ml) induced thrombin generation in FXI-deficient plasma quite similarly to 1.9 microg/ml (a concentration that is achieved in patients with haemophilia A and inhibitor after infusion of 80 microg/kg). Based on this finding, a protocol was designed for four patients with severe FXI deficiency and an inhibitor or immunoglobulin A deficiency who underwent five major surgical procedures. This included administration of tranexamic acid from two hours before surgery until seven to 14 days after, and single infusion of low-dose rFVIIa. No excessive bleeding or thrombosis were observed. In conclusion, a single low dose of rFVIIa and tranexamic acid secure normal haemostasis in patients with severe FXI deficiency who can not receive blood products.

Low Concentrations of Recombinant Factor VIIa May Improve the Impaired Thrombin Generation of Glanzmann Thrombasthenia Patients.

INTRODUCTION: Glanzmann thrombasthenia (GT) is a rare bleeding disorder. The disease is caused by the lack or dysfunction of platelet membrane glycoprotein IIb/IIIa (integrin αIIbß3) which is essential for platelet aggregation. Bleeding episodes are usually managed by platelet transfusions. Recombinant activated factor VII (rFVIIa) is a common adjunct or alternative treatment option. OBJECTIVE: This article evaluates GT patients' response to increasing concentrations of rFVIIa using an ex vivo thrombin generation assay to elaborate the knowledge in which rFVIIa treats a platelet dysfunction for bleeding episodes and preoperative management. MATERIALS AND METHODS: Twenty-four GT patients in a non-bleeding state were enrolled into the study. Thrombin generation was measured in platelet-rich plasma (PRP) in the presence of 0.7 to 7.0 µg/mL rFVIIa. Clinical data of rFVIIa used to treat GT patients' bleeding episodes was collected, and patients' follow-up course was documented. RESULTS: Thrombin generation was significantly decreased in GT patients compared with controls. An individual response to rFVIIa spiking was noted in GT patients' PRP. In the majority of patients, a significant improvement in thrombin generation was already demonstrated with low concentrations (0.7 µg/mL) of rFVIIa. CONCLUSION: Thrombin generation is improved in the majority of GT patients following ex vivo spiking with rFVIIa. The magnitude of this improvement is individual and was noted at low concentrations of rFVIIa. There is a need for a prospective clinical trial to find the optimal doses or rFVIIa for treatment of GT patients.

Simultaneous genotyping of coagulation factor XI type II and type III mutations by multiplex real-time polymerase chain reaction to determine their prevalence in healthy and factor XI-deficient Italians.

BACKGROUND: Factor XI deficiency is a rare autosomal recessive coagulopathy, which is, however, common among Ashkenazi Jews, in whom the so-called type II (E117X) and type III (F283L) mutations account for 98% of alleles. In non-Jewish populations, a higher level of allelic heterogeneity has been reported. However, the type II mutation was found in individuals from England, Portugal, and Italy, and haplotype analysis confirmed its Jewish origin. The aims of this study were to develop a rapid and accurate assay for the simultaneous detection of type II/type III mutations and to determine the frequency of these mutations in a large Italian population of healthy individuals and in a cohort of factor XI-deficient Italian patients. DESIGN AND METHODS: Type II and III mutations were detected using a newly developed multiplex four-color real-time polymerase chain reaction assay. Haplotype analysis was performed by either DNA sequencing or fragment-length analysis. RESULTS: Both type II and type III mutations were found among 3879 healthy Italians with an allele frequency of 0.00064 and 0.00051, respectively. Among the 31 analyzed factor XI-deficient patients, the type II mutation was found in three individuals in the homozygous state and in eight individuals in the heterozygous state (one compound heterozygote type II/III). Haplotype analysis revealed the Jewish origin of both mutations. CONCLUSIONS: The newly developed assay is highly specific and reliable (0.02% false positives); and offers a useful means for the molecular diagnosis of factor XI deficiency. Type II and III mutations are present in the Italian population and should be searched for first in factor XI-deficient patients.

Disulfide bond disruption by a beta 3-Cys549Arg mutation in six Jordanian families with Glanzmann thrombasthenia causes diminished production of constitutively active alpha IIb beta 3.

Alpha IIb beta 3 integrin mediates platelet aggregation following its activation. Its absence or dysfunction causes Glanzmann thrombasthenia (GT), an inherited bleeding disorder that is rare worldwide but relatively frequent in several populations with high rates of consanguinity, including Arabs in Israel and Jordan. Cysteine residues in the beta 3 epidermal growth factor (EGF) domains are involved in alpha IIb beta 3 formation and activation. In this study we present a novel Cys549Arg mutation in beta 3 identified in six Jordanian families, which in the homozygous state is manifested by severe GT. The mutation is located in EGF-3 of beta 3 predicting disruption of a conserved disulfide bond between Cys549 and Cys558. Haplotype analysis disclosed a common founder whose age estimate was 120-150 years. Flow cytometry revealed 1-14% of normal alpha IIb beta 3 expression at the patients' platelet surface. The Cys549Arg or artificial Cys549Ser mutations were introduced into a beta 3 expression vector. Co-transfection of baby hamster kidney cells with normal or mutant beta 3 along with normal alpha IIb demonstrated reduced surface expression of alpha IIb beta 3 by both mutants. The mutants were constitutively active as demonstrated by 20-fold increased binding of the ligand-mimetic antibody PAC-1. Immunoblotting and immunoprecipitation experiments showed reduced beta 3 and alpha IIb beta 3 expression and a higher than normal ratio of pro-alpha IIb to mature alpha IIb. Immunofluorescence experiments showed that beta 3 and alpha IIb beta 3 were mostly retained in the endoplasmic reticulum. In conclusion, the novel ancestral mutation found in a cluster of Jordanian GT patients disrupts a conserved Cys549-Cys558 bond which results in reduced production of constitutively active alpha IIb beta 3.

Characterization of seven novel mutations causing factor XI deficiency.

BACKGROUND AND OBJECTIVES: Factor XI (FXI) deficiency is a rare autosomal recessive disorder, the main manifestation of which is injury-related bleeding. The disorder is rare in most populations, but common among Jews in whom two mutations, E117X and F283L, account for 98% of cases. Other mutations, C38R and C128X, are prevalent in French Basques and Britons, respectively. Additional sporadic mutations have been described in most parts of the world. The objective of this study was to identify the mutations in 15 unrelated FXI-deficient patients and characterize missense mutations by expression in baby hamster kidney (BHK) cells. DESIGN AND METHODS: Clinical and laboratory information and DNA samples were obtained from the patients and mutations were identified by sequencing. Missense mutations were expressed in BHK cells and their effect on FXI secretion and dimerization was assessed using enzyme-linked immunosorbent assay and immunoblotting. RESULTS: Of 16 mutations detected, seven are novel including two deletions, one splice site and four missense mutations. Expression of the four novel missense mutations (C58Y, Y427C, C527Y and V20A) in cells revealed no secretion of FXI-C58Y, Y427C and C527Y and secretion of only 22% of normal in the medium for FXI-V20A. Secretion of FXI from BHK cells harboring a previously reported E297K substitution cells was also impaired (4.5% of wild-type). Homodimerization was normal for all five mutants. INTERPRETATION AND CONCLUSIONS: Defective homodimerization of FXI was previously recognized as a major mechanism for defective secretion of FXI from producing cells. In this study, five FXI missense mutations (four novel) were associated with impaired secretion albeit normal dimerization, underscoring the existence of other mechanisms for defective secretion.

The interval between prothrombin time tests and the quality of oral anticoagulants treatment in patients with chronic atrial fibrillation.

BACKGROUND: The incidence of stroke in patients with atrial fibrillation (AF) can be significantly reduced with warfarin therapy especially if optimally controlled. OBJECTIVES: To evaluate the effect of the interval between consecutive prothrombin time measurements on the time in therapeutic range (INR 2-3) in a cohort of patients with AF on chronic warfarin treatment in the community. METHODS: All INR measurements available from a relatively large cohort of patients with chronic AF were reviewed and the mean interval between consecutive INR tests of each patient was correlated with the time in therapeutic range (TTR). RESULTS: Altogether 251,916 INR measurements performed in 4408 patients over a period of seven years were reviewed. Sixty percent of patients had their INR measured on average every 2 to 3 weeks and most others were followed at intervals of 4 weeks or longer. A small proportion (3.6%) had their INR measured on average every week. A significant decline in the time in therapeutic range was observed as the intervals between tests increased. At one to three weeks interval the TTR was 48%, at 4 weeks interval 45% and at 5 weeks 41% (P<0.0005). A five percent increment in TTR was observed if more tests were performed at multiplications of exactly 7 days (43% vs 48% P<0.0001). CONCLUSIONS: A better control with an increase in the TTR was observed in patients with atrial fibrillation if prothrombin time tests are performed at regular intervals of no longer than 3 weeks.

Age estimates of ancestral mutations causing factor VII deficiency and Dubin-Johnson syndrome in Iranian and Moroccan Jews are consistent with ancient Jewish migrations.

Factor VII (FVII) deficiency and Dubin-Johnson syndrome (DJS) are rare autosomal recessive disorders caused by mutations in F7 and MRP2 genes, respectively. Both disorders are relatively frequent among Iranian and Moroccan Jews. FVII deficiency in both populations is caused by a founder A244V mutation in the F7 gene and DJS is caused by two founder mutations, I1173F and R1150H in the MRP2 gene that are specific for Iranian and Moroccan Jewish patients, respectively. We estimated the age of FVII A244V and MRP2 I1173F by analysis of microsatellite markers flanking F7 and MRP2 genes, respectively, in 13 Iranian Jewish homozygotes for the I1173F mutation and 21 Iranian and Moroccan Jewish homozygotes for the A244V mutation. Dating of the mutations was estimated by the DMLE+2.0 program employing observed linkage disequilibria of multiple genetic markers. The estimated age of the I1173F mutation was approximately 1500 years, and the age of the A244V mutation was approximately 2600 years. These estimates suggest that I1173F causing DJS in Iranian Jews occurred after the separation of Iranian Jews from Moroccan Jews 2000-2600 years ago, while A244V causing FVII deficiency in Iranian and Moroccan Jews occurred prior to the divergence of these two populations.

Molecular diversity of Glanzmann thrombasthenia in southern India: new insights into mRNA splicing and structure-function correlations of alphaIIbbeta3 integrin (ITGA2B, ITGB3).

The molecular basis of Glanzmann thrombasthenia (GT) was studied in 40 families from southern India. Of 23 identified mutations (13 in the alphaIIb (ITGA2B) gene and 10 in the beta3 (ITGB3) gene), 20 were novel and three were described previously. Three mutations in the beta3 gene-p.Leu143Trp (Leu117Trp), p.Tyr307Stop (Tyr281Stop), and p.Arg119Gln (Arg93Gln)-were detected in 12, three, and two families, respectively, with definite founder effects observed for the first two mutations. Alternative splicing was predicted in silico for the normal variant and a missense variant of the beta3 gene, and for 10/11 frameshift or nonsense mutations in alphaIIb or beta3. The prediction was confirmed experimentally for a c.2898_2902dupCCCCT mutation in exon 28 of the alphaIIb gene that induced exon skipping. Seven out of nine missense mutations substituted highly conserved amino acids buried in the proteins' cores, predicting structural abnormalities. Among these, a beta3 substitution, p.Cys39Gly (Cys13Gly) was found to cause intracellular degradation of the beta3 subunit, in contrast to previous findings that mutations at Cys435, the partner of Cys13 in a disulfide bond, cause constitutive activation of alphaIIbbeta3. The two patients with a beta3 Arg93Gln mutation had normal clot retraction, consistent with a recent finding that this substitution is associated with normal surface expression of alphaIIbbeta3. In conclusion, this study demonstrates that a variety of mutations account for GT in southern Indian patients, provides new insights into mRNA splicing, and highlights the role of specific amino acids in structure-function correlations of alphaIIbbeta3.

Inhibitors to Factor XI in patients with severe Factor XI deficiency.

Factor XI (FXI) deficiency is a rare bleeding disorder that may arise from any of a number of missense, nonsense, splice site, insertion, and deletion mutations within the FXI gene. Severely affected patients are at considerable risk of developing inhibitors to FXI and, although spontaneous bleeding is uncommon in such patients, bleeding after surgery or trauma can be severe. As treatment with fresh frozen plasma (FFP) or FXI concentrates is ineffective in patients with inhibitors, other therapies must be sought. Traditionally, such patients have been treated with various agents and methods, including plasma exchange, cyclophosphamide, intravenous immunoglobulin, and prothrombin complex concentrates. However, emerging data indicate that recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark) may also be effective in FXI deficiency with inhibitors. Further work is required to determine the optimal dosing schedules of the agent in this indication.