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.
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).
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.
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.
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 μg/ml) induced thrombin generation in FXI-deficient plasma quite similarly to 1.9 μg/ml (a concentration that is achieved in patients with haemophilia A and inhibitor after infusion of 80 μg/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.
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 ∼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.
Integrins αvβ3 and αvβ5 have been long considered as proangiogenic receptors, yet genetic ablation studies demonstrated enhanced angiogenesis in mice lacking αvβ3 and αvβ5 integrins, which was ...attributed to increased expression of vascular endothelial growth factor receptor-2 (VEGFR-2). In this study, we determined the effect of αvβ3 and αvβ5 suppression in endothelial cells (EC) on vascular endothelial growth factor (VEGF) and VEGFR-2 expression. αv was suppressed by shRNA in HUVEC (venous endothelial cells) and HMVEC (microvascular endothelial cells). VEGFR-2 was significantly upregulated by αv suppression in HUVEC and downregulated in HMVEC at both mRNA and protein levels, as assessed by real-time PCR and flow cytometry, respectively. HMVEC displayed completely abolished Sp1 binding to the VEGFR-2 promoter, and HUVEC exhibited enhanced binding to the –170 E-Box element in the VEGFR-2 promoter, assessed by electrophoretic mobility shift assay. Realtime PCR also revealed opposite effects on the expression of 5 additional important genes involved in angiogenesis in the two cell types. VEGF mRNA expression was enhanced in HUVEC and reduced in HMVEC; however, these alterations were not statistically significant. VEGF-induced proliferation was upregulated in HUVEC and reduced in HMVEC following αv suppression. No tube formation on Matrigel was observed in αv suppressed cells, regardless of their origin. These results indicate that suppression of αv integrins can either augment or inhibit VEGFR-2 levels and VEGF-induced proliferation in EC from different vascular beds. Hence, therapeutic antiangiogenic intervention by siRNA- mediated suppression of αv integrins should take into account variable and potentially hazardous responses in different vascular beds.
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.
Dubin-Johnson syndrome (DJS) is an inherited disorder characterized by conjugated hyperbilirubinemia and is caused by a deficiency of the multidrug resistance protein 2 (MRP2) located in the apical ...membrane of hepatocytes. The aim of this study was to identify the mutations in two previously characterized clusters of patients with Dubin-Johnson syndrome among Iranian and Moroccan Jews and determine the consequence of the mutations on MRP2 expression and function by expression studies. All 32 exons and adjacent regions of the MRP2 gene were screened by polymerase chain reaction and DNA sequencing. Two novel mutations were identified in exon 25. One mutation, 3517A→T, predicting a I1173F substitution, was found in 22 homozygous Iranian Jewish DJS patients from 13 unrelated families and a second mutation, 3449G→A, predicting a R1150H substitution, was found in 5 homozygous Moroccan Jewish DJS patients from 4 unrelated families. Use of four intragenic dimorphisms and haplotype analyses disclosed a specific founder effect for each mutation. The mutations were introduced into an MRP2 expression vector by site-directed mutagenesis, transfected into HEK-293 cells, and analyzed by a fluorescence transport assay, immunoblot, and immunocytochemistry. Continuous measurement of probenecid-sensitive carboxyfluorescein efflux revealed that both mutations impaired the transport activity of MRP2. Immunoblot analysis and immunocytochemistry showed that MRP2 (R1150H) matured properly and localized at the plasma membrane of transfected cells. In contrast, expression of MRP2 (I1173F) was low and mislocated to the endoplasmic reticulum of the transfected cells. These findings provide an explanation for the DJS phenotype in these two patient groups. Furthermore, the close localization of the two mutations identify this region of MRP2 as important for both activity and processing of the protein.
Summary
αIIbβ3 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 β3 epidermal growth factor (EGF) domains are involved in αIIbβ3 formation and activation. In this study we present a novel Cys549Arg mutation in β3 identified in six Jordanian families, which in the homozygous state is manifested by severe GT. The mutation is located in EGF-3 of β3 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 αIIbβ3 expression at the patients' platelet surface. The Cys549Arg or artificial Cys549Ser mutations were introduced into a β3 expression vector. Co-transfection of baby hamster kidney cells with normal or mutant β3 along with normal αIIb demonstrated reduced surface expression of αIIbβ3 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 β3 and αIIbβ3 expression and a higher than normal ratio of pro-αIIb to mature αIIb. Immunofluorescence experiments showed that β3 and αIIbβ3 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 αIIbβ3.
Combined deficiency of factors V and VIII is an autosomal recessive bleeding disorder resulting from alterations in an unknown gene on chromosome 18q, distinct from the factor V and factor VIII ...genes. ERGIC-53, a component of the ER–Golgi intermediate compartment, was mapped to a YAC and BAC contig containing the critical region for the combined factors V and VIII deficiency gene. DNA sequence analysis identified two different mutations, accounting for all affected individuals in nine families studied. Immunofluorescence and Western analysis of immortalized lymphocytes from patients homozygous for either of the two mutations demonstrate complete lack of expression of the mutated gene in these cells. These findings suggest that ERGIC-53 may function as a molecular chaperone for the transport from ER to Golgi of a specific subset of secreted proteins, including coagulation factors V and VIII.
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