Pregnant women show a low level of protein S (PS) in plasma,which is known to be a risk for deep venous thrombosis. 17-Estradiol (E 2 ), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E 2 in HepG2-ER␣ cells, which stably express ER␣, and also the genomic ER signaling pathway, which modulates the liganddependent repression of the PS␣ gene (PROS1). We observed that E 2 repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E 2 down-regulated PROS1 promoter activity and that this E 2 -dependent repression disappeared upon the deletion or mutation of two adjacent GCrich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ER␣. In a chromatin immunoprecipitation assay, we found ER␣-Sp protein-promoter interaction involved in the E 2 -dependent repression of PROS1 transcription. Furthermore, we demonstrated that E 2 treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E 2 might repress PROS1 transcription depending upon ER␣-Sp1 recruiting transcriptional repressors in HepG2-ER␣ cells and, consequently, that high levels of E 2 leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women.
Female carriers of haemophilia B are usually asymptomatic; however, the disease resulting from different pathophysiological mechanisms has rarely been documented in females. In this study, we investigated the mechanisms responsible for haemophilia B in fraternal female twins. We sequenced the factor IX gene (F9) of the propositus, her father, a severe haemophilia B patient and the other family members. X chromosome inactivation was assessed by the methylation-sensitive HpaII-PCR assay using X-linked polymorphisms in human phosphoglycerate kinase 1 gene (PGK1) and glutamate receptor ionotropic AMPA 3 gene (GRIA3). The twins were found to be heterozygotes with a nonsense mutation (p.Arg384X) inherited from their father. The propositus, more severely affected twin, exhibited a significantly higher percentage of inactivation in the maternally derived X chromosome carrying a normal F9. The other twin also showed a skewed maternal X inactivation, resulting in a patient with mild haemophilia B. Thus, the degree of skewing of maternal X inactivation is closely correlated with the coagulation parameters and the clinical phenotypes of the twins. Furthermore, we identified a crossing-over in the Xq25-26 region of the maternal X chromosome of the more severely affected twin. This crossing-over was absent in the other twin, consistent with their fraternal state. Differently skewed X inactivation in the fraternal female twins might cause moderately severe and mild haemophilia B phenotypes, respectively.
Combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is an inherited bleeding disorder characterized by a reduction in plasma concentrations of FV and FVIII. F5F8D is genetically linked to mutations in either LMAN1 or MCFD2. Here, we investigated the molecular basis of F5F8D in a Japanese patient, and identified a novel missense mutation (p.Trp67Ser, c.200G>C) in the LMAN1, but no mutation in the MCFD2. The amount of LMAN1 in Epstein-Barr virus-immortalized lymphoblasts from the patient was found to be almost the same as that in cells from a normal individual. Interestingly, an anti-MCFD2 antibody did not co-immunoprecipitate the mutant LMAN1 with MCFD2 in lymphoblasts from the patient, suggesting the affinity of MCFD2 for the mutant LMAN1 is weak or abolished by the binding of the anti-MCFD2 antibody. In addition, a Myc/63His-tagged recombinant form of wild-type LMAN1 could bind to D-mannose, but that of the mutant could not. The p.Trp67Ser mutation was located in the carbohydrate recognition domain (CRD), which is thought to participate in the selective binding of LMAN1 to the D-mannose of glycoproteins as well as the EF-motif of MCFD2. Taken together, it was suggested that the p.Trp67Ser mutation might affect the molecular chaperone function of LMAN1, impairing affinity for D-mannose as well as for MCFD2, which may be responsible for F5F8D in the patient. This is the first report of F5F8D caused by a qualitative defect of LMAN1 due to a missense mutation in LMAN1. Am. J. Hematol. 84:738-742, 2009. V V C 2009 WileyLiss, Inc. IntroductionCoagulation factor V (FV) and factor VIII (FVIII) are both essential in the blood coagulation cascade, as cofactors for the proteases factor X and factor IX, respectively. Combined deficiency of FV and FVIII (F5F8D) is an autosomal recessive bleeding disorder first described by Oeri et al. in 1954 [1], and a distinct clinical entity from chance co-inheritance of hemophilia A (FVIII deficiency) and parahemophilia (FV deficiency). F5F8D is extremely rare (1:2,000,000) in the general population [2], and characterized by a mild-to-moderate bleeding tendency manifested after surgical trauma, abortion, and delivery. Menorrhagia is also common, but hematuria and gastrointestinal bleeding are infrequent, and hemarthrosis is rare [3]. Generally, patients with F5F8D show plasma levels of FV and FVIII in the range of 5-30% of normal [4].Positional cloning has identified two genes, LMAN1 (lectin, mannose-binding, 1; also known as ERGIC-53) and MCFD2 (multiple coagulation factor deficiency gene 2), associated with F5F8D [4,5] LMAN1 is a Type-1 transmembrane protein that cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC) [6,7]. It contains a mannose-specific carbohydrate recognition domain (CRD) on the ER luminal side, and ER exit and retrieval motifs on the cytoplasmic side [8]. MCFD2 has an EF-hand domain that interacts with LMAN1 in a Ca 21 -dependent manner [5]. The LMAN1-MCFD2 protein complex functions as a cargo ...
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