Polymorphisms and mutations of human TMPRSS6 in iron deficiency anemia.

Male subjects with iron deficiency from the general population were examined for polymorphisms or sporadic mutations in TMPRSS6 to identify genetic risk factors for iron deficiency anemia. Three uncommon non-synonymous polymorphisms were identified, G228D, R446W, and V795I (allele frequencies 0.0074, 0.023 and 0.0074 respectively), of which the R446W polymorphism appeared to be overrepresented in the anemic population. In addition, three children with iron refractory iron deficiency anemia, and one sibling with iron responsive iron deficiency anemia were also examined for polymorphisms or sporadic mutations in TMPRSS6. Two children (family 1) were compound heterozygotes for a L674F mutation and a previously described splicing defect predicted to cause skipping of exon 13 (IVS13+1 G>A). One child from the second family was homozygous for a deletion (497T) causing a frameshift (L166X+36) and premature termination. The sibling and mother from the second family were compound heterozygotes for the L166X mutation and the uncommon R446W polymorphism. Although in vitro expression studies demonstrated that the R446W isoform was biologically similar to wildtype Tmprss6, clinical data indicate that the R446W produces a milder disease when carried in trans with severe mutation in Tmprss6. The four children carrying mutations in TMPRSS6 all exhibited inappropriately high urinary hepcidin levels for the degree of iron deficiency.

Chronic non-spherocytic hemolytic anemia associated with severe neurological disease due to gamma-glutamylcysteine synthetase deficiency in a patient of Moroccan origin.

A previously undescribed mutation of hereditary gamma-glutamylcysteine synthetase (GCS) deficiency was found in a 5 year old boy of Moroccan origin. He presented with chronic haemolytic anaemia, delayed psychomotor development and progressive motor sensitive neuropathy of lower extremities. The parents were third degree relatives. The activity of glycolytic enzymes were found to be normal in the propositus, his parents and a sister, but and a complete lack of GSH was found in the propositus. Accordingly, the measurement of de novo GSH synthetic enzymes was undertaken, and severe GCS deficiency was found in the propositus. Both parents and his sister presented GCS activity ranging from 69% to 90% of normal. GCS gene sequencing showed that the propositus was homozygous for a 1241C>T mutation in exon 11 and both parents and his sister were heterozygous. This mutation predicts a Pro414Leu amino acid substitution. Even though the homology between GCS and crystallographically solved, functionally related proteins is not very high, a three-dimensional model of GCS was derived using Modeller Software. GCS deficiency is a very rare autosomal recessive disorder reported so far in only 8 unrelated probands with severe haemolytic anaemia. In only 3 of these was the anaemia associated with severe neurological dysfunction. We report here the fourth case of GCS deficiency presenting neuropathy, giving further support to the eventual relationship between this enzymopathy and neurological damage.

Thrombotic phenotype of mice with a combined deficiency in plasminogen activator inhibitor 1 and vitronectin.

OBJECTIVE: The role of vitronectin (VN) in thrombosis is not fully understood, primarily because this adhesive glycoprotein not only stabilizes plasminogen activator inhibitor 1 (PAI-1) and thus protects fibrin from premature lysis, but also because it binds to platelet integrins and may influence platelet aggregation. The absence of quantitative approaches to characterize the thrombi formed in animal models under different conditions further complicates this analysis. METHODS: In this report, we describe a more comprehensive approach to assess the stability of thrombi formed in mice deficient in PAI-1 (PAI-1(-/-)), VN (VN(-/-)) or both (PAI-1(-/-)/VN(-/-)). RESULTS: We observed that all deficient mice developed unstable thrombi compared with wild type (WT) mice. Thus, only 31% of the thrombi formed in WT mice were unstable compared with 74% of PAI-1(-/-), 80% of VN(-/-), and 87% of PAI-1(-/-)/VN(-/-) mice. In this regard, the average number of emboli per WT mouse was significantly lower (0.55) compared with VN(-/-) (2.66), PAI-1(-/-) (2.1), and VN(-/-)/PAI-1(-/-) (2.35) mice. Finally, the total duration of complete vascular occlusion was higher and the rate of vascular patency was lower in the WT mice compared with the deficient mice. CONCLUSIONS: Taken together, these observations indicate that the thrombotic phenotype of mice with a combined deficiency in PAI-1 and VN does not differ significantly from the phenotype of mice with deficiencies in only PAI-1 or VN. This observation suggests that PAI-1 and VN may influence thrombus stability by regulating a common pathway.

Tissue distribution of factor VIII gene expression in vivo--a closer look.

Previous studies have shown that factor VIII (FVIII) is expressed by multiple tissues. However, little is known about its cellular origin or its level of expression in different organs. In the present study, we examined FVIII gene expression in different tissues on a quantitative basis. Most of the tissues, especially liver and kidney, expressed high levels of FVIII mRNA compared to their level of expression of other hemostatic proteins, including von Willebrand factor (VWF). This was unexpected since FVIII is a trace protein. In situ hybridization analysis confirmed that liver and kidney were rich in FVIII mRNA. In the liver, a clear hybridization signal was detected in cells lining the sinusoids. FVIII mRNA analysis of purified liver cells confirmed the expression of FVIII mRNA by sinusoidal endothelial cells and Kupffer cells. Low but significant levels of FVIII mRNA were also detected in the hepatocytes. VWF mRNA was not detectable in these cells. Similarly, immunohistochemical staining of liver tissue revealed that FVIII protein is primarily associated with sinusoidal cells. VWF protein was predominantly located in the endothelium of larger vessels. In the kidney, FVIII synthesis was localized to the glomeruli and to tubular epithelial cells. Taken together, these results suggest that besides hepatocytes, non-parenchymal cells (e.g. sinusoidal endothelial cells) contribute to FVIII synthesis. VWF synthesis is primarily confined to extra-hepatic tissues.

Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin.

Plasminogen activator inhibitor 1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin (VN), a domain present in at least seven other proteins. In this study, we investigate the PAI-1 binding activity of these SMB homologs and attempt to more specifically localize the PAI-1 binding site within this domain. SMBVN and several of its homologs were expressed in Escherichia coli, purified, and tested for PAI-1 binding activity in a competitive ligand binding assay. Although recombinant SMBVN was fully active in this assay, none of the homologs bound to PAI-1 or competed with VN for PAI-1 binding. These inactive homologs are structurally related to SMBVN, having 33-45% sequence identity and containing all 8 cysteines at conserved positions. Thus, homolog-scanning experiments were conducted by exchanging progressively larger portions of the NH2- or COOH-terminal regions of active SMBVN with the corresponding regions of the inactive homologs. These experiments revealed that the minimum PAI-1-binding sequence was present in the central region (residues 12-30) of SMBVN. Alanine scanning mutagenesis further demonstrated that each of the 8 cysteines as well as Gly12, Asp22, Leu24, Try27, Tyr28, and Asp34 were critical for PAI-1 binding and were required to stabilize PAI-1 activity. These results indicate that the PAI-1 binding motif is localized to residues 12-30 of SMBVN and suggest that this motif is anchored in the active conformation by disulfide bonds.

Vitronectin gene expression in vivo. Evidence for extrahepatic synthesis and acute phase regulation.

A competitive polymerase chain reaction (PCR) assay was developed to quantitate vitronectin (Vn) mRNA in murine tissues using a synthetic RNA as an external standard. Although the liver contained the highest concentration of Vn mRNA, significant levels were also detected in the brain (25-fold less) and in adipose tissue, heart, and skeletal muscle (100-fold less than liver). Lower concentrations also were detected in the lung, uterus, testis, and thymus, and little or no Vn mRNA could be detected in kidney, spleen, and blood. These results indicate that significant amounts of Vn mRNA are produced in extrahepatic organs. The regulation of Vn gene expression in vivo was studied in a murine model system in which acute systemic inflammation was induced by endotoxin administration. Plasma Vn levels increased 2- to 3-fold within 16 h after endotoxin administration and remained elevated for up to 72 h. This increase appeared to result from increased synthesis in the liver since the steady-state level of hepatic Vn mRNA increased 4-fold after endotoxin administration. Moreover, Vn mRNA levels in heart, lung, and brain were not significantly increased by endotoxin. These results suggest that Vn gene expression in vivo is regulated in a tissue-specific manner and identify Vn as a novel acute phase reactant.

A computer model for evaluating the costs of cholecystectomy or ursodiol treatment in the management of cholelithiasis.

Over 500,000 cholecystectomies are performed each year in the United States. The procedure has its risks (mortality is 1.2% in the general population and 3% in patients aged 75 years or over) and it is expensive: the average direct cost of each cholecystectomy is about $10,000, for a national total of $5 billion annually. An alternative to surgery is to dissolve the stone with ursodiol, a naturally occurring bile acid, which has been successfully used for cholesterol gallstones with a diameter less than 20 mm. A computer model was developed to compare the costs of surgery for gallstones and treatment with ursodiol. In a hypothetical health maintenance organization with one million enrollees, use of ursodiol treatment rather than cholecystectomy would result in cumulative savings of $8.5 million after one year, $30.7 million after five years, and $57.8 million after ten years.

Holliday junctions in FLP recombination: resolution by step-arrest mutants of FLP protein.

The FLP "recombinase" of the 2-micron circle yeast plasmid can resolve synthetic FLP site-Holliday junctions. Mutants of the FLP protein that are blocked in recombination but are normal in substrate cleavage can also mediate resolution. The products of resolution by these mutants are almost exclusively nicked molecules with a protein-bound 3' end. There is no significant asymmetry in strand cleavage (top versus bottom) by the mutants in linear or in circular FLP substrates; nor is there a bias in resolution (toward parentals or toward recombinants) of Holliday junctions (corresponding to top- or to bottom-strand exchange) by wild-type FLP. During normal FLP recombination, a small amount of the expected Holliday intermediate can be detected.