Elevated preoperative von Willebrand factor is associated with perioperative thrombosis in infants and neonates with congenital heart disease.

Essentials Perioperative thrombosis is a major cause of morbidity and mortality in congenital heart disease. Neonates and infants undergoing repair of congenital heart lesions were prospectively followed. Elevated von Willebrand factor (VWF) to ADAMTS-13 activity ratios typified the postoperative period. Thrombosis was associated with preoperative VWF activity and cryoprecipitate transfusion SUMMARY: Background The surgical repair of congenital heart malformations is frequently complicated by perioperative thrombosis of unclear etiology. An imbalance between von Willebrand factor (VWF) and ADAMTS-13 is an emerging variable in thrombosis. Objectives To describe perioperative changes to VWF, ADAMTS-13 and NETosis, and evaluate clinical and biochemical associations with postoperative thrombosis. Methods Neonates and infants undergoing palliation or definitive surgical repair of congenital heart malformations were recruited (n = 133). Preoperative and postoperative plasma levels of VWF, ADAMTS-13 and markers of NETosis were determined. Patients were followed for up to 30 days for the occurrence of thrombosis. Univariate and multivariate logistic regression analyses were conducted to identify variables associated with thrombosis. Results We identified significant postoperative increases in VWF activity, VWF level, DNA-histone complexes and cell-free DNA with an overall decrease in ADAMTS-13 activity. Patients experiencing postoperative thrombotic events (9%) were characterized by surgery performed at a lower intraoperative temperature, higher preoperative lactic acid levels, and higher preoperative VWF activity and level. A multivariate logistic regression model identified preoperative VWF activity (odds ratio (OR) 8.39 per IU mL-1 , 95% confidence interval [CI] 1.73-40.55) and transfusion of cryoprecipitate (OR 1.10 per mL kg-1 , 95% CI 1.03-1.17) as being associated with thrombosis. Conclusions Pediatric patients undergoing surgical repair of congenital heart malformations are exposed to high levels of VWF with diminished or minimal change to ADAMTS-13 in the immediate postoperative period. Elevated preoperative VWF activity is associated with postoperative thrombosis in pediatric congenital heart disease.

The importance of mRNA structure in determining the pathogenicity of synonymous and non-synonymous mutations in haemophilia.

INTRODUCTION: Mutational analysis is commonly used to support the diagnosis and management of haemophilia. This has allowed for the generation of large mutation databases which provide unparalleled insight into genotype-phenotype relationships. Haemophilia is associated with inversions, deletions, insertions, nonsense and missense mutations. Both synonymous and non-synonymous mutations influence the base pairing of messenger RNA (mRNA), which can alter mRNA structure, cellular half-life and ribosome processivity/elongation. However, the role of mRNA structure in determining the pathogenicity of point mutations in haemophilia has not been evaluated. AIM: To evaluate mRNA thermodynamic stability and associated RNA prediction software as a means to distinguish between neutral and disease-associated mutations in haemophilia. METHODS: Five mRNA structure prediction software programs were used to assess the thermodynamic stability of mRNA fragments carrying neutral vs. disease-associated and synonymous vs. non-synonymous point mutations in F8, F9 and a third X-linked gene, DMD (dystrophin). RESULTS: In F8 and DMD, disease-associated mutations tend to occur in more structurally stable mRNA regions, represented by lower MFE (minimum free energy) levels. In comparing multiple software packages for mRNA structure prediction, a 101-151 nucleotide fragment length appears to be a feasible range for structuring future studies. CONCLUSION: mRNA thermodynamic stability is one predictive characteristic, which when combined with other RNA and protein features, may offer significant insight when screening sequencing data for novel disease-associated mutations. Our results also suggest potential utility in evaluating the mRNA thermodynamic stability profile of a gene when determining the viability of interchanging codons for biological and therapeutic applications.

Genetic determinants of immunogenicity to factor IX during the treatment of haemophilia B.

Inhibitors are an impediment to the effective management of haemophilia B (HB), but there is limited understanding of the underlying genetic risk factors. In this study we aim to understand the role of F9 gene mutations on inhibitor development in patients with HB. Mutations in the F9 gene were identified and HLA typing performed for five boys with severe HB. Data from the CDC Haemophilia B Mutation Project (CHBMP) database were used to assess association between F9 gene mutation type and inhibitor development. Analysis of the CHBMP database showed that larger disruptions in the F9 gene are associated with a higher life-time prevalence of inhibitors. We detected the following mutations in the five subjects, including four novel mutations: Nonsense in three patients (c.223 C>T; p.Arg75* in two siblings, c.553 C>T; p.Glu185*); Splice site in two patients (c.723 + 1 G>A, c.278-27 A>G); Missense in one patient (c.580 A>G, p.Thr194Ala; c.723 G>T; p.Gln241His). Of the two siblings only one responded to immune tolerance induction (ITI). These siblings have identical F9 gene mutations but differ with respect to the HLA alleles. Interestingly, an analysis of peptide-MHC binding affinities shows a significantly higher (one-sided unpaired t-test, P = 0.0018) median affinity for FIX-derived peptides in the sibling that responded to ITI. We conclude that the nature of the F9 gene mutation may be an important risk factor for the development of inhibitors. In addition, the HLA alleles of the individual patients, in conjunction with the mutation type, could be a predictor for the development of inhibitors as well as the response to ITI.

Factor IX oligomerization underlies reduced activity upon disruption of physiological conditions.

Coagulation factor IX (FIX) is a serine protease that plays a pivotal role in the blood coagulation cascade. FIX deficiency leads to a blood clotting disorder known as haemophilia B. FIX, synthesized as a prepro-peptide of 461 amino acids, is processed and secreted into plasma. The protein undergoes numerous modifications, including, but not limited to glycosylation, γ-carboxylation and disulphide bond formation. Upon processing and limited proteolysis, the protein is converted into an active protease. Under physiological conditions, the FIX zymogen is a monomer. The purpose of this work was to analyse the conditions that may affect FIX monomeric state and promote and/or reduce oligomerization. Using native gel electrophoresis and size exclusion chromatography, we found that under decreased pH and ionic strength conditions, the FIX zymogen can oligomerize, resulting in the formation of higher molecular weight species, with a concomitant reduction in specific activity. Similarly, FIX oligomers formed readily with low bovine serum albumin (BSA) concentrations; however, increased BSA concentrations impeded FIX oligomerization. We hypothesize that normal blood physiological conditions are critical for maintaining active FIX monomers. Under conditions of stress associated with acidosis, electrolyte imbalance and low albumin levels, FIX oligomerization is expected to take place thus leading to compromised activity. Furthermore, albumin, which is commonly used as a drug stabilizer, may enhance the efficacy of FIX biological drugs by reducing oligomerization.

Analysis of F9 point mutations and their correlation to severity of haemophilia B disease.

Haemophilia B is an X-linked recessive disorder caused by deficiency of functional coagulation factor IX, which results almost exclusively from mutations in the F9 gene. We sought to determine features, which could distinguish between mutations that cause severe disease symptoms from those that cause non-severe disease symptoms. Towards this objective, we have performed a statistical analysis of reported point mutations in F9. These include: potential local changes in mRNA free energy, codon usage, charge and type of mutated amino acid, location of the mutation with regard to protein secondary structure and functional domain and amino acids' evolutionary conservation scores. Wilcoxon signed-rank tests showed highly significant differences between severe and non-severe disease causing mutations in their effect on free energy of small mRNA fragments and evolutionarily conserved amino acids. Our results suggest that information at the mRNA level as well as conservation of the amino acid correlate well with disease severity. This study demonstrates that computational tools may be used to characterize the severity of haemophilia B associated with point mutations and suggests their utility in predicting the outcome of sequence changes in recombinant proteins.

Cyclosporin A-dependent downregulation of the Na+/Ca2+ exchanger expression.

Cyclosporin A (CsA) is an immunosuppressive drug commonly given to transplant patients. Its application is accompanied by severe side effects related to calcium, among them hypertension and nephrotoxicity. The Na+/Ca2+ exchanger (NCX) is a major calcium regulator expressed in the surface membrane of all excitable and many nonexcitable tissues. Three genes, NCX1, NCX2, and NCX3 code for Na+/Ca2+ exchange activity. NCX1 gene products are the most abundant. We have shown previously that exposure of NCX1-transfected HEK 293 cells to CsA, leads to concentration-dependent reduction of Na+/Ca2+ exchange activity and surface expression, without a reduction in total cell-expressed NCX1 protein. We show now that the effect of CsA on NCX1 protein expression is not restricted to transfected cells overexpressing the NCX1 protein but exhibited also in cells expressing endogenously the NCX1 protein (L6, H9c2, and primary smooth muscle cells). Exposure of NCX2- and NCX3-transfected cells to CsA results also in reduction of Na+/Ca2+ exchange activity and surface expression, though the sensitivity to the drug was lower than in NCX1-transfected cells. Studying the molecular mechanism of CsA-NCX interaction suggests that cyclophilin (Cyp) is involved in NCX1 protein expression and its modulation by CsA. Deletion of 426 amino acids from the large cytoplasmic loop of the protein retains the CsA-dependent downregulation of the truncated NCX1 suggesting that CsA-Cyp-NCX interaction involves the remaining protein domains.

SV40 Pseudovirion gene delivery of a toxin to treat human adenocarcinomas in mice.

SV40 vectors packaged in vitro (pseudovirions) are an efficient delivery system for plasmids up to 17.7 kb, with or without SV40 sequences. A truncated Pseudomonas exotoxin gene (PE38) was delivered into various human cells (HeLa, KB-3-1, human lymphoblastoids, and erythroleukemia cells), in vitro using pseudovirions. The number of viable cells was reduced significantly in the PE38-transduced cells. Human KB adenocarcinomas growing in mice were treated with intratumoral injection of PE38 packaged in vitro, and tumor size decreased significantly. Intraperitoneal treatments were as effective in reducing tumor size as intratumoral treatments. To check the viability of mock- or PE38-treated mice, every 4 days they were weighed, their blood was tested, and various tissues were screened for pathology. All parameters showed that the in vitro-packaged vectors, injected into tumors or intraperitoneally, caused no abnormalities in mice. The combined treatment of doxorubicin with in vitro-packaged PE38 reduced tumor size slightly more than each of the treatments separately. However, the combined treatment did not cause the weight loss seen with doxorubicin alone. These results indicate that SV40 in vitro packaging is an effective system for cancer gene delivery using two different routes of injection and in combination with chemotherapy.

Relative abundance of growth hormone receptor isoforms in rhesus monkey tissues and human transformed lymphocytes.

The growth hormone receptor (GHR) is expressed as one active, full-sequence isoform and one truncated, inactive one that lacks the intracellular signaling domain. The aim of this study was to investigate the variation in the tissue expression of the full and truncated mRNA and protein. Epstein-Barr virus-transformed human B lymphocyte lines were established from 9 normal individuals with a height standard deviation score (SDS) of - 0.1 +/- 1.1 (mean +/- SD). Tissues were also collected from 3 Rhesus monkeys, whose GHR has 94.1 % homology with the human molecule. mRNA quantitation was determined by Real Time Quantitative PCR. Growth hormone receptor expression in transformed lymphocytes was also studied by fluorescence-activated cell sorter analysis. Both isoforms were expressed in transformed lymphocytes, but individual variation in the relative mRNA expression was small (truncated isoform percentage of total receptor mRNA: 17.1 +/- 4.4, mean +/- SD). There was no correlation between donors' height SDS and the expression of either isoform or the ratio between them. Protein expression by FACS analysis showed wider variation among the subjects; however, the relative ratio was similar in all the subjects. In monkey tissues, the truncated receptor showed a tissue-specific distribution. In conclusion, the expression of both isoforms in transformed lymphocytes from normal subjects shows small differences at the RNA or protein levels, and does not correlate with height SDS. Growth hormone splice isoforms show tissue specificity, suggesting local regulation of splicing. Tissues with relatively high expression of the truncated isoform are likely to be more resistant to the effects of GH due to the dominant negative effect of this isoform. In addition, the differential tissue expression might influence the levels of growth hormone binding protein in the immediate milieu of each tissue.

Transcriptional and translational regulation of the splicing isoforms of the growth hormone receptor by glucocorticoids.

Glucocorticoids exert multiple effects on the growth hormone IGF-I axis. The GH receptor is expressed as an active, full-sequence molecule and a truncated, inactive one that lacks the intracellular signaling domain. We used the HuH7 human hepatoma cell line to investigate the effect of glucocorticoids on growth hormone receptor mRNA and protein expression. cDNA quantification was performed by Real Time Quantitative PCR. Growth hormone receptor expression at the protein level was studied by fluorescence-activated cell sorter analysis using specific polyclonal antibodies raised against the two isoform unique C-terminal sequences. Cells were treated with pharmacologic doses of dexamethasone (Dex 10 -7 - 10 -5 M) to assess its acute (1 hour or overnight) and chronic effects (7 days). Dex induced a dose-dependent increase of both the full (427 %) and truncated (180 %) mRNAs. At the protein level, Dex upregulated the full sequence more (183 %) than the truncated (126 %) protein. For a better understanding of this regulation system, cells were incubated with Dex 10 -6 M for 24 h in the absence or presence of a transcriptional inhibitor, actinomycin D, or a translational inhibitor, cycloheximide. Actinomycin D had no effect on Dex-induced upregulation, while cycloheximide blocked the truncated mRNA but not the full sequence mRNA upregulation, suggesting that this effect of glucocorticoids is a post-transcriptional event. After 7 days of chronic treatment, Dex induced a dose-dependent downregulation of the active receptor without any changes in the expression of the truncated isoform either at the mRNA or protein levels. We conclude that short-term glucocorticoid treatment results in an enhancement of the growth hormone receptor expression, while long-term treatment has a suppressive effect, through both transcriptional and translational mechanisms ultimately influencing both isoforms of the receptor.

Differences in rhodamine-123 efflux in B-type chronic lymphocytic leukemia suggest possible gender and stage variations in drug-resistance gene activity.

Peripheral blood samples from 61 patients (36 male, 25 female) with all stages of B-type chronic lymphocytic leukemia (CLL) were studied for MDR1 phenotype using monoclonal antibodies and rhodamine-123 dye exclusion, a functional assay of MDR1 expression. The duration of the disease varied from 1 month to 22 years at the time of initial study. Overall, 74% of the patients were positive for rhodamine-123 exclusion. When analyzed by gender, significantly more men than women were positive (89% versus 48%, p<0.001). There were more positive men than women for every stage of the disease. Female patients were found to be either MDR1 phenotype positive or negative at any stage of the disease. In contrast, all male patients with early (stages 0-II) disease were MDR1 phenotype positive. One early-stage (stage II) male patient converted from rhodamine-efflux positive to rhodamine-efflux negative as he progressed from stage-II to stage-IV disease. We suggest that some of the differences in disease biology of male versus female CLL patients (women having a more benign course) may be due to gender-dependent differences in drug-resistance gene activity, including MDR1. Our results also emphasize the need to take into account gender in evaluating the clinical course of patients with CLL.

Efficient transduction of human hematopoietic cells with the human multidrug resistance gene 1 via SV40 pseudovirions.

Transduction of MDR1 may be of use in chemoprotection of normal bone marrow (BM) cells during treatment of malignancies, or as a selectable marker for the transfer of other genes into the BM, a critical target for the cure of many diseases. To that aim, the human multidrug resistance gene MDR1 was cloned into an SV40 pseudoviral vector containing the SV40 origin of replication (ori) and encapsidation signal (ses), and the plasmid was encapsidated in COS cells as SV40/MDR1 pseudovirions. Expression of the human MDR1 gene was demonstrated in murine MEL cells infected with SV40/MDR1 pseudovirions, using a monoclonal antibody (MPK16) specific for the human 170-kD P-glycoprotein. Functional P-glycoprotein was demonstrated by resistance to colchicine in NIH-3T3 cells infected with SV40/MDR1 pseudovirions. Activity of P-glycoprotein was assayed by rhodamine-123 dye exclusion and fluorescence-activated cell sorter analysis (FACS) in various cell types including hematopoietic cells. Highly efficient gene transfer and expression was demonstrated in all murine and human cell types tested, including primary human BM cells. Using multiplicities of infection (moi) of 1-2, over 95% of cells were found to become MDR1+. The percent of MDR1+ cells was proportional to the moi. We conclude that the SV40 pseudoviral vector is efficient for gene transmission into human hematopoietic cells.

Paracentric inversion X(q21.2q24) associated with mental retardation in males and normal ovarian function in females.

We describe a familial paracentric inversion (X)(q21.2 q24) in a family with 2 male and 2 female carriers. The males were mentally retarded and the females were normal with normal ovarian function. It is suggested that a recessive mental retardation (MR) gene was disrupted by one of the inversion breakpoints, although an X-linked MR gene which by chance is linked to the inv(X) could not be ruled out. In the female carriers of the paracentric inversion a random X-inactivation was demonstrated. The normal ovarian function is an exception to the concept of "critical region" at Xq13-q26.

Endoreduplication and polyploidy in fragile X cells induced by methotrexate and fluorodeoxyuridine: implications for diagnosis.

Lymphoblastoid cell lines from fragile X patients and amniotic cells from fragile X embryos, when cultured with methotrexate (MTX) or fluorodeoxyuridine (FUdR), showed a significant increase in endoreduplication and polyploidy. This phenomenon was not observed in fragile X lymphocytes or in lymphoblastoid cell lines and amniotic cells of normal control individuals. The relationship between the inducible fragile site at Xq27.3 and the inducible endoreduplication is discussed. The induction of endoreduplication and polyploidy in fragile X lymphoblasts and amniocytes is evaluated as a possible diagnostic test.