Pediatric Diamond-Blackfan anemia in the Netherlands: An overview of clinical characteristics and underlying molecular defects.

INTRODUCTION: Diamond-Blackfan anemia (DBA) is characterized by hypoplastic anemia, congenital anomalies, and a predisposition for malignancies. Most of our understanding of this disorder stems from molecular studies combined with extensive data input from international patient registries. OBJECTIVES: To create an overview of the pediatric DBA population in the Netherlands. METHODS: Forty-three patients diagnosed with DBA from all Dutch university pediatric hospitals were included in this study, and their clinical and genetic characteristics were collected from patient records. RESULTS: Congenital malformations were present in 24 of 43 patients (55.8%). An underlying genetic defect was identified in 26 of 43 patients (60.5%), the majority of which were found in the RPS19 gene (12 of 43, 27.9%) with 1 patient carrying a mutation in a novel DBA candidate gene, RPL9. In 31 of 35 (88.6%) patients, an initial response to glucocorticoid treatment was observed. Six patients (14.0%) underwent hematopoietic stem cell transplantation, and eleven patients (11 of 43, 25.6%) became treatment-independent spontaneously. CONCLUSION: In agreement with previous reports, the Dutch pediatric DBA population is both clinically and genetically heterogeneous. National and international registries, together with more extensive genetic testing, are crucial to increase our understanding of genotype and phenotype correlations of this intriguing disorder.

Role of SHP2 (PTPN11) in glycoprotein VI-dependent thrombus formation: Improved platelet responsiveness by the allosteric drug SHP099 in Noonan syndrome patients.

INTRODUCTION: The protein tyrosine phosphatase SHP2 (PTPN11) is a negative regulator of glycoprotein VI (GPVI)-induced platelet signal under certain conditions. Clinical trials with derivatives of the allosteric drug SHP099, inhibiting SHP2, are ongoing as potential therapy for solid cancers. Gain-of-function mutations of the PTPN11 gene are observed in part of the patients with the Noonan syndrome, associated with a mild bleeding disorder. Assessment of the effects of SHP2 inhibition in platelets from controls and Noonan syndrome patients. MATERIALS AND METHODS: Washed human platelets were incubated with SHP099 and stimulated with collagen-related peptide (CRP) for stirred aggregation and flow cytometric measurements. Whole-blood microfluidics assays using a dosed collagen and tissue factor coating were performed to assess shear-dependent thrombus and fibrin formation. Effects on clot formation were evaluated by thromboelastometry. RESULTS: Pharmacological inhibition of SHP2 did not alter GPVI-dependent platelet aggregation under stirring, but it enhanced integrin αIIbß3 activation in response to CRP. Using whole-blood microfluidics, SHP099 increased the thrombus buildup on collagen surfaces. In the presence of tissue factor and coagulation, SHP099 increased thrombus size and reduced time to fibrin formation. Blood from PTPN11-mutated Noonan syndrome patients, with low platelet responsiveness, after ex vivo treatment with SHP099 showed a normalized platelet function. In thromboelastometry, SHP2 inhibition tended to increase tissue factor-induced blood clotting profiles with tranexamic acid, preventing fibrinolysis. CONCLUSION: Pharmacological inhibition of SHP2 by the allosteric drug SHP099 enhances GPVI-induced platelet activation under shear conditions with a potential to improve platelet functions of Noonan syndrome patients.

[Disorders of the central lymphatic system]. / Stoornissen in het centrale lymfestelsel.

The central lymphatic system consists of the thoracic duct, cisterna chyli and the retroperitoneal lymphatics through which lymph and chyle flows. Disorders of the central lymphatic system can for instance lead to leakage (chylothorax), accumulation of fluid (lymphedema) and retrograde flow (protein losing enteropathy). Abnormalities in the central lymphatic system were overlooked for years, followed by lack of diagnostic and therapeutic options. This has changed, as the technique of intranodal contrast injection in inguinal lymph nodes brought renewed interest in the central lymphatic system. In this article, the importance of intranodal contrast injection in diagnosis and treatment of disorders of the central lymphatic system will be presented through 3 clinical cases.

Influence of genetic variants in TPMT and COMT associated with cisplatin induced hearing loss in patients with cancer: two new cohorts and a meta-analysis reveal significant heterogeneity between cohorts.

Treatment with cisplatin-containing chemotherapy regimens causes hearing loss in 40-60% of cancer patients. It has been suggested that genetic variants in the genes encoding thiopurine S-methyltransferase (TPMT) and catechol O-methyltransferase (COMT) can predict the development of cisplatin-induced ototoxicity and may explain interindividual variability in sensitivity to cisplatin-induced hearing loss. Two recently published studies however, sought to validate these findings and showed inconsistent results. The aim of this study was to evaluate the role of polymorphisms in the TPMT and COMT genes in cisplatin-induced ototoxicity. Therefore we investigated two independent cohorts of 110 Dutch and 38 Spanish patients with osteosarcoma and performed a meta-analysis including all previously published studies resulting in a total population of 664 patients with cancer. With this largest meta-analysis performed to date, we show that the influence of TPMT and COMT on the development of cisplatin-induced hearing loss may be less important than previously suggested.

Production of hemopexin by TNF-alpha stimulated human mesangial cells.

BACKGROUND: Plasma hemopexin has been shown to induce proteinuria after intrarenal infusion in rats, as well as glomerular alterations identical to those seen in corticosteroid-responsive nephrotic syndrome (CRNS). The question emerged whether also renal cells are potentially able to release hemopexin. METHODS: Normal human mesangial cells (HMC) were incubated overnight in serum-free medium with or without tumor necrosis factor-alpha (TNF-alpha) (10 ng/mL). Parallel cultures were supplemented with prednisolone (10-3 mol/L). Concentrated supernatants were analyzed by Western blotting, using antihemopexin immunoglobulin G (IgG). Antitransferrin IgG served as control antibody. In addition, cytospins were stained using polyclonal or monoclonal antihemopexin IgG. A part of the cells was used for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR), to study hemopexin mRNA. RESULTS: Eighty five kD bands were exclusively detected by antihemopexin IgG in the Western blots in supernatants from TNF-alpha-stimulated cultures and to a lesser extent in prednisolone-treated cultures. Cells from TNF-alpha-stimulated cultures stain positive for hemopexin in contrast to those from prednisolone-treated or nonstimulated cultures. RT-PCR data suggest that mRNA for hemopexin is up-regulated in TNF-alpha-treated versus prednisolone-treated HMC. CONCLUSION: Stimulated HMC are able to release hemopexin in vitro in a corticosteroid-dependent manner. As preliminary data indicate that mesangial hemopexin is able to affect glomerular anionic sites, it is conceivable that stimulated mesangium may contribute to enhanced glomerular permeability in CRNS through local hemopexin release.

Decrease of thrombomodulin contributes to the procoagulant state of endothelium in hemolytic uremic syndrome.

The typical form of hemolytic uremic syndrome (D+HUS) is a thrombotic microangiopathy that causes acute renal failure in children. The etiology of this disease is a toxin called Shiga-like toxin (Stx), present in certain strains of gram-negative bacteria. Vascular endothelial cell (EC) injury appears to be central in the pathogenesis of D+HUS. Thrombomodulin (TM) is a glycoprotein present in EC with anti-thrombogenic properties. The objective of this study was to investigate the effects of Stx on the surface expression of TM in EC using an in vitro culture of human glomerular microvascular endothelial cells. We also evaluated other inflammatory mediators [tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide], which are known to increase Stx receptor expression and are potentially involved in the pathogenesis of D+HUS. Stx2 induced a significant decrease of TM expression in this cell type after pre-incubation with TNF-alpha. This decrease could not be attributed to the inhibition of protein synthesis only, as cycloheximide, another inhibitor of protein synthesis, did not affect TM surface expression. These results suggest that the Stx2-induced decrease of TM expression in glomerular EC might contribute to the local procoagulant state present in D+HUS.