Dr. Richard van Wijk is leader of the UMC Utrecht Red Blood Cell Center and group leader of the Laboratory for Red Blood Cell Research. The UMC Utrecht Red Blood Cell Center is a national and international Center of Expertise for benign disorders of the red blood cell. The Center is formed by the Laboratory for Red Blood Cell Research in strong collaboration with the Section Advanced Diagnostics in Haematology (Department of Clinical Chemistry and Haematology). Thereby, it involves the close collaboration of clinicians Marc Bierings and Roger Schutgens (Department of Hematology).
Dr. van Wijk is consultant in the diagnosis of hereditary red cell disorders, especially those associated with haemolytic anaemia and erythrocytosis. Furthermore, he directs translational research on these disorders by taking questions from clinical hematology into the research laboratory.
1. Consolidating and expanding the UMC Utrecht Red Blood Cell Center.
Background
Erythroenzymopathies are a group of rare hereditary hemolytic anemias caused by abnormalities in the genes encoding the enzymes of the main metabolic erythrocyte pathways. Over the past few years these disorders have been the object of intensive research which has resulted in a better understanding of their molecular basis. However, erythroenzymopathies remain a rare group of diseases of difficult diagnosis and little knowledge regarding the genotype-to-phenotype correlation. To date there are only few recognized national and international centres for the referral of blood samples or patients, of which UMC Utrecht is one. The main area of expertise is in the field red cell disorders due to defective glycolysis, in particular pyruvate kinase (PK) deficiency. A worldwide database of mutations associated with PK deficiency has been constructed and is constantly being updated (http://www.pklrmutationdatabase.com). It serves as an important tool to study structure-function relationship.
The idiopathic erythrocytosis (IE) group of disorders is defined by an absolute increase in red cell mass and hematocrit without elevation of the megakaryocytic or granulocytic lineages. This rare disorder is associated with a wide range of serum erythropoietin (Epo) levels and broadly falls into groups of raised/inappropriately normal or low/undetectable Epo levels. Recently, a spectrum of molecular defects has been described in association with IE, which reflects the heterogeneity of this disorder. These defects often result in aberrant oxygen sensing and dysregulated Epo production. Our work has recently expanded into this area which is of strong interest by both the clinical and scientific community.
General aims
1. To consolidate and expand the UMC Utrecht Red Blood Cell Center as a national and international Center of Expertise for advanced diagnostics (biochemical and molecular), treatment, and research on patients with benign disorders of the red blood cell.
Internationally, this work is supported by recently acknowledged EU funding (PHEA).
2. As many patients are diagnosed with hemolytic anemia e.c.i. we aim to identify new causes of hemolysis in this important group of patients using state-of-the-art proteomics-based technology.
3. To study the molecular basis of IE. This will provide insights into the control of Epo synthesis and Epo-induced signaling pathways.
2. Patient-based translational research
Regularly, patients and disease-associated clinical problems are encountered that are worth pursuing from a basic science perspective.
General aim
1. In general, this research is aimed at gaining insight into the molecular mechanism of disease and the identification of proteins or genes which potentially modify the phenotypic expression of disease: the genotype-to-phenotype correlation.
Approximately nine such projects are currently running in our laboratory. Three representative examples are:
First mutation in the red blood cell-specific promoter of hexokinase combined with a novel missense mutation cause hexokinase deficiency and mild chronic hemolysis
Hexokinase (HK) deficiency is a very rare cause of hereditary hemolytic anemia. To date, <20 cases have been described. We were the first to identify a regulatory mutation in the erythroid-specific promoter of the HK1 gene in a patient with HK deficiency. Using transient transfection of HK promoter constructs in human K562 erythroleukemia cells, electrophoretic mobility shift assays, chromatin immunoprecipitation assays, (quantitative) RT-PCR, and DNA sequence analysis we aim to establish the precise mechanism by which this mutation causes HK deficiency.
Evaluation of the protein structural context of missense mutations in PK using the 3-dimensional model of PK.
The majority of mutations associated with PK deficiency are point mutations predicting the substitution of a single amino acid. Using the three-dimensional model of tetrameric human erythrocyte PK the protein structural context of mutated residues and the molecular perturbation induced upon mutation is evaluated. The results provide us with a rationale for the observed enzyme deficiency and contribute to both a better understanding of the genotype-to-phenotype correlation in PK deficiency as well as the enzyme’s structure and function. Results are placed in a database and available worldwide via http://www.pklrmutationdatabase.com
Multiple defects in the oxygen-sensing pathway can lead to inappropriate up-regulation of HIF and Epo production, leading to erythrocytosis.
Erythropoietin (Epo) synthesis is tightly regulated by the hypoxia inducible factor (HIF). Three different subunits give rise to three isoforms of HIF. The subunit is proteasomally regulated in the presence of oxygen by hydroxylation of a proline in the oxygen-dependent degradation (ODD) domain of HIF, catalysed by members of the prolyl hydroxylase domain (PHD) family of enzymes. This allows the von Hippel Lindau (VHL) protein to associate with the subunit, which is subsequently tagged with ubiquitin and degraded by the proteasome. Any defect in the oxygen sensing pathway that allows the subunit to escape proteasomal regulation leads to elevated expression of HIF target genes, including Epo. In three patients with unexplained erythrocytosis, novel mutations were identified in the genes encoding VHL, PHD2, and HIF2. The novel mutants will be functionally characterized by binding assays, hydroxylase assays, real-time PCR, and Western blotting of recombinant wild-type and mutant proteins. The results will contribute to the current understanding of proteins implicated in the pathway that senses oxygen and transmits it to signals that eventually propogate erythroid progenitors.
On this project are working under supervision of Prof. Dr. W.W. van Solinge and Dr. R. van Wijk:
- B.A. van Oirschot, technician
- K.M. de Vooght, Ph.D. student
- Ing. A.C.W. van Wesel, technician
Recent publications
Will follow.