Study of (increased) expression of red blood cell pyruvate kinase antigen and ineffective erythropoiesis in patients with red blood cell pyruvate kinase deficiency.

The mature red blood cell is completely dependent on glycolysis for it*s energy
supply. Pyruvate Kinase (PK) is a key enzyme of glycolysis. Deficiency of this
protein is the most frequently occuring glycolytic enzyme disorder and an
important cause of hereditary non-spherocytic haemolytic anaemia (van Wijk and
van Solinge 2005). Clinical symptoms of PK deficiency are usually limited to
patients who are compound heterozygous or homozygous for a mutation in PKLR.
The phenotypic expression is highly variable (Zanella, et al 2007), and
patients with identical genotypes may show a diverse clinical picture (van Wijk
and van Solinge 2006). Also, there is no relationship between the residual
enzymatic activity and the severity of haemolysis. Hence, in PK deficiency the
genotype-to-phenotype correlation is poor. Metabolically, deficiency of PK
results in decreased enzymatic activity and, consequently, ATP depletion and
increased levels of 2,3-diphosphoglycerate. Eventually this leads to red cell
destruction but the precise mechanisms that lead to a shortened lifespan of the
PK-deficient red blood cell are as-yet unknown (Valentine and Paglia 1980).
The number of haematopoietic progenitors, including CFU-GM, BFU-E and CFU-GEMM,
in the spleen of PK-deficient patients are much higher than in spleens of
control subjects (Aizawa, et al 2003). This indicates that increased
extramedullary haematopoiesis takes place in PK-deficient patients. Of
particular interest is the fact that cells undergoing apoptosis have been
detected in splenic red pulp of a PK deficient patient whereas there was an
absence of detectable apoptotic cells in control samples (Aizawa, et al 2003).
This strongly suggests that PK activity is required for the maturation of
erythroid progenitors by preventing these cells from apoptosis. Also in mice it
has been shown that red blood cell PK deficiency is associated with ineffective
erythropoiesis (Aizawa, et al 2005). In addition, further studies have revealed
that over-expression of recombinant wild-type PK in a PK-deficient cell murine
cell line is able to downregulate expression of pro-apoptotic genes, such as
Bad, Bnip3, and Bnip3l (Aisaki, et al 2007).
Recently we have recently shown that loss of PK enzymatic activity is not
accompanied by a quantitative reduction in the amount of PK but, rather, is
associated with (strongly) increased levels of PK antigen in about half of the
patients examined (Van Wijk, et al 2009). This is an intriguing observation in
light of the fact that in many of these patients the underlying mutation in
PKLR predicts instability of the PK monomer and/or tetramer. The results
suggest that the metabolic disturbances induced by PK deficiency lead to an
increased expression of PKLR during erythroid differentiation and maturation.
Consequently, these observations led us to hypothesize that increased
expression of PK may be compensatory, i.e. to correct for the loss of enzymatic
activity, but may also serve to protect the PK-deficient erythroid progenitor
cell from apoptosis by downregulating pro-apoptopic gene expression. PK-antigen
levels may thus be an important determinant of the ultimate clinical phenotype
of patients with PK deficiency and, as such, important for a better
understanding of the complex genotype-to-phenotype correlation in this disease.

Aisaki, K., Aizawa, S., Fujii, H., Kanno, J. & Kanno, H. (2007) Glycolytic
inhibition by mutation of pyruvate kinase gene increases oxidative stress and
causes apoptosis of a pyruvate kinase deficient cell line. Exp Hematol, 35,
1190-1200.
Aizawa, S., Harada, T., Kanbe, E., Tsuboi, I., Aisaki, K., Fujii, H. & Kanno,
H. (2005) Ineffective erythropoiesis in mutant mice with deficient pyruvate
kinase activity. Exp Hematol, 33, 1292-1298.
Aizawa, S., Kohdera, U., Hiramoto, M., Kawakami, Y., Aisaki, K.-I., Kobayashi,
Y., Miwa, M., Fujii, H. & Kanno, H. (2003) Ineffective erythropoiesis in the
spleen of a patient with pyruvate kinase deficiency. Am J Hematol, 74, 68-72.
Valentine, W.N. & Paglia, D.E. (1980) The primary cause of hemolysis in
enzymopathies of anaerobic glycolysis: a viewpoint. Blood Cells, 6, 819-829.
Van Wijk, R., Huizinga, E.G., Van Wesel, A.C.W., Van Oirschot, B.A., Hadders,
M.A. & van Solinge, W.W. (2009) Fifteen novel mutations in PKLR associated with
pyruvate kinase (PK) deficiency: structural implications of amino acid
substitutions in PK. Hum Mutat, 30, 446-453.
van Wijk, R. & van Solinge, W.W. (2005) The energy-less red blood cell is lost:
erythrocyte enzyme abnormalities of glycolysis. Blood, 106, 4034-4042.
van Wijk, R. & van Solinge, W.W. (2006) Pyruvate kinase deficiency: genotype to
phenotype. Hematology (EHA Educ Program), 2, 55-62.
Zanella, A., Fermo, E., Bianchi, P., Chiarelli, L.R. & Valentini, G. (2007)
Pyruvate kinase deficiency: the genotype-phenotype association. Blood Rev, 21,
217-231.

Primary Objective: The here described study aims to strengthen the recently
obtained results regarding increased expression of PK antigen in patients with
PK-deficiency. This will be achieved by increasing the number of investigated
patients and control subjects. The level of PK antigen will be correlated to
the clinical phenotype and the genotype.
Secondary Objective(s): This study further aims to investigate the capability
of human PK-deficient haematopoietic stem cells to differentiate into erythroid
cells, and to study pro-apoptotic gene expression in PK-deficient erythroid
cells.

This study will be conducted as an observationel case control study. Blood
samples will be collected from 25 PK-deficient patients, and 25 control
subjects. These samples will be used to investigate:
1. the level of PK-antigen in mature red blood cells
2. the in-vitro ability of haematopoietic stem cells to form BFU-E*s and CFU-E*s
3. the expression of pro-apoptotic gene expression (e.g. BAD, BNIP3, BNIP3l) in
erythroid progenitor cells.

Patients and control subjects will undergo a single drawing of 35 mL of blood
by
venapunction. In case of children under the age of 12 years, this amount will
be limited to 20 mL. To limit any discomfort, the collection of blood for
this study will be combined with scheduled routine venapunctions for control
visits.