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Prepublished online as a Blood First Edition Paper on September 26, 2002; DOI 10.1182/blood-2002-07-2321.
Submitted August 1, 2002
Accepted September 16, 2002
Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency
Richard van Wijk, Wouter W van Solinge*, Claus Nerlov, Ernest Beutler, Terri Gelbart, Gert Rijksen, and Finn C Nielsen
Department of Clinical Chemistry, University Medical Center Utrecht, Utrecht, The Netherlands
EMBL Mouse Biology Programme, Monterotondo - Scalo, Italy
Department of Molecular & Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA
Department of Hematology, University Medical Center Utrecht, Utrecht, The Netherlands
Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark
* Corresponding author; email: wsolinge{at}lab.azu.nl.
We established the molecular basis for pyruvate kinase deficiency in a male caucasian patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the common PKLR c.1529G>A mutation, known to be associated with PK-deficiency. On the maternal allele, three in cis mutations were identified in the erythroid-specific promoter region of the gene - one deletion of thymine -248 and two single nucleotide substitutions nt -324T>A and nt -83G>C. Analysis of the patient's RNA demonstrated the presence of only the 1529A allele, indicating severely reduced transcription from the allele linked to the mutated promoter region. Transfection of promoter constructs into erythroleukemic K562 cells showed that the most upstream -324T>A and -248delT mutations were non-functional polymorphisms. In contrast, the -83G>C mutation strongly reduced promoter activity. Site directed mutagenesis of the promoter region revealed the presence of a putative regulatory element (PKR-RE1) whose core binding motif CTCTG is located between nts -87 and -83. Electrophoretic mobility shift assay using K562 nuclear extracts indicated binding of an, as yet, unidentified trans-acting factor. This novel element mediates the effects of factors necessary for regulation of pyruvate kinase gene expression during red cell differentiation and maturation.
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