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Prepublished online as a Blood First Edition Paper on September 26, 2002; DOI 10.1182/blood-2002-07-2321.
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Blood, 15 February 2003, Vol. 101, No. 4, pp. 1596-1602
RED CELLS
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
From the Department of Clinical Chemistry and the
Department of Hematology, University Medical Center Utrecht, The
Netherlands; the EMBL Mouse Biology Programme,
Monterotondo-Scalo (RM), Italy; the Department of
Molecular and Experimental Medicine, The Scripps Research Institute, La
Jolla, CA; and the Department of Clinical Biochemistry, Rigshospitalet,
Copenhagen, Denmark.
We established the molecular basis for pyruvate kinase (PK)
deficiency in a white male patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the common
PKLR cDNA sequence (c.) 1529G>A mutation, known to
be associated with PK deficiency. On the maternal allele, 3 in cis
mutations were identified in the erythroid-specific promoter
region of the gene: one deletion of thymine  248 and 2 single
nucleotide substitutions, nucleotide (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 nonfunctional
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 nt 87 and nt 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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