Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency

Abstract

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. Sitedirected 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-yetunidentified 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. (Blood.

Fifteen novel mutations inPKLRassociated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK

“…The erythroid-specific promoter and individual exons of PKLR, including flanking intronic regions, were amplified by PCR as previously described [van Wijk et al, 2003]. DNA sequence analysis was performed using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) on an ABI 310 Genetic Analyzer (Applied Biosystems).…”

“…Haplotype analysis using the erythroid-specific promoter polymorphism -248delT [van Wijk et al, 2003], and polymorphisms in intron 5 (c.507151C4T), intron 11 (variable number of ATT repeats), and exon 12 (c.1705A4C, c.1738C4T, and c.1992T4C) showed an identical haplotype in all families: T-T-16-C-T-C. This suggests a single origin for c.331G4A in the Dutch population; we designated this variant PK Utrecht.…”

Management of Gene Promoter Mutations in Molecular Diagnostics

“…In contrast, the -83G3 C strongly reduces in vitro promoter activity. We concluded that the -83G3 C mutation in the erythroid-specific promoter of PKLR strongly downregulates promoter activity in vitro (35 Fig. 3).…”

Gene promoter analysis in molecular diagnostics: do or don’t?

“…However, the incidence of reported promoter mutations in genes seems to depend on the extensiveness by which genes have been studied in the past and the severity of disease caused by the mutation [3]. Examples of diseases caused by promoter mutations are b-thalassemia [4], Bernard-Soulier syndrome [5], pyruvate kinase deficiency [6], familial hyper cholesterol emia [101] and hemo philia [7] (for a review, see [8]). In general, polymorphic sequence variations are considered to be rather harmless, especially if located in noncoding sections of a gene.…”