BACKGROUND: Hexokinase is one of the key enzymes of glycolysis and catalyzes the phosphorylation of glucose to glucose-6-phosphate. Red blood cell-specific hexokinase is transcribed from HK1 by use of an erythroid-specific promoter. The aim of this study was to investigate the molecular basis for hexokinase deficiency in a patient with chronic hemolysis. DESIGN AND METHODS: Functional studies were performed using transient transfection of HK promoter constructs in human K562 erythroleukemia cells. The DNA-protein interaction at the promoter of hexokinase was studied using electrophoretic mobility shift assays with nuclear extracts from K562 cells. DNA analysis and reverse transcriptase polymerase chain reaction were performed according to standardized procedures. RESULTS: On the paternal allele we identified two novel mutations in cis in the erythroid-specific promoter of HKI: -373A>C and -193A>G. Transfection of promoter reporter constructs showed that the -193A>G mutation reduced promoter activity to 8%. Hence, -193A>G is the first mutation reported to affect red blood cell-specific hexokinase specific transcription. By electrophoretic mobility shift assays we showed that in vitro binding of c-jun to an AP-1 binding site was disrupted by this mutation. Subsequent chromatin-immunoprecipitation assays demonstrated that c-jun binds this region of the promoter in vivo. On the maternal allele we identified a novel missense mutation in exon 3: c.278G>A, encoding an arginine to glutamine substitution at residue 93, affecting both hexokinase-1 and red cell specific-hexokinase. In addition, this missense mutation was shown to compromise normal pre-mRNA processing. CONCLUSIONS: We postulate that reduced erythroid transcription of HK1 together with aberrant splicing of both hexokinase-1 and red cell specific-hexokinase results in hexokinase deficiency and mild chronic hemolysis.
BACKGROUND:Hexokinase is one of the key enzymes of glycolysis and catalyzes the phosphorylation of glucose to glucose-6-phosphate. Red blood cell-specific hexokinase is transcribed from HK1 by use of an erythroid-specific promoter. The aim of this study was to investigate the molecular basis for hexokinase deficiency in a patient with chronic hemolysis. DESIGN AND METHODS: Functional studies were performed using transient transfection of HK promoter constructs in human K562 erythroleukemia cells. The DNA-protein interaction at the promoter of hexokinase was studied using electrophoretic mobility shift assays with nuclear extracts from K562 cells. DNA analysis and reverse transcriptase polymerase chain reaction were performed according to standardized procedures. RESULTS: On the paternal allele we identified two novel mutations in cis in the erythroid-specific promoter of HKI: -373A>C and -193A>G. Transfection of promoter reporter constructs showed that the -193A>G mutation reduced promoter activity to 8%. Hence, -193A>G is the first mutation reported to affect red blood cell-specific hexokinase specific transcription. By electrophoretic mobility shift assays we showed that in vitro binding of c-jun to an AP-1 binding site was disrupted by this mutation. Subsequent chromatin-immunoprecipitation assays demonstrated that c-jun binds this region of the promoter in vivo. On the maternal allele we identified a novel missense mutation in exon 3: c.278G>A, encoding an arginine to glutamine substitution at residue 93, affecting both hexokinase-1 and red cell specific-hexokinase. In addition, this missense mutation was shown to compromise normal pre-mRNA processing. CONCLUSIONS: We postulate that reduced erythroid transcription of HK1 together with aberrant splicing of both hexokinase-1 and red cell specific-hexokinase results in hexokinase deficiency and mild chronic hemolysis.
Authors: Richard van Wijk; Gert Rijksen; Eric G Huizinga; Hendrik K Nieuwenhuis; Wouter W van Solinge Journal: Blood Date: 2002-08-08 Impact factor: 22.113
Authors: Jacques Fellay; Alexander J Thompson; Dongliang Ge; Curtis E Gumbs; Thomas J Urban; Kevin V Shianna; Latasha D Little; Ping Qiu; Arthur H Bertelsen; Mark Watson; Amelia Warner; Andrew J Muir; Clifford Brass; Janice Albrecht; Mark Sulkowski; John G McHutchison; David B Goldstein Journal: Nature Date: 2010-02-21 Impact factor: 49.962
Authors: Volkan Okur; Megan T Cho; Richard van Wijk; Brigitte van Oirschot; Jonathan Picker; Stephanie A Coury; Dorothy Grange; Linda Manwaring; Ian Krantz; Colleen Clark Muraresku; Peter J Hulick; Holley May; Eric Pierce; Emily Place; Kinga Bujakowska; Aida Telegrafi; Ganka Douglas; Kristin G Monaghan; Amber Begtrup; Ashley Wilson; Kyle Retterer; Kwame Anyane-Yeboa; Wendy K Chung Journal: Eur J Hum Genet Date: 2019-02-18 Impact factor: 4.246