title: First mutation in the red blood cell-specific promoter of hexokinase combined with a novel missense mutation causes hexokinase deficiency and mild chronic hemolysis.
abstract: 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.
  
  
  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.affiliation: Department of Clinical Chemistry and Hematology, Laboratory for Red Blood Cell Research, University Medical Center Utrecht, The Netherlands. k.devooght@umcutrecht.nl
authors.forename: Karen M K
authors.initials: KM
authors.lastname: de Vooght
authors.forename: Wouter W
authors.initials: WW
authors.lastname: van Solinge
authors.forename: Annet C
authors.initials: AC
authors.lastname: van Wesel
authors.forename: Sabina
authors.initials: S
authors.lastname: Kersting
authors.forename: Richard
authors.initials: R
authors.lastname: van Wijk
mesh_terms: D000328:Adult; D019943:Amino Acid Substitution; D002908:Chronic Disease; D004912:Erythrocytes / Q000201:enzymology*; D005091:Exons / Q000235:genetics; D030342:Genetic Diseases, Inborn / Q000201:enzymology / Q000235:genetics*; D006461:Hemolysis / Q000235:genetics*; D006593:Hexokinase / Q000172:deficiency* / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D008297:Male; D020125:Mutation, Missense; D012326:RNA Splicing / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: C472074:HK1 protein, human; D006593:Hexokinase