title: A novel c.-22T>C mutation in GALK1 promoter is associated with elevated galactokinase phenotype.
abstract: BACKGROUND
  Many genetic variations of GALK1 have been identified in the patients with galactokinase (GALK1) deficiency. However, the molecular characteristics of GALK1 in individuals with elevated GALK1 activity are relatively unknown.
  
  
  METHODS
  We investigated the relationship between elevated GALK1 activity and the molecular GALK1 gene variations, and the molecular mechanism underlying elevated GALK1 activity. PCR products from 63 subjects, without any attenuation of galactose degradation enzymes, were sequenced to screen for nucleotide alterations in the GALK1 promoter.
  
  
  RESULTS
  Three nucleotide substitutions were identified: c.-179A>G, c.-27A>C, and c.-22T>C. With respect to the c.-22T>C mutation, GALK1 activity in 13 subjects with the T/C or C/C genotype was significantly higher than those in 50 subjects with the T/T genotype (p < 0.001). The dual luciferase reporter assay in Hep3B cells showed that the luciferase activity with the GALK1 promoter with the c.-22C mutant allele increased approximately 2.5-fold, compared to that with the c.-22T. A specific DNA-protein complex was observed in an electrophoretic mobility shift assay, with slightly higher affinity to c.-22C than to c.-22T.
  
  
  CONCLUSIONS
  The c.-22T>C mutation, which was observed frequently in individuals with elevated GALK1 activity, increased the expression of a reporter gene through enhanced binding of a currently unidentified nuclear protein. These results suggest that the elevated GALK1 activity resulted from enhanced gene expression, due to nucleotide variation within GALK1 promoter.
authors.affiliation: Ilsong Institute of Life Science, Hallym University, Anyang, Korea. nayadoo@hanmail.net
authors.forename: Hyung-Doo
authors.initials: HD
authors.lastname: Park
authors.forename: Yoon-Kyoung
authors.initials: YK
authors.lastname: Kim
authors.forename: Kyoung Un
authors.initials: KU
authors.lastname: Park
authors.forename: Jin Q
authors.initials: JQ
authors.lastname: Kim
authors.forename: Young-Han
authors.initials: YH
authors.lastname: Song
authors.forename: Junghan
authors.initials: J
authors.lastname: Song
mesh_terms: D024202:Electrophoretic Mobility Shift Assay; D005686:Galactokinase / Q000235:genetics*; D005693:Galactosemias / Q000175:diagnosis / Q000235:genetics*; D017930:Genes, Reporter; D020022:Genetic Predisposition to Disease; D006801:Humans; D007231:Infant, Newborn; D009154:Mutation; D015997:Neonatal Screening; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011401:Promoter Regions, Genetic
chemical_list: C484715:GALK1 protein, human; D005686:Galactokinase