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A novel c.-22T>C mutation in GALK1 promoter is associated with elevated galactokinase phenotype.

Park HD, Kim YK, Park KU, Kim JQ, Song YH, Song J - BMC Med. Genet. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ilsong Institute of Life Science, Hallym University, Anyang, Korea. nayadoo@hanmail.net

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.

Conclusion: 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.

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Related in: MedlinePlus

c.-22T>C mutation resulted in increased reporter activity in hepatoma cell lines. The promoter region of the GALK1 gene was cloned into pGL3-Basic, and the nucleotides at positions c.-179, c.-27, and c.-22 are indicated. Each construct was transfected into HepG2 and Hep3B cells five times in duplicate, and dual luciferase assays were performed. The mean luciferase activities, normalized for cell transfection efficiencies, were calculated relative to the activity of the wild type construct (wt, set as 100). Standard deviations are indicated. *p < 0.05 and **p < 0.01, significantly different between transfected cell lines and wild type by paired t-tests.
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Figure 3: c.-22T>C mutation resulted in increased reporter activity in hepatoma cell lines. The promoter region of the GALK1 gene was cloned into pGL3-Basic, and the nucleotides at positions c.-179, c.-27, and c.-22 are indicated. Each construct was transfected into HepG2 and Hep3B cells five times in duplicate, and dual luciferase assays were performed. The mean luciferase activities, normalized for cell transfection efficiencies, were calculated relative to the activity of the wild type construct (wt, set as 100). Standard deviations are indicated. *p < 0.05 and **p < 0.01, significantly different between transfected cell lines and wild type by paired t-tests.

Mentions: To test if c.-22T>C affected transcription, reporter analysis was performed using the promoter region of GALK1 gene (-441 to -1), with nucleotide substitutions at position -179, -27, and -22. The HepG2 cells transfected with the c.-22C mutant allele showed an elevated luciferase activity approximately two-fold over those with the wild type sequence. This phenomenon appeared more clearly in the Hep3B cells showing a two and a half fold increase (Fig. 3). In terms of c.-179A>G and c.-27A>C, the relative luciferase activity was not significantly different from that of the wild type.


A novel c.-22T>C mutation in GALK1 promoter is associated with elevated galactokinase phenotype.

Park HD, Kim YK, Park KU, Kim JQ, Song YH, Song J - BMC Med. Genet. (2009)

c.-22T>C mutation resulted in increased reporter activity in hepatoma cell lines. The promoter region of the GALK1 gene was cloned into pGL3-Basic, and the nucleotides at positions c.-179, c.-27, and c.-22 are indicated. Each construct was transfected into HepG2 and Hep3B cells five times in duplicate, and dual luciferase assays were performed. The mean luciferase activities, normalized for cell transfection efficiencies, were calculated relative to the activity of the wild type construct (wt, set as 100). Standard deviations are indicated. *p < 0.05 and **p < 0.01, significantly different between transfected cell lines and wild type by paired t-tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667498&req=5

Figure 3: c.-22T>C mutation resulted in increased reporter activity in hepatoma cell lines. The promoter region of the GALK1 gene was cloned into pGL3-Basic, and the nucleotides at positions c.-179, c.-27, and c.-22 are indicated. Each construct was transfected into HepG2 and Hep3B cells five times in duplicate, and dual luciferase assays were performed. The mean luciferase activities, normalized for cell transfection efficiencies, were calculated relative to the activity of the wild type construct (wt, set as 100). Standard deviations are indicated. *p < 0.05 and **p < 0.01, significantly different between transfected cell lines and wild type by paired t-tests.
Mentions: To test if c.-22T>C affected transcription, reporter analysis was performed using the promoter region of GALK1 gene (-441 to -1), with nucleotide substitutions at position -179, -27, and -22. The HepG2 cells transfected with the c.-22C mutant allele showed an elevated luciferase activity approximately two-fold over those with the wild type sequence. This phenomenon appeared more clearly in the Hep3B cells showing a two and a half fold increase (Fig. 3). In terms of c.-179A>G and c.-27A>C, the relative luciferase activity was not significantly different from that of the wild type.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Ilsong Institute of Life Science, Hallym University, Anyang, Korea. nayadoo@hanmail.net

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus