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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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Oligonucleotide encompassing c.-22 formed DNA-protein complex detected by electrophoretic mobility shift assay (EMSA). A) The radiolabeled oligonucleotide probes containing c.-22T or c.-22C of the GALK1 gene were incubated with Hep3B nuclear extract. When c.-22C was used as a probe, increasing amounts of cold competitors (1, 2, and 5 pmol of c.-22C and c.-22T) or anti-HEN1 antibody were included as indicated. The DNA-protein complex is indicated with an arrow. B) The intensity of the DNA-protein complex in the absence and presence of cold probes, c-22T (□) and c.-22C (■), was measured using ImageJ. The graph represents means and standard deviations of three independent experiments taking the intensity of DNA-protein complex without competitor as 100%.
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Figure 4: Oligonucleotide encompassing c.-22 formed DNA-protein complex detected by electrophoretic mobility shift assay (EMSA). A) The radiolabeled oligonucleotide probes containing c.-22T or c.-22C of the GALK1 gene were incubated with Hep3B nuclear extract. When c.-22C was used as a probe, increasing amounts of cold competitors (1, 2, and 5 pmol of c.-22C and c.-22T) or anti-HEN1 antibody were included as indicated. The DNA-protein complex is indicated with an arrow. B) The intensity of the DNA-protein complex in the absence and presence of cold probes, c-22T (□) and c.-22C (■), was measured using ImageJ. The graph represents means and standard deviations of three independent experiments taking the intensity of DNA-protein complex without competitor as 100%.

Mentions: EMSA was performed to determine if the elevated luciferase activity of c.-22T>C was due to alterations in the binding of a nuclear protein. Thirty-three-bp oligonucleotides with c.-22T and c.-22C were used as a probe, and incubation of these radio-labeled probes with Hep3B nuclear extracts produced a DNA-protein complex (Fig. 4A lanes 2 and 4). When the c-22C DNA-protein complex was challenged with increasing amounts of cold probes, the competition was slightly more efficient with c-22C, than with c-22T (Fig. 4A). The competition analysis was performed three times and the relative band intensity of the DNA-protein complex was consistently higher when competed with c-22T than with c-22C (Fig 4B). This result suggests that the Hep3B nuclear proteins interacted with the c-22C oligonucleotides with slightly higher affinity than with the c-22T. MatInspector was utilized to identify a transcription factor that might differentially interact with c-22C and c-22T, and HEN1 was identified as a potential candidate. However, addition of anti-HEN1 antibody did not affect the mobility of the DNA-protein complex (Fig 1A, lanes 11 and 12). In addition, no supershift was observed with anti-Egr1 and anti-Sp1 antibodies (data not shown).


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)

Oligonucleotide encompassing c.-22 formed DNA-protein complex detected by electrophoretic mobility shift assay (EMSA). A) The radiolabeled oligonucleotide probes containing c.-22T or c.-22C of the GALK1 gene were incubated with Hep3B nuclear extract. When c.-22C was used as a probe, increasing amounts of cold competitors (1, 2, and 5 pmol of c.-22C and c.-22T) or anti-HEN1 antibody were included as indicated. The DNA-protein complex is indicated with an arrow. B) The intensity of the DNA-protein complex in the absence and presence of cold probes, c-22T (□) and c.-22C (■), was measured using ImageJ. The graph represents means and standard deviations of three independent experiments taking the intensity of DNA-protein complex without competitor as 100%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Oligonucleotide encompassing c.-22 formed DNA-protein complex detected by electrophoretic mobility shift assay (EMSA). A) The radiolabeled oligonucleotide probes containing c.-22T or c.-22C of the GALK1 gene were incubated with Hep3B nuclear extract. When c.-22C was used as a probe, increasing amounts of cold competitors (1, 2, and 5 pmol of c.-22C and c.-22T) or anti-HEN1 antibody were included as indicated. The DNA-protein complex is indicated with an arrow. B) The intensity of the DNA-protein complex in the absence and presence of cold probes, c-22T (□) and c.-22C (■), was measured using ImageJ. The graph represents means and standard deviations of three independent experiments taking the intensity of DNA-protein complex without competitor as 100%.
Mentions: EMSA was performed to determine if the elevated luciferase activity of c.-22T>C was due to alterations in the binding of a nuclear protein. Thirty-three-bp oligonucleotides with c.-22T and c.-22C were used as a probe, and incubation of these radio-labeled probes with Hep3B nuclear extracts produced a DNA-protein complex (Fig. 4A lanes 2 and 4). When the c-22C DNA-protein complex was challenged with increasing amounts of cold probes, the competition was slightly more efficient with c-22C, than with c-22T (Fig. 4A). The competition analysis was performed three times and the relative band intensity of the DNA-protein complex was consistently higher when competed with c-22T than with c-22C (Fig 4B). This result suggests that the Hep3B nuclear proteins interacted with the c-22C oligonucleotides with slightly higher affinity than with the c-22T. MatInspector was utilized to identify a transcription factor that might differentially interact with c-22C and c-22T, and HEN1 was identified as a potential candidate. However, addition of anti-HEN1 antibody did not affect the mobility of the DNA-protein complex (Fig 1A, lanes 11 and 12). In addition, no supershift was observed with anti-Egr1 and anti-Sp1 antibodies (data not shown).

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