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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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Distributions of GALK1 activities according to the genotypes at c.-22 of the GALK1 gene are plotted according to patient age. The GALK1 values for the genotype C/C, T/C, and T/T at nucleotide -22 are indicated as ▲, ●, and □, respectively. The GALK1 activities in individuals having at least one C allele (T/C or C/C genotype) were significantly higher than those in individuals with the T/T genotype (p < 0.001 by Mann-Whitney U test).
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Figure 2: Distributions of GALK1 activities according to the genotypes at c.-22 of the GALK1 gene are plotted according to patient age. The GALK1 values for the genotype C/C, T/C, and T/T at nucleotide -22 are indicated as ▲, ●, and □, respectively. The GALK1 activities in individuals having at least one C allele (T/C or C/C genotype) were significantly higher than those in individuals with the T/T genotype (p < 0.001 by Mann-Whitney U test).

Mentions: In terms of c.-22 of GALK1, GALK1 activities, and their normalized values in individuals having at least one C allele (T/C or C/C genotype), were significantly higher than those in individuals with T/T genotype (p < 0.001 by Mann-Whitney U test, respectively) (Table 1 and Fig. 2). There were statistical differences in GALK1 activities between subjects with A/A, and those with A/G or G/G, at c.-179 of the GALK1; however, it disappeared after excluding 13 individuals with the C allele at c.-22 from total subjects. GALK1 activities in 43 individuals with A/A, versus in 7 individuals with A/G or GG genotype at position c.-179 of GALK1, were 98.7 ± 24.1 versus 80.1 ± 20.0 nmol/min/gHb (p = 0.307). This might be because the C allele at c.-22 is linked closely with the A allele at c.179. On the contrary, there was no statistical difference in GALK1 activities between the subjects with A/A and A/C genotypes at position c.-27 of GALK1 (Table 1), which further suggests that c.-27A>C is a nonfunctional polymorphism.


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)

Distributions of GALK1 activities according to the genotypes at c.-22 of the GALK1 gene are plotted according to patient age. The GALK1 values for the genotype C/C, T/C, and T/T at nucleotide -22 are indicated as ▲, ●, and □, respectively. The GALK1 activities in individuals having at least one C allele (T/C or C/C genotype) were significantly higher than those in individuals with the T/T genotype (p < 0.001 by Mann-Whitney U test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Distributions of GALK1 activities according to the genotypes at c.-22 of the GALK1 gene are plotted according to patient age. The GALK1 values for the genotype C/C, T/C, and T/T at nucleotide -22 are indicated as ▲, ●, and □, respectively. The GALK1 activities in individuals having at least one C allele (T/C or C/C genotype) were significantly higher than those in individuals with the T/T genotype (p < 0.001 by Mann-Whitney U test).
Mentions: In terms of c.-22 of GALK1, GALK1 activities, and their normalized values in individuals having at least one C allele (T/C or C/C genotype), were significantly higher than those in individuals with T/T genotype (p < 0.001 by Mann-Whitney U test, respectively) (Table 1 and Fig. 2). There were statistical differences in GALK1 activities between subjects with A/A, and those with A/G or G/G, at c.-179 of the GALK1; however, it disappeared after excluding 13 individuals with the C allele at c.-22 from total subjects. GALK1 activities in 43 individuals with A/A, versus in 7 individuals with A/G or GG genotype at position c.-179 of GALK1, were 98.7 ± 24.1 versus 80.1 ± 20.0 nmol/min/gHb (p = 0.307). This might be because the C allele at c.-22 is linked closely with the A allele at c.179. On the contrary, there was no statistical difference in GALK1 activities between the subjects with A/A and A/C genotypes at position c.-27 of GALK1 (Table 1), which further suggests that c.-27A>C is a nonfunctional polymorphism.

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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