Yonsei Med J.  2017 Nov;58(6):1160-1169. 10.3349/ymj.2017.58.6.1160.

Identification and Functional Characterization of ST3GAL5 and ST8SIA1 Variants in Patients with Thyroid-Associated Ophthalmopathy

• Park HJ
• Kim JH
• Yoon JS
• Choi YJ
• Choi YH
• Kook KH
• Choi JH

• KMID: 2418898
• DOI: http://doi.org/10.3349/ymj.2017.58.6.1160

Abstract

PURPOSE
This study was conducted to identify and to functionally characterize genetic variants in ST3GAL5 and ST8SIA1 in Korean patients with thyroid-associated ophthalmopathy (TAO).
MATERIALS AND METHODS
Genetic analyses were conducted using DNA samples from TAO patients (n=50) and healthy subjects (n=48) to identify TAO-specific genetic variants of ST3GAL5 or ST8SIA1. The effect of each genetic variant on the transcription or expression of these genes was examined. Additionally, correlations between functional haplotypes of ST3GAL5 or ST8SIA1 and clinical characteristics of the patients were investigated.
RESULTS
Six promoter variants and one nonsynonymous variant of ST3GAL5 were identified, and four major promoter haplotypes were assembled. Additionally, three promoter variants and two major haplotypes of ST8SIA1 were identified. All ST3GAL5 and ST8SIA1 variants identified in TAO patients were also found in healthy controls. Promoter activity was significantly decreased in three promoter haplotypes of ST3GAL5 and increased in one promoter haplotype of ST8SIA1. Transcription factors activating protein-1, NKX3.1, and specificity protein 1 were revealed as having roles in transcriptional regulation of these haplotypes. The nonsynonymous variant of ST3GAL5, H104R, did not alter the expression of ST3GAL5. While no differences in clinical characteristics were detected in patients possessing the functional promoter haplotypes of ST3GAL5, exophthalmic values were significantly lower in patients with the ST8SIA1 haplotype, which showed a significant increase in promoter activity.
CONCLUSION
These results from genotype-phenotype analysis might suggest a possible link between the ST8SIA1 functional promoter haplotype and the clinical severity of TAO. However, further studies with larger sample sizes are warranted.

Keyword

Exophthalmos; Korea; sialyltransferases; single nucleotide polymorphism; thyroid-associated ophthalmopathy

MeSH Terms

Adult
Case-Control Studies
Female
Gene Expression Regulation
Genetic Variation
Graves Ophthalmopathy/ethnology/*genetics
Haplotypes
Humans
Male
Middle Aged
Phenotype
Polymorphism, Single Nucleotide
Promoter Regions, Genetic
Sialyltransferases/*genetics/metabolism
Transcription Factors
Transcription Factors
Sialyltransferases

Figure

• 

  Fig. 1 Effect of genetic variants on the promoter activity of ST3GAL5 or ST8SIA1. Luciferase activities were measured 30 hours after transfection of the reporter vectors containing ST3GAL5 (A and B) or ST8SIA1 (C and D) haplotypes (A and C) or variants (B and D) into HCT-116 cells. The luciferase activity of each construct was compared with that of the reference (REF). The data (mean±SD) were obtained from triplicate wells. *p<0.01, †p<0.001.

• 

  Fig. 2 Effect of genetic variants on the binding of the transcription factors to ST3GAL5 (A) or ST8SIA1 (B and C) promoters. (A) 32P-labelled oligonucleotides (lanes 1-3, AP-1 consensus; lanes 4-6, g.-1051G reference; lanes 7-9, g.-1051C variant) were incubated with nuclear protein extracts (25 µg) obtained from HCT-116 cells. Competition assay was performed using 100-fold molar excess of unlabeled AP-1 consensus oligonucleotides (lanes 2, 5, and 8). Supershift assays were performed with an antibody against phosphorylated c-Jun (lanes 3, 6, and 9). (B) 32P-labelled oligonucleotides (lanes 1-3, NKX3.1 consensus; lanes 4-6, g.-2196A reference; lanes 7-9, g.-2196C variant) were incubated with nuclear protein extracts (25 µg) obtained from HCT-116 cells. Competition assay was performed using 100-fold molar excess of unlabeled NKX3.1 consensus (Ref, lanes 2, 5, and 8) or core sequence-mutated (Mut, lanes 3, 6, and 9) oligonucleotides. (C) 32P-labelled oligonucleotides (lanes 1-3, SP1 consensus; lanes 4-6, g.-1984T reference; lanes 7-9, g.-1984C variant) were incubated with nuclear protein extracts (25 µg) obtained from HCT-116 cells. Competition assay was performed using 100-fold molar excess of unlabeled SP1 consensus oligonucleotides (lanes 2, 5, and 8). Supershift assays were performed with an antibody against SP1 (lanes 3, 6, and 9). The arrows indicate the position of the DNA-protein complex.

• 

  Fig. 3 Effect of a nonsynonymous variant, H104R, on ST3GAL5 expression. Immunoblotting assays were performed using cell lysates obtained 48 hours after the transfection of empty vector (EV, pcDNA3.1), ST3GAL5 reference (REF), or variant plasmids into HCT-116 cells. The data (mean±SD) were obtained from three representative experiments. β-actin was used as an internal control.

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