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Carriers of a novel frame-shift insertion in WNT16a possess elevated pancreatic expression of TCF7L2.

Howard EW, Been LF, Lerner M, Brackett D, Lightfoot S, Bullen EC, Sanghera DK - BMC Genet. (2013)

Bottom Line: Prevalence of Wnt16a insertion did not differ among T2D cases (33%) and controls (32%).Our results suggest synergistic effects of WNT16a insertion and the at-risk 'T' allele of TCF7L2 (rs7903146) for elevating the expression of TCF7L2 in human pancreas which may affect the regulation of downstream target genes involved in the development of T2D through Wnt/β-catenin/TCF7L2 signaling pathway.However, further studies would be needed to mechanistically link the two definitively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

ABSTRACT

Background: The discovery of TCF7L2 as a global type 2 diabetes (T2D) gene has sparked investigations to explore the clinical utility of its variants for guiding the development of new diagnostic and therapeutic strategies. However, interpreting the resulting associations into function still remains unclear. Canonical Wnt signaling regulates β-catenin and its binding with TCF7L2, which in turn is critical for the production of glucagon-like peptide-1 (GLP-1). This study examines the role of a novel frame-shift insertion discovered in a conserved region of WNT16a, and it is proposed that this mutation affects T2D susceptibility in conjunction with gene variants in TCF7L2.

Results: Our results predicted that the insertion would convert the upstream open reading frame in the Wnt16a mRNA to an alternative, in-frame translation initiation site, resulting in the prevention of nonsense-mediated decay, leading to a consequent stabilization of the mutated WNT16a message. To examine the role of Wnt16a in the Wnt signaling pathway, DNA and serum samples from 2,034 individuals (48% with T2D) from the Sikh Diabetes Study were used in this investigation. Prevalence of Wnt16a insertion did not differ among T2D cases (33%) and controls (32%). However, there was a 3.2 fold increase in Wnt16a mRNA levels in pancreatic tissues from the insertion carriers and a significant increase (70%, p < 0.0001) in luciferase activity in the constructs carrying the insertion. The expression of TCF7L2 mRNA in pancreas was also elevated (~23-fold) among the insertion carriers (p=0.003).

Conclusions: Our results suggest synergistic effects of WNT16a insertion and the at-risk 'T' allele of TCF7L2 (rs7903146) for elevating the expression of TCF7L2 in human pancreas which may affect the regulation of downstream target genes involved in the development of T2D through Wnt/β-catenin/TCF7L2 signaling pathway. However, further studies would be needed to mechanistically link the two definitively.

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Reporter constructs assembled with the mutated form of the Wnt16a5′ UTR are more efficiently translated than the wild-typeform. The wild-type and mutant Wnt16a 5′ UTR sequenceswere inserted adjacent to a luciferase cDNA, and the resulting plasmidswere used to transfect HEK-293 cells. 48 hours after transfection,cells were lysed, and firefly luciferase (FFL) and Renillaluciferase (RL) levels were measured. Our reporter constructs using thewild-type and the mutant (insertion) sequence of Wnt16a showedsignificantly increased (70%, p<0.0001) levels of luciferase proteinin the constructs carrying the mutant sequence.
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Figure 7: Reporter constructs assembled with the mutated form of the Wnt16a5′ UTR are more efficiently translated than the wild-typeform. The wild-type and mutant Wnt16a 5′ UTR sequenceswere inserted adjacent to a luciferase cDNA, and the resulting plasmidswere used to transfect HEK-293 cells. 48 hours after transfection,cells were lysed, and firefly luciferase (FFL) and Renillaluciferase (RL) levels were measured. Our reporter constructs using thewild-type and the mutant (insertion) sequence of Wnt16a showedsignificantly increased (70%, p<0.0001) levels of luciferase proteinin the constructs carrying the mutant sequence.

Mentions: In order to further evaluate the influence of the CCCA insertion on translationof the Wnt16a message, we assembled reporter constructs driven by thecytomegalovirus (CMV) promoter that included the wild-type and the mutantsequence of the Wnt16a 5′ UTRs. The long uORF was mimicked in ourluciferase construct by the presence of a translation stop site in-frame withthe upstream AUG. If the first AUG in the message was used to initiatetranslation, then no luciferase protein should have been produced. Indeed, whenwe inserted the additional four nucleotides to replicate what occurs in themutant situation, we noted a significantly increased level (~70%) of luciferaseexpression (p=0.0001) (Figure 7). This suggests thatthe upstream AUG can act as an efficient translation initiation site. In thewild-type gene, this would reduce expression of the full-length protein bypreventing initiation at the second AUG. In the presence of the CCCA insertion,both AUGs are in the same reading frame, so full-length protein would beproduced regardless of which AUG was used to initiate translation.


Carriers of a novel frame-shift insertion in WNT16a possess elevated pancreatic expression of TCF7L2.

Howard EW, Been LF, Lerner M, Brackett D, Lightfoot S, Bullen EC, Sanghera DK - BMC Genet. (2013)

Reporter constructs assembled with the mutated form of the Wnt16a5′ UTR are more efficiently translated than the wild-typeform. The wild-type and mutant Wnt16a 5′ UTR sequenceswere inserted adjacent to a luciferase cDNA, and the resulting plasmidswere used to transfect HEK-293 cells. 48 hours after transfection,cells were lysed, and firefly luciferase (FFL) and Renillaluciferase (RL) levels were measured. Our reporter constructs using thewild-type and the mutant (insertion) sequence of Wnt16a showedsignificantly increased (70%, p<0.0001) levels of luciferase proteinin the constructs carrying the mutant sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675375&req=5

Figure 7: Reporter constructs assembled with the mutated form of the Wnt16a5′ UTR are more efficiently translated than the wild-typeform. The wild-type and mutant Wnt16a 5′ UTR sequenceswere inserted adjacent to a luciferase cDNA, and the resulting plasmidswere used to transfect HEK-293 cells. 48 hours after transfection,cells were lysed, and firefly luciferase (FFL) and Renillaluciferase (RL) levels were measured. Our reporter constructs using thewild-type and the mutant (insertion) sequence of Wnt16a showedsignificantly increased (70%, p<0.0001) levels of luciferase proteinin the constructs carrying the mutant sequence.
Mentions: In order to further evaluate the influence of the CCCA insertion on translationof the Wnt16a message, we assembled reporter constructs driven by thecytomegalovirus (CMV) promoter that included the wild-type and the mutantsequence of the Wnt16a 5′ UTRs. The long uORF was mimicked in ourluciferase construct by the presence of a translation stop site in-frame withthe upstream AUG. If the first AUG in the message was used to initiatetranslation, then no luciferase protein should have been produced. Indeed, whenwe inserted the additional four nucleotides to replicate what occurs in themutant situation, we noted a significantly increased level (~70%) of luciferaseexpression (p=0.0001) (Figure 7). This suggests thatthe upstream AUG can act as an efficient translation initiation site. In thewild-type gene, this would reduce expression of the full-length protein bypreventing initiation at the second AUG. In the presence of the CCCA insertion,both AUGs are in the same reading frame, so full-length protein would beproduced regardless of which AUG was used to initiate translation.

Bottom Line: Prevalence of Wnt16a insertion did not differ among T2D cases (33%) and controls (32%).Our results suggest synergistic effects of WNT16a insertion and the at-risk 'T' allele of TCF7L2 (rs7903146) for elevating the expression of TCF7L2 in human pancreas which may affect the regulation of downstream target genes involved in the development of T2D through Wnt/β-catenin/TCF7L2 signaling pathway.However, further studies would be needed to mechanistically link the two definitively.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

ABSTRACT

Background: The discovery of TCF7L2 as a global type 2 diabetes (T2D) gene has sparked investigations to explore the clinical utility of its variants for guiding the development of new diagnostic and therapeutic strategies. However, interpreting the resulting associations into function still remains unclear. Canonical Wnt signaling regulates β-catenin and its binding with TCF7L2, which in turn is critical for the production of glucagon-like peptide-1 (GLP-1). This study examines the role of a novel frame-shift insertion discovered in a conserved region of WNT16a, and it is proposed that this mutation affects T2D susceptibility in conjunction with gene variants in TCF7L2.

Results: Our results predicted that the insertion would convert the upstream open reading frame in the Wnt16a mRNA to an alternative, in-frame translation initiation site, resulting in the prevention of nonsense-mediated decay, leading to a consequent stabilization of the mutated WNT16a message. To examine the role of Wnt16a in the Wnt signaling pathway, DNA and serum samples from 2,034 individuals (48% with T2D) from the Sikh Diabetes Study were used in this investigation. Prevalence of Wnt16a insertion did not differ among T2D cases (33%) and controls (32%). However, there was a 3.2 fold increase in Wnt16a mRNA levels in pancreatic tissues from the insertion carriers and a significant increase (70%, p < 0.0001) in luciferase activity in the constructs carrying the insertion. The expression of TCF7L2 mRNA in pancreas was also elevated (~23-fold) among the insertion carriers (p=0.003).

Conclusions: Our results suggest synergistic effects of WNT16a insertion and the at-risk 'T' allele of TCF7L2 (rs7903146) for elevating the expression of TCF7L2 in human pancreas which may affect the regulation of downstream target genes involved in the development of T2D through Wnt/β-catenin/TCF7L2 signaling pathway. However, further studies would be needed to mechanistically link the two definitively.

Show MeSH
Related in: MedlinePlus