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Mutations in TKT Are the Cause of a Syndrome Including Short Stature, Developmental Delay, and Congenital Heart Defects  Lia Boyle, Mirjam M.C. Wamelink,

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Presentation on theme: "Mutations in TKT Are the Cause of a Syndrome Including Short Stature, Developmental Delay, and Congenital Heart Defects  Lia Boyle, Mirjam M.C. Wamelink,"— Presentation transcript:

1 Mutations in TKT Are the Cause of a Syndrome Including Short Stature, Developmental Delay, and Congenital Heart Defects  Lia Boyle, Mirjam M.C. Wamelink, Gajja S. Salomons, Birthe Roos, Ana Pop, Andrew Dauber, Vivian Hwa, Melissa Andrew, Jessica Douglas, Murray Feingold, Nancy Kramer, Sulagna Saitta, Kyle Retterer, Megan T. Cho, Amber Begtrup, Kristin G. Monaghan, Julia Wynn, Wendy K. Chung  The American Journal of Human Genetics  Volume 98, Issue 6, Pages (June 2016) DOI: /j.ajhg Copyright © 2016 American Society of Human Genetics Terms and Conditions

2 Figure 1 Pentitol Pathway
Yellow highlighting reflects the affected TKT enzyme. Metabolites in excess are in red, and deficient metabolites are in blue. The American Journal of Human Genetics  , DOI: ( /j.ajhg ) Copyright © 2016 American Society of Human Genetics Terms and Conditions

3 Figure 2 Pedigrees and Photographs of Three Families Affected by TKT Variants The American Journal of Human Genetics  , DOI: ( /j.ajhg ) Copyright © 2016 American Society of Human Genetics Terms and Conditions

4 Figure 3 Ectopic Expression of TKT Variants Alters TKT Activity
(A) To study the effect of the TKT variants, we constructed two vectors (pCMV6-AN-GFP-TKT [wild-type, WT] and pCMV6-AN-GFP-TKT Trp257delinsSerThrSerLeuSerSerGly [delins]) and introduced the missense and nonsense variants in the WT construct. HEK293 cells were transfected in triplicate with the different TKT constructs, with Fugene HD reagent (Promega), and harvested by trypsinization 24 hr after transfections. To prove the success of the transient transfections, accumulation of the TKT-GFP fusion protein was studied by SDS-PAGE and western blotting. Immunodetection of the TKT-GFP fusion protein was carried out with rabbit polyclonal anti-GFP primary antibody (Abcam), polyclonal goat anti-rabbit immunoglobulins/HRP secondary antibody (Dako), and enhanced chemiluminescent substrates (Lumi-LightPLUS Western Blotting Substrate; Roche Applied Science). We analyzed all triplicate samples by western blotting for the presence of the GFP-TKT fusion proteins by using an antibody against GFP (indicated by arrows). A representative analysis of the triplicate samples is shown. The fusion protein with an apparent mass of 75 kDa is present in all transfectants except in the mock, the untransfected, and the p.Trp211Ter transfected HEK293 cells. In the latter, degraded TKT fusion proteins are present. (B) TKT activity was measured in both untransfected and transfected HEK cells. After 30 min of incubation at 37°C, 2.5 mM ribose-5-phosphate and 0.5 mM xylulose-5-phosphate was added and the decrease of NADH was photometrically determined. All transfections were performed in triplicate and TKT activity was measured in duplicate per transfectant. The activities are corrected for the basal activity in the mock cells and as the relative activity compared to the WT transfected HEK293 cells. Error bars represent the SE of the triplicate transfections. The American Journal of Human Genetics  , DOI: ( /j.ajhg ) Copyright © 2016 American Society of Human Genetics Terms and Conditions

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