Mutations in TKT Are the Cause of a Syndrome Including Short Stature, Developmental Delay, and Congenital Heart Defects

Abstract

Whole-exome sequencing (WES) is increasingly being utilized to diagnose individuals with undiagnosed disorders. Developmental delay and short stature are common clinical indications for WES. We performed WES in three families, using proband-parent trios and two additional affected siblings. We identified a syndrome due to an autosomal-recessively inherited deficiency of transketolase, encoded by TKT, on chromosome 3p21. Our series includes three families with a total of five affected individuals, ranging in age from 4 to 25 years. Two families of Ashkenazi Jewish ancestry were homozygous for an 18 base pair in-frame insertion in TKT. The third family was compound heterozygous for nonsense and missense variants in TKT. All affected individuals had short stature and were developmentally delayed. Congenital heart defects were noted in four of the five affected individuals, and there was a history of chronic diarrhea and cataracts in the older individuals with the homozygous 18 base pair insertion. Enzymatic testing confirmed significantly reduced transketolase activity. Elevated urinary excretion of erythritol, arabitol, ribitol, and pent(ul)ose-5-phosphates was detected, as well as elevated amounts of erythritol, arabitol, and ribitol in the plasma of affected individuals. Transketolase deficiency reduces NADPH synthesis and nucleic acid synthesis and cell division and could explain the problems with growth. NADPH is also critical for maintaining cerebral glutathione, which might contribute to the neurodevelopmental delays. Transketolase deficiency is one of a growing list of inborn errors of metabolism in the non-oxidative part of the pentose phosphate pathway.

Keywords: TKT; congenital heart disease; neurodevelopmental disability; pentose phosphate pathway; transketolase deficiency.

Figure 1

Pentitol Pathway Yellow highlighting reflects the affected TKT enzyme. Metabolites in excess are in red, and deficient metabolites are in blue.

Figure 2

Pedigrees and Photographs of Three Families Affected by TKT Variants

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-Light^PLUS 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.