Bi-allelic Variants in TKFC Encoding Triokinase/FMN Cyclase Are Associated with Cataracts and Multisystem Disease

Abstract

We report an inborn error of metabolism caused by TKFC deficiency in two unrelated families. Rapid trio genome sequencing in family 1 and exome sequencing in family 2 excluded known genetic etiologies, and further variant analysis identified rare homozygous variants in TKFC. TKFC encodes a bifunctional enzyme involved in fructose metabolism through its glyceraldehyde kinase activity and in the generation of riboflavin cyclic 4',5'-phosphate (cyclic FMN) through an FMN lyase domain. The TKFC homozygous variants reported here are located within the FMN lyase domain. Functional assays in yeast support the deleterious effect of these variants on protein function. Shared phenotypes between affected individuals with TKFC deficiency include cataracts and developmental delay, associated with cerebellar hypoplasia in one case. Further complications observed in two affected individuals included liver dysfunction and microcytic anemia, while one had fatal cardiomyopathy with lactic acidosis following a febrile illness. We postulate that deficiency of TKFC causes disruption of endogenous fructose metabolism leading to generation of by-products that can cause cataract. In line with this, an affected individual had mildly elevated urinary galactitol, which has been linked to cataract development in the galactosemias. Further, in light of a previously reported role of TKFC in regulating innate antiviral immunity through suppression of MDA5, we speculate that deficiency of TKFC leads to impaired innate immunity in response to viral illness, which may explain the fatal illness observed in the most severely affected individual.

Keywords: TKFC; cardiomyopathy; cataracts; cyclic FMN; developmental delay; fructose metabolism; inborn error of metabolism; innate immunity; rapid genome sequencing; triokinase.

Figure 1

Family Pedigrees, Bi-allelic TKFC Variants Observed in Affected Individuals and Brain MRI of P1-1 Representative MRI findings in affected individual P1-1 at 29 months indicating cerebellar hypoplasia. Homozygous TKFC variants segregating in each family were confirmed by Sanger Sequencing.

Figure 2

Multiple Sequence Alignment of TKFC Showing Evolutionary Conservation of Affected Amino Acids, and In Silico Modeling of TKFC Dimer (A) Multiple sequence alignment of TKFC among different representative species and location of homozygous variants within TKFC domains. Homozygous protein variants detected in family 1 (p.Arg543Ile) and family 2 (p.Gly445Ser) are indicated and shown to affect highly conserved amino acid residues. (B) Dimer of TKFC subunits (with K domain shown in yellow and L domain in blue), based on in silico modeling of human TKFC structure. The homozygous TKFC variants identified in the two families are located within the FMN lyase (L) domain (blue). For simplicity, the two mutation sites (red spheres) are shown only for the dimer subunit A. The ATP ligands from both subunits are represented in green sticks. The p.Gly445Ser variant may moderately affect ATP/FAD binding site due to its close proximity. The p.Arg453Ile variant may have a more severe effect due to predominant preference for only Arg among orthologs.

Figure 3

Enzyme Activity of Recombinant Human TKFC Protein Expressed and Purified from Escherichia coli, and Western Blot of TKFC in Subject Fibroblasts (A–C) Enzyme activity was measured using either 10 mmol/L D-glyceraldehyde (A) or 10 mmol/L dihydroxyacetone (B) as substrate. Equal amounts of recombinant protein was used for activity assays as adjusted by polyacrylamide electrophoresis (representative gel in C). Bars show average activity, error bars standard deviation. Recombinant protein was isolated in three replicates in case of wild-type (WT) and the p.Gly445Ser variant and in two replicates for the p.Arg543Ile variant. (D) TKFC protein studies in family 1 show significant reduction of TKFC protein levels in P1-1 and P1-2 compared to metabolic disease control subjects. Protein levels were calculated as a ratio of TKFC levels to vinculin levels. Data are expressed as median with individual data points. One-way ANOVA with Tukey’s multiple comparisons test post hoc was performed to determine significance as indicated by ^∗p < 0.05 and ^∗∗p < 0.01. Representative western blot shows lane 1, protein ladder (L); lane 2, metabolic control (C); lanes 4–6, affected individual P1-1; lanes 7–9: affected individual P1-2.

Figure 4

Functional Studies in Yeast Effect of mutations in wild-type and mutated human and yeast DAK on yeast growth using DHA as sole carbon source. Yeast cells freshly grown on pre-culture plates were inoculated in DHA medium at an initial OD600 nm of 0.2. The cultures were incubated for 4 days at 28°C with vigorous agitation. The OD600 nm were then recorded. The growth experiments were repeated at least twice and the data averaged. Error bars represent standard deviation. Control, cells (Δdak1 or Δdak1&Δdak2) without DAK overexpressing plasmid; yDAK2, cells overexpressing yeast DAK2; hDAK, cells overexpressing the WT human DAK/TKFC gene; hR543I and hG445S, cells overexpressing mutated human DAK/TKFC; yR552I and yG458S, cells overexpressing mutated yeast DAK.