Computational structural genomics and clinical evidence suggest BCKDK gain-of-function may cause a potentially asymptomatic maple syrup urine disease phenotype

Abstract

Maple syrup urine disease (MSUD) is a disorder of branched-chain amino acid metabolism caused by a defect in the branched-chain α-ketoacid dehydrogenase (BCKD) complex (OMIM #248600). The hallmark presentation is encephalopathic crisis in neonates, but can also present with metabolic decompensation, developmental delays, and feeding difficulties. Biochemical evidence for MSUD includes elevated branched-chain amino acids (BCAA) and the pathognomonic presence of alloisoleucine. The BCKD complex contains several subunits associated with autosomal recessive MSUD, while its regulatory proteins have less well-defined disease associations. We report on two families with the same BCKDK variant (c.1115C>G (p.Thr372Arg)). Probands were detected on newborn screening and demonstrated biochemical evidence of MSUD. The variant was identified in reportedly asymptomatic parents and additional family members who had elevated BCAA and alloisoleucine, following an autosomal dominant pattern of inheritance. To better define the functional effect of the variant on the kinase, we completed molecular modeling using sequence-based (2D), structural-based (3D), and dynamic-based (4D) analyses. The BCKDK variant modeling indicated a gain-of-function which leads to impaired BCAA catabolism consistent with the biochemical evidence in this cohort. Combining the evidence gained from molecular modeling with the absence of metabolic decompensation in our patients and several adult family members, despite encountering stressors typically problematic in classic MSUD, we suggest that heterozygous gain-of-function variants in BCKDK may represent a novel biochemical phenotype of MSUD with a benign clinical course.

Keywords: 3D‐genomics; branched‐chain ketoacid dehydrogenase kinase (BCKDK); gain‐of‐function; maple syrup urine disease (MSUD); molecular dynamics simulation; newborn screening.

FIGURE 1

Pedigree of (A) Patient #1 and (B) Patient #2. Plus (+) denotes individuals who carry the BCKDK c.1115C>G (p.Thr372Arg) variant. Shaded icons denote individuals with elevated branched‐chain amino acids. *Amino acid analysis was not completed for Patient #1's 22y maternal half‐sister.

FIGURE 2

Molecular impact of the p.Thr372Arg variant. (A) Domain structure and the locations of the p.Thr372Arg variant and the control variants in the sequence. (B) Sequence conservation within the region of interest among mammals. The Thr372 residue is indicated by a blue arrow. The sequence alignment of the full‐length protein is shown in Figure S1. (C) A dimeric molecular structure of the BCKDK kinase domain in complex with ATP and the metal ions for impact assessment. The kinase domain is further divided into the active site‐containing K‐domain and the regulatory B‐domain. The key functional sites are also indicated. The close‐up insets show the structural comparison of the wild type and the p.Thr372Arg variant at the end of molecular dynamics simulations. New bond formations help the positioning of the ATP cofactor for a more effective “in‐line” transfer of the phosphoryl group to the substrate. The neighboring hydrophobic residues at the B‐K domain interface are shown as a ball‐and‐stick model. (D) Superposition of the wild type and the p.Thr372Arg variant structures highlighting the outward helical movement of the a4‐a5 central helices by the variant. Loosening up the B‐K domain interface allows optimal substrate binding and presentation for catalysis. (E) Comparison of the alterations in ATP and B‐K domain interaction energies for the p.Thr372Arg case variant and the loss‐of‐function control variants compared to the wild type. The p.Arg174Gly BCKDK deficiency autism variant and p.Try331Ala lab control variant are proven to be loss‐function variants (references indicated) while the p.Arg177Trp BCKDK deficiency autism variant is currently annotated as a variant of uncertain significance (VUS) yet predicted to be a loss‐of‐function variant by our analysis. The B‐K domain interaction energy column is indicated by an arrow. The negative free energies indicate stronger interactions.