Adaptive Modelling of Mutated FMO3 Enzyme Could Unveil Unexplored Scenarios Linking Variant Haplotypes to TMAU Phenotypes

Abstract

Background: Trimethylaminuria (TMAU) is a rare genetic disease characterized by the accumulation of trimethylamine (TMA) and its subsequent excretion trough main body fluids, determining the characteristic fish odour in affected patients. We realized an experimental study to investigate the role of several coding variants in the causative gene FMO3, that were only considered as polymorphic or benign, even if the available literature on them did not functionally explain their ineffectiveness on the encoded enzyme.

Methods: Mutational analysis of 26 TMAU patients was realized by Sanger sequencing. Detected variants were, subsequently, deeply statistically and in silico characterized to determine their possible effects on the enzyme activity. To achieve this goal, a docking prediction for TMA/FMO3 and an unbinding pathway study were performed. Finally, a TMAO/TMA urine quantification by 1H-NMR spectroscopy was performed to support modelling results.

Results: The FMO3 screening of all patients highlighted the presence of 17 variants distributed in 26 different haplotypes. Both non-sense and missense considered variants might impair the enzymatic kinetics of FMO3, probably reducing the interaction time between the protein catalytic site and TMA, or losing the wild-type binding site.

Conclusions: Even if further functional assays will confirm our predictive results, considering the possible role of FMO3 variants with still uncertain effects, might be a relevant step towards the detection of novel scenarios in TMAU etiopathogenesis.

Keywords: FMO3; TMAU; genetic variants; in silico; proteomics.

Figure 1

3D structural models of FMO3 proteins in patients carrying missense variants. This panel highlights the predicted tertiary structure of FMO3 in patients 5 (P153L and E158K) (a), 6 (E158K) (b), 7 (V257M) (c), 8 (E158K and E308G) (d), 9 (E158K and R492W (e), 10 (E158K and R238Q) (f), 11 (E158K and G475D) (g) and 12 (D141V and G180V) (h). Green ball-and-stick aa = wild-type aa. Red ball-and-stick aa = mutated aa. Yellow ball-and-stick aa = aa nearest to aa involved in variant.

Figure 2

3D structural models of FMO3 proteins in patients carrying non-sense variants. This panel highlights the predicted tertiary structure of FMO3 in patients 1 (Y331Stop) (a), 2 (P153L, E158K and P380fs) (b), 3 (P380fs) (c), and 4 (E308G and P380fs) (d). The grey tube/ribbons represent the wild-type structures, while the light blue the over imposed mutated ones. The green spheres represent the predicted ligands/coenzymes/cofactors exclusive of wild-type FMO3 (ADP, Na, and TMA), while the red spheres the predicted ones exclusive of different truncated FMO3 (Indole, Mg, and TMA; exceptions are represented by patient 3, who does not present the Mg, and by patient 4, who does not present any exclusive ligand compared to wild-type form of FMO3). Lateral chains of amino acids involved in missense variants are represented in green for the wild-type allele and in red for the mutated one. Yellow chains are characteristics of amino acids nearest to mutated ones.

Figure 3

TMA docking to FMO3 could involve different amino acids in mutated enzymes. The non-sense and missense variants carried by mutated FMO3 (a–o) might shift the TMA binding sites far from the wild-type active site of the enzyme (p). The black arrows indicate the TMA (white spheres) bonded to the active site of FMO3, whose aa are represented as ball-and-stick. The other aa, represented as ball-and-stick, separated from the ones in the catalytic site, are the aa involved in mutations.

Figure 4

The unbinding pathways analyses showed that FMO3 catalytic activity might not be fully performed. The TMA unbinding pathways through the whole FMO3 mutated proteins (a–o) seemed to impair the enzyme functionality when compared to the wild-type one (p). The yellow circles indicate the unbinding pathway produced by pathlines (purple line) of TMA (white spheres) through the FMO3.

Figure 5

500 MHz ¹H spectra of patients included in the study. Here, are represented the spectra resulting from ¹H NMR spectroscopy of the urine collected from patients showing a unique FMO3 variant haplotype. A healthy subject should present no peak for TMA and an intermediate peak for TMAO (not shown). Obtained results mostly confirmed genotyping and modelling analyses. (a) Patient 3; (b) Patient 10; (c) Patient 2; (d) Patient 9; (e) Patient 11; (f) Patient 6; (g) Patient 1; (h) Patient 8; (i) Patient 2; (l) Patient 4; (m) Patient 12; and (n) Patient 7. Metabolites peaks are assigned as follows: Trimethylamine-N-oxide (TMAO): 3.27 ppm; Creatinine (Cr): 3.06 ppm; Creatine (Cn): 3.04 ppm; Trimethylamine (TMA): 2.92 ppm; Dimethylamine (DMA): 2.73 ppm; and Citrulline (cit): 1.62 and 1.92 ppm.