The fructose-1,6-bisphosphatase deficiency and the p.(Lys204ArgfsTer72) variant

Abstract

Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inborn error of fructose metabolism caused by pathogenic variants in the FBP1 gene. As gluconeogenesis is affected, catabolic episodes can induce ketotic hypoglycemia in patients. FBP1 analysis is the most commonly used approach for the diagnosis of this disorder. Herein, a Brazilian patient is reported. The proband, a girl born to a consanguineous couple, presented with severe hypoglycemia crisis in the neonatal period. At the age 17 months, presented a new crisis accompanied by metabolic acidosis associated with a feverish episode. Genetic analysis was performed by next-generation sequencing (NGS), identifying the NM_000507.3:c.611_614del variant in homozygosis in the FBP1 gene. In silico analysis and 3D modeling were performed, suggesting that this variant is associated with a loss of sites for substrate and Mg2+ binding and for posttranslational modifications of FBPase. The c.611_614del variant is located in a repetitive region of the FBP1 gene that appears to be a hotspot for mutational events. This frameshift creates a premature termination codon in the last coding exon which escapes the nonsense-mediated decay mechanism, according to in silico analysis. This variant results in an intrinsically disordered protein with loss of substrate recognition and post-translational modification sites.

Figure 1 -. Sequences analysis of the FBP1 gene and fructose-1,6-bisphosphatase. A) Electropherogram of control, patient and her mother evidencing the affected region in the FBP1 gene. The control sample is a normal homozygous to the analyzed region; in their electropherogram, the gray rectangle indicates the deleted nucleotides in the patient. Highlighted in the second electropherogram, the position of the nucleotides deleted in a homozygous patient to the variant NM_000507.3:c.611_614del in FBP1 gene. The mother's electropherogram evidences that she is a carrier of the variant in analysis. B) Amino acid sequence of the FBPase resulting from the pathogenic variant (mut) in comparison with the wild type (WT). C) Diagram representing FBP1 DNA, FBP1 mRNA, and FBPase variant. In DNA, the blue boxes indicate the coding exons and the region of 50-55 nucleotides upstream of the last intron that is susceptible to scaping NMD is indicated. Red indicates the variant position in DNA and its consequences in mRNA and FBPase protein. Gray arrows refer to the start of the frameshift, PTC (premature terminator codon) and NTC (normal terminator codon) between DNA and mRNA. In the protein diagram, blue dots indicate modified amino acids; yellow dots indicate metal binding sites; in pink the substrate binding regions and in green the active site. RefSeq: NM_000507.3 and P09467. The FBPase domains were named according Pfam.

Figure 2 -. In silico analysis of the NP_000498.2:p.(Lys204ArgfsTer32) variant in the FBP1 gene. A) Disorder prediction for wild-type (blue line) and mutated (orange line) FBPases. B) 3D modeling of fructose-1,6-bisphosphatase. First image, the wild-type FBPase. The region affected in the mutant protein is shown in light blue, the altered substrate binding regions are shown in dark blue, the altered substrate recognition site is shown in magenta, and the altered Mg binding site is shown in green. Right below, the mutant FBPase. The region affected by the frameshift mutation is shown in orange. All sites highlighted in WT are absent in the mutant protein. Lastly, the superposition of WT and mutant FBPase, colored according to the previous. C) The structure of fructose-1,6-bisphosphatase tetramer. Superposition of mutant FBPase monomer (red) onto the functional FBPase tetramer (in different shades of gray). Note the absence of structured elements in the exposed region of the monomer. Tetramer information from PDB ID 2FIE (Lai et al., 2006).