Molecular Insights on Pathogenic Effects of Mutations Causing Phosphoglycerate Kinase Deficiency

Abstract

Phosphoglycerate kinase (PGK) catalyzes an important ATP-generating step in glycolysis. PGK1 deficiency is an uncommon X-linked inherited disorder, generally characterized by various combinations of non-spherocytic hemolytic anemia, neurological dysfunctions, and myopathies. Patients rarely exhibit all three clinical features. To provide a molecular framework to the different pathological manifestations, all known mutations were reviewed and 16 mutant enzymes, obtained as recombinant forms, were functionally and structurally characterized. Most mutations heavily affect thermal stability and to a different extent catalytic efficiency, in line with the remarkably low PGK activity clinically observed in the patients. Mutations grossly impairing protein stability, but moderately affecting kinetic properties (p.I47N, p.L89P, p.C316R, p.S320N, and p.A354P) present the most homogeneous correlation with the clinical phenotype. Patients carrying these mutations display hemolytic anemia and neurological disorders, and,except for p.A354P variant, no myopaty. Variants highly perturbed in both catalytic efficiency (p.G158V, p.D164V, p.K191del, D285V, p.D315N, and p.T378P) and heat stability (all, but p.T378P) result to be mainly associated with myopathy alone. Finally, mutations faintly affecting molecular properties (p.R206P, p.E252A, p.I253T, p.V266M, and p.D268N) correlate with a wide spectrum of clinical symptoms. These are the first studies that correlate the clinical symptoms with the molecular properties of the mutant enzymes. All findings indicate that the different clinical manifestations associated with PGK1 deficiency chiefly depend on the distinctive type of perturbations caused by mutations in the PGK1 gene, highlighting the need for determination of the molecular properties of PGK variants to assist in prognosis and genetic counseling. However, the clinical symptoms can not be understood only on the bases of molecular properties of the mutant enzyme. Different (environmental, metabolic, genetic and/or epigenetic) intervening factors can contribute toward the expression of PGK deficient clinical phenotypes.

A new variant of phosphoglycerate kinase deficiency (p.I371K) with multiple tissue involvement: Molecular and functional characterization

“…The plasmid coding for Ile371Lys mutant enzyme was obtained by subjecting pMM1, a derivative of pET-23b(+) containing the PGK-1 cDNA [20], to site-directed mutagenesis and using Quick Change XL Site-directed Mutagenesis Kit (Stratagene). The following sense and antisense mutagenic oligonucleotides used were: 5′-GCTGCATCACCATCAAAGGTGGTGGAGACA-3′ and 5′-TGTCTCCACCACCTTTGATGGTGATGCAGC-3′, respectively.…”

“…The mutant enzyme was expressed in Escherichia coli BL21(DE3) pLysS cells transformed with the above-mentioned plasmid and induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (25°C, 5 h), and purified to homogeneity using the method previously reported [20]. The enzyme purified from 1 L culture was approximately 14 mg.…”

“…In recent work we have functionally and structurally characterized 16 mutant PGK enzymes obtained as recombinant forms, and compared their molecular properties to those of the recombinant wild-type enzyme, with the final aim to find the reasons of the different pathological dysfunctions exhibited by patients [20]. By this study it has been highlighted that hemolysis associated with neurological dysfunctions is generally a trait of PGK1 deficient patients with unstable variants but only mildly affected in catalytic properties.…”

Clinical Severity of PGK1 Deficiency Due To a Novel p.E120K Substitution Is Exacerbated by Co-inheritance of a Subclinical Translocation t(3;14)(q26.33;q12), Disrupting NUBPL Gene

“…S4) most likely due to an intrinsic propensity of aggregating in a temperature-and timedependent manner (Pey et al 2013). These data led us to conclude that p.E120K is one of the most severely impaired PGK1 variants characterized thus far Chiarelli et al 2012) and consequently the main genetic cause of the phenotype observed in this family.…”

“…PGK1 deficiency (OMIM #300653) is an X-linked recessive condition characterized by variable clinical manifestations involving up to three different tissues, i.e., red blood cells (RBC), skeletal muscle, and neurological tissue. However, patients rarely show all three clinical features (Beutler 2007;Chiarelli et al 2012). To date, 22 different mutations have been identified in PGK1, and 17 of them have been characterized at the protein level Fermo et al 2012;Tamai et al 2014).…”