Fatal congenital heart glycogenosis caused by a recurrent activating R531Q mutation in the gamma 2-subunit of AMP-activated protein kinase (PRKAG2), not by phosphorylase kinase deficiency

Abstract

Fatal congenital nonlysosomal cardiac glycogenosis has been attributed to a subtype of phosphorylase kinase deficiency, but the underlying genes and mutations have not been identified. Analyzing four sporadic, unrelated patients, we found no mutations either in the eight genes encoding phosphorylase kinase subunits or in the two genes encoding the muscle and brain isoforms of glycogen phosphorylase. However, in three of five patients, we identified identical heterozygous R531Q missense mutations of the PRKAG2 gene, which encodes the gamma 2-subunit of AMP-activated protein kinase, a key regulator of energy balance. Biochemical characterization of the recombinant R531Q mutant protein showed >100-fold reduction of binding affinities for the regulatory nucleotides AMP and ATP but an enhanced basal activity and increased phosphorylation of the alpha -subunit. Other PRKAG2 missense mutations were previously identified in patients with autosomal dominant hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome, characterized by juvenile-to-adult clinical onset, moderate cardiac glycogenosis, disturbed excitation conduction, risk of sudden cardiac death in midlife, and molecular perturbations that are similar to--but less severe than--those observed for the R531Q mutation. Thus, recurrent heterozygous R531Q missense mutations in PRKAG2 give rise to a massive nonlysosomal cardiac glycogenosis of fetal symptomatic onset and rapidly fatal course, constituting a genotypically and clinically distinct variant of hypertrophic cardiomyopathy with Wolff-Parkinson-White syndrome. R531Q and other PRKAG2 mutations enhance the basal activity and alpha -subunit phosphorylation of AMP-activated protein kinase, explaining the dominant nature of PRKAG2 disease mutations. Since not all cases displayed PRKAG2 mutations, fatal congenital nonlysosomal cardiac glycogenosis seems to be genetically heterogeneous. However, the existence of a heart-specific primary phosphorylase kinase deficiency is questionable, because no phosphorylase kinase mutations were found.

Figure 1

Heterozygous R531Q missense mutation underlying fatal congenital nonlysosomal heart glycogenosis. A typical sequencer readout is shown (top). The high conservation of the mutant R531 residue is illustrated (middle) by alignments with the corresponding partial sequences from CBS4 of the human (“Hs”) γ1 and γ3 isoforms, pig γ3, and the Drosophila (“Dros”) and yeast AMPK paralogs. Alignments with partial sequences from other CBS domains of γ2 and γ3, in which mutations were identified in the corresponding arginine residues or the adjacent histidine residue, are shown (bottom). In the course of analysis of the PRKAG2 gene and cDNA, the following additional sequence features were noted: (1) RT-PCR product sequences displayed partial deletion of codon 251 (apparently leaky splicing) at the beginning of exon 6 in all five patients and a normal control individual and (2) SNPs include CDS-26C→T (allele frequencies: C, 0.89; T, 0.11 [n=190]), Ex6+36insA (allele frequency: A, 0.83 [n=12]), and Ex10-42C→T (allele frequencies: C, 0.83; T, 0.17 [n=12]).

Figure 2

Image from electron microscopy of glycogen storage in myocardial fibers of R531Q-mutation–positive patient E. Large amounts of free monodispersed β-glycogen (“Gl”) accumulate between the myofibrils and beneath the sarcolemma. Many sarcomeres appear to be destroyed (arrows), and some fibers are totally degenerated (star). In most mitochondria (“M”), solitary vacuoles and electron-dense, thickened, and disoriented cristae can be observed. Numerous endomembranaceous vacuoles (“V”), some of them containing lipid, are seen in fibers with advanced degeneration (star). The scale bar is 1 μm.

Figure 3

Binding of AMP (panels A and C) and ATP (panels B and D) by fusion proteins between GST and the Bateman domain B of γ2 (“GST-B” [panels A and B]) or between GST and both Bateman domains (“GST-A/B” [panels C and D]). In panels A and B, data were fitted to the binding model: Y = L/(K_d+L). In panels C and D, data were fitted to a two-site Hill plot model: Y = 2 × L^h/(B_0.5^h + L^h). “Y” represents fractional saturation, “L” represents ligand concentration, “B_0.5” is the concentration of nucleotide that gives half-maximal binding, and “h” is the Hill coefficient. Best-fit parameters were estimated using GraphPad Prism (GraphPad software), and the curves were generated using the equation with those binding parameters.

Figure 4

Activation of heterotrimeric α1β1γ2 complexes expressed in HEK-293 cells, with extracts made using the slow lysis procedure that causes maximal phosphorylation (Hardie et al. 2000). The complexes contained either wild-type (“WT”) γ2 or R531G and R531Q mutations of γ2 and were assayed in anti-myc immunoprecipitates at various concentrations of AMP. Data were fitted to the following equation: , where “basal” is the activity in the absence of AMP, “stimulation” is the maximal stimulation by AMP, and “A_0.5” is the concentration of AMP that gives half-maximal activation. Best-fit parameters were estimated, and curves were generated as for figure 3.

Figure 5

AMPK activity (A), phosphorylation of Thr-172 on the AMPK α-subunit (B), and phosphorylation of the downstream target ACC (C) in HEK-293 cells expressing heterotrimeric α1β1γ2 complexes, with or without γ2 mutations, as in figure 4. Cells were harvested by both the rapid and the slow lysis procedures, and AMPK activities were measured in anti-myc immunoprecipitates in the absence of AMP. Data are means ± SE of the mean, for results from duplicate culture plates.