Identification, clinical manifestation and structural mechanisms of mutations in AMPK associated cardiac glycogen storage disease

Abstract

Background: Although 21 causative mutations have been associated with PRKAG2 syndrome, our understanding of the syndrome remains incomplete. The aim of this project is to further investigate its unique genetic background, clinical manifestations, and underlying structural changes.

Methods: We recruited 885 hypertrophic cardiomyopathy (HCM) probands and their families internationally. Targeted next-generation sequencing of sudden cardiac death (SCD) genes was performed. The role of the identified variants was assessed using histological techniques and computational modeling.

Findings: Twelve PRKAG2 syndrome kindreds harboring 5 distinct variants were identified. The clinical penetrance of 25 carriers was 100.0%. Twenty-two family members died of SCD or heart failure (HF). All probands developed bradycardia (HRmin, 36.3 ± 9.8 bpm) and cardiac conduction defects, and 33% had evidence of atrial fibrillation/paroxysmal supraventricular tachycardia (PSVT) and 67% had ventricular preexcitation, respectively. Some carriers presented with apical hypertrophy, hypertension, hyperlipidemia, and renal insufficiency. Histological study revealed reduced AMPK activity and major cardiac channels in the heart tissue with K485E mutation. Computational modelling suggests that K485E disrupts the salt bridge connecting the β and γ subunits of AMPK, R302Q/P decreases the binding affinity for ATP, T400N and H401D alter the orientation of H383 and R531 residues, thus altering nucleotide binding, and N488I and L341S lead to structural instability in the Bateman domain, which disrupts the intramolecular regulation.

Interpretation: Including 4 families with 3 new mutations, we describe a cohort of 12 kindreds with PRKAG2 syndrome with novel pathogenic mechanisms by computational modelling. Severe clinical cardiac phenotypes may be developed, including HF, requiring close follow-up.

Keywords: Arrhythmia; Cardiomyopathy; Genetics; Heart failure; PRKAG2 syndrome; Sudden cardiac death.

Fig. 1

Clinical records of R302Q mutant carriers. (a-g) Family pedigrees of Proband 1–7. +/- indicates a heterozygous mutation, circles/squares represent female/male subjects. The arrow indicates the proband. (h) ECG of Proband 2 (deceased) displaying preexcitation, complete left bundle branch block, left ventricular hypertrophy, PR interval 70 ms, and QRS 145 ms.

Fig. 2

Clinical records of novel PRKAG2 mutation carriers. (a-c) 12-lead ECG of PRKAG2-R302P, L341S and H401D carrier at baseline (Proband 8–10). (d-e) Echo images of Proband 7 with R302P in a short axis view of chord and papillary muscle level of LV, showing increased thickness of LV. (f-g) Echo image of Proband 9 with H401D in a long axis view of the LV and a four chambers view, showing increased thickness of the interventricular septum and lateral wall of LV.

Fig. 3

Genetic information of novel mutations. (a) Schematic of AMPK γ2 subunit and PRKAG2 mutations discovered thus far. Novel mutations are shown in red. (b-c) Pedigree of the PRKAG2-L341S and H401D mutation carriers (Proband 9&10). (d-f) DNA chromatogram of L341S, H401D and R302P. (g) Histopathology of ventricular sections with Congo-Red staining from proband 8. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

The macroscopic image and AMPK quantification of the original heart from Proband 12. (a-c) The front/back/side view of the original heart. The apex is blunt, and the fat is slightly increased. The left ventricular wall is obviously thickened, and the subendocardial fibers are increased. (d-i): Phosphorylated AMPK of myocardial tissue (× 400 fold). In d&g/e&h/f&i, nucleuses/phosphorylated AMPK/the overlap are stained in blue/red/both. j: Quantification of phosphorylated AMPK (control myocardium: n = 461; patient myocardium: n = 450). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

RMSD comparison of AMPK grouped by (a) residue location and (b) ligand identity. Structures were aligned on the gamma subunit prior to all RMSD calculations. (c) RMSF plots for the seven simulated variants. (d) RMSF values for the mutated residues at positions 485 and 302; the simulations from which the data were obtained are shown along the x-axis. Values with significant difference from the WT are indicated with an asterisk (see details of p-value at Supplementary Table 4).

Fig. 6

Electrostatic surfaces of different mutations compared with WT. (a&b) AMPK-WT vs. R302Q. (c-e) AMPK-WT vs. L341S and H401D. Change in the orientation of the nucleotide binding pocket in H401 (f) induced by the H401D mutation (g). Positively/negatively-charged regions are shown in red/blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Entropy transfer from each residue (a), and from R302 (b), K485 (c), H401 (d), and L341 (e) to the rest of the protein.

Fig. 8

Unique genetic background, clinical manifestation and underlying mechanisms, as well as management options of PRKAG2 cardiac syndrome. ECG, electrocardiogram; MRI, magnetic resonance imaging; Echo, echocardiogram; ICD, implantable cardioverter-defibrillator.