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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: Genetics, Arrhythmia, PRKAG2 syndrome, Cardiomyopathy, Heart failure, Sudden cardiac death

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus–like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1pdn concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region–integral membrane proteins cycle through ER in interphase cells.

Human erythrocytes are terminally differentiated, nonendocytic cells that lack all intracellular organelles. Here we show that their plasma membranes contain detergent-resistant membrane rafts that constitute a small fraction (4%) of the total membrane protein, with a complex mixture of proteins that differentially associate with rafts. Depletion of raft-cholesterol abrogates association of all proteins with no significant effect on cholesterol:protein ratios in the rest of the membrane, lipid asymmetry, deformability, or transport properties of the bilayer, indicating that cholesterol is critical for protein assembly into rafts and suggesting that rafts have little influence on several erythrocyte functions. Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, which lack glycosylphosphatidylinositol-anchored proteins, show significant elevation in raft-cholesterol but no increase in raft protein association, suggesting that raft assembly does not require glycosylphosphatidylinositol-anchored proteins, raft proteins do not bind directly to cholesterol, and only threshold levels of raft-cholesterol are critical for protein recruitment. Loss of glycosylphosphatidylinositol-anchored proteins had no effect on erythrocytic infection by malarial parasite or movement of raft markers into the parasite's vacuole. However, infection is blocked following raft-cholesterol disruption, suggesting that erythrocyte rafts can be functionally exploited and providing the first evidence for the involvement of host rafts in an apicomplexan infection.