Holleboom, Adriaan G.

Background and Aims Vacuolar H+‐ATP complex (V‐ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V‐ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X‐linked mutations lead to autophagic myopathy. Approach and Results Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low‐density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V‐ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element‐binding protein–mediated cholesterol synthesis pathways. Conclusions Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V‐ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.

Background: The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders of glycosylation (CDGs) with defective N-glycosylation. Methods: We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)–deficiency CDG and PMM2 (phosphomannomutase 2)–deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models—gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells—by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied. Results: We report hypobetalipoproteinemia (LDL cholesterol [LDL-C] and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator. Conclusions: Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor–mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.