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Protein O-glucosyltransferase 1 (POGLUT1) activity is critical for the Notch signaling pathway, being one of the main enzymes responsible for the glycosylation of the extracellular domain of Notch receptors. A biallelic mutation in the POGLUT1 gene has been reported in one family as the cause of an adult-onset limb-girdle muscular dystrophy (LGMD R21; OMIM# 617232). As the result of a collaborative international effort, we have identified the first cohort of 15 patients with LGMD R21, from nine unrelated families coming from different countries, providing a reliable phenotype–genotype and mechanistic insight. Patients carrying novel mutations in POGLUT1 all displayed a clinical picture of limb-girdle muscle weakness. However, the age at onset was broadened from adult to congenital and infantile onset. Moreover, we now report that the unique muscle imaging pattern of “inside-to-outside” fatty degeneration observed in the original cases is indeed a defining feature of POGLUT1 muscular dystrophy. Experiments on muscle biopsies from patients revealed a remarkable and consistent decrease in the level of the NOTCH1 intracellular domain, reduction of the pool of satellite cells (SC), and evidence of α-dystroglycan hypoglycosylation. In vitro biochemical and cell-based assays suggested a pathogenic role of the novel POGLUT1 mutations, leading to reduced enzymatic activity and/or protein stability. The association between the POGLUT1 variants and the muscular phenotype was established by in vivo experiments analyzing the indirect flight muscle development in transgenic Drosophila, showing that the human POGLUT1 mutations reduced its myogenic activity. In line with the well-known role of the Notch pathway in the homeostasis of SC and muscle regeneration, SC-derived myoblasts from patients’ muscle samples showed decreased proliferation and facilitated differentiation. Together, these observations suggest that alterations in SC biology caused by reduced Notch1 signaling result in muscular dystrophy in LGMD R21 patients, likely with additional contribution from α-dystroglycan hypoglycosylation. This study settles the muscular clinical phenotype linked to POGLUT1 mutations and establishes the pathogenic mechanism underlying this muscle disorder. The description of a specific imaging pattern of fatty degeneration and muscle pathology with a decrease of α-dystroglycan glycosylation provides excellent tools which will help diagnose and follow up LGMD R21 patients., This work was supported in part by the Instituto de Salud Carlos III and FEDER (FIS PI16-01843 to C. Paradas and JR15/00042 to M. Cabrera-Serrano), the Consejería de Salud, Junta de Andalucía (PI-0085-2016 and PE-S1275 to C. Paradas, and B-0005-2017 to M. Cabrera-Serrano), NIH/NIGMS (R01GM084135 and R35GM130317 to H. Jafar-Nejad, and R01GM061126 to R.S. Haltiwanger), JSPS KAKENHI Grants-in-Aid for Research Activity Start-up and Scientific Research (B) (JP17H06743 and JP19H03176 to H. Takeuchi), and Takeda Science Foundation and Daiichi Sankyo Foundation of Life Science (to H. Takeuchi). MYO-SEQ has been supported by Sanofi Genzyme, Ultragenyx, the LGMD2I Research Fund, Samantha J Brazzo Foundation, LGMD2D Foundation, Kurt + Peter Foundation, Muscular Dystrophy UK and Coalition to Cure Calpain 3. Work in CGB’s group is supported by NINDS/NIH intramural funds.