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Abstract
α-dystroglycan (α-DG) is a peripheral membrane protein that is an integral component of the dystrophin-glycoprotein complex. In an inherited subset of muscular dystrophies known as dystroglycanopathies, α-DG has reduced glycosylation which results in lower affinity binding to several extracellular matrix proteins including laminins. The glycosylation status of α-DG is normally assessed by the binding of the α-DG antibody IIH6 to a specific glycan epitope on α-DG involved in laminin binding. Immunocytochemistry and immunoblotting are two of the most widely used methods to detect the amount of α-DG glycosylation in muscle. While the interpretation of the presence or absence of the epitope on muscle using these techniques is straightforward, the assessment of a mild defect can be challenging. In this study, flow cytometry was used to compare the amount of IIH6-reactive glycans in fibroblasts from dystroglycanopathy patients with defects in genes known to cause α-DG hypoglycosylation to the amount in fibroblasts from healthy and pathological control subjects. A total of twenty one dystroglycanopathy patient fibroblasts were assessed, as well as fibroblasts from three healthy controls and seven pathological controls. Control fibroblasts have clearly detectable amounts of IIH6-reactive glycans, and there is a significant difference in the amount of this glycosylation, as measured by the mean fluorescence intensity of an antibody recognising the epitope and the percentage of cells positive for the epitope, between these controls and dystroglycanopathy patient fibroblasts (p<0.0001 for both). Our results indicate that the amount of α-DG glycosylation in patient fibroblasts is comparable to that in patient skeletal muscle. This method could complement existing immunohistochemical assays in skeletal muscle as it is quantitative and simple to perform, and could be used when a muscle biopsy is not available. This test could also be used to assess the pathogenicity of variants of unknown significance in genes involved in dystroglycanopathies.
Highlights
The congenital muscular dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders with varying degrees of clinical severity, broadly characterised by progressive muscle degeneration, weakness, and often central nervous system involvement
They are caused by mutations in several genes involved in the glycosylation of a-DG; Protein O-mannosyltransferase [1] (POMT1; MIM 607423), Protein O-mannosyltransferase 2 [2] (POMT2; MIM 607439), Protein O-mannose ß-1,2-N-acetylglucosaminyltransferase [3] (POMGNT1; MIM 606822), Fukutin [4] (FKTN; MIM 607440), Fukutin-related protein [5] (FKRP; MIM 606596), like-acetylglucosaminyltransferase [6]
Subjects and Clinical Features A total of twenty one dystroglycanopathy patient fibroblasts were assessed in this study; six with mutations in FKRP, three in POMGNT1, four in POMT1, two in POMT2, two in ISPD, two in B3GALNT2, and two in GDP-mannose pyrophosphorylase B (GMPPB)
Summary
The congenital muscular dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders with varying degrees of clinical severity, broadly characterised by progressive muscle degeneration, weakness, and often central nervous system involvement. 603590), Dolichyl-phosphate mannosyltransferase 2 [7] (DPM2: MIM 603564), Dolichyl-phosphate mannosyltransferase 3 [8] (DPM3; MIM 605951), Dolichol Kinase [9] (DOLK; MIM 610746), Isoprenoid Synthase Domain Containing [10,11,12] (ISPD; MIM 614631), Glycosyltransferase-like domain containing 2 [13] (GTDC2; MIM 147730), b-1,3-N-acetylgalactosaminyltransferase 2 [14] (B3GALNT2; MIM 610194), Transmembrane protein 5 (TMEM5; MIM 605862) [15], b-1,3-N-acetylglucosaminyltransferase 1 (B3GNT1; MIM 605517) [16], GDP-mannose pyrophosphorylase B (GMPPB) [17], and protein kinase-like protein SgK196 (SGK196) [18] Mutations in these genes account for the majority of dystroglycanopathy patients not all carry mutations in any of these genes [10,19,20]. This phenotypic classification system is described in more detail in Godfrey et al, 2011 [22]
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