The dystroglycanopathies are a novel group of human muscular dystrophies due to mutations in known or putative glycosyltransferase enzymes. They share the common pathological feature of a hypoglycosylated form of alpha-dystroglycan, diminishing its ability to bind extracellular matrix ligands. The LARGE glycosyltransferase is mutated in both the myodystrophy mouse and congenital muscular dystrophy type 1D (MDC1D). We have transfected various cell lines with a variety of LARGE expression constructs in order to characterize their subcellular localization and effect on alpha-dystroglycan glycosylation. Wild-type LARGE co-localized with the Golgi marker GM130 and stimulated the production of highly glycosylated alpha-dystroglycan (hyperglycosylation). MDC1D mutants had no effect on alpha-dystroglycan glycosylation and failed to localize correctly, confirming their pathogenicity. The two predicted catalytic domains of LARGE contain three conserved DxD motifs. Systematically mutating each of these motifs to NNN resulted in the mislocalization of one construct, while all failed to have any effect on alpha-dystroglycan glycosylation. A construct lacking the transmembrane domain also failed to localize at the Golgi apparatus. These results indicate that LARGE needs to both physically interact with alpha-dystroglycan and function as a glycosyltransferase in order to stimulate alpha-dystroglycan hyperglycosylation. We have also cloned and overexpressed a homologue of LARGE, glycosyltransferase-like 1B (GYLTL1B). Like LARGE it localized to the Golgi apparatus and stimulated alpha-dystroglycan hyperglycosylation. These results suggest that GYLTL1B may be a candidate gene for muscular dystrophy and that its overexpression could compensate for the deficiency of both LARGE and other glycosyltransferases.

Background: Limb-girdle muscular dystrophy type 2I is caused by mutations in the fukutin-related protein gene (FKRP). FKRP encodes a putative glycosyltransferase protein that is involved in ␣-dystroglycan glycosylation.Objectives: To identify patients with limb-girdle muscular dystrophy type 2I and to derive genotypephenotype correlations.Design: Two hundred fourteen patients who showed muscle histopathologic features consistent with muscular dystrophy or myopathy of unknown etiology were studied. The entire 1.5-kilobase FKRP coding sequence from patient DNA was analyzed using denaturing highperformance liquid chromatography of overlapping polymerase chain reaction products, followed by direct sequencing of heteroduplexes.Results: Thirteen patients with limb-girdle muscular dystrophy type 2I (6% of all patients tested) were identified by FKRP mutation analysis, and 7 additional patients were identified by family screening. Six missense mutations (1 novel) were identified. The 826CϾA nucleotide change was a common mutation, present in 35% of the mutated chromosomes. Clinical presentations included asymptomatic hyperCKemia, severe early-onset muscular dystrophy, and mild late-onset muscular dystrophy. Dilated cardiomyopathy and ventilatory impairment were frequent features. Significant intrafamilial and interfamilial clinical variability was observed.Conclusions: FKRP mutations are a frequent cause of limb-girdle muscular dystrophies. The degree of respiratory and cardiac insufficiency in patients did not correlate with the severity of muscle involvement. The finding of 2 asymptomatic patients with FKRP mutations suggests that modulating factors may ameliorate the clinical phenotype.

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Limb girdle muscular dystrophy type 2I (LGMD2I) is due to mutations in the fukutin-related protein gene (FKRP), encoding a putative glycosyltransferase involved in alpha-dystroglycan processing. To further characterize the molecular pathogenesis of LGMD2I, we conducted a histological, immunohistochemical, ultrastructural and molecular analysis of ten muscle biopsies from patients with molecularly diagnosed LGMD2I. Hypoglycosylation of alpha-dystroglycan was observed in all FKRP-mutated patients. Muscle histopathology was consistent with either severe muscular dystrophy or myopathy with a mild inflammatory response consisting of up-regulation of class I major histocompatibility complex in skeletal muscle fibers and small foci of mononuclear cells. At the ultrastructural level, muscle fibers showed focal thinning of basal lamina and swollen endoplasmic reticulum cisternae with membrane re-arrangement. The pathways of the unfolded protein response (UPR; glucose-regulated protein 78 and CHOP) were significantly activated in LGMD2I muscle tissue. Our data suggest that the UPR response is activated in LGMD2I muscle biopsies, and the observed histopathological and ultrastructural alterations may be related to sarcoplasmic structures involved in FKRP and alpha-dystroglycan metabolism and malfunctioning.