Transgenic overexpression of LARGE induces α-dystroglycan hyperglycosylation in skeletal and cardiac muscle.

BACKGROUND: LARGE is one of seven putative or demonstrated glycosyltransferase enzymes defective in a common group of muscular dystrophies with reduced glycosylation of α-dystroglycan. Overexpression of LARGE induces hyperglycosylation of α-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, restoring functional glycosylation. Viral delivery of LARGE to skeletal muscle in animal models of dystroglycanopathy has identical effects in vivo, suggesting that the restoration of functional glycosylation could have therapeutic applications in these disorders. Pharmacological strategies to upregulate Large expression are also being explored. METHODOLOGY/PRINCIPAL FINDINGS: In order to asses the safety and efficacy of long term LARGE over-expression in vivo, we have generated four mouse lines expressing a human LARGE transgene. On observation, LARGE transgenic mice were indistinguishable from the wild type littermates. Tissue analysis from young mice of all four lines showed a variable pattern of transgene expression: highest in skeletal and cardiac muscles, and lower in brain, kidney and liver. Transgene expression in striated muscles correlated with α-dystroglycan hyperglycosylation, as determined by immunoreactivity to antibody IIH6 and increased laminin binding on an overlay assay. Other components of the dystroglycan complex and extracellular matrix ligands were normally expressed, and general muscle histology was indistinguishable from wild type controls. Further detailed muscle physiological analysis demonstrated a loss of force in response to eccentric exercise in the older, but not in the younger mice, suggesting this deficit developed over time. However this remained a subclinical feature as no pathology was observed in older mice in any muscles including the diaphragm, which is sensitive to mechanical load-induced damage. CONCLUSIONS/SIGNIFICANCE: This work shows that potential therapies in the dystroglycanopathies based on LARGE upregulation and α-dystroglycan hyperglycosylation in muscle should be safe.

Muscular dystrophies due to glycosylation defects.

In the last few years, muscular dystrophies due to reduced glycosylation of alpha-dystroglycan (ADG) have emerged as a common group of conditions, now referred to as dystroglycanopathies. Mutations in six genes (POMT1, POMT2, POMGnT1, Fukutin, FKRP and LARGE) have so far been identified in patients with a dystroglycanopathy. Allelic mutations in each of these genes can result in a wide spectrum of clinical conditions, ranging from severe congenital onset with associated structural brain malformations (Walker Warburg syndrome; muscle-eye-brain disease; Fukuyama muscular dystrophy; congenital muscular dystrophy type 1D) to a relatively milder congenital variant with no brain involvement (congenital muscular dystrophy type 1C), and to limb-girdle muscular dystrophy (LGMD) type 2 variants with onset in childhood or adult life (LGMD2I, LGMD2L, and LGMD2N). ADG is a peripheral membrane protein that undergoes multiple and complex glycosylation steps to regulate its ability to effectively interact with extracellular matrix proteins, such as laminin, agrin, and perlecan. Although the precise composition of the glycans present on ADG are not known, it has been demonstrated that the forced overexpression of LARGE, or its paralog LARGE2, is capable of increasing the glycosylation of ADG in normal cells. In addition, its overexpression is capable of restoring dystroglycan glycosylation and laminin binding properties in primary cell cultures of patients affected by different genetically defined dystroglycanopathy variants. These observations suggest that there could be a role for therapeutic strategies to overcome the glycosylation defect in these conditions via the overexpression of LARGE.

Mild POMGnT1 mutations underlie a novel limb-girdle muscular dystrophy variant.

BACKGROUND: Mutations in protein-O-mannose-beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) have been found in muscle-eye-brain disease, a congenital muscular dystrophy with structural eye and brain defects and severe mental retardation. OBJECTIVE: To investigate whether mutations in POMGnT1 could be responsible for milder allelic variants of muscular dystrophy. DESIGN: Screening for mutations in POMGnT1. SETTING: Tertiary neuromuscular unit. PATIENT: A patient with limb-girdle muscular dystrophy phenotype, with onset at 12 years of age, severe myopia, normal intellect, and decreased alpha-dystroglycan immunolabeling in skeletal muscle. RESULTS: A homozygous POMGnT1 missense mutation (c.1666G>A, p.Asp556Asn) was identified. Enzyme studies of the patient's fibroblasts showed an altered kinetic profile, less marked than in patients with muscle-eye-brain disease and in keeping with the relatively mild phenotype in our patient. CONCLUSIONS: Our findings widen the spectrum of disorders known to result from mutations in POMGnT1 to include limb-girdle muscular dystrophy with no mental retardation. We propose that this condition be known as LGMD2M. The enzyme assay used to diagnose muscle-eye-brain disease may not detect subtle abnormalities of POMGnT1 function, and additional kinetic studies must be carried out in such cases.

Refining genotype phenotype correlations in muscular dystrophies with defective glycosylation of dystroglycan.

Muscular dystrophies with reduced glycosylation of alpha-dystroglycan (alpha-DG), commonly referred to as dystroglycanopathies, are a heterogeneous group of autosomal recessive conditions which include a wide spectrum of clinical severity. Reported phenotypes range from severe congenital onset Walker-Warburg syndrome (WWS) with severe structural brain and eye involvement, to relatively mild adult onset limb girdle muscular dystrophy (LGMD). Specific clinical syndromes were originally described in association with mutations in any one of six demonstrated or putative glycosyltransferases. Work performed on patients with mutations in the FKRP gene has identified that the spectrum of phenotypes due to mutations in this gene is much wider than originally assumed. To further define the mutation frequency and phenotypes associated with mutations in the other five genes, we studied a large cohort of patients with evidence of a dystroglycanopathy. Exclusion of mutations in FKRP was a prerequisite for participation in this study. Ninety-two probands were screened for mutations in POMT1, POMT2, POMGnT1, fukutin and LARGE. Homozygous and compound heterozygous mutations were detected in a total of 31 probands (34 individuals from 31 families); 37 different mutations were identified, of which 32 were novel. Mutations in POMT2 were the most prevalent in our cohort with nine cases, followed by POMT1 with eight cases, POMGnT1 with seven cases, fukutin with six cases and LARGE with only a single case. All patients with POMT1 and POMT2 mutations had evidence of either structural or functional central nervous system involvement including four patients with mental retardation and a LGMD phenotype. In contrast mutations in fukutin and POMGnT1 were detected in four patients with LGMD and no evidence of brain involvement. The majority of patients (six out of nine) with mutations in POMT2 had a Muscle-Eye-Brain (MEB)-like condition. In addition we identified a mutation in the gene LARGE in a patient with WWS. Our data expands the clinical phenotypes associated with POMT1, POMT2, POMGnT1, fukutin and LARGE mutations. Mutations in these five glycosyltransferase genes were detected in 34% of patients indicating that, after the exclusion of FKRP, the majority of patients with a dystroglycanopathy harbour mutations in novel genes.

Molecular mechanisms and phenotypic variation in RYR1-related congenital myopathies.

Dominant mutations in the skeletal muscle ryanodine receptor (RYR1) gene are well-recognized causes of both malignant hyperthermia susceptibility (MHS) and central core disease (CCD). More recently, recessive RYR1 mutations have been described in few congenital myopathy patients with variable pathology, including multi-minicores. Although a clinical overlap between patients with dominant and recessive RYR1 mutations exists, in most cases with recessive mutations the pattern of muscle weakness is remarkably different from that observed in dominant CCD. In order to characterize the spectrum of congenital myopathies associated with RYR1 mutations, we have investigated a cohort of 44 patients from 28 families with clinical and/or histopathological features suggestive of RYR1 involvement. We have identified 25 RYR1 mutations, 9 of them novel, including 12 dominant and 13 recessive mutations. With only one exception, dominant mutations were associated with a CCD phenotype, prominent cores and predominantly occurred in the RYR1 C-terminal exons 101 and 102. In contrast, the 13 recessive RYR1 mutations were distributed evenly along the entire RYR1 gene and were associated with a wide range of clinico-pathological phenotypes. Protein expression studies in nine cases suggested a correlation between specific mutations, RyR1 protein levels and resulting phenotype: in particular, whilst patients with dominant or recessive mutations associated with typical CCD phenotypes appeared to have normal RyR1 expression, individuals with more generalized weakness, multi-minicores and external ophthalmoplegia had a pronounced depletion of the RyR1 protein. The phenomenon of protein depletion was observed in some patients compound heterozygous for recessive mutations at the genomic level and silenced another allele in skeletal muscle, providing additional information on the mechanism of disease in these patients. Our data represent the most extensive study of RYR1-related myopathies and indicate complex genotype-phenotype correlations associated with mutations differentially affecting assembly and function of the RyR1 calcium release channel.

Epigenetic allele silencing unveils recessive RYR1 mutations in core myopathies.

Epigenetic regulation of gene expression is a source of genetic variation, which can mimic recessive mutations by creating transcriptional haploinsufficiency. Germline epimutations and genomic imprinting are typical examples, although their existence can be difficult to reveal. Genomic imprinting can be tissue specific, with biallelic expression in some tissues and monoallelic expression in others or with polymorphic expression in the general population. Mutations in the skeletal-muscle ryanodine-receptor gene (RYR1) are associated with malignant hyperthermia susceptibility and the congenital myopathies central core disease and multiminicore disease. RYR1 has never been thought to be affected by epigenetic regulation. However, during the RYR1-mutation analysis of a cohort of patients with recessive core myopathies, we discovered that 6 (55%) of 11 patients had monoallelic RYR1 transcription in skeletal muscle, despite being heterozygous at the genomic level. In families for which parental DNA was available, segregation studies showed that the nonexpressed allele was maternally inherited. Transcription analysis in patients' fibroblasts and lymphoblastoid cell lines indicated biallelic expression, which suggests tissue-specific silencing. Transcription analysis of normal human fetal tissues showed that RYR1 was monoallelically expressed in skeletal and smooth muscles, brain, and eye in 10% of cases. In contrast, 25 normal adult human skeletal-muscle samples displayed only biallelic expression. Finally, the administration of the DNA methyltransferase inhibitor 5-aza-deoxycytidine to cultured patient skeletal-muscle myoblasts reactivated the transcription of the silenced allele, which suggests hypermethylation as a mechanism for RYR1 silencing. Our data indicate that RYR1 undergoes polymorphic, tissue-specific, and developmentally regulated allele silencing and that this unveils recessive mutations in patients with core myopathies. Furthermore, our data suggest that imprinting is a likely mechanism for this phenomenon and that similar mechanisms could play a role in human phenotypic heterogeneity.

Fukutin gene mutations in steroid-responsive limb girdle muscular dystrophy.

OBJECTIVE: Defects in glycosylation of alpha-dystroglycan are associated with several forms of muscular dystrophy, often characterized by congenital onset and severe structural brain involvement, collectively known as dystroglycanopathies. Six causative genes have been identified in these disorders including fukutin. Mutations in fukutin cause Fukuyama congenital muscular dystrophy. This is the second most common form of muscular dystrophy in Japan and is invariably associated with mental retardation and structural brain defects. The aim of this study was to determine the genetic defect in two white families with a dystroglycanopathy. METHODS: The six genes responsible for dystroglycanopathies were studied in three children with a severe reduction of alpha-dystroglycan in skeletal muscle. RESULTS: We identified pathogenic fukutin mutations in these two families. Affected children had normal intelligence and brain structure and shared a limb girdle muscular dystrophy (LGMD) phenotype, had marked elevation of serum creatine kinase, and were all ambulant with remarkable steroid responsiveness. INTERPRETATION: Our data suggest that fukutin mutations occur outside Japan and can be associated with much milder phenotypes than Fukuyama congenital muscular dystrophy. These findings significantly expand the spectrum of phenotypes associated with fukutin mutations to include this novel form of limb girdle muscular dystrophy that we propose to name LGMD2L.

Spectrum of brain changes in patients with congenital muscular dystrophy and FKRP gene mutations.

OBJECTIVES: To report the spectrum of brain magnetic resonance imaging findings in 13 patients with congenital muscular dystrophy and FKRP gene mutations and to explore possible genotype-phenotype correlations. DESIGN: We retrospectively reviewed brain magnetic resonance imaging in patients with congenital muscular dystrophy and FKRP gene mutations. PATIENTS: Thirteen patients with congenital muscular dystrophy and mutations in the FKRP gene. RESULTS: Five of the 13 patients had the typical phenotype originally described for congenital muscular dystrophy (MDC1C) with normal intelligence and normal brain magnetic resonance imaging while 3 other patients had isolated cerebellar cysts and mental retardation without any other sign of posterior fossa of supratentorial abnormalities. In the remaining 5 patients cerebellar cysts were associated with structural brain changes involving the posterior fossa and the cortex, ranging from focal unilateral periventricular nodular heterotopia to marked cerebellar dysplasia and pontine hypoplasia. In 2 of these 5 patients the severity and distribution of changes resembled muscle-eye-brain disease in 1 patient who had mild Walker-Warburg syndrome. The distribution of FKRP gene mutations identified in this group of patients did not reveal any obvious association with the severity of central nervous system involvement. CONCLUSIONS: The severity of central nervous system involvement observed in our patients in contrast broadly reflected the severity of the disruption of alpha-dystroglycan glycosylation. In particular, dystroglycan expression was almost absent in the patients with muscle-eye-brain diseaselike phenotype and less severely reduced in the patients with congenital muscular dystrophy (MDC1C) with or without cerebellar cysts. This study further highlights the central role that dystroglycan has in neuronal migration.

Mutations in SIL1 cause Marinesco-Sjögren syndrome, a cerebellar ataxia with cataract and myopathy.

SIL1 (also called BAP) acts as a nucleotide exchange factor for the Hsp70 chaperone BiP (also called GRP78), which is a key regulator of the main functions of the endoplasmic reticulum. We found nine distinct mutations that would disrupt the SIL1 protein in individuals with Marinesco-Sjögren syndrome, an autosomal recessive cerebellar ataxia complicated by cataracts, developmental delay and myopathy. Identification of SIL1 mutations implicates Marinesco-Sjögren syndrome as a disease of endoplasmic reticulum dysfunction and suggests a role for this organelle in multisystem disorders.

Sub-cellular localisation of fukutin related protein in different cell lines and in the muscle of patients with MDC1C and LGMD2I.

MDC1C and LGMD2I are two allelic forms of muscular dystrophies caused by mutations in the gene encoding for fukutin related protein (FKRP). FKRP encodes for a putative glycosyltransferase, the precise function of which is unknown. However, the marked reduction of alpha-dystroglycan glycosylation in the muscle of MDC1C and LGMD2I patients suggests a role for FKRP in dystroglycan processing. Using a polyclonal antibody raised against FKRP we now show that endogenous FKRP locates to the Golgi apparatus of neuronal, oligodendroglial, and the cardiac muscle cell line H9c2. In differentiated C2C12 myotubes and in transverse sections of normal skeletal and cardiac muscle, endogenous FKRP surrounded the myonuclei. This localisation was unaffected in the skeletal muscle of patients with MDC1C and LGMD2I carrying various FKRP mutations. These observations imply a specific role for FKRP during striated muscle, neuronal and glial development and suggest that protein mis-localisation is not a common mechanism of disease in FKRP-related dystrophies.

Prenatal diagnosis in laminin alpha2 chain (merosin)-deficient congenital muscular dystrophy: a collective experience of five international centers.

The congenital muscular dystrophies (CMD) are clinically and genetically heterogeneous. The merosin (laminin alpha2 chain) deficient form (MDC1A), is characterized clinically by neonatal hypotonia, delayed motor milestones and associated contractures. It is caused by deficiency in the basal lamina of muscle fibers of the alpha2 chain of laminins 2 and 4 (LAMA2 gene at 6q22-23). Laminin alpha2 chain is also expressed in fetal trophoblast, which provides a suitable tissue for prenatal diagnosis in families where the index case has total deficiency of the protein. This article reports the collective experience of five centers over the past 10 years in 114 prenatal diagnostic studies using either protein analysis of the chorionic villus (CV) of the trophoblast plus DNA molecular studies with markers flanking the 6q22-23 region and intragenic polymorphisms (n=58), or using only DNA (n=44) or only protein (n=12) approaches. Of the 102 fetuses studied by molecular genetics, 27 (26%) were predicted to be affected while 75 (74%) were considered as unaffected, with 52 (51%) being heterozygous, thus conforming closely to an autosomal recessive inheritance. In 18 of the 27 affected fetuses, the trophoblast was studied by immunocytochemistry and there was a total or only traces deficiency of the protein in CV basement membrane in all. In 10 cases material from the presumably affected fetus was available for analysis after termination of the pregnancy and immunohistochemical study confirmed the diagnosis in all of them. Prenatal studies of 'at risk' pregnancies in the five centers produced neither false negative (merosin-deficiency in CVs in a normal fetus), nor false positive (normal merosin expression in CVs and affected child), indicating the reliability of the technique, when all the necessary controls are done. Our experience suggests that protein and DNA analysis can be used either independently or combined, according to the facilities of each center, to provide accurate prenatal diagnosis of the MDC1A, and have an essential role in genetic counseling.

Localization and functional analysis of the LARGE family of glycosyltransferases: significance for muscular dystrophy.

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.

Antenatal and postnatal brain magnetic resonance imaging in muscle-eye-brain disease.

BACKGROUND: Muscle-eye-brain disease (MEB) is a rare autosomal recessive disorder characterized by congenital muscular dystrophy, structural eye abnormalities, and type II lissencephaly. Previous reports of brain abnormalities on magnetic resonance images (MRIs) in MEB have been in children older than 1 year. OBJECTIVE: To describe serial antenatal and postnatal brain MRIs in a child with MEB. DESIGN: Case report. PATIENT: We report a 2-year-old white boy with genetically confirmed MEB. Antenatal MRIs at 25 and 35 weeks' gestation showed posterior ventriculomegaly but no cortical dysplasia. A postnatal brain MRI at age 1 week showed frontal cortical dysplasia and abnormal signal intensity within the frontal white matter. A brain MRI at 8 months showed bilateral frontoparietal polymicrogyria. All images demonstrated flattening of the pons and mild hypoplasia of the inferior vermis. The child had no weakness, and muscle involvement was only suspected when the serum creatine kinase level was found to be elevated at age 8 months. CONCLUSION: Cortical dysplasia in MEB may not be evident until several postnatal months; therefore, if MEB is suspected, brain MRI performed in the first few months of life should be interpreted with caution.

Prenatal diagnosis of Ullrich congenital muscular dystrophy using haplotype analysis and collagen VI immunocytochemistry.

OBJECTIVES: Ullrich congenital muscular dystrophy (UCMD) is a recessively inherited condition characterised by proximal joint contractures, marked distal joint hyperextensibility, rigidity of the spine and early respiratory failure. Recently, mutations in the genes encoding the subunits of collagen VI have been identified in this disease. We undertook two prenatal diagnoses for UCMD in a consanguineous family where the disease was consistent with linkage to the COL6A3 locus and immunolabelling of collagen VI in the proband's skeletal muscle was severely reduced. METHODS: Both haplotype analysis and collagen VI immunolabelling were used to determine the status of the fetuses. RESULTS: Haplotype analysis of DNA extracted from chorionic villus samples (CVS) from the initial at-risk pregnancy with markers encompassing COL6A3 demonstrated that this fetus had inherited the same haplotypes as the affected child, and immunolabelling of the at-risk CVS demonstrated the virtual absence of collagen VI. A second latter fetus inherited neither of the at-risk haplotypes and collagen VI expression in the CVS was normal. During the second pregnancy, a homozygous G > A change in the last nucleotide of exon 27 of COL6A3 was identified in the proband, substantiating the results obtained from haplotype analysis and collagen VI immunolabelling. CONCLUSION: These findings demonstrate that haplotype analysis in combination with immunocytochemistry is a rapid and reliable method for prenatal diagnosis of UCMD, provided the family is genetically informative and reduced collagen VI expression in the proband has been demonstrated.

Defective glycosylation in congenital muscular dystrophies.

PURPOSE OF REVIEW: The recent identification of mutations in five genes coding for proteins with putative or demonstrated glycosyltransferase activity has shed light on a novel mechanism responsible for muscular dystrophy. Abnormal glycosylation of alpha-dystroglycan appears to be a common finding in all these conditions. Surprisingly, the disease severity due to mutations in several of these genes is extremely variable. This article provides an overview of the clinical, biochemical and genetic advances that have been made over the last year in this field. RECENT FINDINGS: Mutations in the human LARGE gene, a putative glycosyltransferase mutated in the myodystrophy mouse, have now been identified in a form of human muscular dystrophy. In addition, the clinical variability of patients with mutations in the genes encoding fukutin, protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1 and the fukutin-related protein has been significantly expanded. Disease severity in patients with mutations in the gene encoding the fukutin-related protein varies from a severe prenatal form of congenital muscular dystrophy with cobblestone lissencephaly and structural eye defects to a mild form of limb-girdle muscular dystrophy with onset in adult life and neither brain nor eye involvement. SUMMARY: Glycosylation disorders represent a rapidly growing and common group of muscular dystrophies. Accurate genetic diagnosis can now be made for five forms, and it is anticipated that several other variants will eventually fall into these categories.

Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies.

We recently identified mutations in the fukutin related protein (FKRP) gene in patients with congenital muscular dystrophy type 1C (MDC1C) and limb girdle muscular dystrophy type 2I (LGMD2I). The sarcolemma of these patients typically displays an immunocytochemical reduction of alpha-dystroglycan. In this report we extend these observations and report a clear correlation between the residual expression of alpha-dystroglycan and the phenotype. Three broad categories were identified. Patients at the severe end of the clinical spectrum (MDC1C) were compound heterozygote between a null allele and a missense mutation or carried two missense mutations and displayed a profound depletion of alpha-dystroglycan. Patients with LGMD with a Duchenne-like severity typically had a moderate reduction in alpha-dystroglycan and were compound heterozygotes between a common C826A (Leu276Ileu) FKRP mutation and either a missense or a nonsense mutation. Individuals with the milder form of LGMD2I were almost invariably homozygous for the Leu276Ile FKRP mutation and showed a variable but subtle alteration in alpha-dystroglycan immunolabeling. Our data therefore suggest a correlation between a reduction in alpha-dystroglycan, the mutation and the clinical phenotype in MDC1C and LGMD2I which supports the hypothesis that dystroglycan plays a central role in the pathogenesis of these disorders.

A novel form of recessive limb girdle muscular dystrophy with mental retardation and abnormal expression of alpha-dystroglycan.

The limb girdle muscular dystrophies are a heterogeneous group of conditions characterized by proximal muscle weakness and disease onset ranging from infancy to adulthood. We report here eight patients from seven unrelated families affected by a novel and relatively mild form of autosomal recessive limb girdle muscular dystrophy (LGMD2) with onset in the first decade of life and characterized by severe mental retardation but normal brain imaging. Immunocytochemical studies revealed a significant selective reduction of alpha-dystroglycan expression in the muscle biopsies. Linkage analysis excluded known loci for both limb girdle muscular dystrophy and congenital muscular dystrophies in the consanguineous families. We consider that this represents a novel form of muscular dystrophy with associated brain involvement. The biochemical studies suggest that it may belong to the growing number of muscular dystrophies with abnormal expression of alpha-dystroglycan.

Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan.

The congenital muscular dystrophies (CMD) are a heterogeneous group of autosomal recessive disorders. A new pathomechanism has recently been identified in a group of these disorders in which known or putative glycosyltransferases are defective. Common to all these conditions is the hypoglycosylation of alpha-dystroglycan. Fukuyama CMD, muscle-eye-brain disease and Walker-Warburg syndrome, each associated with eye abnormalities and neuronal migration defects, result from mutations in fukutin, POMGnT1 and POMT1, respectively, while mutations in the fukutin-related protein (FKRP) gene cause congenital muscular dystrophy 1C, typically lacking brain involvement. Another putative glycosyltransferase, Large, is mutated in the myodystrophy mouse. The human homologue of this gene is therefore a strong candidate for involvement in novel forms of muscular dystrophy. We studied 36 patients with muscular dystrophy and either mental retardation, structural brain changes or abnormal alpha-dystroglycan immunolabelling, unlinked to any reported CMD loci. Linkage analysis in seven informative families excluded involvement of LARGE but sequencing of this gene in the remaining 29 families identified one patient with a G1525A (Glu509Lys) missense mutation and a 1 bp insertion, 1999insT. This 17-year-old girl presented with congenital muscular dystrophy, profound mental retardation, white matter changes and subtle structural abnormalities on brain MRI. Her skeletal muscle biopsy showed reduced immunolabelling of alpha-dystroglycan. Immunoblotting with an antibody to a glycosylated epitope demonstrated a reduced molecular weight form of alpha-dystroglycan that retained some laminin binding activity. This is the first description of mutations in the human LARGE gene and we propose to name this new disorder MDC1D.

Profound skeletal muscle depletion of alpha-dystroglycan in Walker-Warburg syndrome.

Walker-Warburg syndrome (WWS) is an autosomal recessive disorder characterized by the combined involvement of the central nervous and skeletal muscle systems. Although the molecular basis of WWS remains unknown, defects in the muscle fibre basal lamina are characteristic of other forms of congenital muscular dystrophy (CMD). In agreement with this, some forms of CMD, due to glycosyltransferase defects, display a reduction in the immunolabelling of alpha-dystroglycan, whilst beta-dystroglycan labelling appears normal. Here we describe an almost complete absence of alpha-dystroglycan using both immunohistochemistry and immunoblotting in two patients with WWS. In addition, there was a mild reduction of laminin-alpha 2. In contrast, immunohistochemical labelling of perlecan and collagen VI was normal. Linkage analysis excluded the recently identified POMT1 locus, responsible for a proportion of WWS cases. These results confirm that WWS is a genetically heterogeneous condition and suggest that disruption of the alpha-dystroglycan/laminin-alpha 2 axis in the basal lamina may play a role in the degeneration of muscle fibres in WWS-also in cases not due to POMT1 defects.