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.     

Glycosylation defects in muscular dystrophies

PURPOSE OF REVIEW: Congenital disorders of glycosylation are caused by defects in the synthesis of the glycan moiety of glycoproteins or other glycoconjugates. There has been a great explosion in the number of neuromuscular diseases caused by mutations in genes that affect carbohydrate metabolism or protein glycosylation. A common defect in these disorders is the defective processing of alpha-dystroglycan. RECENT FINDINGS: Recent advances demonstrating mutations in glycosyltransferases and dysfunction of the alpha-beta dystroglycan axis causing different forms of muscular dystrophy, especially with brain involvement, shows clearly that muscle integrity is dependent on glycosylation. We first review the newly identified muscular dystrophies, with a focus on the hypoglycosylation of alpha-dystroglycan, from a clinical, biochemical and genetic standpoint, and second hereditary inclusion body myopathies caused by mutations in the gene that encodes an enzyme responsible for the protein's posttranslational modification that cause sialidation defects. It is shown very recently that molecular recognition of dystroglycan by LARGE is a key determinant in the biosynthetic pathway to produce mature and functional dystroglycan. Gene transfer of LARGE into the cells of individuals with congenital muscular dystrophies restores alpha-dystroglycan function. SUMMARY: The clinical spectrum of congenital disorders of glycosylation is becoming increasingly broad. A demonstration of mutations in glycosyltransferases will further help to design diagnostic tools and therapeutic approaches. Recent findings which show that molecular recognition by LARGE is essential for expression of functional dystroglycan and LARGE can functionally bypass alpha-dystroglycan glycosylation defects in distinct congenital muscular dystrophies, indicate a new therapeutic strategy.     

The dystroglycanopathies: the new disorders of O-linked glycosylation

It has become clear in the past half decade that a number of forms of congenital muscular dystrophy are in fact congenital disorders of glycosylation. Genes for Walker Warburg syndrome, muscle-eye-brain disease, Fukuyama congenital muscular dystrophy, congenital muscular dystrophy 1C and 1D, and limb girdle muscular dystrophy 21 have been identified, and gene mutations resulting in these diseases all cause the underglycosylation of alpha dystroglycan with O-linked carbohydrates. Unlike congenital disorders of glycosylation involving the N-linked pathway, these O-linked disorders possess distinctive muscle, eye, and brain phenotypes. Studies using mice and patient tissues strongly suggest that underglycosylation of dystroglycan inhibits the binding extracellular matrix proteins, effectively divorcing this important cell adhesion molecule from its extracellular environment. Moreover, defects in dystroglycan alone can account for most, if not all, cellular pathology. Thus, these disorders are now collectively referred to as dystroglycanopathies.     

Mechanisms of disease: congenital muscular dystrophies-glycosylation takes center stage

Recent studies have defined a group of muscular dystrophies, now termed the dystroglycanopathies, as novel disorders of glycosylation. These conditions include Walker-Warburg syndrome, muscle-eye-brain disease, Fukuyama-type congenital muscular dystrophy, congenital muscular dystrophy types 1C and 1D, and limb-girdle muscular dystrophy type 2I. Although clinical findings can be highly variable, dystroglycanopathies are all characterized by cortical malformations and ocular defects at the more severe end of the clinical spectrum, in addition to muscular dystrophy. All of these disorders are defined by the underglycosylation of alpha-dystroglycan. Defective glycosylation of dystroglycan severs the link between this important cell adhesion molecule and the extracellular matrix, thereby contributing to cellular pathology. Recent experiments indicate that glycosylation might not only define forms of muscular dystrophy but also provide an avenue to the development of therapies for these disorders.     

Fukutin-related protein localizes to the Golgi apparatus and mutations lead to mislocalization in muscle in vivo

Mutations in the fukutin-related protein gene (FKRP) are associated with a spectrum of diseases from mild limb-girdle muscular dystrophy type 2I to severe congenital muscular dystrophy type 1C, muscle-eye-brain disease (MEB), and Walker-Warburg syndrome (WWS). The effect of mutations on the transportation of the mutant proteins may constitute the underlying mechanisms for the pathogenesis of these diseases. Here we examined the subcellular localization of mouse and human normal and mutant FKRP proteins in cells and in muscle in vivo. Both normal human and mouse FKRPs localize in part of the Golgi apparatus in muscle fibers. Mutations in the FKRP gene invariably altered the localization of the protein, leading to endoplasmic reticulum retention within cells and diminished Golgi localization in muscle fibers. Our results therefore suggest that an individual missense point mutation can confer at least two independent effects on the protein, causing (1) reduction or loss of the presumed glycosyltransferase activity directly and (2) mislocalization that could further alter the function of the protein. The complexity of the effect of individual missense point mutations may partly explain the wide variation of the FKRP-related myopathies.     

Fukutin gene mutations in an Italian patient with early onset muscular dystrophy but no central nervous system involvement

Hypoglycosylation of α‐dystroglycan characterizes a subgroup of muscular dystrophies of variable severity, including Fukuyama congenital muscular dystrophy. We found fukutin gene mutations in a 4.5‐year‐old Italian patient, with reduced α‐dystroglycan expression, dystrophic features on muscle biopsy, hypotonia since birth, mild myopathy, but no brain involvement. Mutations in the fukutin gene can be associated with much milder phenotypes than classical Fukuyama congenital muscular dystrophy, and, although rare, can occur in non‐Japanese. Muscle Nerve, 2009     

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 -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.     

Recent advances in congenital muscular dystrophy research

Congenital muscular dystrophy (CMD) is a group of heterogeneous disorders characterized clinically by delayed milestones due to generalized muscle weakness and dystrophic muscle pathology. The discovery of fukutin, responsible gene for Fukuyama CMD (FCMD) and defective glycosylation in its muscle biopsy has lead significant advances in CMD researches, especially disorders with glycosylation defects to a dystroglycan (alphaDG). The highly glycosylated a DG is one of the major dystrophin-associated proteins anchored a basement membrane protein, laminin 2 to the dystrophin molecule. The disorders with the defective glycosylation are now categorized as a dystroglycanopathies which include FCMD, muscle-eye-brain (MEB) disease, Walker-Warburg syndrome (WWS) and diseases with mutations in fukutin-related protein (FKRP) and LARGE genes. Among them, MEB and WWS were proven to have mutations in the glycosyltransferase genes, POMGnT1 (protein O-mannose beta 1,2-N-acetylglucosaminyl/transferase 1) and POMT1 (protein O-mannosyltransferase 1), respectively, though others are still unknown how the glycosylation defect is induced. Although the disease with FKRP mutation has variable phenotypes from CMD to limb-girdle muscular dystrophy, others with defective to decreased a DG show CMD, central nervous system involvement with migration disorder (polymicrogyria) and ocular abnormalities.     

Proteolysis of beta-dystroglycan in muscular diseases

Alpha-dystroglycan is a cell surface peripheral membrane protein which binds to the extracellular matrix (ECM), while beta-dystroglycan is a type I integral membrane protein which anchors alpha-dystroglycan to the cell membrane via the N-terminal extracellular domain. The complex composed of alpha-and beta-dystroglycan is called the dystroglycan complex. We reported previously a matrix metalloproteinase (MMP) activity that disrupts the dystroglycan complex by cleaving the extracellular domain of beta-dystroglycan. This MMP creates a characteristic 30 kDa fragment of beta-dystroglycan that is detected by the monoclonal antibody 43DAG/8D5 directed against the C-terminus of beta-dystroglycan. We also reported that the 30 kDa fragment of beta-dystroglycan was increased in the skeletal and cardiac muscles of cardiomyopathic hamsters, the model animals of sarcoglycanopathy, and that this resulted in the disruption of the link between the ECM and cell membrane via the dystroglycan complex. In this study, we investigated the proteolysis of beta-dystroglycan in the biopsied skeletal muscles of various human muscular diseases, including sarcoglycanopathy, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Fukuyama congenital muscular dystrophy, Miyoshi myopathy, LGMD2A, facioscapulohumeral muscular dystrophy, myotonic dystrophy and dermatomyositis/polymyositis. We show that the 30 kDa fragment of beta-dystroglycan is increased significantly in sarcoglycanopathy and DMD, but not in the other diseases. We propose that the proteolysis of beta-dystroglycan may contribute to skeletal muscle degeneration by disrupting the link between the ECM and cell membrane in sarcoglycanopathy and DMD.     

Clinical and molecular characterization of patients with limb-girdle muscular dystrophy type 2I

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 alpha-dystroglycan glycosylation. OBJECTIVES: To identify patients with limb-girdle muscular dystrophy type 2I and to derive genotype-phenotype 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 high-performance 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 826C>A 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.     

Inhibition of dystroglycan cleavage causes muscular dystrophy in transgenic mice

Dystroglycan (DG) is an essential component of the dystrophin-glycoprotein complex, a molecular scaffold that links the extracellular matrix to the actin cytoskeleton. Dystroglycan protein is post-translationally cleaved into alpha dystroglycan, a highly glycosylated peripheral membrane protein, and beta dystroglycan, a transmembrane protein. Despite clear evidence of the importance of dystroglycan and its associated proteins in muscular dystrophy, the purpose of dystroglycan proteolysis is unclear. By introducing a point mutation at the normal site of proteolysis (serine 654 to alanine, DGS654A), we have created a dystroglycan protein that is severely inhibited in its cleavage. Transgenic expression of DGS654A in mouse skeletal muscles inhibited the expression of endogenously cleaved dystroglycan, while overexpression of wild type dystroglycan by similar amounts did not. DGS654A animals had increased serum creatine kinase activity and most muscles had increased numbers of central nuclei. Overexpression of wild type dystroglycan, by contrast, caused no dystrophy by these measures. Dystrophy in DGS654A muscles correlated with reduced binding of antibodies that recognize glycosylated forms of alpha dystroglycan. Lastly, neuromuscular junctions in DGS654A muscles were aberrant in structure. These data show that aberrant processing of the dystroglycan polypeptide causes muscular dystrophy and suggest that dystroglycan processing is important for the proper glycosylation of alpha dystroglycan.     

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.     

Absence of the basilar pons in mice lacking a functional Large glycosyltransferase gene suggests a defect in pontine neuron migration

Several forms of congenital muscular dystrophy result from mutations in glycosyltransferases that modify alpha-dystroglycan. As pontine hypoplasia has been reported in some clinical cases of congenital muscular dystrophy, we have begun to examine whether these glycosyltransferases are required for the normal development of the basilar pons, one of several precerebellar nuclei of the hindbrain. In veils (Large(vls)) mice, which carry a loss-of-function mutation in the Large glycosyltransferase gene, the basilar pons is absent. Instead, ectopic clusters of pontine neurons are found lateral to their normal site, suggesting that these neurons are unable to migrate to their appropriate site. Two other precerebellar nuclei, the lateral reticular nucleus and the inferior olive, are present in Large(vls) mice. In addition, the basilar pons forms normally in dystrophin-deficient mice. These results demonstrate that the Large glycosyltransferase but not dystrophin is required for normal basilar pontine development.     

Molecular diagnosis of muscular dystrophies

Dystrophin, the protein product of the DMD/BMD (Duchenne muscular dystrophy/Becker muscular dystrophy) gene, is associated with dystrophin-associated proteins (DAPs), which are classified into three groups: the dystroglycan complex, the sarcoglycan complex and the syntrophin complex. There is a connecting axis between subsarcolemmal actin filaments and laminin, one of the main components of the extracellular matrix through dystrophin and dystroglycan. This system may play an important role in protecting the sarcolemma during contraction and relaxation of muscle fibers. In this paper, the abnormalities of DAPs and laminin as a cause of muscular dystrophies are reviewed. While there are no reports on the role of mutations of dystroglycan and the syntrophin gene as being a cause of muscular dystrophies, the immunostaining intensities of these complexes are reduced as a secondary phenomenon of defects of dystrophin in DMD. The sarcoglycan complex, which is comprised of membrane-integrated proteins, contains at least four components, each of which is encoded by a separate gene. This complex plays a crucial role in the development of severe childhood autosomal recessive muscular dystrophy (SCARMD). In this disease, the absence of any single component may result in a loss of the complex function. Therefore, SCARMD develops irrespective of any mechanism involving a defect of individual genes. As such SCARMD is collectively referred to as sarcoglycanopathy. Laminin, a heterotrimer and genetic defect of the α2 subunit, has been shown to be the cause of the classical type of congenital muscular dystrophy. This disease is characterized by floppy infants with severe muscular dystrophy, dysmyelinating neuropathy and white matter changes in the brain. In the clinical setting and in the mouse model of this disease a defect of the laminin α2 subunit in skeletal muscle has been demonstrated. α2 subunit-null mutant mice also exhibit the muscular dystrophy phenotype and a muscle pathology compatible with dystrophia muscularis (dy) mice. A final common mechanism of muscle-cell necrosis in many of the muscular dystrophies is associated with the destabilization of the sarcolemma.     

Dystroglycan: important player in skeletal muscle and beyond

Dystroglycan is a transmembrane protein that connects the extracellular matrix to the cytoskeleton. Given the ubiquitous tissue expression of dystroglycan, different functional roles in various organ systems have been characterized during the past decade. More recently, aberrant glycosylation of dystroglycan has been identified as a novel pathogenetic mechanism in several forms of congenital and late onset muscular dystrophy syndromes. The current review summarizes the recent scientific achievements as they relate to the function of dystroglycan under normal and pathophysiological conditions.     

Homozygous dystroglycan mutation associated with a novel muscle–eye–brain disease-like phenotype with multicystic leucodystrophy

Defects in dystroglycan post-translational modification result in congenital muscular dystrophy with or without additional eye and brain involvement, are referred to as secondary dystroglycanopathies and have been associated with mutations in 11 different genes encoding glycosyltransferases or associated proteins. However, only one patient with a mutation in the dystroglycan encoding gene DAG1 itself has been described before. We here report a homozygous novel DAG1 missense mutation c.2006G>T predicted to result in the amino acid substitution p.Cys669Phe in the β-subunit of dystroglycan in two Libyan siblings. The affected girls presented with a severe muscle–eye–brain disease-like phenotype with distinct additional findings of macrocephaly and extended bilateral multicystic white matter disease, overlapping with the cerebral findings in patients with megalencephalic leucoencephalopathy with subcortical cysts. This novel clinical phenotype observed in our patients further expands the clinical spectrum of dystroglycanopathies and suggests a role of DAG1 not only for dystroglycanopathies but also for some forms of more extensive and multicystic leucodystrophy.     

A case of Fukuyama-type congenital muscular dystrophy with a very mild mental deficit

A boy had the clinical features of congenital muscular dystrophy with a very mild mental deficit. A muscle biopsy at one year of age showed the typical findings of Fukuyama-type congenital muscular dystrophy, including selective loss of immunoreactions for alpha dystroglycan. Magnetic resonance imaging showed no findings suggestive of migration disorders. The diagnosis of Fukuyama-type congenital muscular dystrophy was confirmed by a molecular assay at 8 years of age, and his haplotype analysis was heterozygous. At 9 years of age, his FIQ on the Wechsler Scale for Children revealed 69, while his IQ on the Tanaka Binnet scale of intelligence was 97. In this report the relationship between mild clinical condition of the studied case and its genotype is discussed.     

Heart transplantation in a child with LGMD2I presenting as isolated dilated cardiomyopathy

Limb-girdle muscle dystrophy type 2I is associated with mutations in the gene encoding Fukutin-related protein. Clinical phenotypes are heterogeneous, ranging from isolated hyperCkemia to severe congenital muscular dystrophy. Affected patients frequently develop dilated cardiomyopathy, depending on evolution of their skeletal myopathy. We report on an 8 years-old boy presenting a severe dilated cardiomyopathy requiring heart transplantation. The child harbored a homozygous p.Leu276Ile mutation in Fukutin-related protein gene (FKRP). At the current age of 20 years, the patient shows persistent hyperCKemia but no clinical muscle weakness, CT scan showing very mild features of muscle involvement. Our findings add to the array of clinical presentations of FKRP mutations.     

Fukutin mutations in non-Japanese patients with congenital muscular dystrophy: less severe mutations predominate in patients with a non-Walker-Warburg phenotype

Six genes including POMT1, POMT2, POMGNT1, FKRP, Fukutin (FKTN) and LARGE encode proteins involved in the glycosylation of α-dystroglycan (α-DG). Abnormal glycosylation of α-DG is a common finding in Walker-Warburg syndrome (WWS), muscle-eye-brain disease (MEB), Fukuyama congenital muscular dystrophy (FCMD), congenital muscular dystrophy types 1C and 1D and some forms of autosomal recessive limb-girdle muscular dystrophy (LGMD2I, LGMD2K, LGMD2M), and is associated with mutations in the above genes. FCMD, caused by mutations in Fukutin (FKTN), is most frequent in Japan, but an increasing number of FKTN mutations are being reported outside of Japan. We describe four new patients with FKTN mutations and phenotypes ranging from: severe WWS in a Greek-Croatian patient, to congenital muscular dystrophy and cobblestone lissencephaly resembling MEB-FCMD in two Turkish patients, and limb-girdle muscular dystrophy and no mental retardation in a German patient. Four of the five different FKTN mutations have not been previously described.     

Alpha-dystroglycan, the usual suspect?

An increasing number of congenital muscular dystrophies might originate from genetic abnormalities of glycosyltransferases genes which are believed to target the subunit of the dystroglycan (DG) adhesion complex as their major enzymatic substrate. -DG is highly glycosylated and peripherally associated with the sarcolemma of skeletal muscle and the plasma membrane in a wide variety of cells. Several lines of evidence indicate that -DG hypoglycosylation might represent the primary molecular event characterizing congenital dystrophies, since it is likely to alter -DG high-affinity binding to laminin and other extracellular molecules, thus negatively influencing the basement-membrane/cytoskeleton axis and eventually leading to sarcolemmal instability, infiltration of myofibers and congenital weakness. For this reason, congenital diseases such as Walker-Warburg Syndrome or Muscle–Eye–Brain disease, have been recently denominated ‘secondary dystroglycanopathies’. However, some crucial points need to be fully addressed in order to finally assess the degree of involvement of -DG in congenital muscular diseases, for example: the possibility that mutations hitting the DG gene might lead to primary dystroglycanopathies; the putative functional or pathological role of hypoglycosylated – or even hyperglycosylated – -DG molecules; or also the compensatory role played by the recently identified paralogue glycosyltransferases in -DG sugar decoration.