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.     

Hint and luck for identification of a gene for Fukuyama muscular dystrophy, fukutin]

Fukuyama type congenital muscular dystrophy (FCMD), the second most common form of childhood muscular dystrophy in Japan, is an autosomal recessive severe muscular dystrophy, associated with brain anomalies due to neuronal overmigration. By taking advantages of the presence of a consanguineous patient with both FCMD and xeroderma pigmentosum group A, we performed homozygosity mapping using consanguineous FCMD families mainly, and localized the FCMD locus to chromosome 9q31-33. Subsequently, we have identified the gene responsible for FCMD on 9q31, which encodes a novel 461-amino-acid protein termed fukutin. Most FCMD-bearing chromosomes are derived from a single ancestral founder (87%), and a 3kb-retrotransposal insertion was found to be a founder mutation. Two independent point mutations in this gene have also been detected on chromosomes carrying the non-founder haplotype. FCMD is the first human disease to be caused by an ancient retrotransposal integration. We further identified the gene for muscle-eye-brain (MEB) disease, which encodes POMGnT1. Recent studies have revealed that posttranslational modification of alpha-dystroglycan is associated with congenital muscular dystrophy with brain malformations. Since hypoglycosylation of alpha-dystroglycan is common amongst several other disorders, a new clinical entity called alpha-dystroglycanopathy is proposed. However, only POMGnT1 (MEB) and POMT1 (WWS) are shown to have a definite enzymatic activity, and no enzymatic activity has been detected in fukutin. We show positive interactions between fukutin and POMGnT1. Fukutin may form a protein complex with POMGnT1 and modulate POMGnT1's enzymatic activity. Through cDNA microarray, we also show aberrant neuromuscular junction formation and delayed muscle fiber maturation in alpha-dystroglycanopathies, suggesting a new pathomechanism.     

Alpha-dystroglycanopathy (FCMD, MEB, etc): abnormal glycosylation and muscular dystrophy]

Fukuyama congenital muscular dystrophy (FCMD), Walker-Warburg syndrome (WWS), and muscle-eye-brain (MEB) disease are similar disorders characterized by congenital muscular dystrophy, brain and eye anomalies. We previously identified the genes for FCMD and MEB, which encode fukutin and POMGnT1. Recent studies have revealed that posttranslational modification of alpha-dystroglycan is associated with congenital muscular dystrophy with brain malformations. Since hypoglycosylation of alpha-dystroglycan is common amongst several other disorders, a new clinical entity called alpha-dystroglycanopathy is proposed. However, only POMGnT1 (MEB) and POMT1 (WWS) are shown to have a definite enzymatic activity, and no enzymatic activity has been detected in fukutin. We show positive interactions between fukutin and POMGnT1. Fukutin may form a protein complex with POMGnT1 and modulate POMGnT1's enzymatic activity. Through cDNA microarray, we also show aberrant neuromuscular junction formation and delayed muscle fiber maturation in alpha-dystroglycanopathies, suggesting a new pathomechanism.     

Fukutin gene mutations cause dilated cardiomyopathy with minimal muscle weakness

OBJECTIVE: The fukutin gene (FKTN) is the causative gene for Fukuyama-type congenital muscular dystrophy, characterized by rather homogeneous clinical features of severe muscle wasting and hypotonia from early infancy with mental retardation. In contrast with the severe dystrophic involvement of skeletal muscle, cardiac insufficiency is quite rare. Fukuyama-type congenital muscular dystrophy is one of the disorders associated with glycosylation defects of alpha-dystroglycan, an indispensable molecule for intra-extra cell membrane linkage. METHODS: Protein and functional analyses of alpha-dystroglycan and mutation screening of FKTN and other associated genes were performed. RESULTS: Surprisingly, we identified six patients in four families showing dilated cardiomyopathy with no or minimal limb girdle muscle involvement and normal intelligence, associated with a compound heterozygous FKTN mutation. One patient died by rapid progressive dilated cardiomyopathy at 12 years old, and the other patient received cardiac implantation at 18 years old. Skeletal muscles from the patients showed minimal dystrophic features but have altered glycosylation of alpha-dystroglycan and reduced laminin binding ability. One cardiac muscle that underwent biopsy showed altered glycosylation of alpha-dystroglycan similar to that observed in a Fukuyama-type congenital muscular dystrophy patient. INTERPRETATION: FKTN mutations could cause much wider spectrum of clinical features than previously perceived, including familial dilated cardiomyopathy and mildest limb girdle muscular dystrophy.     

Selective deficiency of alpha-dystroglycan in Fukuyama-type congenital muscular dystrophy.

BACKGROUND: Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive disorder characterized by severe dystrophic muscle wasting from birth or early infancy with structural brain abnormalities. The gene for FCMD is located on chromosome 9q31, and encodes a novel protein named fukutin. The function of fukutin is not known yet, but is suggested to be an enzyme that modifies the cell-surface glycoprotein or glycolipids. OBJECTIVE: To elucidate the roles of fukutin gene mutation in skeletal and cardiac muscles and brain. METHODS: Immunohistochemical and immunoblot analyses were performed in skeletal and cardiac muscles and brain tissue samples from patients with FCMD and control subjects. RESULTS: The authors found a selective deficiency of highly glycosylated alpha-dystroglycan, but not beta-dystroglycan, on the surface membrane of skeletal and cardiac muscle fibers in patients with FCMD. Immunoblot analyses also showed no immunoreactive band for alpha-dystroglycan, but were positive for beta-dystroglycan in FCMD in skeletal and cardiac muscles. CONCLUSION: The current findings suggest a critical role for fukutin gene mutation in the loss or modification of glycosylation of the extracellular peripheral membrane protein, alpha-dystroglycan, which may cause a crucial disruption of the transmembranous molecular linkage of muscle fibers in patients with FCMD.     

Basement membrane fragility underlies embryonic lethality in fukutin-null mice

Fukuyama-type congenital muscular dystrophy (FCMD), associated with brain malformation due to defects in neuronal migration, is caused by mutations in fukutin. Several lines of evidence suggest that the fukutin protein plays a pivotal role in synthesis of O-mannosyl sugar moieties of alpha-dystroglycan, a cell surface laminin receptor. Here, through targeted disruption of the orthologous mouse fukutin gene, we show that the fukutin protein is essential, as homozygous-null embryos die by E9.5 of gestation. Fukutin-null embryos show phenotypic diversity, features of which include growth retardation, folding of the egg cylinder, leakage of maternal red blood cells into the yolk sac cavity, and an increased number of apoptotic cells in the ectoderm. Loss of immunoreactivity against sugar moieties in alpha-dystroglycan suggests a reduced laminin-binding capacity. Ultrastructural analysis shows thin and breached basement membranes (BMs). BM fragility may underlie all of these abnormal phenotypes, and maintenance of BM function may require fukutin-mediated glycosylation of alpha-dystroglycan early in embryonic development.     

A novel FKRP mutation in congenital muscular dystrophy disrupts the dystrophin glycoprotein complex

Mutations in the gene encoding fukutin related protein (FKRP) produce a spectrum of disease including congenital muscular dystrophy and limb girdle muscular dystrophy. FKRP is one member of a class of molecules thought to be glycosyltransferases that mediate O-linked glycosylation. The primary target of these glycosyltransferases is thought to be dystroglycan. We now report two unrelated Mexican children with congenital muscular dystrophy who each have the identical, novel 1387A>G, N463D mutation. Muscle biopsies from these children show a reduction of alpha-dystroglycan and also show reduction of beta-dystroglycan, and alpha-, beta-, and gamma-sarcoglycan, suggesting that FKRP mutations can perturb membrane associated proteins beyond dystroglycan.     

Fukuyama-type congenital muscular dystrophy]

Fukuyama-type congenital muscular dystrophy (FCMD) is characterized by congenital muscular dystrophy in combination with cortical dysgenesis and ocular abnormality. We identified on chromosome 9 q31 the gene for FCMD, which encodes a novel 461-amino-acid protein (fukutin). Most FCMD-bearing chromosomes have been derived from a single ancestral founder, whose mutation consisted of a 3-kb retrotransposal insertion in the 3' non-coding region of the fukutin gene. Some point mutations causing premature termination were found. Amino acid sequence and transfection experiments suggest that fukutin may be an extracellular protein. Pathological study on the brain of the FCMD fetuses revealed that the glia-limitans and basement-membrane complex had frequent breaks. Because of this, developing neurons were shown to overmigrate in the cerebrum. Electron microscopy of the skeletal muscle in FCMD showed that the basal lamina has a disrupted appearance. Thus, a structural alteration of the basal lamina appears to play a key role in the pathophysiology of FCMD. The spectrum of clinical variability of FCMD is much wider than recognized previously. Point mutations have been seen to render the FCMD phenotype rather severe. FCMD could give rise to only in the Japanese who have a milder retrotransposon mutation.     

Co-localization of fukutin and alpha-dystroglycan in the mouse central nervous system

Hypoglycosylation of alpha-dystroglycan (alpha-DG) has been identified in several human diseases associated with muscular dystrophy and brain malformations, including Fukuyama-type congenital muscular dystrophy (FCMD) caused by mutations in the fukutin gene. Although disruption of the intra-extra membrane linkage in the sarcolemma via the dystroglycan (DG) has been hypothesized as a possible underlying mechanism, little is known about the pathogenesis of brain anomalies in these conditions. In this study, we examined the patterns of expression of fukutin and alpha-DG in developing and adult mouse brains. Antisera against fukutin and alpha-DG identified neurons of the fetal cerebral and cerebellar cortex and the subpial pontine migratory stream. In adult mice, fukutin and alpha-DG were extensively co-expressed in neurons of the cerebral and cerebellar cortex, hippocampus, basal ganglia and olfactory bulb, as well as in the pontine nucleus and the cranial nerve nuclei. These results support the hypothesis that fukutin is involved in the glycosylation process of alpha-DG and that a defect in this process plays an essential role in the pathogenesis of FCMD. Further research into the physiological function of alpha-DG in migrating and mature neurons is required.     

A new mutation of the fukutin gene in a non-Japanese patient

Fukuyama-type congenital muscular dystrophy (FCMD), Walker-Warburg syndrome, and muscle-eye-brain disease are clinically similar autosomal recessive disorders characterized by congenital muscular dystrophy, cobblestone lissencephaly, and eye anomalies. FCMD is frequent in Japan, but no FCMD patient with confirmed fukutin gene mutations has been identified in a non-Japanese population. Here, we describe a Turkish CMD patient with severe brain and eye anomalies. Sequence analysis of the patient's DNA identified a homozygous 1bp insertion mutation in exon 5 of the fukutin gene. To our knowledge, this is the first case worldwide in which a fukutin mutation has been found outside the Japanese population. This report emphasizes the importance of considering fukutin mutations for diagnostic purposes outside of Japan.     

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.     

Walker-Warburg Syndrome with POMT1 mutations can be associated with cleft lip and cleft palate

Walker-Warburg Syndrome (WWS) is an alpha-dystroglycan deficient congenital muscular dystrophy that is associated with brain and eye abnormalities. Patients present with hypotonia, weakness, developmental delay, mental retardation and occasional seizures. Other abnormalities were also described including cleft lip and palate. Mutations in POMT1, POMT2, fukutin, FKRP and LARGE genes are found in 20-30% of children with WWS. We report a novel mutation in POMT1 gene and provide further evidence that WWS with cleft lip and palate is associated with POMT1 mutations. We recommend POMT1 analysis in WWS cases associated with cleft lip and palate when considering which gene to sequence first.     

Dystroglycan linkage and muscular dystrophy]

Dystroglycan is a key complex between basal lamina laminin, extracellularly and membrano-cytoskeleton, intracellularly. The damage of this linkage is turned out to cause muscular dystrophies. Dystroglycan knockout is lethal. Dystroglycan-associated intracellular proteins such as dystrophin, dystrobrevin, sarcoglycans, plectin and caveolin-3 are responsible for causing severe (Duchenne type) and moderate forms (Becker, LGMDs). Laminin, dystroglycan-binding extracellular protein, is deficient in the most severe form of congenital muscular dystrophy with normal intelligence and eye. Recently, a remarkable progress is made in most severe forms of congenital muscular dystrophy with anomalies of brain and eye such as Fukuyama type (Japan) and muscle-eye-brain disease (Finland). The gene product for Fukuyama type, fukutin, belongs to a family of glycosylation enzymes in bacteria and yeast. Since alpha-dystroglycan contains 14-15 o-glycans, ser/thr-mannose 2-1 GlcNAc 4-1 Gal 3-2 Sial in the middle third mucin-domain and the sial-o-glycan is essential for laminin-binding, and since alpha-dystroglycan is defective in Fukuyama type sarcolemma with anti both sugar moiety- and peptide-antidodies, defective fukutin causes incomplete o-glycosylation of alpha-dystroglycan. In '02, it is clarified that a glycosylation enzyme, POMGnT1 which modifies GlcNAc onto ser/thr-mannose, is defective in 6 MEB patients. The loss of the enzyme activity is turned out to lose alpha-dystroglycan from sarcolemma of MEB. These data strongly suggests that o-glycosylation defect of alpha-dystroglycan causes the most severe congenital muscular dystrophy such as Fukuyama type, MEB and Walker Warburg syndrome.     

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.     

Expression of genes related to muscular dystrophy with lissencephaly

There is a group of congenital muscular dystrophies accompanying the brain lesions termed cobblestone lissencephaly. Abnormal glia limitans could be considered the major pathogenesis of cobblestone lissencephaly. In this group, protein-O-linked mannose-beta1,2-N-acetylglucosaminyltransferase and protein-O-mannosyltransferase 1 are considered to be responsible for muscle-eye-brain disease and Walker-Warburg syndrome, respectively, by glycosylation of alpha-dystroglycan. However, the functions of fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy, are still unclear. In this study, expression of the three aforementioned genes was compared by in situ hybridization in control cases to elucidate the functions of fukutin.Immunohistochemistry of fukutin and alpha-dystroglycan was also performed. In the central nervous system, all three genes were expressed in astrocytes and in immature neurons. A few mature neurons expressed fukutin, but many expressed the other two genes. All genes were expressed in various non-nervous tissues including tissues relating to secretion. Fukutin and alpha-dystroglycan were generally colocalized, but localization was not always the same, especially in the liver. Fukutin may be associated with the glycosylation of alpha-dystroglycan, and expression in astrocytes may indicate a relation to glia limitans. The roles of fukutin in mature neurons may be less critical compared with the other two genes. Additional functions of fukutin, especially in the liver, are suspected.     

An autosomal recessive limb girdle muscular dystrophy (LGMD2) with mild mental retardation is allelic to Walker-Warburg syndrome (WWS) caused by a mutation in the POMT1 gene

Mutations of the protein O-mannosyltransferase (POMT1) gene affect glycosylation of alpha-dystroglycan, leading to Walker-Warburg syndrome, a lethal disorder in early life with severe congenital muscular dystrophy, and brain and eye malformations. Recently, we described a novel form of recessive limb girdle muscular dystrophy with mild mental retardation, associated with an abnormal alpha-dystroglycan pattern in the muscle, suggesting a glycosylation defect. Here, we present evidence that this distinct phenotype results from a common mutation (A200P) in the POMT1 gene. Our findings further expand the phenotype of glycosylation disorders linked to POMT1 mutations. Furthermore, the A200P mutation is part of a conserved core haplotype, indicating an ancestral founder mutation.     

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.     

Four Caucasian patients with mutations in the fukutin gene and variable clinical phenotype

Fukuyama congenital muscular dystrophy (FCMD) is frequent in Japan, due to a founder mutation of the fukutin gene (FKTN). Outside Japan, FKTN mutations have only been reported in a few patients with a wide spectrum of phenotypes from Walker–Warburg syndrome to limb-girdle muscular dystrophy (LGMD2M). We studied four new Caucasian patients from three unrelated families. All showed raised serum CK initially isolated in one case and muscular dystrophy. Immunohistochemical studies and haplotype analysis led us to search for mutations in FKTN. Two patients (two sisters) presented with congenital muscular dystrophy, mental retardation, and posterior fossa malformation including cysts, and brain atrophy at Brain MRI. The other two patients had normal intelligence and brain MRI. Sequencing of the FKTN gene identified three previously described mutations and two novel missense mutations. Outside Japan, fukutinopathies are associated with a large spectrum of phenotypes from isolated hyperCKaemia to severe CMD, showing a clear overlap with that of FKRP.     

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.