Elevated Serum Creatine Kinase and Small Cerebellum Prompt Diagnosis of Congenital Muscular Dystrophy due to FKRP Mutations
Rosanna TrovatoGuja AstreaLaura BartalenaPaolo GhirriJacopo BaldacciMatteo GiampietriRoberta BattiniFilippo M. SantorelliChiara Fiorillo
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Abstract:
Fukutin-related protein (FKRP) is a putative glycosyltransferase that mediate O-linked glycosylation of the α-dystroglycan. Mutations in the FKRP gene cause a spectrum of diseases ranging from a limb girdle muscular dystrophy 2I (LGMD2I), to severe Walker-Warburg or muscle-eye-brain forms and a congenital muscular dystrophy (with or without mental retardation) termed MDC1C. This article reports on a Moroccan infant who presented at birth with moderate floppiness, high serum creatine kinase (CK) levels, and brain ultrasonograph suggestive of widening of the posterior fossa. Muscle biopsy displayed moderate dystrophic pattern with complete absence of α-distroglycan and genetic studies identified a homozygous missense variant in FKRP. Mutations in FKRP should be looked for in forms of neonatal-onset hyperCKaemia with floppiness and small cerebellum.Keywords:
Congenital muscular dystrophy
Creatine kinase
Dystroglycan
Dystroglycan
Perlecan
Alpha (finance)
Congenital muscular dystrophy
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Dystroglycan
Agrin
Perlecan
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Abstract Background Congenital muscular dystrophies (CMDs) result from genetically inherited defects in the biosynthesis and/or the posttranslational modification (glycosylation) of laminin-α2 and α-dystroglycan (α-DG), respectively. The interaction between both proteins is responsible for the stability and integrity of the muscle cell. We aimed to study the expression profiles of both proteins in two classes of CMDs. Subjects and methods Whole-exome sequencing (WES) was done for four patients with neuromuscular manifestations. The expression of core α-DG and laminin-α2 subunit in skin fibroblasts and MCF-7 cells was assessed by western blot. Results WES revealed two cases with nonsense mutations; c.2938G > T and c.4348 C > T, in LAMA2 encodes laminin-α2. It revealed also two cases with mutations in POMGNT1 encode protein O-mannose beta-1,2-N-acetylglucosaminyltransferase mutations. One patient had a missense mutation c.1325G > A, and the other had a synonymous variant c.636 C > T. Immunodetection of core α-DG in skin fibroblasts revealed the expression of truncated forms of core α-DG accompanied by reduced expression of laminin-α2 in POMGNT1-CMD patients and one patient with LAMA2-CMD. One patient with LAMA2-CMD had overexpression of laminin-α2 and expression of a low level of an abnormal form of increased molecular weight core α-DG. MCF-7 cells showed truncated forms of core α-CDG with an absent laminin-α2. Conclusion A correlation between the expression pattern/level of core α-DG and laminin-α2 could be found in patients with different types of CMD.
Congenital muscular dystrophy
Dystroglycan
Nonsense mutation
Collagen VI
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Dystroglycan
Congenital muscular dystrophy
Cleavage (geology)
Transferase
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Congenital muscular dystrophy
Nonsense mutation
Mutation Testing
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Gene therapy holds great promise for treating many genetic diseases, including muscular dystrophies. Mutations in the gene encoding laminin alpha2 chain ? an extracellular protein prominently expressed in the neuromuscular system ? cause a severe neuromuscular disorder: congenital muscular dystrophy type 1A (MDC1A). Currently, there is no treatment for MDC1A. Preclinical studies are the first step in testing genetic approaches for future gene therapy in humans. In the course of my research, I focused on a genetically manipulated mouse model of MDC1A, investigating whether the transgenic introduction of laminin ?1 chain into laminin alpha2 chain deficient tissues would prevent the development of disease symptoms. The overexpression of laminin alpha1 chain greatly improved overall health and normalized the life span of laminin alpha2 chain deficient animals. Laminin alpha1 chain, which in the adult body is expressed only in a few epithelial tissues, functionally compensated for laminin alpha2 chain loss in muscle, peripheral nervous system and testis, correcting their morphology and restoring their function. Moreover, laminin alpha1 chain proved to be essential for the normal expression levels of laminin receptors dystroglycan and integrin alpha7 in laminin alpha2 chain deficient muscle. I suggest that our preclinical studies with laminin alpha1 chain transgene may serve as a paradigm for gene therapy of congenital muscular dystrophy in patients. (Less)
Congenital muscular dystrophy
Dystroglycan
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Alpha-dystroglycan (αDG) is an extracellular peripheral glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin globular domains and certain arenaviruses. An important enzyme, known as Like-acetylglucosaminyltransferase (LARGE), has been shown to transfer repeating units of -glucuronic acid-β1,3-xylose-α1,3- (matriglycan) to αDG that is required for functional receptor as an extracellular matrix protein scaffold. The reduction in the amount of LARGE-dependent matriglycan result in heterogeneous forms of dystroglycanopathy that is associated with hypoglycosylation of αDG and a consequent lack of ligand-binding activity. Our aim was to investigate whether LARGE expression showed correlation with glycosylation of αDG and histopathological parameters in different types of muscular dystrophies, except for dystroglycanopathies. The expression level of LARGE and glycosylation status of αDG were examined in skeletal muscle biopsies from 26 patients with various forms of muscular dystrophy [Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), sarcoglycanopathy, dysferlinopathy, calpainopathy, and merosin and collagen VI deficient congenital muscular dystrophies (CMDs)] and correlation of results with different histopathological features was investigated. Despite the fact that these diseases are not caused by defects of glycosyltransferases, decreased expression of LARGE was detected in many patient samples, partly correlating with the type of muscular dystrophy. Although immunolabelling of fully glycosylated αDG with VIA4–1 was reduced in dystrophinopathy patients, no significant relationship between reduction of LARGE expression and αDG hypoglycosylation was detected. Also, Merosin deficient CMD patients showed normal immunostaining with αDG despite severe reduction of LARGE expression. Our data shows that it is not always possible to correlate LARGE expression and αDG glycosylation in different types of muscular dystrophies and suggests that there might be differences in αDG processing by LARGE which could be regulated under different pathological conditions.
Congenital muscular dystrophy
Dystroglycan
ITGA7
Dysferlin
Collagen VI
Immunostaining
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Laminin-α2 deficient congenital muscular dystrophy, classified as MDC1A, is a severe
progressive muscle-wasting disease that leads to death in early childhood. MDC1A is caused by
mutations in lama2, the gene encoding the laminin-α2 chain being part of laminin-2, the main
laminin isoform present in the extracellular matrix of muscles and peripheral nerves. Via selfpolymerization,
laminin-2 forms the primary laminin scaffold and binds with high affinity to α-
dystroglycan on the cell surface, providing a connection to the cytoskeleton via the
transmembranous protein β-dystroglycan. Deficiency in laminin-α2 leads to absence of laminin-2
and to upregulation of laminin-8, a laminin isoform that cannot self-polymerize and does not bind
to α-dystroglycan. Therefore, in laminin α2-deficient muscle the chain of proteins linking the
intracellular contractile apparatus via the plasma membrane to the extracellular matrix is
interrupted. Consequently, muscle fibers loose their stability and degenerate what finally leads to
a progressive muscle wasting.
In previous studies, we have shown that a miniaturized form of the extracellular matrix protein
agrin, which is not related to the disease-causing lama2 gene and was designed to contain highaffinity
binding sites for the laminins and for α-dystroglycan, was sufficient to markedly improve
muscle function and overall health in the dyW-/- mouse model of MDC1A. In a follow-up study we
provided additional evidence that mini-agrin, both increases the tolerance to mechanical load but
also improves the regeneration capacity of the dystrophic muscle.
We now report on our progress towards further testing the use of this approach for the treatment
of MDC1A. To test whether mini-agrin application after onset of the disease would still ameliorate
the dystrophic symptoms, we have established the inducible tetracycline-regulated “tet-off”
expression system in dyW-/- mice to temporally control mini-agrin expression in skeletal muscles.
We show that mini-agrin slows down the progression of the dystrophy when applied at birth or in
advanced stages of the disease. However, the extent of the amelioration depends on the
dystrophic condition of the muscle at the time of mini-agrin application. Thus, the earlier miniagrin
is applied, the higher is the profit of its beneficial properties.
In addition to gene therapeutical approaches, the increase of endogenous agrin expression levels
in skeletal muscles by pharmacologically active compounds would be a safe and promising
strategy for the treatment of MDC1A. To evaluate the potential and pave the way to further
expand on the development of such a treatment, we determined whether full-length agrin
ameliorates the dystrophic phenotype to a comparable extent as it was observed by application of
mini-agrin. We provide evidence that constitutive overexpression of chick full-length agrin in dyW-/-
muscle ameliorates the dystrophic phenotype, although not as pronounced as mini-agrin does.
In conclusion, our results are conceptual proof that linkage of laminin to the muscle fiber
membrane is a means to treat MDC1A at any stage of the disease. Our findings definitely
encourage to further expanding on this therapeutic concept, especially in combination with
treatment using functionally different approaches. Moreover, these experiments set the basis for
further developing clinically feasible and relevant application methods such as gene therapy4
and/or the screening of small molecules able to upregulate production of agrin in muscle.
Congenital muscular dystrophy
Dystroglycan
ITGA7
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Congenital muscular dystrophy
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Congenital muscular dystrophies (CMD) are a clinically heterogeneous group of muscular disorders characterized by hypotonia, muscle weakness and early or congenital joint contractures. Electromyography reveals a myopathic pattern, creatine-kinase (CK) may be moderately elevated and muscle biopsy shows pathological changes consistent with a dystrophic process.Report the cases of two brothers with 'Occidental type cerebro-muscular dystrophy' versus 'merosin-deficient CMD'.Two children, a boy and a girl, of a first consanguineous parents. In the first case, the diagnosis of Occidental type cerebro-muscular dystrophy was made in 1983, at the age of 4 years, according to clinical, biochemical, electromyographic, pathological and neuroradiological data. In the second case, the diagnosis of merosindeficient form of CMD was made with the same criteria and with immunohistochemistry and Western blot techniques in 1997, when she was 6 months old.Occidental type cerebro-muscular dystrophy, described 13 years ago by one member of our group, corresponds with merosin-deficient form of CMD.
Congenital muscular dystrophy
Muscle contracture
Muscle Hypotonia
Creatine kinase
Muscle disorder
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