Onset Limb-girdle Muscular Dystrophy Research Articles

A novel noncoding FKRP mutation in early onset limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophy 2I (LGMD2I, LGMD R9) (OMIM: muscular dystrophy—dystroglycanopathy [limb-girdle], type C, 5) is an autosomal recessive disorder caused by mutations in the fukutin-related protein ( FKRP ) gene.1 The phenotypic spectrum associated with FKRP is heterogeneous; the most severe phenotype, Walker-Warburg-like syndrome, is characterized by congenital hypotonia, progressive muscle weakness and atrophy, ocular and brain malformations, severe motor developmental delay, and profound intellectual disability. Milder phenotypes include the onset of disease from infancy to adulthood and variable clinical course including asymptomatic elevated serum creatinine kinase (CK), early onset rapidly progressive course with loss of ambulation in teenage years, late onset with slow progression, and primary cardiomyopathy with minimal skeletal muscle weakness.1,2 LGMD2I is typically characterized by proximal muscle weakness, calf hypertrophy, hypotonia, elevated CK level, normal cognition, and no structural brain abnormalities. Dilated cardiomyopathy and respiratory muscle weakness can be seen.1 Mutations in the FKRP gene reduce specific O-mannose-linked glycosylation of alpha-dystroglycan, leading to the instability of the linkage between the dystrophin–glycoprotein complex and laminin alpha 2 in the basement membrane of skeletal muscle.3 The most common FKRP mutation seen in LGMD2I is c.826C>A (p.Leu276Ile). Patients who are homozygous for this mutation typically have a milder phenotype, whereas compound heterozygotes have a relatively severe clinical course. In patients heterozygous for c.826C>A, clinical severity may correspond best to the mutation in the second allele.1,4 We describe a 9-year-old boy with LGMD2I who has the c.826C>A mutation in FKRP on 1 allele and a novel variant on the second allele. The authors acknowledge Ms. Terese Nelson who performed the histology and immunostaining and Mr. Joel Carl for assembling the figure

Adult onset limb-girdle muscular dystrophy — A recessive titinopathy masquerading as myositis

Rarely, inflammation can be present in genetic myopathies, such as dysferlinopathies, facioscapulohumeral muscular dystrophy and GNE-myopathy (hereditary inclusion body myopathy). This may lead to erroneous initial diagnosis and unnecessary therapy which bear serious side effects. We report on an unusual case of mutations in the TTN gene presenting with inflammatory infiltrates in the muscle biopsy. Only after intensive immune-modulating therapies failed, a genetic myopathy was considered. Exome sequencing and search for mutated muscle protein-encoding genes disclosed compound heterozygous mutations in TTN: K26320T and A6135G. The parents carry one each of the mutations. Titinopathy could be considered also in patients presenting with inflammatory infiltrates resistant to therapy.

A translational approach to dystroglycanopathies: a frequent type of muscular dystrophy

Meeting abstract The dystroglycanopathies (DGpathies) are a clinically and genetically diverse group of recessively inherited conditions ranging from the most severe Walker-Warburg syndrome (WWS), to mild forms of adult onset limb girdle muscular dystrophy (LGMD). The LGMD2I caused by the L276I mutation in fukutin-related protein (FKRP) is common in the Caucasians population. Their hallmark is a reduction in the functional O-glycosylation of a-dystroglycan. Unfortunately in about 50% of the patient’s disease genes are unknown. Curative treatments are not available. To this end, we pursued a translational approach: By whole exome sequencing, we have discovered 3 new genes (ISPD, B3GALNT2 and GMPPB). The biochemical function of isoprenoid synthase domain containing (ISPD) in mammals remain unknown. Remarkably, we identified a novel DGpathy phenotype harbouring mutations in ISPD characterized by LGMD, oculomotor apraxia, myopia and cerebellar hypoplasia. To explore the possible effects of missense mutations, we have mapped them onto the homology model of human ISPD derived from the structure of a related bacterial protein (CDP-ME synthase; 1VPA). Of the four mutations identified in catalytic domain, two (A53TD and R126H) are predicted to significantly affect the catalytic activity of human ISPD whereas the mutations P149L and Y226C are likely to perturb the nearby secondary structure leading to destabilization of the mutant protein. We have expressed recombinant His-tagged wt and mut. ISPD protein in E.coli and purified Co-IMAC affinity chromatography. By the use of Thermofluor we could confirm the differences in the stability between wt and mut. ISPD proteins. To enable clinical trials, we performed a pilot study for the discovery of serum biomarkers in LGMD2I and could identify various candidates which we grouped into a) myofibrillar proteins b) glycolytic enzymes, c) extracellular matrix and d) other muscle specific proteins.

L.I.3: Novel disease mechanisms in myopathies

The great majority of congenital dystrophies myopathies and adult onset limb girdle muscular dystrophies are often associated to mutations in genes encoding proteins associated with well-defined structural components of muscle fibres. Typical examples are mutations of genes encoding for proteins involved in sarcomeric structure and assembly. A more recent category of proteins are those involved in posttranslational modification of structural proteins, such as those involved in glycosylation of alpha dystroglycan. The identification of mutant genes and defective proteins over the years has followed a rational path, based on identification of plausible candidates often after indication of the involvement of a specific pathway by traditional genetic and pathological approaches. The power of the new generation of DNA sequencing techniques, and improved protein biochemistry, are however leading to the identification of genes by completely unbiased approaches. This is challenging, as demonstration of pathogenicity for genes of unknown function, involved in rare neuromuscular diseases is not simple. However this unbiased approach is uncovering entirely novel disease mechanisms which are of great interest to understand how muscle functions, and the pathogenesis of rare but also more common diseases. In my presentation I will concentrate on a restricted number of novel genes recently found mutated in myopathies. Some genes implicated in calcium handling clearly link excitation contraction coupling to muscular dystrophies; others provide insight on the importance of tight regulation of alternative splicing for muscle function. The full understanding of the mechanism of disease in these conditions will not only improve our understanding of pathophysiology of individual muscle diseases, but also shed new light on how muscle functions under physiological conditions.

G.P.312: Intrafamilial variability in GMPPB associated alpha-dystroglycanopathy and broadening of the clinical phenotype

Alpha-dystroglycanopathies (αDGs) represent a heterogeneous group of muscular dystrophies. There are currently 18 known genes leading to forms of alphaDG, with mutations of GDP-mannose pyrophosphorylase B (GMPPB) being one of the most recently described in one case series. The spectrum of reported GMPPB patients spans from severe neonatal to a mild later onset limb-girdle muscular dystrophy (LGMD) phenotype with or without intellectual disability. GMPPB is important for synthesis of GDP-mannose, a mannosyl donor in 4 glycosylation pathways. We present a 3 affected siblings with compound heterozygous mutations (previously described p.Asp27His and novel p.Gln264TER) in GMPPB presenting as LGMD with variable degrees of muscle weakness but a broader spectrum of early intellectual impairment, epilepsy (1 of 3 patients), and ocular findings. Onset and degree of weakness was variable. Patient P1 has severe intellectual disability, cataracts, glaucoma, and a history of intractable epilepsy, but minimal weakness; while patient P2 has severe weakness with near full time wheelchair use since 16 years old. CK level was elevated in all patients (3015–18,685). Muscle biopsy in P2 revealed dystrophic changes with reduced αDG staining. Brain MRI showed mild posterior cortical thinning in P1. Neuropsychological testing revealed variable intellectual disability in all three patients. Muscle imaging revealed variable changes consistent with areas of fatty infiltration. These siblings further contribute to and expand the spectrum of patients with mutations of GMPPB highlighting that motor and cognitive phenotypes associated with GGMPPB related αDGpathy may not directly correlate. Cognitive deficits and epilepsy can precede recognition of muscle symptoms, thus delaying more targeted testing. Simple CK testing may identify a larger cohort of patients with αDGpathy presenting with minimal motor but more severe cognitive or seizure phenotype whose muscle disease had remained undetected.

Puerto Rican Founder Mutation G787A in the SGCG Gene

We describe 2 siblings who are homozygous for the G787A mutation in the γ-sarcoglycan gene (SGCG), who presented with a severe childhood onset limb-girdle muscular dystrophy, and share a similar clinical phenotype and disease course consistent with LGMD 2C. The siblings' mother is asymptomatic and is heterozygous for the same mutation. The father is estranged but presumably was also an asymptomatic heterozygous carrier as the father's sister (siblings' aunt) died of complications related to a muscular dystrophy at the age of 14. The paternal grandparents of these siblings were first cousins. All members of the family are of Puerto Rican ancestry supporting the theory that this is a founder mutation, as has been previously suggested by Duncan et al The clinical presentation, workup, and course of our patients are described in detail. These 2 cases effectively double the reported cases of this founder mutation.

P.8.9 Progressive muscular dystrophy in a mouse model of FKRP deficiency

Mutations in fukutin related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement, including Muscle Eye Brain Disease. The key defining feature of this group of disorders is the hypoglycosylation of αdystroglycan and its inability to effectively bind extracellular matrix ligands such as laminin α2. We recently generated a mouse with a knock-down in FKRP in the muscle but not the central nervous system (FKRPMD). This mouse shows evidence of muscle fibre degeneration by 6 weeks of age. Despite the hypoglycosylation of α dystroglycan in all muscles examined, the soleus was only mildly affected relative to the other limb muscles. In order to investigate this aspect further we have now undertaken a detailed characterisation of the pattern of muscle involvement in this model, together with a study of the basement membrane changes that accompany the disease process. These analyses should assist in the design of future therapeutic strategies for the FKRP related group of diseases.

G.P.6 Assessing the long term expression of LARGE as a potential therapy in a mouse model of LGMD2I

Mutations in at least eight genes, including fukutin related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement including Walker–Warburg syndrome. We have now generated a mouse with a knock-down in Fkrp expression in the skeletal muscle, but not the central nervous system (FKRPMD). Analysis of skeletal muscle from this mouse demonstrates hypoglycosylation of α-dystroglycan and a clear muscle pathology by 12weeks of age. Previous work has shown that LARGE overexpression induces hyperglycoyslation of α-dystroglycan in wildtype cells and additionally cells from dystroglycanopathy patients with various primary gene defects, suggesting LARGE could be an important therapeutic approach in these disorders. To test this strategy for FKRP associated disorders, we have crossed our FKRPMD mouse line with a second line overexpressing LARGE. Here we present our histological and physiological evaluation of these mice (FKRPMDLARGE).

Mutation in the Fukutin-Gene (FKTN) as cause of a mild congenital muscular dystrophy

Aims: Defective glycosylation of alpha-dystroglycan as a cause of congenital or limb-girdle muscular dystrophies trigger a broad clinical spectrum varying from mild adult onset limb-girdle muscular dystrophies without brain malformation and cognitive deficits (LGMD2M) to severe congenital muscular dystrophies including brain malformation and central nervous dysfunction. Seven genes, in which mutations can cause defective glycosylation are currently known; FKRP, POMT1, POMT2, POMGnT1, FKTN, LARGE and DAG1. Outside of Japan only isolated cases of fukutin-related muscular dystrophies (LGMD2M) with childhood onset have been described. A steroid therapy seems to have a positive effect. We report on an 8 year old boy suffering from a mild congenital muscular dystrophy type C4 (MDDGC4) with mutation in the Fukutin-Gene. To our knowledge this has not been described before Methods: Our patient exhibited delayed motor development and muscular hypotonia, both commencing in infancy. Free walking at the age of 23 months. At the age of 18 months a elevated CK was revealed (2985U/l), as well as elevated AST (151U/l), ALT (223U/l) and LDH (1270U/l). With a muscle biopsy showing a dystrophic histologic pattern and the immunehistochemistry illustrating an absent sarcolemnal expression of alpha dystroglycan the genetic analysis of the FKRP gene was initiated, showing a normal genotype. A subsequent analysis of the Fukutin gene identified a stop mutation. A steroid treatmend was recommenden to the parents recently, who rejected this option for now. Results: A subsequent analysis of the Fukutin gene identified a heterozygous in-frame duplication within Exon 5 as well as a heterozygous stop mutation in Exon 6 of the fukutin gene Conclusion: In patients showing motor development delay, as well as significantly elevated CK and a dystrophic histologic pattern in the muscle biopsy while cognitive development and cerebral imaging are normal a fukutin-related myopathy should be considered after exlusion of a mutation of the FKRP gene; particularly, because the diagnosis has therapeutic consequence in terms of steroid treatment.

P2.12 Generation of a new mouse model for therapeutic testing in the dystroglycanopathies

Mutations in fukutin related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement, including Muscle Eye Brain Disease. We have previously generated a mouse with a knock-down in Fkrp expression levels (FKRPKD) due to insertion of a floxed neomycin cassette in intron 2 of the mouse Fkrp gene. Since this mouse dies at birth due to central nervous involvement we have now replaced FKRP activity in the developing neural tube by crossing this line with one expressing Cre recombinase under the Sox-1 promoter. This has resulted in a viable mouse model in which glycosylated αdystroglycan and laminin alpha 2 has been restored at the pial basement membrane in the brain. These mice display a marked reduction in the glycosylation of skeletal muscle α-dystroglycan (ADG) throughout postnatal life and develop a clear muscle phenotype (fibre degeneration and regeneration) in both the limb muscles and the diaphragm by 12 weeks of age. This new model should prove invaluable in the design and testing of future therapeutic strategies in the dystroglycanopathies.

P2.15 Assessing the therapeutic potential of LARGE in a new mouse model of dystroglycanopathy

Mutations in fukutin related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms. We have now generated a mouse with a knock-down in Fkrp expression levels in the skeletal muscle but not the central nervous system (Sox1 Cre FKRPKD). The skeletal muscle of this mouse shows a marked reduction in glycosylated αdystroglycan and develops a clear muscle phenotype by 12weeks of age. Previous work has shown that the over-expression of LARGE induces the hyperglycosylation of α-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, strongly suggesting that LARGE could be an important therapeutic approach in these disorders.

P2.60 Distal myopathy caused by a homozygous mutation in the titin gene

Muscle disease causing mutations in the titin gene (TTN) were first identified in Finland. They are responsible for two main phenotypes: the AD late onset distal myopathy and the AR early onset limb-girdle muscular dystrophy. We report a Portuguese patient with the clinical phenotype of distal myopathy caused by a homozygous mutation in the TTN gene. The patient is a 26year old Caucasian female, the only child of a clinically normal first degree consanguineous couple. At the age of 22, she started to complain of weakness in the lower limbs.

Fukutin-Related Protein Alters the Deposition of Laminin in the Eye and Brain

Mutations in fukutin-related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement. The defining feature of this group of disorders is the hypoglycosylation of α-dystroglycan and its inability to effectively bind extracellular matrix ligands such as laminin α2. However, α-dystroglycan has the potential to interact with a number of laminin isoforms many of which are basement membrane/tissue specific and developmentally regulated. To further investigate this we evaluated laminin α-chain expression in the cerebral cortex and eye of our FKRP knock-down mouse (FKRP(KD)). These mice showed a marked disturbance in the deposition of laminin α-chains including α1, α2, α4, and α5, although only laminin α1- and γ1-chain mRNA expression was significantly upregulated relative to controls. Moreover, there was a diffuse pattern of laminin deposition below the pial surface which correlated with an abrupt termination of many of the radial glial cells. This along with the pial basement membrane defects, contributed to the abnormal positioning of both early- and late-born neurons. Defects in the inner limiting membrane of the eye were associated with a reduction of laminin α1 demonstrating the involvement of the α-dystroglycan:laminin α1 axis in the disease process. These observations demonstrate for the first time that a reduction in Fkrp influences the ability of tissue-specific forms of α-dystroglycan to direct the deposition of several laminin isoforms in the formation of different basement membranes.

P1.28 Adult onset limb-girdle muscular dystrophy 2I in a Chinese family with novel compound heterozygous mutation of the FKRP gene

P1.28 Adult onset limb-girdle muscular dystrophy 2I in a Chinese family with novel compound heterozygous mutation of the FKRP gene

A homozygous FKRP start codon mutation is associated with Walker–Warburg syndrome, the severe end of the clinical spectrum

Dystroglycanopathies are a heterogeneous group of disorders caused by defects in the glycosylation pathway of alpha-dystroglycan. The clinical spectrum ranges from severe congenital muscular dystrophy with structural brain and eye involvement to a relatively mild adult onset limb-girdle muscular dystrophy without brain abnormalities and normal intelligence. Mutations have been identified in one of six putative or demonstrated glycosyltransferases. Many different FKRP mutations have been identified, which cover the complete clinical spectrum of dystroglycanopathies. In contrast to the other known genes involved in these disorders, genotype-phenotype correlations are not obvious for FKRP mutations. To date, no homozygous or compound heterozygous null mutations have been identified in FKRP, suggesting that null mutations in FKRP could result in embryonic lethality. We report a family with two siblings carrying a homozygous mutation in the start codon of FKRP that is likely to result in a loss of functional FKRP protein. The clinical phenotype of the patients was consistent with Walker-Warburg syndrome, the most severe disorder in the disease spectrum of dystroglycanopathies.

EM.P.2.10 Deficiency of multiple alpha dystroglycan ligand interactions underlie the phenotype of a FKRP-deficient mouse model for muscle eye brain disease

Mutations in fukutin related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement, including Muscle Eye Brain Disease. A common feature of these disorders is a variable reduction in the glycosylation of skeletal muscle α-dystroglycan (ADG). Glycosylated ADG is an essential component of the dystrophin-related glycoprotein complex (DGC) whose known ECM ligands include laminin alpha 2, perlecan, agrin and neurexin.

Further evidence of Fukutin mutations as a cause of childhood onset limb-girdle muscular dystrophy without mental retardation

The dystroglycanopathies comprise a clinically and genetically heterogeneous group of muscular dystrophies characterized by deficient glycosylation of α-dystroglycan. Mutations in the fukutin ( FKTN) gene have primarily been identified among patients with classic Fukuyama congenital muscular dystrophy (FCMD), a severe form of dystroglycanopathy characterized by CMD, cobblestone lissencephaly and ocular defects. We describe two brothers of Caucasian and Japanese ancestry with normal intelligence and limb-girdle muscular dystrophy (LGMD) due to compound heterozygous FKTN mutations. Muscle biopsy showed a dystrophy with selectively reduced α-dystroglycan glycoepitope immunostaining. Immunoblots revealed hypoglycosylation of α-dystroglycan and loss of laminin binding. FKTN gene sequencing identified two variants: c.340G>A and c.527T>C, predicting missense mutations p.A114T and p.F176S, respectively. Our results provide further evidence for ethnic and allelic heterogeneity and the presence of milder phenotypes in FKTN-dystroglycanopathy despite a substantial degree of α-dystroglycan hypoglycosylation in skeletal muscle.

Atypical manifestation of late onset limb girdle muscular dystrophy presenting with recurrent falling and shoulder dysfunction: a case report

IntroductionMyopathies can be sub-classified into congenital, hereditary, mitochondrial, and secondary myopathies.Congenital myopathies are usually diagnosed post partal or in early childhood. Manifestation in adolescence is uncommon and most cases occur as sporadic mutations. Therefore, there is a risk of under diagnosing this disease in middle-aged patients showing pain, dysfunction, recurrent trauma or falls, where muscle atrophy is seen as a secondary injury.Case presentationOur report is about a 54 year old Caucasian woman with an extended history of pain, loss of function and weakness in her right shoulder. The clinical picture showed a frozen right shoulder. The main finding was a marked limb-muscle atrophy of both delta- und biceps-muscles and a rotator cuff tear that had developed over years. Previous medical consultations attributed the atrophy to recurrent falls, shoulder dysfunction and pain. Conservative treatment (analgesics, physiotherapy, training) had failed.The familiar anamnesis was free of any neurological diseases or other genetic diseases.MRI showed a sub-total proximal muscular limb atrophy and a rotator cuff tear in both shoulders. An incision-biopsy of the right delta- and biceps-muscle revealed a chronical myopathy. The level of creatinkinasis was expected to be high but measurements showed values only slightly above normal. Immunohistochemistry, eventually revealed a mild form of LGMD (type 2I). Due to the pattern of symptoms and diagnostic results we described the case as atypical LGMD.ConclusionOur case presents a phenotype of a late onset of limb girdle muscular dystrophy syndrome associated with shoulder pain and dysfunction and recurrent falls. This kind of disease is not very common. In particular, muscle atrophy in the elderly is generally seen as a secondary injury. This case should remind us of the importance of a differential diagnosis of a late onset of muscular dystrophy-syndrome in the elderly, since an early diagnosis offers more treatment options, therefore preventing a rapid progression.

Reduced expression of fukutin related protein in mice results in a model for fukutin related protein associated muscular dystrophies

Mutations in fukutin related protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement, including Muscle Eye Brain disease. A common feature of these disorders is the variable reduction in the glycosylation of skeletal muscle alpha-dystroglycan. In order to gain insight into the pathogenesis and clinical variability, we have generated two lines of mice, the first containing a missense mutation and a neomycin cassette, FKRP-Neo(Tyr307Asn) and the second containing the FKRP(Tyr307Asn) mutation alone. We have previously associated this missense mutation with a severe muscle-eye-brain phenotype in several families. Homozygote Fkrp-Neo(Tyr307Asn) mice die soon after birth and show a reduction in the laminin-binding epitope of alpha-dystroglycan in muscle, eye and brain, and have reduced levels of FKRP transcript. Homozygous Fkrp(Tyr307Asn) mice showed no discernible phenotype up to 6 months of age, contrary to the severe clinical course observed in patients with the same mutation. These results suggest the generation of a mouse model for FKRP related muscular dystrophy requires a knock-down rather than a knock-in strategy in order to give rise to a disease phenotype.

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.