Thomas L. Winder

ISPD loss-of-function mutations disrupt dystroglycan O-mannosylation and cause Walker-Warburg syndrome

Walker-Warburg syndrome (WWS) is clinically defined as congenital muscular dystrophy that is accompanied by a variety of brain and eye malformations. It represents the most severe clinical phenotype in a spectrum of diseases associated with abnormal post-translational processing of α-dystroglycan that share a defect in laminin-binding glycan synthesis. Although mutations in six genes have been identified as causes of WWS, only half of all individuals with the disease can currently be diagnosed on this basis. A cell fusion complementation assay in fibroblasts from undiagnosed individuals with WWS was used to identify five new complementation groups. Further evaluation of one group by linkage analysis and targeted sequencing identified recessive mutations in the ISPD gene (encoding isoprenoid synthase domain containing). The pathogenicity of the identified ISPD mutations was shown by complementation of fibroblasts with wild-type ISPD. Finally, we show that recessive mutations in ISPD abolish the initial step in laminin-binding glycan synthesis by disrupting dystroglycan O-mannosylation. This establishes a new mechanism for WWS pathophysiology.

Limb-Girdle Muscular Dystrophy in the United States

Limb-girdle muscular dystrophy (LGMD) has been linked to 15 chromosomal loci, 7 autosomal-dominant (LGMD1A to E) and 10 autosomal-recessive (LGMD2A to J). To determine the distribution of subtypes among patients in the United States, 6 medical centers evaluated patients with a referral diagnosis of LGMD. Muscle biopsies provided histopathology and immunodiagnostic testing, and their protein abnormalities along with clinical parameters directed mutation screening. The diagnosis in 23 patients was a disorder other than LGMD. Of the remaining 289 unrelated patients, 266 had muscle biopsies sufficient for complete microscopic evaluation; 121 also underwent Western blotting. From this combined evaluation, the distribution of immunophenotypes is 12% calpainopathy, 18% dysferlinopathy, 15% sarcoglycanopathy, 15% dystroglycanopathy, and 1.5% caveolinopathy. Genotypes distributed among 2 dominant and 7 recessive subtypes have been determined for 83 patients. This study of a large racially and ethnically diverse population of patients with LGMD indicates that establishing a putative subtype is possible more than half the time using available diagnostic testing. An efficient approach to genotypic diagnosis is muscle biopsy immunophenotyping followed by directed mutational analysis. The most common LGMDs in the United States are calpainopathies, dysferlinopathies, sarcoglycanopathies, and dystroglycanopathies.

Amyloidosis and exercise intolerance in ANO5 muscular dystrophy.

Anoctamin 5 and dysferlin mutations can result in myopathies with similar clinical phenotype. Amyloid deposits can occur in the muscle of patients with dysferlinopathy. We describe a 53-year-old woman with exercise intolerance since childhood, recurrent rhabdomyolysis and late-onset weakness. Muscle biopsy showed amyloid deposits within the blood vessel walls and around muscle fibers. Mutation analysis identified two pathogenic heterozygous mutations in anoctamin 5 and no mutations in dysferlin. To our knowledge this is the first report of muscle amyloidosis in anoctamin 5 muscular dystrophy. This finding suggests that patients with amyloid in muscle should be screened for anoctamin 5 muscular dystrophy.

Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies.

The β-tropomyosin gene encodes a component of the sarcomeric thin filament. Rod-shaped dimers of tropomyosin regulate actin-myosin interactions and β-tropomyosin mutations have been associated with nemaline myopathy, cap myopathy, Escobar syndrome and distal arthrogryposis types 1A and 2B. In this study, we expand the allelic spectrum of β-tropomyosin-related myopathies through the identification of a novel β-tropomyosin mutation in two clinical contexts not previously associated with β-tropomyosin. The first clinical phenotype is core-rod myopathy, with a β-tropomyosin mutation uncovered by whole exome sequencing in a family with autosomal dominant distal myopathy and muscle biopsy features of both minicores and nemaline rods. The second phenotype, observed in four unrelated families, is autosomal dominant trismus-pseudocamptodactyly syndrome (distal arthrogryposis type 7; previously associated exclusively with myosin heavy chain 8 mutations). In all four families, the mutation identified was a novel 3-bp in-frame deletion (c.20_22del) that results in deletion of a conserved lysine at the seventh amino acid position (p.K7del). This is the first mutation identified in the extreme N-terminus of β-tropomyosin. To understand the potential pathogenic mechanism(s) underlying this mutation, we performed both computational analysis and in vivo modelling. Our theoretical model predicts that the mutation disrupts the N-terminus of the α-helices of dimeric β-tropomyosin, a change predicted to alter protein-protein binding between β-tropomyosin and other molecules and to disturb head-to-tail polymerization of β-tropomyosin dimers. To create an in vivo model, we expressed wild-type or p.K7del β-tropomyosin in the developing zebrafish. p.K7del β-tropomyosin fails to localize properly within the thin filament compartment and its expression alters sarcomere length, suggesting that the mutation interferes with head-to-tail β-tropomyosin polymerization and with overall sarcomeric structure. We describe a novel β-tropomyosin mutation, two clinical-histopathological phenotypes not previously associated with β-tropomyosin and pathogenic data from the first animal model of β-tropomyosin-related myopathies.

Cardiac pathology exceeds skeletal muscle pathology in two cases of limb-girdle muscular dystrophy type 2I†

Limb‐girdle muscular dystrophy type 2I (LGMD‐2I) is caused by mutations in the fukutin‐related protein gene (FKRP) that lead to abnormal glycosylation of α‐dystroglycan in skeletal muscle. Heart involvement in LGMD‐2I is common, but little is known about a underlying cardiac pathology. Herein we describe two patients with LGMD‐2I (homozygous FKRP mutation c.826C>A, p.Leu276Ile) who developed severe congestive heart failure that required cardiac transplantation. The dystrophic pathology and impairment of α‐dystroglycan glycosylation were severe in the heart but mild in skeletal muscle, underscoring the lack of correlation between cardiac and skeletal muscle involvement in some LGMD‐2I patients. Muscle Nerve, 2009

ANO5‐muscular dystrophy: clinical, pathological and molecular findings

Anoctamin 5 (ANO5) is a putative intracellular calcium‐activated chloride channel. Recessive mutations in ANO5 cause primary skeletal muscle disorders (limb‐girdle muscular dystrophy 2L and distal muscular dystrophy), which are phenotypically similar to dysferlinopathy, a muscular dystrophy due to dysferlin‐encoding gene (DYSF) mutations.

Myoglobinuria and muscle pain are common in patients with limb-girdle muscular dystrophy 2I

Fukutin-related protein ( FKRP; OMIM #606596) is critical for the appropriate glycosylation of α-dystroglycan, a component of the dystrophin-glycoprotein complex. The 12-kb FKRP gene is composed of 3 noncoding exons and 1 exon encompassing the entire open reading frame.1 Mutations in FKRP cause autosomal recessive muscular dystrophy with a wide range of clinical severity. A common missense mutation, c.826C>A (p.L276I), has been identified. Generally, individuals homozygous for this mutation have a mild form of limb-girdle muscular dystrophy (LGMD) 2I, compound heterozygotes with 1 c.826C>A allele have a more severe form of LGMD 2I, and those with 2 unique alleles are most severely affected, including some with congenital muscular dystrophy.1,2 We report a high incidence of myoglobinuria and muscle pain in a retrospective study of patients with LGMD 2I. ### Methods. #### Standard protocol approvals, registrations, and patient consents. Institutional Review Board approval was obtained for all recruitment and data collection. Written informed consent was obtained from all participants or their legal guardians. This study was posted on clinicaltrials.gov (NCT00313677). All patients with FKRP mutations were eligible for enrollment from 2006 to the present. #### Genetic testing. FKRP mutations were confirmed in the University of Iowa …

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 alpha-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 alpha-dystroglycan glycoepitope immunostaining. Immunoblots revealed hypoglycosylation of alpha-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 alpha-dystroglycan hypoglycosylation in skeletal muscle.

Clinical, Pathologic, and Mutational Spectrum of Dystroglycanopathy Caused by LARGE Mutations

Dystroglycanopathies are a subtype of congenital muscular dystrophy of varying severity that can affect the brain and eyes, ranging from Walker-Warburg syndrome with severe brain malformation to milder congenital muscular dystrophy presentations with affected or normal cognition and later onset. Mutations in dystroglycanopathy genes affect a specific glycoepitope on α-dystroglycan; of the 14 genes implicated to date, LARGE encodes the glycosyltransferase that adds the final xylose and glucuronic acid, allowing α-dystroglycan to bind ligands, including laminin 211 and neurexin. Only 11 patients with LARGE mutations have been reported. We report the clinical, neuroimaging, and genetic features of 4 additional patients. We confirm that gross deletions and rearrangements are important mutational mechanisms for LARGE. The brain abnormalities overshadowed the initially mild muscle phenotype in all 4 patients. We present the first comprehensive postnatal neuropathology of the brain, spinal cord, and eyes of a patient with a homozygous LARGE mutation at Cys443. In this patient, polymicrogyria was the predominant cortical malformation; densely festooned polymicrogyria were overlaid by a continuous agyric surface. In view of the severity of these abnormalities, Cys443 may be a functionally important residue in the LARGE protein, whereas the mutation p.Glu509Lys of Patient 1 in this study may confer a milder phenotype. Overall, these results expand the clinical and genetic spectrum of dystroglycanopathy.

GMPPB‐Associated Dystroglycanopathy: Emerging Common Variants with Phenotype Correlation

Mutations in GDP‐mannose pyrophosphorylase B (GMPPB), a catalyst for the formation of the sugar donor GDP‐mannose, were recently identified as a cause of muscular dystrophy resulting from abnormal glycosylation of α‐dystroglycan. In this series, we report nine unrelated individuals with GMPPB‐associated dystroglycanopathy. The most mildly affected subject has normal strength at 25 years, whereas three severely affected children presented in infancy with intellectual disability and epilepsy. Muscle biopsies of all subjects are dystrophic with abnormal immunostaining for glycosylated α‐dystroglycan. This cohort, together with previously published cases, allows preliminary genotype–phenotype correlations to be made for the emerging GMPPB common variants c.79G>C (p.D27H) and c.860G>A (p.R287Q). We observe that c.79G>C (p.D27H) is associated with a mild limb‐girdle muscular dystrophy phenotype, whereas c.860G>A (p.R287Q) is associated with a relatively severe congenital muscular dystrophy typically involving brain development. Sixty‐six percent of GMPPB families to date have one of these common variants.