Beril Talim

Muscular Dystrophy and Neuronal Migration Disorder Caused by Mutations in a Glycosyltransferase, POMGnT1

Muscle-eye-brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, and lissencephaly. Mammalian O-mannosyl glycosylation is a rare type of protein modification that is observed in a limited number of glycoproteins of brain, nerve, and skeletal muscle. Here we isolated a human cDNA for protein O-mannose beta-1,2-N-acetylglucosaminyltransferase (POMGnT1), which participates in O-mannosyl glycan synthesis. We also identified six independent mutations of the POMGnT1 gene in six patients with MEB. Expression of most frequent mutation revealed a great loss of the enzymatic activity. These findings suggest that interference in O-mannosyl glycosylation is a new pathomechanism for muscular dystrophy as well as neuronal migration disorder.

A Dystroglycan Mutation Associated with Limb-Girdle Muscular Dystrophy

Dystroglycan, which serves as a major extracellular matrix receptor in muscle and the central nervous system, requires extensive O-glycosylation to function. We identified a dystroglycan missense mutation (Thr192→Met) in a woman with limb-girdle muscular dystrophy and cognitive impairment. A mouse model harboring this mutation recapitulates the immunohistochemical and neuromuscular abnormalities observed in the patient. In vitro and in vivo studies showed that the mutation impairs the receptor function of dystroglycan in skeletal muscle and brain by inhibiting the post-translational modification, mediated by the glycosyltransferase LARGE, of the phosphorylated O-mannosyl glycans on α-dystroglycan that is required for high-affinity binding to laminin.

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.

Clinical and genetic distinction between Walker–Warburg syndrome and muscle–eye–brain disease

Background: Three rare autosomal recessive disorders share the combination of congenital muscular dystrophy and brain malformations including a neuronal migration defect: muscle–eye-brain disease (MEB), Walker–Warburg syndrome (WWS), and Fukuyama congenital muscular dystrophy (FCMD). In addition, ocular abnormalities are a constant feature in MEB and WWS. Lack of consistent ocular abnormalities in FCMD has allowed a clear clinical demarcation of this syndrome, whereas the phenotypic distinction between MEB and WWS has remained controversial. The MEB gene is located on chromosome 1p32-p34. Objectives: To establish distinguishing diagnostic criteria for MEB and WWS and to determine whether MEB and WWS are allelic disorders. Methods: The authors undertook clinical characterization followed by linkage analysis in 19 MEB/WWS families with 29 affected individuals. With use of clinical diagnostic criteria based on Finnish patients with MEB, each patient was categorized as having either MEB or WWS. A linkage and haplotype analysis using 10 markers spanning the MEB locus was performed on the entire family resource. Results: Patients in 11 families were classified as having MEB and in 8 families as WWS. Strong evidence in favor of genetic heterogeneity was obtained in the 19 families. There was evidence for linkage to 1p32-p34 in all but 1 of the 11 pedigrees segregating the MEB phenotype. In contrast, linkage to the MEB locus was excluded in seven of eight of the WWS families. Conclusion: These results allow the classification of MEB and WWS as distinct disorders on both clinical and genetic grounds and provide a basis for the mapping of the WWS gene(s).

Use of Whole-Exome Sequencing to Determine the Genetic Basis of Multiple Mitochondrial Respiratory Chain Complex Deficiencies

IMPORTANCE Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease. OBJECTIVE To determine the molecular basis of multiple respiratory chain complex deficiencies. DESIGN, SETTING, AND PARTICIPANTS We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005 and 2012. All had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. Whole-exome sequencing was performed using 62-Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family. RESULTS Presumptive causal variants were identified in 28 patients (53%; 95% CI, 39%-67%) and possible causal variants were identified in 4 (8%; 95% CI, 2%-18%). Together these accounted for 32 patients (60% 95% CI, 46%-74%) and involved 18 different genes. These included recurrent mutations in RMND1, AARS2, and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1, and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1 and cardiomyopathy with AARS2 and MTO1. However, atypical clinical features were present in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%; 95% CI, 26%-54%). CONCLUSIONS AND RELEVANCE Exome sequencing enhances the ability to identify potential nuclear gene mutations in patients with biochemically defined defects affecting multiple mitochondrial respiratory chain complexes. Additional study is required in independent patient populations to determine the utility of this approach in comparison with traditional diagnostic methods.

A homozygous nonsense mutation in the Fukutin gene causes a Walker-Warburg syndrome phenotype

Neuronal migration is a key process in the development of the cerebral cortex. During neocortex lamination new sets of neurones proliferate at the subventricular zone and migrate alongside specialised radial glial fibres to occupy their final destinations in an “inside-out” fashion.1 More than 25 neuronal migration disorders resulting in death or improper positioning of the cortical neurones have been described in humans.2 In the cobblestone neocortex the postmitotic neurones do not respond to their stop signals, and, crossing through the neocortex, bypass the glia limitans and invade the subarachnoid space. The resulting cortex is chaotically structured, consisting of an irregular lissencephalic surface and absence of lamination. Cobblestone lissencephalies are often seen in association with additional features, such as eye malformations and congenital muscular dystrophy. Walker-Warburg syndrome (WWS, OMIM:236670), muscle-eye-brain (MEB, OMIM:253280), and Fukuyama congenital muscular dystrophy (FCMD, OMIM:253800) are the three major entities of this group. Patients are classified into these three entities on the basis of the severity of the phenotype and the presence of syndrome specific symptoms (table 1). WWS is the most severe syndrome of the group, especially with regard to the brain phenotype. The WWS brain manifests cobblestone lissencephaly with agenesis of the corpus callosum, fusion of hemispheres, hydrocephalus, dilatation of the fourth ventricle, cerebellar hypoplasia, hydrocephalus, and sometimes encephalocele.3,4 View this table: Table 1 Clinical features of patient 1 compared with cobblestone lissencephalies ### Key points

Assignment of the muscle-eye-brain disease gene to 1p32-p34 by linkage analysis and homozygosity mapping.

Muscle-eye-brain disease (MEB) is an autosomal recessive disease of unknown etiology characterized by severe mental retardation, ocular abnormalities, congenital muscular dystrophy, and a polymicrogyria-pachygyria-type neuronal migration disorder of the brain. A similar combination of muscle and brain involvement is also seen in Walker-Warburg syndrome (WWS) and Fukuyama congenital muscular dystrophy (FCMD). Whereas the gene underlying FCMD has been mapped and cloned, the genetic location of the WWS gene is still unknown. Here we report the assignment of the MEB gene to chromosome 1p32-p34 by linkage analysis and homozygosity mapping in eight families with 12 affected individuals. After a genomewide search for linkage in four affected sib pairs had pinpointed the assignment to 1p, the MEB locus was more precisely assigned to a 9-cM interval flanked by markers D1S200 proximally and D1S211 distally. Multipoint linkage analysis gave a maximum LOD score of 6.17 at locus D1S2677. These findings provide a starting point for the positional cloning of the disease gene, which may play an important role in muscle function and brain development. It also provides an opportunity to test other congenital muscular dystrophy phenotypes, in particular WWS, for linkage to the same locus.

Clinical and imaging findings in six cases of congenital muscular dystrophy with rigid spine syndrome linked to chromosome 1p (RSMD1).

We report clinical and imaging findings in six cases from five families affected by the form of congenital muscular dystrophy with rigid spine linked to the locus rigid spine muscular dystrophy 1 on chromosome 1p35-36. All cases showed rigidity of the spine, predominant neck and trunk weakness and frequent and severe thoracic scoliosis. Respiratory impairment was always observed in the first decade. Muscle imaging showed a marked involvement of adductors, sartorius and biceps femoris while rectus femoris and gracilis were relatively spared. This pattern of selective muscle involvement was consistent in all six cases and could be easily observed on either computerised tomography or magnetic resonance imaging. The results of this study suggest that muscle imaging, in combination with clinical assessment can help to identify the rigid spine muscular dystrophy 1 form of congenital muscular dystrophy and can help to target the appropriate genetic investigations.

Brain involvement in muscular dystrophies with defective dystroglycan glycosylation

To assess the range and severity of brain involvement, as assessed by magnetic resonance imaging, in 27 patients with mutations in POMT1 (4), POMT2 (9), POMGnT1 (7), Fukutin (4), or LARGE (3), responsible for muscular dystrophies with abnormal glycosylation of dystroglycan (dystroglycanopathies).

Mutations in KLHL40 Are a Frequent Cause of Severe Autosomal-Recessive Nemaline Myopathy

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.