Ralf Herrmann

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.

Mutations in the FKRP gene can cause muscle-eye-brain disease and Walker–Warburg syndrome

The hypoglycosylation of α-dystroglycan is a new disease mechanism recently identified in four congenital muscular dystrophies (CMDs): Walker–Warburg syndrome (WWS), muscle-eye-brain disease (MEB), Fukuyama CMD (FCMD), and CMD type 1C (MDC1C).1 The underlying genetic defects in these disorders are mutations in known or putative glycosyltransferase enzymes, which among their targets probably include α-dystroglycan. FCMD (MIM: 253800) is caused by mutations in fukutin2; MEB (MEB [MIM 236670]) is due to mutations in POMGnT13; and in WWS (WWS [MIM: 236670]) POMT1 is mutated.4 In addition to the brain abnormalities, both MEB and WWS have structural eye involvement. In FCMD, eye involvement is more variable, ranging from myopia to retinal detachment, persistent primary vitreous body, persistent hyaloid artery, or microphthalmos.5 WWS, MEB, and FCMD display type II or cobblestone lissencephaly, in which the main abnormality is different degrees of brain malformation secondary at least in part to the overmigration of heterotopic neurones into the leptominenges through gaps in the external (pial) basement membrane.6,7 Whereas there are broad similarities between WWS and MEB, clear diagnostic criteria differentiating between these two conditions have been proposed8 and are shown as clinical features in table 1. A similar combination of muscular dystrophy and cobblestone lissencephaly is also found in the myodystrophy mouse (myd, renamed Largemyd), in which the Large gene is mutated.6,9,10 Our group has very recently identified mutations in the human LARGE gene in a patient with a novel form of CMD (MDC1D).11 View this table: Table 1 Clinical features of patients 1 and 2, compared with MEB and WWS patients with confirmed mutations in POGnT1 and POMT1, respectively The gene encoding the fukutin related protein (FKRP, [MIM 606612]) is mutated in a severe form of CMD (MDC1C, [OMIM 606612]).12 Clinical features of MDC1C are …

Mutations in SIL1 cause Marinesco-Sjogren syndrome, a cerebellar ataxia with cataract and myopathy

SIL1 (also called BAP) acts as a nucleotide exchange factor for the Hsp70 chaperone BiP (also called GRP78), which is a key regulator of the main functions of the endoplasmic reticulum. We found nine distinct mutations that would disrupt the SIL1 protein in individuals with Marinesco-Sjögren syndrome, an autosomal recessive cerebellar ataxia complicated by cataracts, developmental delay and myopathy. Identification of SIL1 mutations implicates Marinesco-Sjögren syndrome as a disease of endoplasmic reticulum dysfunction and suggests a role for this organelle in multisystem disorders.

Phenotypic spectrum associated with mutations in the fukutin-related protein gene

We describe 22 patients with mutations in the fukutin‐related protein (FKPR) gene. Four patients had congenital muscular dystrophy (MDC1C), with presentation at birth, severe weakness and inability to stand unsupported. The other 18 had limb girdle muscular dystrophy (LGMD2I). Eleven showed a Duchenne‐like course with loss of ambulation in the early teens while 7 had a milder phenotype. Muscle biopsy invariably showed abnormal expression of a‐dystroglycan. MDC1C patients either carried 2 missense or 1 missense and 1 nonsense mutations. Patients with LGMD2I shared a common mutation (C826A,Leu276Ileu) and their phenotypic severity was correlated with the second allelic mutation. Ann Neurol 2003;53:537–542

Intragenic deletion in the LARGE gene causes Walker-Warburg syndrome.

Intragenic homozygous deletions in the Large gene are associated with a severe neuromuscular phenotype in the myodystrophy (myd) mouse. These mutations result in a virtual lack of glycosylation of α-dystroglycan. Compound heterozygous LARGE mutations have been reported in a single human patient, manifesting with mild congenital muscular dystrophy (CMD) and severe mental retardation. These mutations are likely to retain some residual LARGE glycosyltransferase activity as indicated by residual α-dystroglycan glycosylation in patient cells. We hypothesized that more severe LARGE mutations are associated with a more severe CMD phenotype in humans. Here we report a 63-kb intragenic LARGE deletion in a family with Walker-Warburg syndrome (WWS), which is characterized by CMD, and severe structural brain and eye malformations. This finding demonstrates that LARGE gene mutations can give rise to a wide clinical spectrum, similar as for other genes that have a role in the post-translational modification of the α-dystroglycan protein.

Hexosamine biosynthetic pathway mutations cause neuromuscular transmission defect.

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.

Secondary reduction of α7B integrin in laminin α2 deficient congenital muscular dystrophy supports an additional transmembrane link in skeletal muscle

The integrins are a large family of heterodimeric transmembrane cellular receptors which mediate the association between the extracellular matrix (ECM) and cytoskeletal proteins. The α7β1 integrin is a major laminin binding integrin in skeletal and cardiac muscle and is thought to be involved in myogenic differentiation and migration processes. The main binding partners of the α7 integrin are laminin-1 (α1-β1-γ1), laminin-2 (α2-β1-γ1) and laminin-4 (α2-β2-γ1). Targeted deletion of the gene for the α7 integrin subunit (ITGA7) in mice leads to a novel form of muscular dystrophy. In the present study we have investigated the expression of two alternative splice variants, the α7B and β1D integrin subunits, in normal human skeletal muscle, as well as in various forms of muscular dystrophy. In normal human skeletal muscle the expression of the α7 integrin subunit appeared to be developmentally regulated: it was first detected at 2 years of age. In contrast, the β1D integrin could be detected in immature and mature muscle in the sarcolemma of normal fetal skeletal muscle at 18 weeks gestation. The expression of α7B integrin was significantly reduced at the sarcolemma in six patients with laminin α2 chain deficient congenital muscular dystrophy (CMD) (age >2 years). However, this reduction was not correlated with the amount of laminin α2 chain expressed. In contrast, the expression of the laminin α2 chain was not altered in the skeletal muscle of the α7 knock-out mice. These data argue in favor that there is not a tight correlation between the expression of the α7 integrin subunit and that of the laminin α2 chain in either human or murine dystrophic muscle. Interestingly, in dystrophinopathies (Duchenne and Becker muscular dystrophy; DMD/BMD) expression of α7B was upregulated irrespective of the level of dystrophin expression as shown by a strong sarcolemmal staining pattern even in young boys (age <2 years). The expression of the β1D integrin subunit was not altered in any of our patients with different types of muscular dystrophy. In contrast, sarcolemmal expression of β1D integrin was significantly reduced in the α7 integrin knock-out mice, whereas the expression of the components of the DGC was not altered. The secondary loss of α7B in laminin α2 chain deficiency defines a biochemical change in the composition of the plasma membrane resulting from a primary protein deficiency in the basal lamina. These findings, in addition to the occurrence of a muscular dystrophy in α7 deficient mice, implies that the α7B integrin is an important laminin receptor within the plasma membrane which plays a significant role in skeletal muscle function and stability.

ISPD gene mutations are a common cause of congenital and limb-girdle muscular dystrophies

Dystroglycanopathies are a clinically and genetically diverse group of recessively inherited conditions ranging from the most severe of the congenital muscular dystrophies, Walker–Warburg syndrome, to mild forms of adult-onset limb-girdle muscular dystrophy. Their hallmark is a reduction in the functional glycosylation of α-dystroglycan, which can be detected in muscle biopsies. An important part of this glycosylation is a unique O-mannosylation, essential for the interaction of α-dystroglycan with extracellular matrix proteins such as laminin-α2. Mutations in eight genes coding for proteins in the glycosylation pathway are responsible for ∼50% of dystroglycanopathy cases. Despite multiple efforts using traditional positional cloning, the causative genes for unsolved dystroglycanopathy cases have escaped discovery for several years. In a recent collaborative study, we discovered that loss-of-function recessive mutations in a novel gene, called isoprenoid synthase domain containing (ISPD), are a relatively common cause of Walker–Warburg syndrome. In this article, we report the involvement of the ISPD gene in milder dystroglycanopathy phenotypes ranging from congenital muscular dystrophy to limb-girdle muscular dystrophy and identified allelic ISPD variants in nine cases belonging to seven families. In two ambulant cases, there was evidence of structural brain involvement, whereas in seven, the clinical manifestation was restricted to a dystrophic skeletal muscle phenotype. Although the function of ISPD in mammals is not yet known, mutations in this gene clearly lead to a reduction in the functional glycosylation of α-dystroglycan, which not only causes the severe Walker–Warburg syndrome but is also a common cause of the milder forms of dystroglycanopathy.

Congenital myasthenic syndrome with tubular aggregates caused by GFPT1 mutations.

Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous group of inherited disorders of the neuromuscular junction. A difficult to diagnose subgroup of CMS is characterised by proximal muscle weakness and fatigue while ocular and facial involvement is only minimal. DOK7 mutations have been identified as causing the disorder in about half of the cases. More recently, using classical positional cloning, we have identified mutations in a previously unrecognised CMS gene, GFPT1, in a series of DOK7-negative cases. However, detailed description of clinical features of GFPT1 patients has not been reported yet. Here we describe the clinical picture of 24 limb-girdle CMS (LG-CMS) patients and pathological findings of 18 of them, all carrying GFPT1 mutations. Additional patients with CMS, but without tubular aggregates, and patients with non-fatigable weakness with tubular aggregates were also screened. In most patients with GFPT1 mutations, onset of the disease occurs in the first decade of life with characteristic limb-girdle weakness and fatigue. A common feature was beneficial and sustained response to acetylcholinesterase inhibitor treatment. Most of the patients who had a muscle biopsy showed tubular aggregates in myofibers. Analysis of endplate morphology in one of the patients revealed unspecific abnormalities. Our study delineates the phenotype of CMS associated with GFPT1 mutations and expands the understanding of neuromuscular junction disorders. As tubular aggregates in context of a neuromuscular transmission defect appear to be highly indicative, we suggest calling this condition congenital myasthenic syndrome with tubular aggregates (CMS-TA).

Kelch-like homologue 9 mutation is associated with an early onset autosomal dominant distal myopathy

Distal myopathies are a heterogeneous group of disorders characterized by progressive weakness and muscular atrophy, beginning in distal limb muscles and affecting proximal limb muscles at a later stage. We studied a large German kindred with 10 affected members. Weakness and atrophy of the anterior tibial muscles started between the ages of 8 and 16 years, followed by atrophy of intrinsic hand muscles. Progression was slow, and patients retained the ability to walk until the seventh decade. Serum creatinine kinase levels were increased in the range of 150–1400 U/l. Muscle biopsies showed myopathic changes, whereas immunohistochemistry showed normal expression of marker proteins for muscular dystrophies. Patients had reduced sensation with stocking-glove distribution in the distal limbs in later life. Nerve conduction studies revealed no evidence of neuropathy. Genome-wide linkage analysis in this family revealed a new locus for distal myopathy at 9p21.2-p22.3 (multipoint logarithm of the odds ratio = 4.21). By positional cloning we found a heterozygous mutation L95F in the Kelch-like homologue 9 gene, encoding a bric-a-brac Kelch protein. Molecular modelling indicated that the mutation may interfere with the interaction of the bric-a-brac domain with Cullin 3. Coimmunoprecipitation experiments confirmed that the mutation reduces association with Cullin 3 in the Kelch-like homologue 9-Cullin 3–E3 ubiquitin ligase complex, which is involved in ubiquitin-dependent protein degradation. We identified a unique form of early onset autosomal dominant distal myopathy which is associated with a Kelch-like homologue 9 mutation and interferes with normal skeletal muscle through a novel pathogenetic mechanism.