A CASQ1 founder mutation in three Italian families with protein aggregate myopathy and hyperCKaemia.

BACKGROUND: Protein aggregate myopathies are increasingly recognised conditions characterised by a surplus of endogenous proteins. The molecular and mutational background for many protein aggregate myopathies has been clarified with the discovery of several underlying mutations. Familial idiopathic hyperCKaemia is a benign genetically heterogeneous condition with autosomal dominant features in a high proportion of cases. METHODS: In 10 patients from three Italian families with autosomal dominant benign vacuolar myopathy and hyperCKaemia, we performed linkage analysis and exome sequencing as well as morphological and biochemical investigations. RESULTS AND CONCLUSIONS: We show, by Sanger and exome sequencing, that the protein aggregate myopathy with benign evolution and muscle inclusions composed of excess CASQ1, affecting three Italian families, is due to the D244G heterozygous missense mutation in the CASQ1 gene. Investigation of microsatellite markers revealed a common haplotype in the three families indicating consanguinity and a founder effect. Results from immunocytochemistry, electron microscopy, biochemistry and transfected cell line investigations contribute to our understanding of pathogenetic mechanisms underlining this defect. The mutation is common to other Italian patients and is likely to share a founder effect with them. HyperCKaemia in the CASQ1-related myopathy is common and sometimes the sole overt manifestation. It is likely that CASQ1 mutations may remain undiagnosed if a muscle biopsy is not performed, and the condition could be more common than supposed.

Variable disease severity in Saudi Arabian and Sudanese families with c.3924 + 2 T > C mutation of LAMA2.

BACKGROUND: Congenital muscular dystrophy type 1A is caused by mutations in the LAMA2 gene that encodes the laminin α2 chain, a component of the skeletal muscle extracellular matrix protein laminin-211. The clinical spectrum of the disease is more heterogeneous than previously thought, particularly in terms of motor achievement and disease progression. We investigated clinical findings and performed molecular genetic analysis in 3 families from Saudi Arabia and 1 from Sudan in whom congenital muscular dystrophy 1A was suspected based on homozygosity mapping and laminin α2 chain deficiency. METHODS: We investigated 9 affected individuals from 1 Sudanese and 3 Saudi families in whom MDC1A was suggested by clinical, neuroimaging and/or pathological findings and by homozygosity mapping at the LAMA2 locus. Morphological and immunohistochemical analysis were performed in 3 patients from the 3 Saudi families. SSCP analysis, DNA sequencing and microsatellite analysis were carried out in the 4 index cases. RESULTS: A previously described mutation in the LAMA2 gene, a homozygous T > C substitution at position +2 of the consensus donor splice site of exon 26, was found in the 4 index patients. Clinical evaluation of 9 patients from the 4 families revealed variable disease severity particularly as regards motor achievement and disease progression. Microsatellite analysis showed an identical mutation-associated haplotype in the 4 index cases indicating a founder effect of the mutation in all 4 families. CONCLUSIONS: Our data provide further evidence that the clinical spectrum of MDC1A due to a single mutation is heterogeneous, particularly in terms of motor achievement and disease progression, making it difficult to give a reliable prognosis even in patients with identical LAMA2-associated haplotype. The c.3924 + 2 T > C mutation to date has been found only in patients originating from the Middle East or Sudan; therefore laminin 2 chain deficiency in patients from those regions should initially prompt a search for this mutation.

Osteopontin is highly expressed in severely dystrophic muscle and seems to play a role in muscle regeneration and fibrosis.

AIMS: To increase our understanding of profibrotic mechanisms in dystrophic muscle. METHODS AND RESULTS: Extracellular matrix, fibrosis-related molecules and histopathology were assessed in skeletal muscle of patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy type 1A (MDC1A).Osteopontin expression was much higher in DMD and MDC1A than in BMD and control muscle. Osteopontin was expressed in mononuclear cell infiltrates, on some muscle fibre surfaces, in regenerating fibres, and in calcified fibres. In all pathological muscles, matrix metalloproteinase (MMP)-1 was increased around groups of fibres that were also characterized by absence of collagen 1. The amounts of MMP-2, MMP-9 and tissue inhibitor of MMP -1 transcripts were also increased, whereas their proteins were variably expressed in muscle fibres (surface or cytoplasm) and at foci of necrosis and regeneration. Inflammatory cells, fibroblasts and myofibroblasts were more numerous in DMD and MDC1A than in BMD muscle. CONCLUSIONS: Several fibrosis-related factors are greatly altered in severely dystrophic skeletal muscle. Osteopontin was the most conspicuously upregulated, both as transcript and as protein, in muscle fibres and infiltrating cells, indicating an intimate involvement in fibrosis, and also in inflammation and muscle regeneration, although its precise roles in these processes remain to be elucidated.

Calsequestrin and junctin immunoreactivity in hexagonally cross-linked tubular arrays myopathy.

We report on a patient with muscle pain not associated with muscle weakness. Microscopic examination of the muscle biopsy revealed rod-like cytoplasmic bodies in many fast fibres. By electron microscopy these had a crystalloid structure identical to the hexagonally cross-linked caveolin 3-positive tubular arrays, previously described in patients with similarly benign myopathy. We found that these inclusions were positive for calsequestrin and for the calsequestrin-binding protein junctin, as well as for caveolin 3. However, the genes coding for these proteins were not mutated. For diagnostic purposes calsequestrin and caveolin 3 positivity should be checked when rods are encountered in muscle biopsy for mild myopathy.

LAMA2 stop-codon mutation: merosin-deficient congenital muscular dystrophy with occipital polymicrogyria, epilepsy and psychomotor regression.

Merosin-deficient congenital muscular dystrophy (MD) type 1A (MDC1A) is one of the most frequent forms of CMD in Western countries. The classical form, characterized by a total lack of laminin alpha2 chain expression, usually shows severe clinical features; cases with complete laminin alpha2 deficiency and mild phenotype have also been reported, although the mechanisms underlying the lack of genotype-phenotype correlation have not been elucidated. Epilepsy and focal cortical dysplasia-in addition to the classical diffuse white matter abnormalities-have been described in some of these patients associated with cognitive deterioration. We report on a patient with total laminin alpha2 deficiency due to a homozygous stop-codon mutation in the LAMA2 gene, with mild evolution. When 6.9 years old, she developed focal occipital seizures and absence-like status when awake, with probable relation to an extensive bilateral occipital micropolygyria. Soon afterwards she lost ambulation and developed cognitive deterioration. Our case confirms that the clinical spectrum of MDC1A is more heterogeneous than previously thought.

Danon disease: a novel LAMP2 mutation affecting the pre-mRNA splicing and causing aberrant transcripts and partial protein expression.

LAMP2, the causative gene of Danon disease, located on chromosome Xq24, encodes the lysosome-associated membrane protein-2 (LAMP-2). We describe clinical features and molecular data in an Italian patient with Danon disease. The patient had hyperCKemia, hypertrophic cardiomyopathy, no muscle weakness and slight mental impairment. Muscle biopsy revealed autophagic vacuoles with sarcolemmal features and glycogen storage. Immunohistochemistry and immunoblot revealed traces of LAMP-2 protein in skeletal muscle. Molecular analysis of the LAMP2 gene revealed a novel hemizygous mutation affecting the invariant +1 position of the splice site of intron 8, resulting in aberrant transcripts with skipping of exon 8 in all three LAMP-2 isoforms, skipping of exons 7 and 8 in LAMP-2A and 2C, and a 15 bp deletion in exon 8 of LAMP-2B. Low levels of normal LAMP-2B transcript were also present. Danon disease is an under-recognized and frequently fatal condition, treatable by heart transplantation. Investigation of the primary molecular defect is important for cardiac surveillance and genetic counseling.

Clinical, molecular, and protein correlations in a large sample of genetically diagnosed Italian limb girdle muscular dystrophy patients.

Limb girdle muscular dystrophies (LGMD) are characterized by genetic and clinical heterogeneity: seven autosomal dominant and 12 autosomal recessive loci have so far been identified. Aims of this study were to evaluate the relative proportion of the different types of LGMD in 181 predominantly Italian LGMD patients (representing 155 independent families), to describe the clinical pattern of the different forms, and to identify possible correlations between genotype, phenotype, and protein expression levels, as prognostic factors. Based on protein data, the majority of probands (n=72) presented calpain-3 deficiency; other defects were as follows: dysferlin (n=31), sarcoglycans (n=32), alpha-dystroglycan (n=4), and caveolin-3 (n=2). Genetic analysis identified 111 different mutations, including 47 novel ones. LGMD relative frequency was as follows: LGMD1C (caveolin-3) 1.3%; LGMD2A (calpain-3) 28.4%; LGMD2B (dysferlin) 18.7%; LGMD2C (gamma-sarcoglycan) 4.5%; LGMD2D (alpha-sarcoglycan) 8.4%; LGMD2E (beta-sarcoglycan) 4.5%; LGMD2F (delta-sarcoglycan) 0.7%; LGMD2I (Fukutin-related protein) 6.4%; and undetermined 27.1%. Compared to Northern European populations, Italian patients are less likely to be affected with LGMD2I. The order of decreasing clinical severity was: sarcoglycanopathy, calpainopathy, dysferlinopathy, and caveolinopathy. LGMD2I patients showed both infantile noncongenital and mild late-onset presentations. Age at disease onset correlated with variability of genotype and protein levels in LGMD2B. Truncating mutations determined earlier onset than missense substitutions (20+/-5.1 years vs. 36.7+/-11.1 years; P=0.0037). Similarly, dysferlin absence was associated with an earlier onset when compared to partial deficiency (20.2+/-standard deviation [SD] 5.2 years vs. 28.4+/-SD 11.2 years; P=0.014).

Severe congenital muscular dystrophy in a LAMA2-mutated case.

Clinical features and molecular data are described for a patient with undetectable expression of laminin alpha2 chain (merosin) and severe congenital muscular dystrophy. Molecular analysis of the LAMA2 gene revealed two previously un-described mutations. The patient achieved independent sitting at age 2, but lost head balance at age 7; he was never able to stand unsupported. Cerebral magnetic resonance imaging revealed diffuse hypomyelination in both cerebral hemispheres; electrophysiological assessment revealed progressive sensorimotor axonal polyneuropathy. Investigation of the primary molecular defect in congenital muscular dystrophy patients is important for genetic counseling, because the clinical features of the various forms overlap, and because significant laminin alpha2 chain reduction may occur in patients with primary defects in other genes.

LAMA2 gene analysis in congenital muscular dystrophy: new mutations, prenatal diagnosis, and founder effect.

OBJECTIVE: To determine if laminin-alpha2 deficiency is due to mutations in the LAMA2 gene or secondary to mutations in other congenital muscular dystrophy genes. METHODS: We performed molecular analysis of LAMA2, by single-strand conformation polymorphism and sequencing, in 15 patients with undetectable or greatly reduced laminin-alpha2 expression. We also performed 4 prenatal diagnoses and investigated a founder effect. RESULTS: We found 1 known and 9 previously undescribed LAMA2 mutations spanning all protein domains. These were nonsense or frameshifts causing laminin-alpha2 absence or, in 1 case, a homozygous missense mutation producing partial protein expression and milder phenotype. LAMA2 mutations were undetected in 5 patients, in 2 of whom FKRP mutations explained the phenotype. In 3 prenatal cases, the fetus was heterozygous for the mutation of interest and pregnancy continued; in 1 case, the fetus was affected and aborted. In 2 patients, the Cys967Stop mutation and identical haplotypes flanking the LAMA2 gene indicated a founder effect. CONCLUSIONS: The clinical phenotype was severe in most patients with LAMA2 mutations and associated with undetectable protein expression. One case with no protein and another with partial expression had milder phenotypes. Typical white matter alterations on magnetic resonance imaging were found in all patients with LAMA2 mutations, supporting the utility of magnetic resonance imaging in differential diagnosis. The founder mutation (Cys967Stop) probably originated in Albania. Genetic characterization of affected families is mainly of use for prenatal diagnosis.

Decorin and biglycan expression is differentially altered in several muscular dystrophies.

Biglycan and decorin are small extracellular proteoglycans that interact with cytokines, whose activity they may modulate, and with matrix proteins, particularly collagens. To better understand their role in muscle fibrosis, we investigated expression of decorin and biglycan transcripts and protein in muscle of several forms of muscular dystrophy, and also expression of perlecan, an extracellular proteoglycan unrelated to collagen deposition. In Duchenne muscular dystrophy (DMD) and LAMA2-mutated congenital muscular dystrophy (MDC1A) we also quantitated transcript levels of the profibrotic cytokine TGF-beta1. We examined muscle biopsies from nine DMD patients, aged 2-8 years; 14 BMD (Becker muscular dystrophy) patients (nine aged 1-5 years; five aged 30-37 years); four MDC1A patients (aged 2-7 years); six dysferlin-deficient patients (aged 19-53 years) with mutation ascertained in two, and normal expression of proteins related to limb girdle muscular dystrophies in the others; 10 sarcoglycan-deficient patients: seven with alpha-sarcoglycan mutation, two with beta-sarcoglycan mutation and one with gamma-sarcoglycan mutation (five aged 8-15 years; five aged 26-43 years); and nine children (aged 1-6 years) and 12 adults (aged 16-61 years) suspected of neuromuscular disease, but who had normal muscle on biopsy. Biglycan mRNA levels varied in DMD and MDC1A depending on the quantitation method, but were upregulated in BMD, sarcoglycanopathies and dysferlinopathy. Decorin mRNA was significantly downregulated in DMD and MDC1A, whereas TGF-beta1 was significantly upregulated. Decorin mRNA was normal in paediatric BMD, but upregulated in adult BMD, sarcoglycanopathies and dysferlinopathy. Perlecan transcript levels were similar to those of age-matched controls in all disease groups. By immunohistochemistry, decorin and biglycan were mainly localized in muscle connective tissue; their presence increased in relation to increased fibrosis in all dystrophic muscle. By visual inspection, decorin bands on immunoblot did not differ from those of age-matched controls in all patient groups. However, when the intensity of the bands was quantitated against vimentin and normalized against sarcomeric actin, in DMD and MDC1A the ratio of band intensities was significantly lower than in age-matched controls. Variations in the transcript and protein levels of these proteoglycans in different muscular dystrophies probably reflect the variable disruption of extracellular matrix organization that occurs in these diseases. The significantly lowered decorin levels in DMD and MDC1A may be related to the increased TGF-beta1 levels, suggesting a therapeutic role of decorin in these severe dystrophies.

Abnormal lysosomal and ubiquitin-proteasome pathways in 19p13.3 distal myopathy.

We describe a second large Italian kindred with autosomal dominant vacuolar myopathy characterized by variable severity, adult-onset weakness of distal limb muscles, and no cardiac involvement. At least 19 individuals over four generations are affected. Histopathological and immunochemical features of the vacuoles, present in many fibers, indicate protein degradation abnormalities with dysregulation of the lysosomal pathway and activation of the ubiquitin-proteasomal pathway. Linkage analysis localized the defect to the 19p13.3 locus in a region with no known genes. We speculate that the primary defect may be an abnormality in the lysosomal degradation pathway or related components.