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M Fanin

Publications and source records attributed to M Fanin.

At least 19 recordsLinked to original sources
PubMed2006

Screening of calpain-3 autolytic activity in LGMD muscle: a functional map of CAPN3 gene mutations.

BACKGROUND: The diagnosis of calpainopathy is obtained by identifying calpain-3 protein deficiency or CAPN3 gene mutations. However, in many patients with limb girdle muscular dystrophy type 2A (LGMD2A), the calpain-3 protein quantity is normal because loss-of-function mutations cause its enzymatic inactivation. The identification of such patients is difficult unless a functional test suggests pursuing a search for mutations. MATERIALS AND METHODS: A functional in vitro assay, which was able to test calpain-3 autolytic function, was used to screen a large series of muscle biopsy specimens from patients with unclassified LGMD/hyperCKaemia who have previously shown normal calpain-3 protein quantity. RESULTS: Of 148 muscle biopsy specimens tested,17 samples (11%) had lost normal autolytic function. CAPN3 gene mutations were identified in 15 of 17 patients (88%), who account for about 20% of the total patients with LGMD2A diagnosed in our series. CONCLUSIONS: The loss of calpain-3 autolytic activity is highly predictive of primary calpainopathy, and the use of this test as part of calpainopathy diagnosis would improve the rate of disease detection markedly. This study provides the first evidence of the pathogenetic effect of specific CAPN3 gene mutations on the corresponding protein function in LGMD2A muscle and offers new insights into the structural-functional relationship of the gene and protein regions that are crucial for the autolytic activity of calpain-3.

Age of Onset↗
PubMed2006

Epilepsy and limb girdle muscular dystrophy type 2A: double trouble, serendipitous finding or new phenotype?

Autosomal recessive limb girdle muscular dystrophies (LGMD) type 2A are a group of disorders characterised by progressive involvement of proximal limb girdle muscles and caused by changes in the CAPN3 gene. Involvement of tissues other than the skeletal muscle has not been reported so far. Here we describe the unusual association of LGMD2A and idiopathic generalised epilepsy in a 14-year-old girl.

Adolescent↗
PubMed2006

Early onset calpainopathy with normal non-functional calpain 3 level.

Limb girdle muscular dystrophy 2A (LGMD2A), caused by calpain 3 deficiency, is currently diagnosed through the immunodetection of muscle protein by Western blot (WB) analysis . However, WB may provide normal results in patients with LGMD2A. The case of a female (3y 6mo of age) is described. She was found to be affected by asymptomatic hypercreatine-kinaesaemia during routine biochemical analysis at 10 months of age and had developed myopathic signs at the last neurological assessment. The WB of muscle biopsy performed at 28 months of age showed a normal quantity and pattern of bands for calpain 3. Despite this finding, on molecular analysis she was found to be a compound heterozygote for two mutations of the calpain 3 (CAPN3) gene (R110X and G222R). Autocatalytic activity assay showed a loss of function of calpain 3. This is the first genetically confirmed case of very early onset calpainopathy with a normal amount of protein at WB. Molecular analysis is also suggested in very young patients with normal WB.

Blotting, Western↗
PubMed2005

Decreased expression of DMPK: correlation with CTG repeat expansion and fibre type composition in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a trinucleotide repeat-expansion, cytosine-thymine-guanine (CTG)n, in the 3' untranslated region of a gene encoding the myotonic dystrophy protein kinase (DMPK). To correlate CTG expansion and protein expression, we studied muscle specimens from 16 adult DM1 patients using three anti-DMPK antibodies for immunoblotting. We estimated the amount of the full-length DMPK (85 kDa) in muscle biopsies from normal controls and from DM1 patients carrying different (CTG)n expansions. We found that DMPK concentration was decreased to about 50% in DM patients' muscles; the protein decrease did not seem correlated with the CTG repeat length. However, the fibre type composition in skeletal muscle seemed somehow to affect DMPK decrease, as the lowest level of the enzyme was found in patients with the lowest content of type 1 fibre.

Adult↗
PubMed2005

Ultrastructural changes in dysferlinopathy support defective membrane repair mechanism.

BACKGROUND: The dysferlin gene has recently been shown to be involved in limb girdle muscular dystrophy type 2B and its allelic disease, Miyoshi myopathy, both of which are characterised by an active muscle degeneration and regeneration process. Dysferlin is known to play an essential role in skeletal muscle fibre repair, but the process underlying the pathogenetic mechanism of dysferlinopathy is not completely understood. AIMS: To define both specific alterations of muscle fibres and a possible sequential mechanism of myopathy development. METHODS: A histological, immunohistochemical, and ultrastructural analysis of 10 muscle biopsies from patients with molecularly diagnosed dysferlinopathy. RESULTS: An inflammatory response was seen in most of the muscle biopsies. The immunohistochemical pattern demonstrated active regeneration and inflammation. Non-necrotic fibres showed alterations at different submicroscopic levels, namely: the sarcolemma and basal lamina, subsarcolemmal region, and sarcoplasmic compartment. In the subsarcolemmal region there were prominent aggregations of small vesicles, probably derived from the Golgi apparatus, which consisted of empty, swollen cisternae. In the sarcolemma there were many gaps and microvilli-like projections, whereas the basal lamina was multilayered. CONCLUSIONS: The histopathological, immunohistochemical, and ultrastructural data show that dysferlinopathy is characterised by a very active inflammatory/degenerative process, possibly associated with an inefficient repair and regenerative system. The presence of many crowded vesicles just beneath the sarcolemma provides submicroscopical proof of a defective resealing mechanism, which fails to repair the sarcolemma.

Adolescent↗
PubMed2005

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes.

BACKGROUND: The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. OBJECTIVE: To obtain unbiased information on the consequences of CAPN3 mutations. PATIENTS: 530 subjects with different grades of symptoms and 300 controls. METHODS: High throughput denaturing HPLC analysis of DNA pools. RESULTS: 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. CONCLUSIONS: A non-invasive and cost-effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103.

Adult↗
PubMed2004

LAMA2 loss-of-function mutation in a girl with a mild congenital muscular dystrophy.

The authors report a girl with autosomal recessive congenital muscular dystrophy linked to chromosome 6 (MDC1A) who carries a homozygous out-of-frame deletion in exon 56 of the LAMA2 gene but has a mild phenotype. She is still ambulant at age 13 years, shows white matter abnormalities on MRI, and traces of laminin alpha2 in her muscle biopsy with one of three antibodies used. This patient suggests that modulating factors can be associated with a less severe clinical phenotype in MDC1A.

Adolescent↗
PubMed2004

Molecular diagnosis in LGMD2A: mutation analysis or protein testing?

Limb girdle muscular dystrophy (LGMD) type 2A (LGMD2A) is caused by mutations in the CAPN3 gene encoding for calpain-3, a muscle specific protease. While a large number of CAPN3 gene mutations have already been described in calpainopathy patients, the diagnosis has recently shifted from molecular genetics towards biochemical assay of defective protein. However, an estimate of sensitivity and specificity of protein analysis remains to be established. Thus, we first correlated protein and molecular data in our large LGMD2A patient population. By a preliminary immunoblot screening for calpain-3 protein of 548 unclassified patients with various phenotypes (LGMD, myopathy, or elevated levels of serum creatine kinase [hyperCKemia]), we selected 208 cases for CAPN3 gene mutation analysis: 69 had protein deficiency and 139 had normal expression. Mutation search was conducted using SSCP, denaturing high performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS-PCR), and direct sequencing methods. We identified 58 LGMD2A mutant patients: 46 (80%) had a variable degree of protein deficiency and 12 (20%) had normal amount of calpain-3. We calculated that the probability of having LGMD2A is very high (84%) when patients show a complete calpain-3 deficiency and progressively decreases with the amount of protein; this new data offers an important tool for genetic counseling when only protein data are available. A total of 37 different CAPN3 gene mutations were detected, 10 of which are novel. In our population, 87% of mutant alleles were concentrated in seven exons (exons 1, 4, 5, 8, 10, 11, and 21) and 61% correspond to only eight mutations, indicating the regions where future molecular analysis could be restricted. This study reports the largest collection of LGMD2A patients so far in which both protein and gene mutations were obtained to draw genotype-protein-phenotype correlations and provide insights into a critical protein domain.

Adolescent↗
PubMed2003

LGMD2E patients risk developing dilated cardiomyopathy.

Sarcoglycan gene mutations cause various limb-girdle muscular dystrophies. The sarcoglycans are expressed both in skeletal and cardiac muscle but, surprisingly, so far only a few sarcoglycanopathy patients have had documented cardiomyopathy. We studied six patients with beta-sarcoglycanopathy. Immunohistochemical and immunoblot analysis performed on skeletal muscle biopsies from five patients, showed the loss of all sarcoglycans in three cases and marked reduction in two patients. Non-invasive cardiac examinations revealed that three patients had cardiac involvement: one had a severe Duchenne-like dystrophy, lethal dilated cardiomyopathy, and shared the same mutation reported in another cardiomyopathic patient; the other two patients had limb-girdle dystrophy and moderate cardiac involvement (one of them has a novel gene mutation). Given the age profile of the patients studied, the 50% cardiac involvement found in our LGMD2E patients is likely to be a conservative estimate. Careful cardiac monitoring should be carried out in beta-sarcoglycanopathy patients who are at high risk of developing cardiomyopathy.

Adolescent↗
PubMed2002

Defective assembly of sarcoglycan complex in patients with beta-sarcoglycan gene mutations. Study of aneural and innervated cultured myotubes.

Mutations in the sarcoglycan (SG) genes cause autosomal recessive muscular dystrophies. The absence of each SG complex component in muscle impairs the proper assembly of the entire SG complex, resulting in sarcolemmal damage. We investigated the consequences of beta-SG gene mutations in cultured muscle from two beta-SG mutated patients, and analysed each individual SG protein expression by cross-sectional immunocytochemistry and Western blot in aneural and innervated myotubes. Patients' muscle biopsy showed total loss of SG complex; however, a limited amount of beta-SG was detected in aneural and innervated myotubes, where the protein was localized to the plasma membrane. This paradoxical beta-SG expression can be attributable to antibody cross-reaction or to the expression of an unknown SG isoform specific of immature muscle. In our cultured myotubes, the other components of the SG complex were absent, suggesting that beta-SG gene mutations result in a defective assembly of the entire SG complex in early stages of muscle development, and that the role of beta-SG is crucial for the normal structure and/or function of the SG complex in the sarcolemma.

Base Sequence↗
PubMed2002

Muscle pathology in dysferlin deficiency.

Dysferlin deficiency is being increasingly recognized in limb-girdle dystrophy and distal myopathy but its role in the development of muscle pathology is still poorly understood. For this purpose, 26 muscle biopsies from 25 dysferlinopathy patients were analysed by routine histochemistry and by immunohistochemistry with eight different antibodies, and scored for inflammatory response and type of cell infiltrate, fibre degeneration and regeneration, fibre type composition and severity of histopathological changes. In cases with an advanced-stage dystrophic pattern we observed type 1 fibre predominance exceeding 80%, suggesting a selective loss of type 2 fibres or a conversion process. The extent of muscle fibre regeneration and degeneration in dysferlinopathy was intermediate between sarcoglycanopathy and Duchenne dystrophy or myositis, suggesting a rather aggressive course of the disease. An increased inflammatory response was observed in the majority of our patients (16/26), who also showed an active dystrophic pattern. Type and localization of cellular infiltrates suggest that inflammatory reaction is secondary to necrosis. Major histocompatibility complex (MHC) class I molecules were overexpressed in dysferlinopathy, mainly in association with fibre phagocytosis and regeneration; their occasional expression in non-necrotic fibres might represent a marker of ongoing necrosis. Muscle inflammation might be triggered by the structurally altered membrane consequent to dysferlin defect.

Adolescent↗
PubMed2001

Calpain-3 and dysferlin protein screening in patients with limb-girdle dystrophy and myopathy.

BACKGROUND: Mutations in the genes encoding for calpain-3 and dysferlin are responsible for limb-girdle muscular dystrophy (LGMD) type 2A and 2B, the most common forms of autosomal recessive LGMD. OBJECTIVE: To identify calpain-3 or dysferlin deficiency in a large cohort of patients with as yet unclassified LGMD and myopathy through candidate protein analysis. METHODS: The authors' muscle biopsy database search identified 407 candidate muscle biopsies with normal dystrophin and sarcoglycan. Calpain-3 and dysferlin were studied by Western blotting and immunohistochemistry. RESULTS: Combined calpain-3 and dysferlin Western blot analysis identified calpain-3 deficiency in 66 (16%) muscle biopsies. In 31 cases (47%), the protein was absent, and in 35 (53%), it was severely reduced in amount (3 to 50% of control). Dysferlin deficiency was found in 26 (6.5%) muscle biopsies. In 9, the protein was absent (35%), and in 17 (65%), it was severely reduced in amount (traces to 20% of control). Twenty-eight percent (53/191) of patients with LGMD phenotype had calpain-3 deficiency. Sixty percent (21/35) of patients with distal myopathy had dysferlin deficiency. Dysferlin immunohistochemistry showed, in the completely dysferlin-deficient patients, absent reaction at the sarcolemma but positive nuclear membrane labeling and, in the partially dysferlin-deficient patients, scattered granular positive cytoplasmic areas and diffuse reaction in regenerating fibers. CONCLUSION: About 25% of previously unclassified dystrophy/myopathy cases are due to calpain-3 or dysferlin protein deficiency. These results suggest that immunoblot analysis may be used to define patients for calpain-3 and dysferlin gene mutation studies.

Biopsy↗
PubMed2000

A novel laminin alpha2 isoform in severe laminin alpha2 deficient congenital muscular dystrophy.

OBJECTIVES: Laminin alpha2 deficiency presents at birth with muscle weakness, hypotonia, and usually asymptomatic white matter signal on MRI. Few patients with laminin alpha2 deficiency have been described with seizures and structural brain abnormalities. The reason for the variation in the severity of the clinical phenotype in congenital muscular dystrophy (CMD) with laminin alpha2 deficiency is not known. METHODS: A patient with CMD with partial laminin alpha2 presenting with brain structural abnormalities and untreatable generalized and partial complex seizure was studied. Alternative laminin alpha2 splicing was studied by single-strand conformational polymorphism/sequencing analysis. RESULTS: A novel laminin alpha2 isoform was identified. Nonsense laminin alpha2 mutations (stop codons) were inherited from both parents; however, one of the nonsense mutations was in a region of exon 31, which is alternatively spliced. The alternatively spliced isoform excluded one of the stop codon mutations, and was thus able to produce normal laminin alpha2 corresponding to this isoform. Laminin alpha2 immunofluorescence showed that this isoform was not evenly distributed at the muscle fiber basal lamina, but preferentially localized in discrete areas. Laminin alpha5, beta1, gamma1, and nidogen showed decreased expression by immunofluorescence. CONCLUSIONS: The severity of this patient's phenotype may be due to overexpression of the exon 31-spliced laminin alpha2 isoform. Exon 31 lies in the IIIA domain of the laminin alpha2 protein, just proximal to the triple coil-coiled region. It is possible that chain assembly is impaired by this isoform, resulting in a loss of possible rescue mechanisms.

Adolescent↗
PubMed2000

Private beta- and gamma-sarcoglycan gene mutations: evidence of a founder effect in Northern Italy.

Autosomal recessive muscular dystrophies called "sarcoglycanopathies" result from mutations in the genes encoding alpha-, beta-, gamma-, or delta-sarcoglycan complex components. The present study involved six unrelated families from Northern Italy showing mutations in the beta- or gamma-sarcoglycan genes. An 8 bp duplication in the beta-sarcoglycan gene and 1 bp insertion in the gamma-sarcoglycan gene occur with high frequency in our population. These mutations have never been reported thus far in other countries. Many patients are homozygotes for a single mutation, although they derived from non-consanguineous marriages. We suggest that these alleles are "private" mutations of this geographical region. A panel of highly informative microsatellite markers that map in the beta- and gamma-sarcoglycan gene locus was used to assess the haplotypes among affected patients and control population, in order to test the presence of linkage disequilibrium. We found that the 8 bp duplication in the beta-sarcoglycan gene and the 1 bp insertion in the gamma-sarcoglycan gene are in linkage disequilibrium with neighbouring polymorphisms. The recurrence of specific sarcoglycan mutations in Northern Italy is probably due to a founder effect, combined with a relative genetic isolation.

Alleles↗
PubMed2000

Neuromuscular damage after hyperthermic isolated limb perfusion in patients with melanoma or sarcoma treated with chemotherapeutic agents.

PURPOSE: To evaluate the incidence and entity of muscle damage after hyperthermic limb perfusion (HLP) with doxorubicin or melphalan, two widely used chemotherapeutic agents. METHODS: We collected muscle biopsies from eleven patients with lower limb sarcoma or melanoma immediately before and at a variable time after the chemotherapeutic procedure (mean = 49.4 days). Biopsy specimens were stained with standard histochemical and immunohistochemical methods on cryostat sections and the grade of fiber atrophy was calculated. RESULTS: Clear neurogenic alterations were present in pre-HLP biopsies of seven patients related to age and previous therapy. In six patients, the comparison between biopsies before and after HLP demonstrated worsening of preexisting neurogenic condition and appearance of mitochondrial-related damage. Reduction in type I or type II fiber diameter was present in nine patients, but no relation to doxorubicin or melphalan treatment was clear. An unexpected, large accumulation of desmin was detected in the muscle biopsy of one patient receiving doxorubicin, probably related to the mechanism of doxorubicin-induced myotoxicity. CONCLUSIONS: The observed neuromuscular toxic effects could be related to the physical or chemical conditions of HLP, in particular perfusion temperature; in addition, the present study demonstrates that preexisting neuromuscular changes, i.e. neuropathy, modulates the degree of further damage following HLP.

Adult↗
PubMed2000

Acute quadriplegic myopathy in a 17-month-old boy.

Acute quadriplegic myopathy is a rare condition associated with the use of nondepolarizing muscle-blocking agents and corticosteroids in the course of severe systemic illness. A 17-month-old boy underwent liver transplantation for fulminant hepatitis. He was intubated for 24 days and treated with vecuronium bromide and high-dose methylprednisolone. The child was weaned from the ventilator and presented extreme weakness in the upper limbs and total paralysis of the lower limbs. Serum creatine kinase level was normal and electromyography showed myopathic abnormalities. Muscle biopsy showed severe type-1 fiber atrophy and selective loss of myosin thick filaments was seen on electron microscopy. Scattered regenerating fetal myosin-positive fibers were present, mu calpain was absent, while m calpain was diffusely expressed. Physical therapy was immediately started and the child recovered even though corticosteroids were not discontinued. The pathogenesis of acute quadriplegic myopathy is still unknown. We suggest that it could be due to abnormal protein turnover in the muscle. Several independent factors, such as corticosteroid treatment, immobilization, or cytokines, could take part in a cascade of events that leads to an excessive yet selective degradation of proteins involving myosin thick filaments and possibly components of sarcolemma, causing muscle inexcitability.

Anti-Inflammatory Agents↗
PubMed1999

Regeneration in sarcoglycanopathies: expression studies of sarcoglycans and other muscle proteins.

We have studied the immunohistochemical expression of 14 different muscle proteins of the basal lamina, sarcolemma and cytoskeleton in primary sarcoglycanopathies (13 cases) and compared it with Duchenne dystrophy (6 cases) and myositis (5 cases). Sarcolemmal proteins (i.e. 4 sarcoglycans, beta-dystroglycan, dystrophin, beta-spectrin) were reduced both in sarcoglycanopathies and Duchenne dystrophy, because of structural and functional impairment of the plasma membrane. Sarcolemmal proteins are poorly expressed in regenerating fibers of all muscle disorders, due to a developmental delay or to an abnormal assembly. Laminins (alpha2 and beta chains) were preserved in all cases while utrophin was expressed in Duchenne muscle but not in sarcoglycanopathies. Regenerating fibers were studied with different markers (i.e. fetal myosin, desmin, vimentin, laminin alpha1). Fetal myosin positive fibers (as well as desmin and laminin alpha1), were significantly higher in Duchenne dystrophy (25%) than in age-matched sarcoglycanopathies (7%). Vimentin, a marker of early regeneration, was expressed at higher level in sarcoglycanopathies than in Duchenne dystrophy, suggesting in the former a lower extent of regeneration or a shorter regeneration cycle.

Adolescent↗