Elisabetta Tasca

Frequency of LGMD gene mutations in Italian patients with distinct clinical phenotypes.

Background: The frequency of various limb-girdle muscular dystrophy (LGMD) molecular diagnoses has previously been investigated only in cohorts of patients presenting LGMD phenotype. Methods: A total of 550 muscle biopsies underwent multiple protein screening (including calpain-3 functional assay) and extensive gene mutation analysis to examine the frequency of LGMD subtypes in patients with distinct clinical phenotypes (severe childhood-onset LGMD, adult-onset LGMD, distoproximal myopathy, and asymptomatic hyperCKemia). Results: The percentage of molecularly ascertained cases directly relates with the degree of clinical involvement: 60% of total LGMD (77% of childhood-onset, 46% of adult-onset, 66% of distoproximal myopathy) and 14% of hyperCKemia. The higher number of molecular diagnoses in severe phenotypes might suggest that genes selected for our screening are those more frequently associated with severe LGMD, and that the hyperCKemia group includes heterogeneous diagnoses. The probability of obtaining a molecular diagnosis increases when a protein defect is found in a muscle biopsy: in such cases, we diagnosed 87% of LGMD and 76% of hyperCKemia. Conclusions: Diagnosing 77% of childhood-onset limb-girdle muscular dystrophy (LGMD) and 60% of total LGMD is an important result. The missing identification of gene mutations in about 40% of patients with typical LGMD phenotype suggests that unknown genetic or nongenetic etiologies are still to be recognized. Dysferlin, caveolin-3, and emerin protein defects invariably corresponded to primary disorders (100%), whereas a lower correlation was found for sarcoglycans (77%) and calpain-3 (84%). The different efficiency of genetic diagnosis after the identification of a protein defect in the various disorders is possibly due to different pathogenetic effects of mutations.

Molecular pathology and enzyme processing in various phenotypes of acid maltase deficiency

Objectives: To examine at molecular, biochemical, and muscle pathology level the striking clinical heterogeneity resulting from acid α-glucosidase deficiency. Methods: We investigated 23 patients with infantile-onset or late-onset glycogen storage disease type II by enzyme activity, protein expression by immunoblotting, GAA gene mutations, and muscle pathology including immunolabeling for Golgi and sarcolemmal proteins. Results: The enzyme activity was absent or minimal in infantile-onset cases and variably reduced in late-onset patients. Genotype-phenotype correlation (seven novel mutations were found) showed that most late-onset patients had the heterozygous IVS1 leaky splicing mutation (one patient was homozygous), but the course of the disease was often difficult to predict on the basis of the mutations alone. All patients showed an abnormal pattern of enzyme protein processing, with increased amounts of the inactive forms and very low or absent amounts of the mature forms. The molecular weight of the mature and the intermediate forms appeared higher in patients’ samples than in the control muscle. We observed a Golgi proliferation in muscle fibers possibly caused by the retention of inactive forms of enzyme protein that cannot be correctly targeted from Golgi to lysosomes. The vacuolar membranes expressed sarcolemmal proteins in late-onset but not in infantile-onset patients, suggesting an extensive autophagy and vacuolar membrane remodeling in late-onset patients. Conclusions: The different protein molecular weight between patients and controls could be due to an excessive sialylation of mutant enzyme: this might be possibly caused by a delayed transport and longer transit of the inactive proteins in the Golgi apparatus.

Fatigue in muscular dystrophies

Fatigue is a frequent complaint in muscular dystrophies but it is yet not well defined or studied. We have examined the issue of muscle fatigue in a series of molecularly defined muscular dystrophies. A greater fatigability is seen in muscular dystrophy patients and can be an acute or chronic status. In Duchenne Muscular Dystrophy and beta-sarcoglycanopathy besides the alteration of dystrophin and/or sarcoglycan complex, a neuronal nitric oxide synthase depletion is frequently found and might correlate with post-exercise fatigability as well as with cardiac involvement. Therefore, it might be an important modulating factor of the severity of myopathy. In myotonic dystrophy, fatigue is a common complaint: muscle is involved and type 1 atrophy is a frequent feature; brain involvement and depressed mood might likely explain the extent of fatigue and daytime sleepiness commonly observed in these patients. Furthermore, in our observation in a series of 24 cases, muscle and brain can be independently involved in DM1 patients. These observations have profound impact on the type of physical therapy to be prescribed in such patients.

How to tackle the diagnosis of limb-girdle muscular dystrophy 2A

Limb-girdle muscular dystrophy (LGMD) 2A (calpainopathy) is the most frequent form of LGMD in many European countries. The increasing demand for a molecular diagnosis makes the identification of strategies to improve gene mutation detection crucial. We conducted both a quantitative analysis of calpain-3 protein in 519 muscles from patients with unclassified LGMD, unclassified myopathy and hyperCKemia, and a functional assay of calpain-3 autolytic activity in 108 cases with LGMD and normal protein quantity. Subsequently, screening of CAPN3 gene mutations was performed using allele-specific tests and simplified SSCP analysis. We diagnosed a total of 94 LGMD2A patients, carrying 66 different mutations (six are newly identified). The probability of diagnosing calpainopathy was very high in patients showing either a quantitative (80%) or a functional calpain-3 protein defect (88%). Our data show a high predictive value for reduced-absent calpain-3 or lost autolytic activity. These biochemical assays are powerful tools for otherwise laborious genetic screening of cases with a high probability of being primary calpainopathy. Our multistep diagnostic approach is rational and highly effective. This strategy has improved the detection rate of the disease and our extension of screening to presymptomatic phenotypes (hyperCKemia) has allowed us to obtain early diagnoses, which has important consequences for patient care and genetic counseling.

The clinical course of calpainopathy (LGMD2A) and dysferlinopathy (LGMD2B).

Abstract Objective: Autosomal recessive limb girdle muscular dystrophies (LGMD type 2) are a clinically and genetically heterogeneous group of disorders, characterized by progressive involvement and wasting of limb girdle muscles. In order to describe the peculiar clinical features of LGMD2A (calpainopathy) and LGMD2B (dysferlinopathy), the most frequent forms of LGMD in European countries, we analysed and compared the phenotype and the clinical course in two relatively large groups of these patients. Methods: We selected 22 patients with a molecular diagnosis of LGMD2A and 21 patients with LGMD2B and reported their clinical data collected from both clinical history and during periodical neuromuscular examinations: age and distribution of muscle involvement at onset, clinical functional score by the use of ten-point modified scale of Gardner–Medwin and Walton at onset and at last clinical examination, and the rate of disease progression. Results: LGMD2A group included patients with different ages at onset (early-onset or late-onset), different phenotypes (upper girdle in Erb LGMD or lower girdle in Leyden–Moebius LGMD) and different disease progressions (rapid or slow course). LGMD2B patients differed for pattern of muscle involvement at onset (distal in Miyoshi dystrophy or proximal in Leyden–Moebius LGMD) but they had a rather homogeneous age at onset (in the second/third decade) and rate of disease progression. Discussion: Our data show that besides the clinical differences within each group of patients, the two forms of LGMD present distinctive clinical features. The various phenotypes and courses can be attributed to specific pathogenetic mechanisms and might suggest differential therapeutic strategies.

Correlations between clinical severity, genotype and muscle pathology in limb girdle muscular dystrophy type 2A

Background: Limb girdle muscular dystrophy type 2A (LGMD2A) is characterised by wide variability in clinical features and rate of progression. Patients with two null mutations usually have a rapid course, but in the remaining cases (two missense mutations or compound heterozygote mutations) prognosis is uncertain. Methods: We conducted what is to our knowledge the first systematic histopathological, biochemical and molecular investigation of 24 LGMD2A patients, subdivided according to rapid or slow disease progression, to determine if some parameters could correlate with disease progression. Results: We found that muscle histopathology score and the extent of regenerating and degenerating fibres could be correlated with the rate of disease course when the biochemical and molecular data do not offer sufficient information. Comparison of clinical and muscle histopathological data between LGMD2A and four other types of LGMD (LGMD2B–E) also gave another important and novel result. We found that LGMD2A has significantly lower levels of dystrophic features (ie degenerating and regenerating fibres) and higher levels of chronic changes (ie lobulated fibres) compared with other LGMDs, particularly LGMD2B. These results might explain the observation that atrophic muscle involvement seems to be a clinical feature peculiar to LGMD2A patients. Conclusions: Distinguishing patterns of muscle histopathological changes in LGMD2A might reflect the effects of a disease-specific pathogenetic mechanism and provide clues complementary to genetic data.

Dominant muscular dystrophy with a novel SYNE1 gene mutation

The synaptic nuclear envelope protein-1 (SYNE1) gene encodes nesprin-1, a protein characterized by the presence of multiple spectrin repeats and highly expressed in striated muscles. In addition to autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) type 4, mutations in the SYNE1 gene cause spinocerebellar ataxia type 8, myogenic multiplex arthrogryposis congenita with features of EDMD, intellectual disability with spastic paraplegia, and axonal neuropathy. We report a family including 3 patients (mother and 2 sons) affected with a dominant form of muscular dystrophy with joint contractures without significant cardiac abnormalities (“EDMD-like” phenotype), in which we identified a novel mutation in the SYNE1 gene. Patient 1 (I-2, Fig. 1) is a woman who, at age 5 years, underwent surgery for congenital pulmonary-valve stenosis; she was subsequently free from heart disturbances. She had onset of progressive muscle weakness at age 6-7 years; foot and elbow joint contractures were present in adolescence. At age 19 she underwent bilateral Achilles tenotomy. She died at age 44 years due to melanoma; she was still able to walk for short distances, but was unable to climb stairs. Patient 2 (II-1, Fig. 1) is a 28-year-old man who had onset of proximal weakness in early childhood, with clumsy gait, and Gowers sign; he had foot, elbow, and knee contractures when he was evaluated at age 7 years. At age 13 he underwent Achilles tenotomy. He is still ambulant but has severe difficulties. Cardiac investigations showed no arrhythmias. Patient 3 (II-2, Fig. 1) is a 26-year-old man who, since age 3 years, had proximal progressive muscle weakness with joint contractures in the foot, elbow, and upper spine. At age 9 he underwent surgery for equinus foot deformity. Clinical cardiac evaluation was normal. The 2 muscle biopsies studied showed dystrophic changes but no vacuolar changes or abnormal nuclear morphology (Fig. 1). Emerin and lamin A/C (LMNA) immunofluorescence (Fig. 1) and lysosomal-associated membrane protein-2 (LAMP-2) immunoblot (Fig. 1) showed normal protein expression. LMNA gene sequencing showed no mutations. Targeted next generation sequencing (NGS) identified a variation in the LAMP2 gene (p.G221R) and a novel heterozygous mutation in the SYNE1 gene (NM_182961.3, c.323C>T, p.N108S) localized in the actinbinding domain of nesprin-1 (isoform 1, http://www.dmd. nl). In silico analysis of the mutations predicted both LAMP2 and SYNE1 gene variations to be damaging. Danon disease in these patients was excluded because the clinical phenotype was not typical, they had normal LAMP-2 protein expression (Danon males usually display absent protein), and the LAMP2 variation we identified was reported as a polymorphism (it may be a genetic modifier). The only potentially causative mutation identified by NGS in our family was the novel mutation in the SYNE1 gene, which has been recognized to cause EDMD in a few kindreds. Although many SYNE1 gene polymorphisms have been identified in individuals at risk for bipolar disorder, ovarian cancer, and autism, and in a reference population, a series of concordant clues suggest a primary role of the SYNE1 mutation identified in our family. These include its predicted deleterious effect, its localization in a functionally crucial domain, its absence in a large series of control chromosomes, and the finding of no other mutations by NGS in this family. Altogether, this SYNE1 gene mutation seems to cause an “EDMD-like” phenotype, characterized by progressive muscular dystrophy with joint contractures, but without heart involvement. One patient with a similar clinical phenotype but a different SYNE1 gene mutation has been reported previously. Table 1. The incidence rate (per million person-years) and prevalence rate (per million persons) of myasthenia gravis in Arab countries.

Transcriptional and Translational Effects of Intronic CAPN3 Gene Mutations

Variants of unknown significance in the CAPN3 gene constitute a significant challenge for genetic counselling. Despite the frequency of intronic nucleotide changes in this gene (15‐25% of all mutations), so far their pathogenicity has only been inferred by in‐silico analysis, and occasionally, proven by RNA analysis. In this study, 5 different intronic variants (one novel) that bioinformatic tools predicted would affect RNA splicing, underwent comprehensive studies which were designed to prove they are disease‐causing. Muscle mRNA from 15 calpainopathy patients was analyzed by RT‐PCR and splicing‐specific‐PCR tests. We established the previously unrecognized pathogenicity of these mutations, which caused aberrant splicing, most frequently by the activation of cryptic splicing sites or, occasionally, by exon skipping. The absence or severe reduction of protein demonstrated their deleterious effect at translational level. We concluded that bioinformatic tools are valuable to suggest the potential effects of intronic variants; however, the experimental demonstration of the pathogenicity is not always easy to do even when using RNA analysis (low abundance, degradation mechanisms), and it might not be successful unless splicing‐specific‐PCR tests are used. A comprehensive approach is therefore recommended to identify and describe unclassified variants in order to offer essential data for basic and clinical geneticists.

Sarcolemmal Neuronal Nitric Oxide Synthase Defect in Limb-Girdle Muscular Dystrophy: An Adverse Modulating Factor in the Disease Course?

Reduction of neuronal nitric oxide synthase (nNOS) has been associated with the pathogenesis and clinical expression of inherited myopathies. To determine whether a defect in nNOS might be an adverse modulating factor in the course of limb-girdle muscular dystrophy, we investigated cytosolic and sarcolemmal nNOS expression in muscle biopsies from 32 patients with 7 forms of limb-girdle muscular dystrophy. Primary calpainopathy, dysferlinopathy, and caveolinopathy biopsies showed normal levels of cytosolic nNOS and preserved sarcolemmal nNOS immunoreactivity. By contrast, the cytosolic nNOS levels in sarcoglycanopathy muscles were variably reduced. Sarcolemmal nNOS immunoreactivity varied from absent to reduced, depending on the integrity of the sarcoglycan complex. In muscles with loss of the entire sarcoglycan complex, sarcolemmal nNOS was absent; it otherwise depended on the specific sarcoglycan gene and type of mutation. The integrity of the entire sarcoglycan complex is, therefore, essential for the stabilization of nNOS to the sarcolemma. Absence of sarcolemmal nNOS in sarcoglycanopathy muscle was always associated with severe muscular dystrophy and sometimes with dilated cardiomyopathy, supporting the hypothesis that nNOS defect might contribute to skeletal and cardiac muscle disease progression. These results emphasize the value of nNOS immunohistochemical analysis in limb-girdle muscular dystrophy and provide additional insights for future therapeutic interventions in these disorders.

Novel missense mutations in PNPLA2 causing late onset and clinical heterogeneity of neutral lipid storage disease with myopathy in three siblings

Neutral lipid storage disease with myopathy (NLSD-M) is a rare autosomal recessive disorder characterised by an abnormal accumulation of triacylglycerol into cytoplasmic lipid droplets (LDs). NLSD-M patients are mainly affected by progressive myopathy, cardiomyopathy and hepatomegaly. Mutations in the PNPLA2 gene cause variable phenotypes of NLSD-M. PNPLA2 codes for adipose triglyceride lipase (ATGL), an enzyme that hydrolyses fatty acids from triacylglycerol. This report outlines the clinical and genetic findings in a NLSD-M Italian family with three affected members. In our patients, we identified two novel PNPLA2 missense mutations (p.L56R and p.I193F). Functional data analysis demonstrated that these mutations caused the production of ATGL proteins able to bind to LDs, but with decreased lipase activity. The oldest brother, at the age of 38, had weakness and atrophy of the right upper arm and kyphosis. Now he is 61 years old and is unable to raise arms in the horizontal position. The second brother, from the age of 44, had exercise intolerance, cramps and pain in lower limbs. He is currently 50 years old and has an asymmetric distal amyotrophy. One of the two sisters, 58 years old, presents the same PNPLA2 mutations, but she is still oligo-symptomatic on neuromuscular examination with slight triceps muscle involvement. She suffered from diabetes and liver steatosis. This NLSD-M family shows a wide range of intra-familial phenotypic variability in subjects carrying the same mutations, both in terms of target-organs and in terms of rate of disease progression.