Enrico Peterle

Next-generation sequencing identifies transportin 3 as the causative gene for LGMD1F.

Limb-girdle muscular dystrophies (LGMD) are genetically and clinically heterogeneous conditions. We investigated a large family with autosomal dominant transmission pattern, previously classified as LGMD1F and mapped to chromosome 7q32. Affected members are characterized by muscle weakness affecting earlier the pelvic girdle and the ileopsoas muscles. We sequenced the whole exome of four family members and identified a shared heterozygous frame-shift variant in the Transportin 3 (TNPO3) gene, encoding a member of the importin-β super-family. The TNPO3 gene is mapped within the LGMD1F critical interval and its 923-amino acid human gene product is also expressed in skeletal muscle. In addition, we identified an isolated case of LGMD with a new missense mutation in the same gene. We localized the mutant TNPO3 around the nucleus, but not inside. The involvement of gene related to the nuclear transport suggests a novel disease mechanism leading to muscular dystrophy.

Pilot trial of clenbuterol in spinal and bulbar muscular atrophy

Objective: To test the efficacy and tolerability of clenbuterol in patients with spinal and bulbar muscular atrophy (SBMA). Methods: Twenty patients with a diagnosis of SBMA were given oral clenbuterol (0.04 mg/d) for 12 months. The primary efficacy end point was the change from baseline of the walking distance covered in 6 minutes at 12 months. Secondary end points included the change over time in muscle strength assessed with the Medical Research Council scale, the revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R), and forced vital capacity values. Safety was assessed by a series of laboratory and instrumental tests, as well as reporting of adverse events. Results: Sixteen patients completed the study. There was a significant and sustained increase in walking distance covered in 6 minutes and forced vital capacity between the baseline and the 12-month assessments (p < 0.001). No differences were recorded in Medical Research Council or ALSFRS-R scores between baseline and follow-up assessments. Serious side effects, including those on heart function, were absent. A significant increase in serum creatine kinase levels was observed. Conclusions: Our findings suggest a positive effect of clenbuterol on SBMA disease progression. Classification of evidence: This study provides Class IV evidence that clenbuterol is effective in improving motor function in SBMA.

Clinical phenotype, muscle MRI and muscle pathology of LGMD1F.

Of the seven autosomal dominant genetically distinct forms of LGMD so far described, in only four the causative gene has been identified (LGMD1A-1D). We describe clinical, histopathological and muscle MRI features of a large Italo-Spanish kindred with LGMD1F presenting proximal-limb and axial muscle weakness. We obtained complete clinical data and graded the progression of the disease in 29 patients. Muscle MRI was performed in seven patients. Three muscle biopsies from two patients were investigated. Patients with age at onset in the early teens, had a more severe phenotype with a rapid disease course; adult onset patients presented a slow course. Muscle MRI showed prominent atrophy of lower limb muscles, involving especially the vastus lateralis. Widening the patients population resulted in the identification of previously unreported features, including dysphagia, arachnodactyly and respiratory insufficiency. Muscle biopsies showed diffuse fibre atrophy, which evolved with time, chronic myopathic changes, basophilic cytoplasmic areas, autophagosomes and accumulation of myofibrillar and cytoskeletal proteins. The LGMD1F is characterized by a selective involvement of limb muscles with respiratory impairment in advanced stages, and by different degrees of clinical progression. Novel clinical features emerged from the investigation of additional patients.

Ultrastructural changes in LGMD1F.

A large Italo‐Spanish kindred with autosomal‐dominant inheritance has been reported with proximal limb and axial muscle weakness. Clinical, histological and genetic features have been described. A limb girdle muscular dystrophy 1F (LGMD1F) disease locus at chromosome 7q32.1–32.2 has been previously identified. We report a muscle pathological study of two patients (mother and daughter) from this family. Muscle morphologic findings showed increased fiber size variability, fiber atrophy, and acid‐phosphatase‐positive vacuoles. Immunofluorescence against desmin, myotilin, p62 and LC3 showed accumulation of myofibrils, ubiquitin binding protein aggregates and autophagosomes. The ultrastructural study confirmed autophagosomal vacuoles. Many alterations of myofibrillar component were detected, such as prominent disarray, rod‐like structures with granular aspect, and occasionally, cytoplasmic bodies. Our ultrastructural data and muscle pathological features are peculiar to LGMD1F and support the hypothesis that the genetic defect leads to a myopathy phenotype associated with disarrangement of the cytoskeletal network.

An intronic mutation causes severe LGMD2A in a large inbred family belonging to a genetic isolate in the Alps

To the Editor : Limb girdle muscular dystrophy 2A (LGMD2A; MIM#253600) is caused by mutations in the calpain3 gene (CAPN3 ) and is one of the most prevalent forms of LGMD (1). The molecular diagnosis of LGMD2A is difficult because of the relatively large size of the gene (2). A further complication derives from nucleotide changes localized in intronic sequences (over 15%), because their pathological significance is difficult to predict and compromises a conclusive genetic counseling (3). We report a large family which includes 17 LGMD patients with a severe form of LGMD, with onset of symptoms in childhood (4–6 years in nine cases) or in late teens (18 years in one case), and rapid progression (loss of ambulation at 20–25 years in five patients or at 39 years in one patient). A mutation in intron 9 (c.1193+6T>A) of the CAPN3 gene (1–4) was identified in homozygote state in five affected individuals and in heterozygote state in six unaffected individuals (Fig. 1). The pathogenetic effect of this mutation is an aberrant splicing by use of an alternative cryptic donor splice site (3), resulting in the insertion of 31 bp at the beginning of intron 9 (Fig. 1). This is the first time in which this mutation segregates in a homozygous state. This rare occurrence offered us the opportunity to obtain genotype–phenotype correlations and, indirectly, infer the protein expression level. In this family no muscle tissue was available for protein testing; however, during the preparation of this manuscript, we identified the same homozygous mutation in another unrelated patient with the absence calpain-3 protein and loss of ambulation at 32 years. As expected for an intronic mutation affecting normal splicing, this mutation was found to be associated with both a deleterious effect on protein synthesis and a clinically severe form of LGMD. The presence of 17 LGMD2A patients in this family is an unusual occurrence in a recessive and severe disorder, unless this event is because of a high coefficient of consanguinity. Three consanguineous marriages have been documented and this occurrence was strongly suspected in at least three additional married couples since the spouses had the same family name. The cases with the same family name include also the only affected parent–child pair, where the affected parent (V-5) and her partner (V-6) share an identical surname. All family members came from a small mountain valley in the Alps (Trentino-Alto Adige Region, Italy), crossed by the Fersina River (‘Bernstol-Fersental’ Valley), and inhabited since medieval times by a population called ‘Mòcheni’ who immigrated from Germany. Because of the small population size and the physical mountain barrier, the ‘Mòcheni’ constitute a genetic, ethnic and linguistic isolate (they speak an ancient Bavarian dialect as a native language and have remained separated from the majority of Germanspeaking populations). According to the census in 2001, the ‘Mòcheno’ language was spoken by 2278 people, while the inhabitants of the valley were about 4190. In 2001, 8 of the 17 patients in this family were alive, so that the prevalence of the disease in this area could be estimated to be 1 in 524 inhabitants (carrier frequency: 1 in 11 inhabitants). The geographic origin of this family suggests that the recurrence of this mutation should be attributed to the existence of a genetic isolate resulting in increased endogamy. People of Germanic origin, inhabiting this area are separated from adjacent populations by physical and linguistic barriers, which have contributed to their isolation and allele frequency divergence. Various genetic studies have showed the consequences of isolation followed by a genetic drift (founder effect) within the ‘Mòcheni’ population (5, 6). Following the identification of this homozygous mutation in the proband, we proposed a molecular diagnosis to several other affected relatives, some of whom have remained without diagnosis for many years. We are now able to offer genetic counseling and heterozygote diagnosis in additional family members and in unrelated individuals originating from the geographic area where this mutation is frequent.

Incomplete penetrance in limb-girdle muscular dystrophy type 1F

Limb-girdle muscular dystrophy (LGMD) type 1F (MIM # 608423) is a rare autosomal dominant disorder whose locus was mapped and gene identified by investigating the same large family. During examination of this family, we expanded the original pedigree and the clinical features of the disease. It is characterized by a variable degree of muscle weakness and functional impairment, with onset of symptoms either before age 15 (juvenile form) or in the third to fourth decades (adult form). The clinical2genetic investigation of this family revealed that, in some patients, the disease was transmitted through apparently unaffected parents (incomplete penetrance). To calculate the exact penetrance rate, we examined both the clinical phenotype and the genotype of 115 family members. The attribution of clinical status (either affected or unaffected) was based on a neuromuscular examination performed by the same physician using a standardized protocol and by a questionnaire that we designed to identify main disease symptoms (i.e., muscle weakness, gait difficulty, and dysphagia). One hundred fifteen individuals (including the 27 subjects investigated in the original search for the gene defect in this family and 88 new individuals previously untested) underwent DNA sample collection (obtained after written consent). The genotype (either mutant or non-mutant) was defined using a mutation-specific test [amplification refractory mutation system2polymerase chain reaction (ARMS-PCR)] and confirmed by DNA sequencing. The mutation segregating in this family (c.2771delA in TPNO3 gene encoding transportin-3) was identified in 45 of 115 individuals (39%) (Fig. 1); among 45 mutant cases, 39 (86.7%) were affected (at a mean age of 47.5 years) and 6 (13.3%) were unaffected. Two unaffected subjects were younger than age 15 years, with a future chance of developing the disease, and 4 were adult “non-penetrant” individuals (at a mean age of 31.5 years). Furthermore, 3 additional unaffected adults whose DNA was unavailable, were obligate carriers of the disease based on the pattern of inheritance. Overall, the penetrance rate was estimated to be 84.7%. We observed that clinical signs and symptoms of the disease were progressively more likely to manifest with increasing age (Fig. 1). This indicates that, in LGMD1F,