Lucia Morandi

Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria

The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

Different mutations in the LMNA gene cause autosomal dominant and autosomal recessive Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EMD) is a condition characterized by the clinical triad of early-onset contractures, progressive weakness in humeroperoneal muscles, and cardiomyopathy with conduction block. The disease was described for the first time as an X-linked muscular dystrophy, but autosomal dominant and autosomal recessive forms were reported. The genes for X-linked EMD and autosomal dominant EMD (AD-EMD) were identified. We report here that heterozygote mutations in LMNA, the gene for AD-EMD, may cause diverse phenotypes ranging from typical EMD to no phenotypic effect. Our results show that LMNA mutations are also responsible for the recessive form of the disease. Our results give further support to the notion that different genetic forms of EMD have a common pathophysiological background. The distribution of the mutations in AD-EMD patients (in the tail and in the 2A rod domain) suggests that unique interactions between lamin A/C and other nuclear components exist that have an important role in cardiac and skeletal muscle function.

Randomized, double-blind, placebo-controlled trial of phenylbutyrate in spinal muscular atrophy

Objective: To assess the efficacy of phenylbutyrate (PB) in patients with spinal muscular atrophy in a randomized, double-blind, placebo-controlled trial involving 10 Italian centers. Methods: One hundred seven children were assigned to receive PB (500 mg/kg/day) or matching placebo on an intermittent regimen (7 days on/7 days off) for 13 weeks. The Hammersmith functional motor scale (primary outcome measure), myometry, and forced vital capacity were assessed at baseline and at weeks 5 and 13. Results: Between January and September 2004, 107 patients aged 30 to 154 months were enrolled. PB was well tolerated, with only one child withdrawing because of adverse events. Mean improvement in functional score was 0.60 in the PB arm and 0.73 in placebo arm (p = 0.70). Changes in the secondary endpoints were also similar in the two study arms. Conclusions: Phenylbutyrate was not effective at the regimen, schedule, and duration used in this study.

Reliability of hand-held dynamometry in spinal muscular atrophy.

We have assessed the reliability of hand‐held myometry in 33 patients with spinal muscular atrophy (SMA), testing elbow flexion, handgrip, three‐point pinch, knee flexion, knee extension, and foot dorsiflexion, and determining intraclass correlation coefficients (ICC). Interrater reliability was high for upper limbs, with an ICC of 0.92 for three‐point pinch and 0.98 for elbow flexion and grip. For lower limbs interrater reliability was good with ICC >0.85 for all measures except foot dorsiflexion. Test–retest results were excellent with ICC >0.91 in all instances. Hand‐held myometry is easily performed in SMA patients of various ages and muscle strengths, is a reliable measure of limb muscle strength, and can be used in longitudinal studies and clinical trials.

Transforming growth factor-β1 and fibrosis in congenital muscular dystrophies

We evaluated transforming growth factor-beta1 (TGF-beta1) expression in the muscle of four laminin alpha2-negative, four laminin alpha2-positive and seven partial laminin alpha2-deficient congenital muscular dystrophy (CMD) patients, and compared it to Duchenne muscular dystrophy (DMD) patients and controls. TGF-beta1 mRNA levels in skeletal muscle from laminin alpha2-negative and laminin alpha2-positive CMD patients were significantly greater than in controls (P < 0.05 and P < 0.005, respectively), while in partial laminin alpha2-deficient muscular dystrophy patients the amount was not significantly higher than in controls (P > 0.1). The TGF-beta1 values were lower than those found in DMD, although the extent of fibrosis was greater in CMD than in DMD and controls. Our findings suggest that TGF-beta1 is involved in CMD muscle fibrosis, but differently from what we observed in DMD muscles as it seems not to be the major player in connective tissue proliferation.

Myasthenia Gravis (MG): Epidemiological Data and Prognostic Factors

Abstract: Data from 756 myasthenic patients were analyzed for diagnostic criteria, clinical aspects, and therapeutic approaches. The patients were followed up at our institution from 1981 to 2001. Clinical evaluation was performed according to the myasthenia gravis score adopted at our clinic. Clinical features of each patient (comprising demographic, clinical, neurophysiological, immunological, radiological, and surgical data, as well as serial myasthenia gravis scores) were filed in a relational database containing more than 7000 records. Clinical efficacy and variables influencing outcome were assessed by life‐table methods and Cox proportional hazards regression analysis. Complete stable remission, as defined by the Task Force of the Medical Scientific Advisory Board of the Myasthenia Gravis Foundation of America, was the end point for good prognosis. Four hundred and ninety‐nine patients (66%) were female and 257 (34%) were male. Mean follow‐up was 55.1 ± 48.1 months. Onset of symptoms peaked in the third decade in females, whereas the male distribution was bimodal with peaks in the third and sixth decades. Modality of myasthenia gravis presentation was as follows: ocular, 39.3%; generalized, 28.5%; bulbar, 31.3%; and respiratory, 0.8%. Thymectomy was carried out on 63.7% of our patients by different approaches: (1) transcervical; (2) transsternal; (3) video‐thoracoscopic mini‐invasive surgery. The last approach has been preferentially used in more recent years and accounted for 62.4% of the thymectomized myasthenia gravis population. Univariate analysis and Kaplan‐Meier analysis showed that variables such as sex (female), age at onset (below 40 years), thymectomy, and histological diagnosis of thymic hyperplasia were significantly associated with complete stable remission, whereas on multivariate analysis only age at onset below 40 years and thymectomy were confirmed.

Muscle inflammation and MHC class I up-regulation in muscular dystrophy with lack of dysferlin: an immunopathological study

Muscle inflammation is characteristic of inflammatory myopathies but also occurs in muscular dystrophy with lack of the sarcolemmal protein dysferlin. We quantified inflammatory cells and major histocompatibility complex (MHC) expression in muscle from 10 patients with dysferlinopathy. Infiltrating cells were always present although numbers varied considerably; macrophages were more common than T cells, T cytotoxicity was absent, and MHC class I was overexpressed on muscle fibers. These findings differ from polymyositis (PM) but are closely similar to those in SJL/J mice (which lack dysferlin) and emphasize the relationship between absence of dysferlin and immune system abnormalities in muscle.

Reliability of the North Star Ambulatory Assessment in a multicentric setting

The aim of this study was to investigate the suitability of the North Star Ambulatory Assessment as a possible outcome measure in multicentric clinical trials. More specifically we wished to investigate the level of training needed for achieving a good interobserver reliability in a multicentric setting. The scale was specifically designed for ambulant children with Duchenne Muscular Dystrophy and includes 17 items that are relevant for this cohort. Thirteen Italian centers participated in the study. In the first phase of the study we provided two training videos and an example of the scale performed on a child. After the first session of training, all the 13 examiners were asked to send a video with an assessment performed in their centre and to score all the videos collected. There were no difficulties in performing the items and in obtaining adequate videos with a hand held camera but the results showed a poor interobserver reliability (<.5). After a second training session with review and discussion of the videos previously scored, the same examiners were asked to score three new videos. The results of this session had an excellent interobserver reliability (.995). The level of agreement was maintained even when the same videos were rescored after a month, showing a significant intra-observer reliability (.95). Our results suggest that the NSAA is a test that can be easily performed, completed in 10 min and can be used in a multicentric setting, providing that adequate training is administered.

Phenotypic clustering of lamin A/C mutations in neuromuscular patients

Background: Mutations in the LMNA gene, encoding human lamin A/C, have been associated with an increasing number of disorders often involving skeletal and cardiac muscle, but no clear genotype/phenotype correlation could be established to date. Methods: We analyzed the LMNA gene in a large cohort of patients mainly affected by neuromuscular or cardiac disease and clustered mutated patients in two groups to unravel possible correlations. Results: We identified 28 variants, 9 of which reported for the first time. The two groups of patients were characterized by clinical and genetic differences: 1) patients with childhood onset displayed skeletal muscle involvement with predominant scapuloperoneal and facial weakness associated with missense mutations; 2) patients with adult onset mainly showed cardiac disorders or myopathy with limb girdle distribution, often associated with frameshift mutations presumably leading to a truncated protein. Conclusions: Our findings, supported by meta-analysis of previous literature, suggest the presence of two different pathogenetic mechanisms: late onset phenotypes may arise through loss of function secondary to haploinsufficiency, while dominant negative or toxic gain of function mechanisms may explain the severity of early phenotypes. This model of patient stratification may help patient management and facilitate future studies aimed at deciphering lamin A/C pathogenesis.

Mutation analysis of the lamin A/C gene (LMNA) among patients with different cardiomuscular phenotypes

Laminopathies represent a heterogeneous group of genetic disorders characterised by mutations in the LMNA gene, which encodes two lamins, A and C, by alternative splicing of the primary transcript.1 Lamins belong to the intermediate filament multigene family and form the nuclear lamina, a mesh-like structure adjacent to the nucleoplasmic side of the inner nuclear membrane.2 They interact with emerin, the proteins encoded by the gene for the X-linked (X EDMD) form of EDMD, with several nuclear envelope proteins and with chromatin. Despite their widespread distribution and their role in nuclear architecture, alterations of lamin A/C are responsible for a number of very specific but quite heterogeneous disorders. The first laminopathy was the autosomal dominant form of Emery-Dreifuss muscular dystrophy (EDMD), a genetic disorder characterised by the clinical triad of early onset contractures, progressive muscular wasting and weakness with humeroperoneal distribution and cardiac conduction defects.3 The finding that emerin, an inner nuclear envelope protein, and LMNA were both involved in EDMD suggested that the lamins may represent specific and relevant factors in cardiac and skeletal muscle and that integrity of the nuclear membrane and associated structures is specifically required for muscle function. However, later on it was found that besides autosomal dominant Emery–Dreifuss muscular dystrophy (AD-EDMD), mutations in LMNA are responsible for six other disorders: limb girdle muscular dystrophy 1B, (LGMD1B),4,5 dilated cardiomyopathy with conduction system disease, (DCM-CD),6 Dunningan-type familial partial lipodystrophy,7–9 one recessive axonal form of Charcot-Marie-Tooth neuropathy,10 mandibuloacral dysplasia,11 and Hutchinson Gilford progeria.12,13 Despite the very different phenotypic consequences of mutations in LMNA , and the quite large number of mutations identified, no genotype/phenotype correlation has been demonstrated, pointing to the role of factors other than lamins A and C in determining the different tissue specific phenotypes. …