WES in a family trio suggests involvement of TECPR2 in a complex form of progressive motor neuron disease.

We have performed whole-exome sequencing in a family trio with a 16-year-old girl suffering of progressive motor neuron disease. There was no family history of the disease and no parental consanguinity. Our exome analysis indicated the proband as a compound heterozygote for two missense variants in the TECPR2 gene according to a recessive mode of inheritance. The TECPR2 gene has been reported as a positive regulator of autophagy which is an essential mechanism for maintaining neuron homeostasis and survival and plays a key role in major adult and pediatric neurodegenerative diseases. Variants in this gene have been found responsible for a recently described form of hereditary spastic paraplegia called SPG49 in two previous reports. We propose that both variants causing amino acid substitution, p.Leu684Val and p.Thr903Met, inherited in trans-phase compound heterozygote form, can be responsible for the phenotype observed in our patient. We also consider the possible contribution of a heterozygous variant in the SPG7 gene. Sanger sequencing confirmed the segregation of variants within the family tree including the patient's unaffected brother.

[Thoracodorsal pedicled perforator flap for chest wall and breast reconstruction in children: Illustration with two clinical cases]. / Utilisation du lambeau perforant thoracodorsal dans les séquelles traumatiques cutanées thoracomammaires de l'enfant : illustration par deux cas cliniques.

Perforator flaps represent a new approach in reconstructive surgery including the thoracodorsal perforator flap. It can be used as a free or pedicled tissue transfer. By exposing two clinical cases, we demonstrate that this flap is an interesting option for children and adolescents chest wall skin coverage with less morbidity compared to myocutaneous latissimus dorsi flap.

Novel FAM126A mutations in hypomyelination and congenital cataract disease.

Hypomyelination and congenital cataract (HCC, OMIM #610532) is a rare autosomal recessive disorder due to FAM126A mutations characterized by congenital cataract, progressive neurologic impairment, and myelin deficiency in the central and peripheral nervous system. We have identified two novel mutations in three affected members of two unrelated families. Two sibs harbouring a microdeletion causing a premature stop in the protein showed the classical clinical and neuroradiologic HCC picture. The third patient carrying a missense mutation showed a relatively mild clinical picture without peripheral neuropathy. A residual amount of hyccin protein in primary fibroblasts was demonstrated by functional studies indicating that missense mutations are likely to have less detrimental effects if compared with splice-site mutations or deletions that cause the full-blown HCC phenotype, including peripheral nervous system involvement.

Early-onset absence epilepsy: SLC2A1 gene analysis and treatment evolution.

BACKGROUND AND PURPOSES: To determine the prevalence of SLC2A1 mutations in children with early-onset absence epilepsy (EOAE) and to investigate whether there were differences in demographic and electroclinical data between patients who became seizure-free with anti-epileptic drug (AED) monotherapy (group I) and those who needed add-on treatment of a second AED (group II). METHODS: We reviewed children with EOAE attending different Italian epilepsy centers. All participants had onset of absence seizures within the first 3 years of life but otherwise conformed to a strict definition of childhood absence epilepsy. Mutation analysis of SLC2A1 was performed in each patient. RESULTS: Eighty-four children (57 in group I, 27 in group II) fulfilled the inclusion criteria. No mutation in SLC2A1 was found. There were no statistical differences between the two groups with regard to F/M ratio, age at onset of EOAE, early history of febrile seizures, first-degree family history for genetic generalized epilepsy, duration of AED therapy at 3 years after enrollment, use of AEDs at 3 years, failed withdrawals at 3 years, terminal remission of EOAE at 3 years, and 6-month follow-up EEG data. Mean duration of seizures/active epilepsy was significantly shorter in group I than in group II (P = 0.008). CONCLUSIONS: We demonstrate that in a large series of children with rigorous diagnosis of EOAE, no mutations in SLC2A1 gene are detected. Except for duration of seizures/active epilepsy, no significant differences in demographic and electroclinical aspects are observed between children with EOAE who responded well to AED monotherapy and those who became seizure-free with add-on treatment of a second AED.

Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy.

Limb-girdle muscular dystrophy (LGMD) is a clinically and genetically heterogeneous group of myopathies, including autosomal dominant and recessive forms. To date, two autosomal dominant forms have been recognized: LGMD1A, linked to chromosome 5q, and LGMD1B, associated with cardiac defects and linked to chromosome 1q11-21. Here we describe eight patients from two different families with a new form of autosomal dominant LGMD, which we propose to call LGMD1C, associated with a severe deficiency of caveolin-3 in muscle fibres. Caveolin-3 (or M-caveolin) is the muscle-specific form of the caveolin protein family, which also includes caveolin-1 and -2. Caveolins are the principal protein components of caveolae (50-100 nm invaginations found in most cell types) which represent appendages or sub-compartments of plasma membranes. We localized the human caveolin-3 gene (CAV3) to chromosome 3p25 and identified two mutations in the gene: a missense mutation in the membrane-spanning region and a micro-deletion in the scaffolding domain. These mutations may interfere with caveolin-3 oligomerization and disrupt caveolae formation at the muscle cell plasma membrane.

Allelic and phenotypic heterogeneity in 49 Italian patients with the muscle form of CPT-II deficiency.

As genotype-phenotype correlations require the study of large patient populations, we investigated 49 Italian patients (33 unreported) with the muscle form of carnitine-palmitoyl-transferase-II (CPT-II) deficiency and CPT2 gene mutations. CPT enzyme activity below 25% of controls would lead to the development of muscle symptoms, and CPT activity below 15% would cause a relatively severe phenotype of the muscle form. Of the 15 different mutations found, 6 are novel (40%). A functional significance of mutations could be derived only for the two homozygous missense mutations found: both the p.S113L and the p.R631C (recurring in four unrelated patients from a genetic isolate) alleles caused a severe CPT enzyme defect (15% and 7%, respectively) and a relatively severe clinical phenotype of the muscle form. We identified three genotypes (homozygous p.R631C, homozygous p.S113L, and heterozygous null mutations) usually associated with a relatively severe and often life-threatening condition, which should be considered both in the clinical management of newly diagnosed patients (to prevent symptoms) and in their possible inclusion in therapeutic trials. We confirmed the existence of symptomatic heterozygous patient(s), through a family study, providing an important issue when offering genetic counseling and suggesting the crucial role of polymorphisms or environmental factors in determining the phenotype.

Inclusion body myopathy, Paget's disease of the bone and frontotemporal dementia: recurrence of the VCP R155H mutation in an Italian family and implications for genetic counselling.

The acronym IBMPFD denotes a syndrome including inclusion body myopathy, Paget's disease of the bone (PDB) and frontotemporal dementia (FTD) as cardinal features, which is caused by missense mutations in the VCP gene. We studied the clinical characteristics and the histopathological features in two siblings and their mother who presented with adult-onset myopathy and presenile, rapidly progressive FTD. One sibling also showed PDB. Light and electron microscopy performed on muscle biopsies demonstrated degenerative changes with inclusion bodies and abnormal aggregates. Mutation analysis of the VCP gene on affected siblings revealed a heterozygous missense mutation (R155H) in a hot spot. This is the first Italian family with multiple individuals diagnosed as having IBMPFD and carrying the recurrent R155H mutation. The implications for genetic counselling were also discussed, with regard to the procedures that may be offered to families suffering from a multisystem disorder with high risk of cognitive decline.

Muscular dystrophies: alterations in a limited number of cellular pathways?

Identification of new genes involved in muscle disorders has dramatically changed the traditional clinical classification of the large and heterogeneous group of the muscular dystrophies. Results obtained in recent years by positional candidate cloning have demonstrated the role of the sarcolemma and of the nuclear envelope in normal muscle function and have elucidated molecular pathways perturbed by mutations that lead to muscular dystrophy.

The Hammersmith functional score correlates with the SMN2 copy number: a multicentric study.

Previous studies showed that SMN2 copy number correlates inversely with the disease severity. Our aim was to evaluate SMN2 copy numbers and the Hammersmith functional motor scale in 87 patients with SMA II in order to establish whether, within SMAII, the number of copies correlates with the severity of functional impairment. Our results showed a relative variability of functional scores, but a significant correlation between the number of SMN2 genes and the level of function.

Neuromuscular forms of glycogen branching enzyme deficiency.

Deficiency of glycogen branching enzyme is causative of Glycogen Storage Disease type IV (GSD-IV), a rare autosomal recessive disorder of the glycogen synthesis, characterized by the accumulation of amylopectin-like polysaccharide, also known as polyglucosan, in almost all tissues. Its clinical presentation is variable and involves the liver or the neuromuscular system and different mutations in the GBE1 gene, located on chromosome 3, have been identified in both phenotypes. This review will addresses the neuromuscular clinical variants, focusing on the molecular genetics aspects of this disorder.

Revisiting mitochondrial ocular myopathies: a study from the Italian Network.

Ocular myopathy, typically manifesting as progressive external ophthalmoplegia (PEO), is among the most common mitochondrial phenotypes. The purpose of this study is to better define the clinical phenotypes associated with ocular myopathy. This is a retrospective study on a large cohort from the database of the "Nation-wide Italian Collaborative Network of Mitochondrial Diseases". We distinguished patients with ocular myopathy as part of a multisystem mitochondrial encephalomyopathy (PEO-encephalomyopathy), and then PEO with isolated ocular myopathy from PEO-plus when PEO was associated with additional features of multisystemic involvement. Ocular myopathy was the most common feature in our cohort of mitochondrial patients. Among the 722 patients with a definite genetic diagnosis, ocular myopathy was observed in 399 subjects (55.3%) and was positively associated with mtDNA single deletions and POLG mutations. Ocular myopathy as manifestation of a multisystem mitochondrial encephalomyopathy (PEO-encephalomyopathy, n = 131) was linked to the m.3243A>G mutation, whereas the other "PEO" patients (n = 268) were associated with mtDNA single deletion and Twinkle mutations. Increased lactate was associated with central neurological involvement. We then defined, among the PEO group, as "pure PEO" the patients with isolated ocular myopathy and "PEO-plus" those with ocular myopathy and other features of neuromuscular and multisystem involvement, excluding central nervous system. The male proportion was significantly lower in pure PEO than PEO-plus. This study reinforces the need for research on the role of gender in mitochondrial diseases. The phenotype definitions here revisited may contribute to a more homogeneous patient categorization, useful in future studies and clinical trials.

Reliability of the Hammersmith functional motor scale for spinal muscular atrophy in a multicentric study.

The aim of this study was to validate the Hammersmith functional motor scale for children with spinal muscular atrophy in a large cohort of 90 non-ambulant children with spinal muscular atrophy type 2 or 3. All had a baseline assessment (T0) and were reassessed either at 3 months (T1) (n = 66) or at 6 months (T2) (n = 24). Inter-observer reliability, tested on 13 children among 3 examiners, was > 95%. Of the 66 children examined after 3 months 4 had adverse effects in between assessments and were excluded from the analysis. Forty-two (68%) of the remaining 62 reassessed had no variation in scores between T0 and T1 and 13 (21%) were within +/- 1 point. 9 (37.5%) of the 24 children reassessed after 6 months had no variation in scores between T0 and T2 and another 9 (37.5%) had variations within +/- 1 point. Our study confirms previous observations of the reliability of the scale and helps to establish a baseline for assessing changes of functional ability over 3 and 6 month intervals. This information can be valuable in view of therapeutic trials.

MYH7-related myopathies: clinical, histopathological and imaging findings in a cohort of Italian patients.

BACKGROUND: Myosin heavy chain 7 (MYH7)-related myopathies are emerging as an important group of muscle diseases of childhood and adulthood, with variable clinical and histopathological expression depending on the type and location of the mutation. Mutations in the head and neck domains are a well-established cause of hypertrophic cardiomyopathy whereas mutation in the distal regions have been associated with a range of skeletal myopathies with or without cardiac involvement, including Laing distal myopathy and Myosin storage myopathy. Recently the spectrum of clinical phenotypes associated with mutations in MYH7 has increased, blurring this scheme and adding further phenotypes to the list. A broader disease spectrum could lead to misdiagnosis of different congenital myopathies, neurogenic atrophy and other neuromuscular conditions. RESULTS: As a result of a multicenter Italian study we collected clinical, histopathological and imaging data from a population of 21 cases from 15 families, carrying reported or novel mutations in MYH7. Patients displayed a variable phenotype including atypical pictures, as dropped head and bent spine, which cannot be classified in previously described groups. Half of the patients showed congenital or early infantile weakness with predominant distal weakness. Conversely, patients with later onset present prevalent proximal weakness. Seven patients were also affected by cardiomyopathy mostly in the form of non-compacted left ventricle. Muscle biopsy was consistent with minicores myopathy in numerous cases. Muscle MRI was meaningful in delineating a shared pattern of selective involvement of tibialis anterior muscles, with relative sparing of quadriceps. CONCLUSION: This work adds to the genotype-phenotype correlation of MYH7-relatedmyopathies confirming the complexity of the disorder.

Successful recovery of the normal electrophysiological properties of PorB (class 3) porin from Neisseria meningitidis after expression in Escherichia coli and renaturation.

Neisseria meningitidis PorB class 3 porins obtained either from native membranes (wild-type) or recovered from inclusion bodies following expression in Escherichia coli (recombinant), have been reconstituted into solvent-free planar phospholipid membranes. The wild-type and recombinant porins exhibited the same single-trimer conductance (1-1.3 nS in 200 mM NaCl), tri-level closure pattern, characteristic of functional channel trimers, and pattern of insertion into planar membranes. Both proteins were open at low voltages and displayed two voltage-dependent closure processes, one at positive and the other at negative potentials. Both showed asymmetric voltage dependence such that one gating process occurred at lower voltages (Vo=15 mV) than the other (Vo=25 mV). The sign of the potential that resulted in closure at low voltages varied from membrane to membrane indicating that they may have the property of auto-directed insertion (in analogy to the mitochondrial channel, VDAC). In the case of the recombinant porin, the steepness of the voltage dependence of one gating process was slightly less (n=1.3) than that observed for the other process or for the wild-type channel (n=1.5-1.7). Both channels have a high (40%) probability of closure even at 0 mV. While both channels show a slight selectivity for Cl- over Na+, the selectivity of the recombinant porin is a bit higher (permeability ratio of 2.8 vs. 1.6) as measured using a 2-fold salt gradient. Thus, the method employed to refold the recombinant porin was successful in not only restoring wild-type structure [H.L. Qi, J.Y. Tai, M.S. Blake, Expression of large amounts of Neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers, Infect. Immun. 62 (1994) 2432-2439; C.A.S.A. Minetti, J.Y. Tai, M.S. Blake, J.K. Pullen, S.M. Liang, D.P. Remeta, Structural and functional characterization of a recombinant PorB class 2 protein from Neisseria meningitidis. Conformational stability and porin activity, J. Biol. Chem. 272 (1997) 10710-10720] but also the overall electrophysiological function.

Localization of the human caveolin-3 gene to the D3S18/D3S4163/D3S4539 locus (3p25), in close proximity to the human oxytocin receptor gene. Identification of the caveolin-3 gene as a candidate for deletion in 3p-syndrome.

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cell types (cardiac and skeletal). Recently, we identified an autosomal dominant form of limb girdle muscular dystrophy in humans that is due to mutations within exon 2 of the caveolin-3 gene (3p25). However, the detailed location of the human caveolin-3 gene and its position with regard to neighboring genes remains unknown. Here, we have isolated three independent BAC clones containing the human caveolin-3 gene. Using a PCR-based approach, we determined that these clones contain both exons 1 and 2 of the human caveolin-3 gene. In addition, we performed microsatellite marker analysis of these BAC clones, using a panel of 13 markers that are known to map within the 3p25 region. Our results indicate that these BAC clones contain the following three markers: D3S18, SHGC-1079 (also known as D3S4163) and D3S4539. Interestingly, D3S18 is a marker for two known human diseases, von Hippel-Lindau disease and 3p-syndrome. As D3S4163 and D3S4539 are known to map in the vicinity of the 3' end of the human oxytocin receptor gene, we determined if these caveolin-3 positive BACs also contain the oxytocin receptor gene. We show that (i) these BACs contain all four exons of the oxytocin receptor gene and (ii) that the genes encoding caveolin-3 and the oxytocin receptor are located approximately 7-10 kb apart and in the opposite orientation. As 3p-syndrome is characterized by cardiac septal defects and caveolin-3 is expressed primarily in the heart and skeletal muscle, caveolin-3 is a candidate gene that may be deleted in 3p-syndrome.

Immunologic study of vinculin in Duchenne muscular dystrophy.

Using immunologic techniques, we studied vinculin, a cytoskeletal protein associated with the membrane-skeleton of the muscle fiber. We examined muscle biopsies from five patients with Duchenne muscular dystrophy (DMD), two with Becker's muscular dystrophy (BMD), three normal human muscle samples, and four biopsies from disease control patients. All DMD patients showed patchy and low-intensity immunostain at the sarcolemma of most fibers and, by immunoblot analysis, the content of vinculin was 42 to 61% of control values. There was no significant vinculin deficiency in samples from patients with BMD and other disease controls. The data suggest that vinculin content is reduced only in muscle where dystrophin is absent or sparse.

Progressive depletion of fast alpha-actinin-positive muscle fibers in Duchenne muscular dystrophy.

In normal human muscle, a monoclonal antibody against alpha-actinin recognizes an isoform that is only expressed in a population of fast fibers histochemically identified as type IIb or fast-twitch glycolytic. Immunohistochemical studies of muscle biopsies from patients with Duchenne muscular dystrophy (DMD) showed that the number of alpha-actinin-positive type IIb fibers was essentially normal in preclinical patients. Symptomatic patients between the ages of 3 and 5 years showed depletion of these fibers, which were not seen in patients older than 5 years. ATPase histochemistry showed that a few type IIb fibers were present in muscle from symptomatic DMD patients but lacked the fast isoform of alpha-actinin. The data suggest that type IIb fibers are affected early in DMD.