I. Nelson

A Missense Mutation of Cytochrome Oxidase Subunit II Causes Defective Assembly and Myopathy

We report the first missense mutation in the mtDNA gene for subunit II of cytochrome c oxidase (COX). The mutation was identified in a 14-year-old boy with a proximal myopathy and lactic acidosis. Muscle histochemistry and mitochondrial respiratory-chain enzymology demonstrated a marked reduction in COX activity. Immunohistochemistry and immunoblot analyses with COX subunit-specific monoclonal antibodies showed a pattern suggestive of a primary mtDNA defect, most likely involving CO II, for COX subunit II (COX II). mtDNA-sequence analysis demonstrated a novel heteroplasmic T-->A transversion at nucleotide position 7,671 in CO II. This mutation changes a methionine to a lysine residue in the middle of the first N-terminal membrane-spanning region of COX II. The immunoblot studies demonstrated a severe reduction in cross-reactivity, not only for COX II but also for the mtDNA-encoded subunit COX III and for nuclear-encoded subunits Vb, VIa, VIb, and VIc. Steady-state levels of the mtDNA-encoded subunit COX I showed a mild reduction, but spectrophotometric analysis revealed a dramatic decrease in COX I-associated heme a3 levels. These observations suggest that, in the COX protein, a structural association of COX II with COX I is necessary to stabilize the binding of heme a3 to COX I.

The mitochondrial DNA G13513A transition in ND5 is associated with a LHON/MELAS overlap syndrome and may be a frequent cause of MELAS.

We report on 4 male patients with clinical, radiological, and muscle biopsy findings typical of the mitochondrial encephalomyopathy with lactic acidosis and stroke‐like episodes (MELAS) phenotype. Skeletal muscle mitochondrial DNA (mtDNA) analysis showed that all patients harbored a heteroplasmic G13513A mutation in the ND5 subunit gene. One of these cases (Patient 1) presented with symptoms characteristic of Lebers hereditary optic neuropathy (LHON) 2 years before the first stroke‐like episode. Quantitative analysis in several postmortem tissue sections showed that the relative proportions of mutant mtDNA were generally lower than those reported with other pathogenic mtDNA mutations. Single‐fiber polymerase chain reaction studies demonstrated significantly higher amounts of mutant mtDNA in ragged red fibers (RRFs) compared with non‐RRFs. This study indicates that the G13513A transition is likely to be pathogenic, that it can cause an LHON/MELAS overlap syndrome, and that it may be a more frequent cause of MELAS than previously recognized.

Six novel connexin32 (GJB1) mutations in X-linked Charcot-Marie-Tooth disease

X-linked Charcot-Marie-Tooth disease (CMTX) is a clinically heterogeneous hereditary motor and sensory neuropathy with X-linked transmission. Common clinical manifestations of CMTX, as in other forms of Charcot-Marie-Tooth disease (CMT), are distal muscle wasting and weakness, hyporeflexia, distal sensory disturbance, and foot deformities. Motor nerve conduction velocity is reduced. In male patients it is often less than 38 m/s in the median nerve (a value often used to distinguish between “demyelinating” and “axonal” forms of CMT), but in female patients conduction velocity may be faster than this or normal. Mutations in the connexin32 (gap junction protein β 1 (GJB1)) gene are responsible for the majority of CMTX cases. This report describes six British CMTX families with six novel mutations (four missense, one nonsense, and one frame shift) of the GJB1 gene. Affected members in these six families had typical signs of CMT but in some affected members of three families there was additional central nervous system involvement or deafness in the absence of any other explanation other than CMT.

Depletion of Mitochondrial DNA by ddC in Untransformed Human Cell Lines

In order to study the interaction between the nuclear and mitochondrial genomes we have developed a non-transformed cell system. It is based upon the complete removal of mtDNA from fibroblasts by treatment with a nucleoside analogue, 2′, 3′ dideoxycytidine (ddC). After exposure to ddC we were able to generate viable fibroblasts devoid of mtDNA and to successfully repopulate them with exogenous mitochondria. This model system will be useful in characterizing nuclear mitochondrial interactions and in studying the effects of different nuclear backgrounds on the expression of different primary defects of mtDNA associated with human disease.

Novel mutations in DNAJB6 cause LGMD1D and distal myopathy in French families

The aim was to determine the genetic background of unknown muscular dystrophy in five French families.

Anti-HMGCR Antibody–Related Necrotizing Autoimmune Myopathy Mimicking Muscular Dystrophy

Introduction Necrotizing autoimmune myopathies (NAMs) are acquired myopathies with myofibrillar necrosis and weak or absent inflammatory component, sometimes associated with anti‐signal recognition particle (SRP) or 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGCR) antibodies. Observation The patient, a girl now aged 20 years, was first assessed at the age of 5 years for abnormal gait revealing frank pelvic deficit. Creatine kinase (CK) levels were as high as 7,500 IU/L. Subsequent muscle biopsy showed some necrosis, fiber regeneration, and fibrosis consistent with muscular dystrophy (MD). Protein immunohistochemistry was normal. The disease course was progressive until wheelchair use at the age of 9 years. At 12 years of age, a second muscle biopsy found an advanced MD with some perivascular inflammatory mononuclear cells. All molecular analyses done through 14 years of follow‐up were negative till anti‐HMGCR antibodies were detected at a significant amount when she was 19 years old. Discussion NAMs begin at a pediatric age and may have a chronic course mimicking MDs. Muscular biopsy can be misleading with a predominantly dystrophic pattern without inflammation. Conclusion This observation should prompt the assessment of NAMs in all MDs, even pediatric, without molecular solutions.

Gene Therapy via Trans-Splicing for LMNA-Related Congenital Muscular Dystrophy

We assessed the potential of Lmna-mRNA repair by spliceosome-mediated RNA trans-splicing as a therapeutic approach for LMNA-related congenital muscular dystrophy. This gene therapy strategy leads to reduction of mutated transcript expression for the benefit of corresponding wild-type (WT) transcripts. We developed 5′-RNA pre-trans-splicing molecules containing the first five exons of Lmna and targeting intron 5 of Lmna pre-mRNA. Among nine pre-trans-splicing molecules, differing in the targeted sequence in intron 5 and tested in C2C12 myoblasts, three induced trans-splicing events on endogenous Lmna mRNA and confirmed at protein level. Further analyses performed in primary myotubes derived from an LMNA-related congenital muscular dystrophy (L-CMD) mouse model led to a partial rescue of the mutant phenotype. Finally, we tested this approach in vivo using adeno-associated virus (AAV) delivery in newborn mice and showed that trans-splicing events occurred in WT mice 50 days after AAV delivery, although at a low rate. Altogether, while these results provide the first evidence for reprogramming LMNA mRNA in vitro, strategies to improve the rate of trans-splicing events still need to be developed for efficient application of this therapeutic approach in vivo.

Loss of Sarcomeric Scaffolding as a Common Baseline Histopathologic Lesion in Titin-Related Myopathies

Titin-related myopathies are heterogeneous clinical conditions associated with mutations in TTN. To define their histopathologic boundaries and try to overcome the difficulty in assessing the pathogenic role of TTN variants, we performed a thorough morphological skeletal muscle analysis including light and electron microscopy in 23 patients with different clinical phenotypes presenting pathogenic autosomal dominant or autosomal recessive (AR) mutations located in different TTN domains. We identified a consistent pattern characterized by diverse defects in oxidative staining with prominent nuclear internalization in congenital phenotypes (AR-CM) (n = 10), ± necrotic/regenerative fibers, associated with endomysial fibrosis and rimmed vacuoles (RVs) in AR early-onset Emery-Dreifuss-like (AR-ED) (n = 4) and AR adult-onset distal myopathies (n = 4), and cytoplasmic bodies (CBs) as predominant finding in hereditary myopathy with early respiratory failure (HMERF) patients (n = 5). Ultrastructurally, the most significant abnormalities, particularly in AR-CM, were multiple narrow core lesions and/or clear small areas of disorganizations affecting one or a few sarcomeres with M-band and sometimes A-band disruption and loss of thick filaments. CBs were noted in some AR-CM and associated with RVs in HMERF and some AR-ED cases. As a whole, we described recognizable histopathological patterns and structural alterations that could point toward considering the pathogenicity of TTN mutations.

EGR2 mutation enhances phenotype spectrum of Dejerine–Sottas syndrome

A 5-year-old patient attended our clinic for progressive proximal, axial, and facial weakness associated to complete bulbar palsy and tongue fasciculations. She was the first child of non-consanguineous healthy parents with a normal motor development. At 3 years, the patient showed complete impossibility to raise from the floor or to sit independently, and needed support to walk. Ambulation was lost at three and a half years. Severe facial weakness and marked tongue fasciculations, absence of deep tendon reflexes, were found as well as mild distal retractions. Oral feeding was compromised. Intelligence was fully conserved, and formal testing of hearing was normal. According to the initial hypothesis of Fazio–Londe syndrome (FLS), despite normal hearing, the child was put on riboflavin for 3 months with no improvement. Facial weakness progressed, causing aspiration pneumonia, and language became hardly understandable. Also, the child presented with obstructive apnea. Given the areflexia and obvious tongue fasciculation, SMN1 gene was investigated, but negative. CK levels were normal. Brain MRI and cardiac ultrasound were normal. Electromyography at age of 3 years demonstrated undetectable compound muscle action potentials of the left and right external peroneal nerve, and absent sensory conduction velocities of the musculocutaneous nerve. Diminished sensory conduction with small amplitude of the left sural nerve was notified (amplitude 4,7 lV, and velocity 25 m/s). At 5 years (Fig. 1a–c), the axial motor deficit had progressed necessitating use of a Garchois brace to maintain sitting position. Whole exome sequencing was performed at the French National Centre for Genotyping. Results revealed a de novo heterozygous mutation in the EGR2 gene (c.1075C[T, p.Arg359Trp), confirmed by Sanger sequencing (Fig. 1d). The variant was not observed in the healthy parents. Bulbospinal muscular atrophy (BMA) may be present in patients with BVVL, Nathalie, Madras, and, in adults, the Kennedy syndrome [2, 5, 8, 9]. BVVL syndrome is due to mutation of SLC52A2 and SLC52A3 genes, encoding for proteins necessary for riboflavin transport [13]. Presentation usually includes progressive BSA with sensorineural deafness, absent in 20 % cases [14, 15]. The severity of proximal weakness, the absence of hearing loss, and the absence of riboflavin response made this syndrome unlikely in our patient, and testing in SLC52A2/A3 genes was negative. It is also rarely associated with Dejerine–Sottas neuropathy (DSN). We consider our case an unusual phenotypic presentation of DSN. DSN is characterized by normal Electronic supplementary material The online version of this article (doi:10.1007/s00415-016-8153-9) contains supplementary material, which is available to authorized users.

G.P.281

Defects in TRIM32 were reported in limb-girdle muscular dystrophy type 2H (LGMD2H). Few cases have been described to date, but this gene is not systematically analysed due to the absence of specific signs and difficulties in protein analysis. By using high-throughput mutation screening techniques, we identified mutations in TRIM32 in two patients presenting nonspecific progressive LGMD. We report the first case of total inactivation by homozygous deletion of the entire TRIM32 gene. Of interest, the deletion removes part of the ASTN2 gene, a large gene in which TRIM32 is nested. Despite the total TRIM32 gene inactivation, the patient does not present a more severe phenotype. However, he developed a mild progressive cognitive impairment that may be related to the loss of function of ASTN2 since association between ASTN2 heterozygous deletions and neurobehavioral disorders was previously reported. Regarding genomic characteristics at breakpoint of the deleted regions of TRIM32, we found a high density of repeated elements, suggesting a possible hotspot. These observations illustrate the importance of high-throughput technologies for identifying molecular defects in LGMD, confirm that total loss of function of TRIM32 is not associated with a specific phenotype and that ASTN2 inactivation could be associated with cognitive impairment.