Laurent Tiret

MTM1 mutation associated with X-linked myotubular myopathy in Labrador Retrievers.

Mutations in the MTM1 gene encoding myotubularin cause X-linked myotubular myopathy (XLMTM), a well-defined subtype of human centronuclear myopathy. Seven male Labrador Retrievers, age 14–26 wk, were clinically evaluated for generalized weakness and muscle atrophy. Muscle biopsies showed variability in fiber size, centrally placed nuclei resembling fetal myotubes, and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions. Ultrastructural studies confirmed the centrally located nuclei, abnormal perinuclear structure, and mitochondrial accumulations. Wild-type triads were infrequent, with most exhibiting an abnormal orientation of T tubules. MTM1 gene sequencing revealed a unique exon 7 variant in all seven affected males, causing a nonconservative missense change, p.N155K, which haplotype data suggest derives from a recent founder in the local population. Analysis of a worldwide panel of 237 unaffected Labrador Retrievers and 59 additional control dogs from 25 other breeds failed to identify this variant, supporting it as the pathogenic mutation. Myotubularin protein levels and localization were abnormal in muscles from affected dogs, and expression of GFP-MTM1 p.N155K in COS-1 cells showed that the mutant protein was sequestered in proteasomes, where it was presumably misfolded and prematurely degraded. These data demonstrate that XLMTM in Labrador Retrievers is a faithful genetic model of the human condition.

The cnm locus, a canine homologue of human autosomal forms of centronuclear myopathy, maps to chromosome 2

Myotubular/centronuclear myopathies are a nosological group of hereditary disorders characterised by severe architectural and metabolic remodelling of skeletal muscle fibres. In most myofibres, nuclei are found at an abnormal central position within a halo devoid of myofibrillar proteins. The X-linked form (myotubular myopathy) is the most prevalent and severe form in human, leading to death during early postnatal life. Maturation of fibres is not completed and fibres resemble myotubes. Linkage analysis in human has helped to identify MTM1 as the morbid gene. MTM1 encodes myotubularin, a dual protein phosphatase. In families in which myotubular myopathy segregates, detected mutations in MTM1 abolish the specific phosphatase activity targeting the second messenger phosphatidylinositol 3-phosphate. Autosomal forms (centronuclear) have a later onset and are often compatible with life. At birth, fibres are normally constituted but progressively follow remodelling with a secondary centralisation of nuclei. Their prevalence is low; hence, no linkage data can be performed and no molecular aetiology is known. In the Labrador Retriever, a spontaneous disorder strikingly mimics the clinical evolution of the human centronuclear myopathy. We have established a canine pedigree and show that the disorder segregates as an autosomal recessive trait in that pedigree. We have further mapped the dog locus to a region on chromosomexa02 that is orthologous to human chromosomexa010p. To date, no human MTM1 gene member has been mapped to this genetic region. This report thus describes the first spontaneous mammalian model of centronuclear myopathy and defines a new locus for this group of diseases.

Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice

Syncytins are envelope genes from endogenous retroviruses, “captured” for a role in placentation. They mediate cell-cell fusion, resulting in the formation of a syncytium (the syncytiotrophoblast) at the fetomaternal interface. These genes have been found in all placental mammals in which they have been searched for. Cell-cell fusion is also pivotal for muscle fiber formation and repair, where the myotubes are formed from the fusion of mononucleated myoblasts into large multinucleated structures. Here we show, taking advantage of mice knocked out for syncytins, that these captured genes contribute to myoblast fusion, with a >20% reduction in muscle mass, mean muscle fiber area and number of nuclei per fiber in knocked out mice for one of the two murine syncytin genes. Remarkably, this reduction is only observed in males, which subsequently show muscle quantitative traits more similar to those of females. In addition, we show that syncytins also contribute to muscle repair after cardiotoxin-induced injury, with again a male-specific effect on the rate and extent of regeneration. Finally, ex vivo experiments carried out on murine myoblasts demonstrate the direct involvement of syncytins in fusion, with a >40% reduction in fusion index upon addition of siRNA against both syncytins. Importantly, similar effects are observed with primary myoblasts from sheep, dog and human, with a 20–40% reduction upon addition of siRNA against the corresponding syncytins. Altogether, these results show a direct contribution of the fusogenic syncytins to myogenesis, with a demonstrated male-dependence of the effect in mice, suggesting that these captured genes could be responsible for the muscle sexual dimorphism observed in placental mammals.

HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth

The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies, yet the underlying cellular and molecular mechanisms remain elusive. In this study, we investigate the role of HACD1/PTPLA, which is involved in the elongation of the very long chain fatty acids, in muscle fibre formation. In humans and dogs, HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscle weakness. Through analysis of HACD1-deficient Labradors, Hacd1-knockout mice, and Hacd1-deficient myoblasts, we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration. We further demonstrate that in normal differentiating myoblasts, expression of the catalytically active HACD1 isoform, which is encoded by a muscle-enriched splice variant, yields decreased lysophosphatidylcholine content, a potent inhibitor of myoblast fusion, and increased concentrations of ≥C18 and monounsaturated fatty acids of phospholipids. These lipid modifications correlate with a reduction in plasma membrane rigidity. In conclusion, we propose that fusion impairment constitutes a novel, non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism.

Assignation of highly polymorphic markers on a canine purebred pedigree.

The descendant of the wolf has been a working and an affective companion for humans for a long time. With the development of molecular genetics, the dog, which was already appreciated in physiological and pharmacological research, has become an even more interesting laboratory animal model. The identification of known mammalian genes of dog mutant alleles confirms that some spontaneous canine disorders are true homologs of human diseases, and provides the opportunity to test potential genetic treatments on animal models (Herzog et al. 1999; Snyder et al. 1999). Also, the analysis of the segregation in a canine pedigree of a disease related to a human syndrome that has not yet been characterized could be an efficient alternative to localize the disease locus (e.g., Acland et al. 1999; Dagenais et al. 1999; van de Sluis et al. 1999). This strategy has recently allowed attribution of the narcoleptic disorder of Doberman pinschers and Labradors to mutations in the Hypocretin receptor 2 gene, making it a promising candidate in humans (Lin et al. 1999). Hence, mapping of a canine disease is directly beneficial to human geneticists. Resources serving the identification of canine disease genes have accumulated over the last 3 years. These have resulted in a linkage map of 341 markers covering 95% of the genome (Werner et al. 1999) and a physical map composed of 218 type I and 182 type II markers (Priat et al. 1998). Recently, the two maps have been integrated into a single map of 724 markers (Mellersh et al. 2000). A BAC library of canine genomic DNA (Li et al. 1999) and reciprocal human-dog chromosome paints (Breen et al. 1999) have also been reported and will facilitate information transfer between the two species. Here, we describe the characterization and mapping of highly polymorphic markers on a genetic map, that will be further integrated into the international panel. In human genetics, markers with three or more alleles in four unrelated individuals were selected for mapping assays (Gyapay et al. 1994). Our threshold for selection criteria was even higher. Indeed, a marker was retained for mapping when four or more alleles were identified in the eight key founders of the purebred pedigree, with the highest allele frequency below 0.625 among the 16 alleles. Our assumption is that markers fulfilling such severe criteria should be polymorphic on other purebred pedigrees, hence being directly informative for most linkage studies of disease loci. We initially screened a genomic library with tetranucleotide repeat probes and sequenced 171 positive clones. In a previous report, we detailed the analysis of 16 microsatellites out of the 72 first clones (Tiret et al. 1999). Among the 99 remaining clones, 39 were discarded because they contained few repeats, 15 could not be amplified under our standardized PCR conditions, 30 did not fit our polymorphism or reading criteria, and 15 were mapped. These selected markers were named DTRcn.17–31 for Dog Tetranucleotide Repeat chromosome number.17 to 31 (see Table 1). The same polymorphism criteria were used to select 39 dinucleotide and 11 tetranucleotide repeat markers from a subset of 156 microsatellites collected from the literature. At the time we chose these markers, most of them had not been mapped. Nine of the 11 tetranucleotide repeat markers, namely FH2016, FH2087U, FH2087L, FH2201, FH2261, FH2293, FH2305, FH2319, and FH2320, were selected because they had been assigned to distinct linkage groups (Mellersh et al. 1997) and could be used as anchoring loci for DTRcn markers. The 137 related Beagles were, therefore, genotyped for 81 type II markers. The mean number of alleles on this pedigree was 6.2 ± 0.2 sem, and the average heterozygosity rate was 0.7 ± 0.01 sem. The percentage of informative meioses was 62%. The linkage results established 38 pairs of linked markers that were ordered by a multipoint analysis in 10 linkage chromosomal groups and 7 unassigned clusters (Fig. 1). CPH6 had been linked to CXX.453 by Ryder et al. (1999). In our analysis, both CXX.453 and CPH6 were linked to C22.277. CPH6 could then be added to CFA22 [by extension, AHTk253 and TF may also be added; see Ryder et al. (1999)], and CXX.453 should be renamed C22.453. CPH9 was linked to DTRcn.29. Thus, INRA2, DTRcn.21, and DTRcn.29 completed the L12 group described by Lingaas et al. (1997), renamed Syntenic Group 3 in Mellersh et al. (2000). C10.404, C10.606, and FH2293 were linked, as described by Werner et al. (1999). However, the recombination estimate of 9 cM between C10.404 and C10.606 exceeded the 2.3-cM value reported by Werner et al. (1999), a hallmark of nonsaturated maps. CXX.617 was linked to C09.474, and we propose to update its name to C09.617. Seven published markers that had not been mapped previously were assigned to CFA, existing syntenic groups (S) and an orphan linkage cluster (OLC). 33 markers remained unlinked. Genotyping of 81 markers on our Beagle pedigree has allowed the mapping of 25 undescribed loci. Altogether, DTRcn.1 to DTRcn.31 and the seven polymorphic markers selected from the literature make a pool of 38 highly polymorphic markers, which can be genotyped either on the radiation hybrid panel or the American canine pedigree, the reference resources now used to build the international integrated map.

A missense mutation in the agouti signaling protein gene (ASIP) is associated with the no light points coat phenotype in donkeys

BackgroundSeven donkey breeds are recognized by the French studbook and are characterized by a black, bay or grey coat colour including light cream-to-white points (LP). Occasionally, Normand bay donkeys give birth to dark foals that lack LP and display the no light points (NLP) pattern. This pattern is more frequent and officially recognized in American miniature donkeys. The LP (or pangare) phenotype resembles that of the light bellied agouti pattern in mouse, while the NLP pattern resembles that of the mammalian recessive black phenotype; both phenotypes are associated with the agouti signaling protein gene (ASIP).FindingsWe used a panel of 127 donkeys to identify a recessive missense c.349xa0Tu2009>u2009C variant in ASIP that was shown to be in complete association with the NLP phenotype. This variant results in a cysteine to arginine substitution at position 117 in the ASIP protein. This cysteine is highly-conserved among vertebrate ASIP proteins and was previously shown by mutagenesis experiments to lie within a functional site. Altogether, our results strongly support that the identified mutation is causative of the NLP phenotype.ConclusionsThus, we propose to name the c.[349xa0Tu2009>u2009C] allele in donkeys, the anlp allele, which enlarges the panel of coat colour alleles in donkeys and ASIP recessive loss-of-function alleles in animals.

Targeted Lipidomic Analysis of Myoblasts by GC-MS and LC-MS/MS

Lipids represent ∼10% of the cell dry mass and play essential roles in membrane composition and physical properties, energy storage, and signaling pathways. In the developing or the regenerating skeletal muscle, modifications in the content or the flipping between leaflets of membrane lipid components can modulate the fusion capacity of myoblasts, thus constituting one of the regulatory mechanisms underlying myofiber growth. Recently, few genes controlling these qualitative and quantitative modifications have started to be unraveled. The precise functional characterization of these genes requires both qualitative and quantitative evaluations of a global lipid profile. Here, we describe a lipidomic protocol using mass spectrometry, allowing assessing the content of fatty acids, glycerophospholipids, and cholesterol in the routinely used C2C12 mouse myoblast cell line, or in primary cultures of mouse myoblasts.

Progressive Structural Defects in Canine Centronuclear Myopathy Indicate a Role for HACD1 in Maintaining Skeletal Muscle Membrane Systems

Mutations in HACD1/PTPLA cause recessive congenital myopathies in humans and dogs. Hydroxyacyl-coA dehydratases are required for elongation of very long chain fatty acids, and HACD1 has a role in early myogenesis, but the functions of this striated muscle-specific enzyme in more differentiated skeletal muscle remain unknown. Canine HACD1 deficiency is histopathologically classified as a centronuclear myopathy (CNM). We investigated the hypothesis that muscle from HACD1-deficient dogs has membrane abnormalities in common with CNMs with different genetic causes. We found progressive changes in tubuloreticular and sarcolemmal membranes and mislocalized triads and mitochondria in skeletal muscle from animals deficient in HACD1. Furthermore, comparable membranous abnormalities in cultured HACD1-deficient myotubes provide additional evidence that these defects are a primary consequence of altered HACD1 expression. Our novel findings, including T-tubule dilatation and disorganization, associated with defects in this additional CNM-associated gene provide a definitive pathophysiologic link with these disorders, confirm that dogs deficient in HACD1 are relevant models, and strengthen the evidence for a unifying pathogenesis in CNMs via defective membrane trafficking and excitation-contraction coupling in muscle. These results build on previous work by determining further functional roles of HACD1 in muscle and provide new insight into the pathology and pathogenetic mechanisms of HACD1 CNM. Consequently, alterations in membrane properties associated withxa0HACD1 mutations should be investigated in humans with related phenotypes.

Feline low-grade alimentary lymphoma: an emerging entity and a potential animal model for human disease

BackgroundLow-grade alimentary lymphoma (LGAL) is characterised by the infiltration of neoplastic T-lymphocytes, typically in the small intestine. The incidence of LGAL has increased over the last ten years and it is now the most frequent digestive neoplasia in cats and comprises 60 to 75% of gastrointestinal lymphoma cases. Given that LGAL shares common clinical, paraclinical and ultrasonographic features with inflammatory bowel diseases, establishing a diagnosis is challenging. A review was designed to summarise current knowledge of the pathogenesis, diagnosis, prognosis and treatment of feline LGAL. Electronic searches of PubMed and Science Direct were carried out without date or language restrictions.ResultsA total of 176 peer-reviewed documents were identified and most of which were published in the last twenty years. 130 studies were found from the veterinary literature and 46 from the human medicine literature. Heterogeneity of study designs and outcome measures made meta-analysis inappropriate. The pathophysiology of feline LGAL still needs to be elucidated, not least the putative roles of infectious agents, environmental factors as well as genetic events. The most common therapeutic strategy is combination treatment with prednisolone and chlorambucil, and prolonged remission can often be achieved. Developments in immunohistochemical analysis and clonality testing have improved the confidence of clinicians in obtaining a correct diagnosis between LGAL and IBD. The condition shares similarities with some diseases in humans, especially human indolent T-cell lymphoproliferative disorder of the gastrointestinal tract.ConclusionsThe pathophysiology of feline LGAL still needs to be elucidated and prospective studies as well as standardisation of therapeutic strategies are needed. A combination of conventional histopathology and immunohistochemistry remains the current gold-standard test, but clinicians should be cautious about reclassifying cats previously diagnosed with IBD to lymphoma on the basis of clonality testing. Importantly, feline LGAL could be considered to be a potential animal model for indolent digestive T-cell lymphoproliferative disorder, a rare condition in human medicine.