X-Linked Myotubular Myopathy: A Novel Mutation Expanding the Genotypic Spectrum of a Phenotypically Heterogeneous Myopathy.

X-linked myotubular myopathy (XLMTM), a centronuclear congenital myopathy secondary to pathogenic variants in the MTM1 gene encoding myotubularin, is typically recognized for its classic and severe phenotype which includes neonatal hypotonia, severe muscle weakness, long-term ventilator dependence, markedly delayed gross motor milestones with inability to independently ambulate, and a high neonatal and childhood mortality. However, milder congenital forms of the condition and other phenotypes are recognized. We describe a 6-year-old boy with a mild XLMTM phenotype with independent gait and no respiratory insufficiency even in the neonatal period. The child has a hemizygous novel splice site variant in the MTM1 gene (c.232-25A > T) whose pathogenicity was confirmed by cDNA studies (exon 5 skipping) and muscle biopsy findings. We also compared the phenotype of our patient with the few reported cases that presented a mild XLMTM phenotype and no respiratory distress at birth, and discussed the potential mechanisms underlying this phenotype such as the presence of residual expression of the normal myotubularin transcript.

Integrating Whole-Genome Sequencing in Clinical Genetics: A Novel Disruptive Structural Rearrangement Identified in the Dystrophin Gene (DMD).

While in most patients the identification of genetic alterations causing dystrophinopathies is a relatively straightforward task, a significant number require genomic and transcriptomic approaches that go beyond a routine diagnostic set-up. In this work, we present a Becker Muscular Dystrophy patient with elevated creatinine kinase levels, progressive muscle weakness, mild intellectual disability and a muscle biopsy showing dystrophic features and irregular dystrophin labelling. Routine molecular techniques (Southern-blot analysis, multiplex PCR, MLPA and genomic DNA sequencing) failed to detect a defect in the DMD gene. Muscle DMD transcript analysis (RT-PCR and cDNA-MLPA) showed the absence of exons 75 to 79, seen to be present at the genomic level. These results prompted the application of low-coverage linked-read whole-genome sequencing (WGS), revealing a possible rearrangement involving DMD intron 74 and a region located upstream of the PRDX4 gene. Breakpoint PCR and Sanger sequencing confirmed the presence of a ~8 Mb genomic inversion. Aberrant DMD transcripts were subsequently identified, some of which contained segments from the region upstream of PRDX4. Besides expanding the mutational spectrum of the disorder, this study reinforces the importance of transcript analysis in the diagnosis of dystrophinopathies and shows how WGS has a legitimate role in clinical laboratory genetics.

LAMA2 gene mutation update: Toward a more comprehensive picture of the laminin-α2 variome and its related phenotypes.

Congenital muscular dystrophy type 1A (MDC1A) is one of the main subtypes of early-onset muscle disease, caused by disease-associated variants in the laminin-α2 (LAMA2) gene. MDC1A usually presents as a severe neonatal hypotonia and failure to thrive. Muscle weakness compromises normal motor development, leading to the inability to sit unsupported or to walk independently. The phenotype associated with LAMA2 defects has been expanded to include milder and atypical cases, being now collectively known as LAMA2-related muscular dystrophies (LAMA2-MD). Through an international multicenter collaborative effort, 61 new LAMA2 disease-associated variants were identified in 86 patients, representing the largest number of patients and new disease-causing variants in a single report. The collaborative variant collection was supported by the LOVD-powered LAMA2 gene variant database (https://www.LOVD.nl/LAMA2), updated as part of this work. As of December 2017, the database contains 486 unique LAMA2 variants (309 disease-associated), obtained from direct submissions and literature reports. Database content was systematically reviewed and further insights concerning LAMA2-MD are presented. We focus on the impact of missense changes, especially the c.2461A > C (p.Thr821Pro) variant and its association with late-onset LAMA2-MD. Finally, we report diagnostically challenging cases, highlighting the relevance of modern genetic analysis in the characterization of clinically heterogeneous muscle diseases.

New massive parallel sequencing approach improves the genetic characterization of congenital myopathies.

Congenital myopathies (CMs) are a heterogeneous group of muscle diseases characterized by hypotonia, delayed motor skills and muscle weakness with onset during the first years of life. The diagnostic workup of CM is highly dependent on the interpretation of the muscle histology, where typical pathognomonic findings are suggestive of a CM but are not necessarily gene specific. Over 20 loci have been linked to these myopathies, including three exceptionally large genes (TTN, NEB and RYR1), which are a challenge for molecular diagnosis. We developed a new approach using massive parallel sequencing (MPS) technology to simultaneously analyze 20 genes linked to CMs. Assay design was based on the Ion AmpliSeq strategy and sequencing runs were performed on an Ion PGM system. A total of 12 patients were analyzed in this study. Among the 2534 variants detected, 14 pathogenic mutations were successfully identified in the DNM2, NEB, RYR1, SEPN1 and TTN genes. Most of these had not been documented and/or fully characterized, hereby contributing to expand the CM mutational spectrum. The utility of this approach was demonstrated by the identification of mutations in 70% of the patients included in this study, which is relevant for CMs especially considering its wide phenotypic and genetic heterogeneity.

Atypical phenotype in two patients with LAMA2 mutations.

Congenital muscular dystrophy type 1A is caused by mutations in the LAMA2 gene, which encodes the α2-chain of laminin. We report two patients with partial laminin-α2 deficiency and atypical phenotypes, one with almost exclusive central nervous system involvement (cognitive impairment and refractory epilepsy) and the second with marked cardiac dysfunction, rigid spine syndrome and limb-girdle weakness. Patients underwent clinical, histopathological, imaging and genetic studies. Both cases have two heterozygous LAMA2 variants sharing a potentially pathogenic missense mutation c.2461A>C (p.Thr821Pro) located in exon 18. Brain MRI was instrumental for the diagnosis, since muscular examination and motor achievements were normal in the first patient and there was a severe cardiac involvement in the second. The clinical phenotype of the patients is markedly different which could in part be explained by the different combination of mutations types (two missense versus a missense and a truncating mutation).

Reviewing Large LAMA2 Deletions and Duplications in Congenital Muscular Dystrophy Patients.

BACKGROUND: Congenital muscular dystrophy (CMD) type 1A (MDC1A) is caused by recessive mutations in laminin-α2 (LAMA2) gene. Laminin-211, a heterotrimeric glycoprotein that contains the α2 chain, is crucial for muscle stability establishing a bond between the sarcolemma and the extracellular matrix. More than 215 mutations are listed in the locus specific database (LSDB) for LAMA2 gene (May 2014). OBJECTIVE: A limited number of large deletions/duplications have been reported in LAMA2. Our main objective was the identification of additional large rearrangements in LAMA2 found in CMD patients and a systematic review of cases in the literature and LSDB. METHODS: In four of the fifty-two patients studied over the last 10 years, only one heterozygous mutation was identified, after sequencing and screening for a frequent LAMA2 deletion. Initial screening of large mutations was performed by multiplex ligation-dependent probe application (MLPA). Further characterization implied several techniques: long-range PCR, cDNA and Southern-blot analysis. RESULTS: Three novel large deletions in LAMA2 and the first pathogenic large duplication were successfully identified, allowing a definitive molecular diagnosis, carrier screening and prenatal diagnosis. A total of fifteen deletions and two duplications previously reported were also reviewed. Two possible mutational "hotspots" for deletions may exist, the first encompassing exons 3 and 4 and second in the 3' region (exons 56 to 65) of LAMA2. CONCLUSIONS: Our findings show that this type of mutation is fairly frequent (18.4% of mutated alleles) and is underestimated in the literature. It is important to include the screening of large deletions/duplications as part of the genetic diagnosis strategy.

Expanding the MTM1 mutational spectrum: novel variants including the first multi-exonic duplication and development of a locus-specific database.

Myotubular myopathy (MIM#310400), the X-linked form of Centronuclear myopathy (CNM) is mainly characterized by neonatal hypotonia and inability to maintain unassisted respiration. The MTM1 gene, responsible for this disease, encodes myotubularin - a lipidic phosphatase involved in vesicle trafficking regulation and maturation. Recently, it was shown that myotubularin interacts with desmin, being a major regulator of intermediate filaments. We report the development of a locus-specific database for MTM1 using the Leiden Open Variation database software (http://www.lovd.nl/MTM1), with data collated for 474 mutations identified in 472 patients (by June 2012). Among the entries are a total of 25 new mutations, including a large deletion encompassing introns 2-15. During database implementation it was noticed that no large duplications had been reported. We tested a group of eight uncharacterized CNM patients for this specific type of mutation, by multiple ligation-dependent probe amplification (MLPA) analysis. A large duplication spanning exons 1-5 was identified in a boy with a mild phenotype, with results pointing toward possible somatic mosaicism. Further characterization revealed that this duplication causes an in-frame deletion at the mRNA level (r.343_444del). Results obtained with a next generation sequencing approach suggested that the duplication extends into the neighboring MAMLD1 gene and subsequent cDNA analysis detected the presence of a MTM1/MAMLD1 fusion transcript. A complex rearrangement involving the duplication of exon 10 has since been reported, with detection also enabled by MLPA analysis. It is thus conceivable that large duplications in MTM1 may account for a number of CNM cases that have remained genetically unresolved.

The import competence of a peroxisomal membrane protein is determined by Pex19p before the docking step.

Biogenesis of the mammalian peroxisomal membrane requires the action of Pex3p and Pex16p, two proteins present in the organelle membrane, and Pex19p, a protein that displays a dual subcellular distribution (peroxisomal and cytosolic). Pex19p interacts with most peroxisomal intrinsic membrane proteins, but whether this property reflects its role as an import receptor for this class of proteins or a chaperone-like function in the assembly/disassembly of peroxisomal membrane proteins has been the subject of much controversy. Here, we describe an in vitro system particularly suited to address this issue. It is shown that insertion of a reporter protein into the peroxisomal membrane is a Pex3p-dependent process that does not require ATP/GTP hydrolysis. The system can be programmed with recombinant versions of Pex19p, allowing us to demonstrate that Pex19p-cargo protein complexes formed in the absence of peroxisomes are the substrates for the peroxisomal docking/insertion machinery. Data suggesting that cargo-loaded Pex19p displays a much higher affinity for Pex3p than Pex19p alone are also provided. These results suggest that soluble Pex19p participates in the targeting of newly synthesized peroxisomal membrane proteins to the organelle membrane and support the existence of a cargo-induced peroxisomal targeting mechanism for Pex19p.

Protein translocation across the peroxisomal membrane.

Peroxisomal matrix proteins are synthesized on free cytosolic ribosomes and posttranslationally imported into the organelle. Translocation of these newly synthesized proteins across the peroxisomal membrane requires the concerted action of many different proteins, the majority of which were already identified. However, not much is known regarding the mechanism of protein translocation across this membrane system. Here, we discuss recent mechanistic and structural data. These results point to a model in which proteins en route to the peroxisomal matrix are translocated across the organelle membrane by their own receptor in a process that occurs through a large membrane protein assembly.

The energetics of Pex5p-mediated peroxisomal protein import.

Most newly synthesized peroxisomal matrix proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with cargo proteins in the cytosol and transports them to the peroxisomal membrane. After delivering the passenger protein into the peroxisomal matrix, Pex5p returns to the cytosol to catalyze additional rounds of transportation. Obviously, such cyclic pathway must require energy, and indeed, data confirming this need are already available. However, the exact step(s) of this cycle where energy input is necessary remains unclear. Here, we present data suggesting that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. This observation raises the possibility that at the peroxisomal membrane ATP is needed predominantly (if not exclusively) downstream of the protein translocation step to reset the Pex5p-mediated transport system.

Characterization of the peroxisomal cycling receptor, Pex5p, using a cell-free in vitro import system.

According to current models of peroxisomal biogenesis, Pex5p cycles between the cytosol and the peroxisome transporting newly synthesized proteins to the organelle matrix. However, little is known regarding the mechanism of this pathway. Here, we show that Pex5p enters and exits the peroxisomal compartment in a process that requires ATP. Insertion of Pex5p into the peroxisomal membrane is blocked by anti-Pex14p IgGs. At the peroxisomal level, two Pex14p-associated populations of Pex5p could be resolved, stage 2 and stage 3 Pex5p, both exposing the majority of their masses into the organelle lumen. Stage 3 Pex5p can be easily detected only under ATP-limiting conditions; in the presence of ATP it leaves the peroxisomal compartment rapidly. Our data suggest that translocation of PTS1-containing proteins across the peroxisomal membrane occurs concomitantly with formation of the Pex5p-Pex14p membrane complex and that this is probably the site from which Pex5p leaves the peroxisomal compartment.

Insertion of Pex5p into the peroxisomal membrane is cargo protein-dependent.

It is now generally accepted that Pex5p, the receptor for most peroxisomal matrix proteins, cycles between the cytosol and the peroxisomal compartment. According to current models of peroxisomal biogenesis, this intracellular trafficking of Pex5p is coupled to the transport of newly synthesized peroxisomal proteins into the organelle matrix. However, direct evidence supporting this hypothesis was never provided. Here, using an in vitro peroxisomal import system, we show that insertion of Pex5p into the peroxisomal membrane requires the presence of cargo proteins. Strikingly the peroxisomal docking/translocation machinery is also able to catalyze the membrane insertion of a Pex5p truncated molecule lacking any known cargo-binding domain. These results suggest that the cytosol/peroxisomal cycle in which Pex5p is involved is directly or indirectly regulated by Pex5p itself and not by the peroxisomal docking/translocation machinery.

Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction.

Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.