Ribosomal protein haploinsufficiency occurs in diverse human diseases including Diamond-Blackfan anemia (DBA), congenital asplenia and T cell leukemia. Yet, how mutations in genes encoding ubiquitously expressed proteins such as these result in cell-type- and tissue-specific defects remains unknown. Here, we identify mutations in GATA1, encoding the critical hematopoietic transcription factor GATA-binding protein-1, that reduce levels of full-length GATA1 protein and cause DBA in rare instances. We show that ribosomal protein haploinsufficiency, the more common cause of DBA, can lead to decreased GATA1 mRNA translation, possibly resulting from a higher threshold for initiation of translation of this mRNA in comparison with other mRNAs. In primary hematopoietic cells from patients with mutations in RPS19, encoding ribosomal protein S19, the amplitude of a transcriptional signature of GATA1 target genes was globally and specifically reduced, indicating that the activity, but not the mRNA level, of GATA1 is decreased in patients with DBA associated with mutations affecting ribosomal proteins. Moreover, the defective hematopoiesis observed in patients with DBA associated with ribosomal protein haploinsufficiency could be partially overcome by increasing GATA1 protein levels. Our results provide a paradigm by which selective defects in translation due to mutations affecting ubiquitous ribosomal proteins can result in human disease.

Our understanding of genes that cause skeletal muscle disease has increased tremendously over the past three decades. Advances in approaches to genetics and genomics have aided in the identification of new pathogenic mechanisms in rare genetic disorders and have opened up new avenues for therapeutic interventions by identification of new molecular pathways in muscle disease. Recent studies have identified mutations of several Kelch proteins in skeletal muscle disorders. The Kelch superfamily is one of the largest evolutionary conserved gene families. The 66 known family members all possess a Kelch-repeat containing domain and are implicated in diverse biological functions. In skeletal muscle development, several Kelch family members regulate the processes of proliferation and/or differentiation resulting in normal functioning of mature muscles. Importantly, many Kelch proteins function as substrate-specific adaptors for Cullin E3 ubiquitin ligase (Cul3), a core component of the ubiquitin-proteasome system to regulate the protein turnover. This review discusses the emerging roles of Kelch proteins in skeletal muscle function and disease.

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

Centronuclear myopathies (CNMs) are characterized by muscle weakness and increased numbers of central nuclei within myofibers. X-linked myotubular myopathy, the most common severe form of CNM, is caused by mutations in MTM1, encoding myotubularin (MTM1), a lipid phosphatase. To increase our understanding of MTM1 function, we conducted a yeast two-hybrid screen to identify MTM1-interacting proteins. Striated muscle preferentially expressed protein kinase (SPEG), the product of SPEG complex locus (SPEG), was identified as an MTM1-interacting protein, confirmed by immunoprecipitation and immunofluorescence studies. SPEG knockout has been previously associated with severe dilated cardiomyopathy in a mouse model. Using whole-exome sequencing, we identified three unrelated CNM-affected probands, including two with documented dilated cardiomyopathy, carrying homozygous or compound-heterozygous SPEG mutations. SPEG was markedly reduced or absent in two individuals whose muscle was available for immunofluorescence and immunoblot studies. Examination of muscle samples from Speg-knockout mice revealed an increased frequency of central nuclei, as seen in human subjects. SPEG localizes in a double line, flanking desmin over the Z lines, and is apparently in alignment with the terminal cisternae of the sarcoplasmic reticulum. Examination of human and murine MTM1-deficient muscles revealed similar abnormalities in staining patterns for both desmin and SPEG. Our results suggest that mutations in SPEG, encoding SPEG, cause a CNM phenotype as a result of its interaction with MTM1. SPEG is present in cardiac muscle, where it plays a critical role; therefore, individuals with SPEG mutations additionally present with dilated cardiomyopathy.

A mutation update on the nebulin gene (NEB) is necessary because of recent developments in analysis methodology, the identification of increasing numbers and novel types of variants, and a widening in the spectrum of clinical and histological phenotypes associated with this gigantic, 183 exons containing gene. Recessive pathogenic variants in NEB are the major cause of nemaline myopathy (NM), one of the most common congenital myopathies. Moreover, pathogenic NEB variants have been identified in core-rod myopathy and in distal myopathies. In this update, we present the disease-causing variants in NEB in 159 families, 143 families with NM, and 16 families with NM-related myopathies. Eighty-eight families are presented here for the first time. We summarize 86 previously published and 126 unpublished variants identified in NEB. Furthermore, we have analyzed the NEB variants deposited in the Exome Variant Server (http://evs.gs.washington.edu/EVS/), identifying that pathogenic variants are a minor fraction of all coding variants (∼7%). This indicates that nebulin tolerates substantial changes in its amino acid sequence, providing an explanation as to why variants in such a large gene result in relatively rare disorders. Lastly, we discuss the difficulties of drawing reliable genotype-phenotype correlations in NEB-associated disease.

Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.

IMPORTANCE: Newer sequencing technologies in combination with traditional gene mapping techniques, such as linkage analysis, can help identify the genetic basis of disease for patients with rare disorders of uncertain etiology. This approach may expand the phenotypic spectrum of disease associated with those genetic mutations. OBJECTIVE: To elucidate the molecular cause of a neuromuscular disease among a family in which 4 members, a mother and her 3 sons, were affected. DESIGN, SETTING, AND PARTICIPANTS: Two of 4 affected members manifested nemaline myopathy, a common subtype of congenital myopathy, while the other 2 had a nonspecific myopathy. Single-nucleotide polymorphism-based linkage analysis was performed on DNA samples from the 4 affected family members, and whole-genome sequencing was performed in the proband. Real-time quantitative reverse transcription-polymerase chain reaction, immunofluorescence, and Western blot analysis were performed on muscle biopsy specimens. MAIN OUTCOMES AND MEASURES: Whole-genome sequencing and linkage analysis identified a variant in a gene that explains the phenotype. RESULTS: We identified a novel neurofilament light polypeptide (NEFL) nonsense mutation in all affected members. NEFL mutations have been previously linked to Charcot-Marie-Tooth disease in humans. This led us to reevaluate the diagnosis, and we recognized that several of the findings, especially those related to the muscle biopsy specimens and electromyography, were consistent with a neurogenic disease. CONCLUSIONS AND RELEVANCE: NEFL mutations are known to cause Charcot-Marie-Tooth disease in humans and motor neuron disease in mice. We report the identification of an NEFL mutation in a family clinically manifesting congenital myopathy. We also describe potential overlap between myopathic and neurogenic findings in this family. These findings expand the phenotypic spectrum of diseases associated with NEFL mutations. This study is an example of the power of genomic approaches to identify potentially pathogenic mutations in unsuspected genes responsible for heterogeneous neuromuscular diseases.

X-linked myotubular myopathy (XLMTM) is a fatal pediatric disease where affected boys display profound weakness of the skeletal muscles. Possible therapies are under development but robust outcome measures in animal models are required for effective translation to human patients. We established a naturally-occurring canine model, where XLMTM dogs display clinical symptoms similar to those observed in humans. The aim of this study was to determine potential endpoints for the assessment of future treatments in this model. Video-based gait analysis was selected, as it is a well-established method of assessing limb function in neuromuscular disease and measures have been correlated to the patient's quality of life. XLMTM dogs (N = 3) and their true littermate wild type controls (N = 3) were assessed at 4-5 time points, beginning at 10 weeks and continuing through 17 weeks. Motion capture and an instrumented carpet were used separately to evaluate spatiotemporal and kinematic changes over time. XLMTM dogs walk more slowly and with shorter stride lengths than wild type dogs, and these differences became greater over time. However, there was no clear difference in angular measures between affected and unaffected dogs. These data demonstrate that spatiotemporal parameters capture functional changes in gait in an XLMTM canine model and support their utility in future therapeutic trials.

Laing early onset distal myopathy and myosin storage myopathy are caused by mutations of slow skeletal/β-cardiac myosin heavy chain encoded by the gene MYH7, as is a common form of familial hypertrophic/dilated cardiomyopathy. The mechanisms by which different phenotypes are produced by mutations in MYH7, even in the same region of the gene, are not known. To explore the clinical spectrum and pathobiology we screened the MYH7 gene in 88 patients from 21 previously unpublished families presenting with distal or generalised skeletal muscle weakness, with or without cardiac involvement. Twelve novel mutations have been identified in thirteen families. In one of these families the grandfather of the proband was found to be a mosaic for the MYH7 mutation. In eight cases de novo mutation appeared to have occurred, which was proven in three. The presenting complaint was footdrop, sometimes leading to delayed walking or tripping, in members of 17 families (81%), with other presentations including cardiomyopathy in infancy, generalised floppiness and scoliosis. Cardiac involvement as well as skeletal muscle weakness was identified in 9 of 21 families. Spinal involvement such as scoliosis or rigidity was identified in 12 (57%). This report widens the clinical and pathological phenotypes, and the genetics of MYH7 mutations leading to skeletal muscle diseases. This article is protected by copyright. All rights reserved.

BACKGROUND: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. RESULTS: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. CONCLUSIONS: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.