Congenital Myopathies and Related Disorders

Histopathology

Fibre size variation occurs but is often mild. Fibre hypertrophy is common, particularly in adults ( Fig. 15.1 ). When fibre typing is retained, the cores have a predilection for type 1 fibres but fibre type uniformity is common, with most fibres staining as type 1 fibres, with the associated properties of slow fibres (see Fig. 15.1 ). A few fibres may co-express fast myosin and there may be a population of very small fibres (‘pinpricks’ < 5 μm in diameter) with fetal myosin scattered throughout the biopsy (see Fig. 6.26 ). The intensity of stain of the type 1 fibres, however, may not always be as strong as in normal muscle. Classical cores may be central or peripheral, single or multiple, but clearly demarcated cores are not always evident in all cases (see Fig. 15.1 ) and some genetically proven cases only show an appearance of fibre type disproportion ( ) (see below). Very young cases, in particular, may only show type 1 uniformity or predominance, suggesting there is an age-related development of the cores (see Fig. 15.1 ; ). This is well illustrated in a mouse model in which an RYR1 mutation caused an age-related appearance of different size cores and ultimately led to the appearance of nemaline rods ( ). Other cases may show only subtle unevenness in oxidative enzyme stains ( Fig. 15.2 ) or multiple focal areas of disruption of variable size, resembling minicores, making a histopathological distinction difficult ( ) ( Fig. 15.1 ; see below). Recessive cases, in particular, may show multiple cores rather than prominent central cores ( ). It is important to remember that core formation is a secondary morphological consequence of disturbances in cellular events that may not itself be the reason for the muscle weakness ( ).

In many cases of central core disease, particularly dominantly inherited cases, the cores are of the ‘structured’ type, as they retain a striated myofibrillar pattern and myofibrillar adenosine triphosphatase (ATPase) activity, although myofibrils of the core are often very contracted ( Fig. 15.3 ). The distinction of ‘structured’ versus ‘unstructured’ cores is not of diagnostic value. In ‘unstructured cores’, myofibrillar ATPase activity is lost ( Fig. 15.4 ) and there is severe myofibrillar disruption with pronounced accumulation of smeared Z line material which may account for the red appearance reported with the Gomori trichrome ( ). The cores in recessive cases are often unstructured, and variable in size, affecting many or only a few sarcomeres; or they may even stretch across the width of a fibre ( ). The length of the cores can be variable but in dominantly inherited cases they are long, extending down an appreciable length of a fibre. The area devoid of mitochondria may be more extensive than the apparent ultrastructural myofibrillar disruption. Sarcoplasmic reticulum and T tubules may also be reduced in cores but some tubular structures may be apparent within them. Cores are often delineated by a rim of periodic acid–Schiff (PAS) stain but lack glycogen within them and consequently also lack phosphorylase (see Ch. 4 ). Immunohistochemistry shows that desmin may accumulate at their perimeter or within them ( Fig. 15.5 ), and other proteins that have been shown to accumulate in cores include αB-crystallin, filamin C, small heat-shock proteins, myotilin, RyR1 and DHPR ( ).

Internal nuclei had not been considered a feature of central core disease in studies of early cases but it is now appreciated that they can be an important indicator of central core disease and defects in the RYR1 gene ( Fig. 15.6a, d ). In some cases they may be numerous, and several may be in a central position, resembling a centronuclear myopathy ( ; see below), but cores may not be obvious. Similarly, an increase in connective tissue was not considered a feature of ‘classical’ cases but can occur, and in some samples there may also be extensive adipose tissue (see Fig. 15.6d ; Fig. 15.6e ). In these samples the separation of fascicles of fibres by adipose tissue and fibrous tissue may cause diagnostic confusion with a muscular dystrophy. Some of these samples may show only subtle unevenness of oxidative enzyme stains, while others show large classical cores or multiple small cores (minicores; see Fig. 15.6b, c, f ).

Although cores are the characteristic feature of central core disease caused by mutations in the gene encoding the ryanodine receptor 1 ( RYR1 ), core formation can also occur following tenotomy, following neurogenic atrophy (see Ch. 9 ). Small focal cores (minicores) can be associated with several gene defects, including SELENON (formerly SEPN1 ) and MYH2 (see below) and the ACTA1, MYH7, TTN, CFL2, KBTBD13, ACTN2 and MEGF10 genes, encoding skeletal α-actin, β-myosin heavy-chain, titin, cofilin2, a protein of the Kelch family, α-actinin 2, and multiple epidermal growth factor-like domains 10, respectively (see Table 15.1 and sections below; ). Cores in association with central nuclei have also been identified in association with the CCDC78 gene ( ). Cores can coexist with rods ( Fig. 15.7 ) and be associated with RYR1, ACTA1 and NEB mutations (see below) ( ). If rods are in a focal aggregate this may appear as a core, as there are no mitochondria and thus no oxidative enzyme stain. In cases described as ‘core-rod myopathy’ the cores and rods are in separate fibres. In some cases with RYR1 mutations, only a few fibres may show rods ( Fig. 15.8 ; ). The coexistence of rods and cores is likely to be genetically heterogeneous as there are examples of cases where linkage to RYR1 and to the loci of nemaline myopathy (see below) has been excluded.

Molecular Genetics

Central core disease is caused by mutations in the gene for the skeletal muscle ryanodine receptor ( RYR1 ) on chromosome 19q. The same gene is also responsible for malignant hyperthermia susceptibility (although additional loci are also linked to this), King–Denborough syndrome and exercise intolerance associated with rhabdomyolysis. The precise association between central core disease and malignant hyperthermia is not clear but, as pointed out previously, all patients with central core disease are considered at risk and appropriate precautions need to be taken.

The RYR1 gene contains 106 exons and encodes the skeletal muscle ryanodine receptor protein (RyR1), named after the fact that it binds ryanodine. The receptor is a large transmembrane, tetrameric structure of the sarcoplasmic reticulum and is involved in the regulation of cytosolic calcium levels and excitation–contraction coupling. Genotype–phenotype correlations suggest that mutations in the cytoplasmic N-terminal domain and the cytoplasmic central domain mostly result in malignant hyperthermia susceptibility rather than in central core disease, while mutations affecting the C-terminal exons of the gene commonly result in central core disease ( ). Mutations in the C-terminal exons (95–102) often result in the classical central core pathology with large cores and account for approximately 66% of cases. Recessive mutations can occur in any region of the gene and are often associated with multiple minicores ( ). The majority of mutations in the RYR1 gene are missense mutations, although deletions have also been detected ( ).

The large size of the RYR1 gene makes molecular analysis laborious, although there are ‘hot spots’ for mutations that can help direct analysis ( ). The phenotypic and histopathological variability associated with the RYR1 gene is broad, but the use of gene panels and next-generation sequencing has considerably assisted diagnosis and the identification of mutations. There are, however, many polymorphisms and variants of unknown significance in this large gene. Studies of the RyR1 protein by immunoblot analysis can reveal a reduction and help to determine if a change is pathogenic ( ). Another complicating factor in the RYR1 gene is that tissue-specific epigenetic silencing of the normal allele, resulting in expression of only the mutant allele, may occur ( ). Although further studies of the cases reported by Zhou et al revealed an additional mutation in four out of the five cases, casting doubt on the proposal of epigenetic silencing, gene silencing could not be excluded in the fifth ( ). This study of Zhou et al highlights the limitation of only studying genomic DNA and the contribution of genetic modifiers is now appreciated ( ).

In summary, the clinical and histopathological spectra associated with mutations in the RYR1 gene are wide. The use of the term ‘central core’ disease for all of them may be confusing, as some biopsies do not show classical cores. They are linked, however, by common clinical features, and similar patterns of muscle involvement, indicating a spectrum of one disorder. For the histopathologist, difficulties in diagnosis may arise if the classical features are not present and because of the histopathological overlap between the various congenital myopathies. In our experience mutations in the RYR1 gene are particularly common and digenic problems should therefore sometimes be considered, particularly if the phenotype is atypical ( ). The pathology associated with one gene can be masked by that of the other ( ). The features that should alert the pathologist to defects in the RYR1 gene are central nuclei, any unevenness in oxidative enzyme stain, be it marked or subtle, and type 1 fibre uniformity or marked predominance. When fibre type disproportion in the absence of cores is present, the RYR1 gene also has to be considered. The coexistence of cores and rods in any fibres also suggests an RYR1 mutation should be considered but can also occur in association with other gene defects such as nebulin and skeletal α-actin (see below).

Histopathology

The defining feature is multiple focal areas of myofibrillar disruption that lack mitochondria. These can be demonstrated with the oxidative enzyme stains reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR), cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) and may appear as punctate or more diffuse areas devoid of stain ( Fig. 15.9 ). The NADH-TR reaction is less specific for absent mitochondria, as unevenness of stain can also relate to general disruption of ultrastructure and loss of myofibrils. Assessment with COX or SDH should therefore also be performed. In some cases, the unevenness of oxidative stain may be subtle and difficult to define as abnormal. Ultrastructurally, minicores show a variable degree of focal disruption of myofibrils that affects only a few sarcomeres. In some fibres, only misalignment of the myofibrils compared with surrounding myofibrils may be seen; in others, focal Z line streaming may occur, while in others the normal sarcomeric structure may be completely disrupted in a varying number of sarcomeres (see Ch. 5 ). Small focal areas adjacent to capillaries resembling minicores are a common non-specific feature. The consistent feature of minicores, however, is the absence of mitochondria, which may extend over a greater area than the disrupted myofibrils. Detailed ultrastructural studies are needed to observe this.

As in central core disease, immunocytochemistry can be helpful in observing the lesions, and proteins such as desmin, myotilin and filamin C can accumulate within them ( ), but there is no specific histopathological method to distinguish the molecular origin of any cores. In contrast to some central cores, minicores are not usually delineated by desmin. Immunolabelling of myosin isoforms can be used to assess the proportion of fast and slow fibres and there are usually only a few, or no, fibres containing fetal myosin in cases with mutations in the SELENON gene (previously known as SEPN1 ) (see below).

Clinical Features

A common phenotype associated with minicores shows marked axial weakness with spinal rigidity, scoliosis, torticollis and respiratory involvement that is often disproportionate to the overall muscle weakness. Patients are clinically similar to cases with the rigid spine syndrome and congenital muscular dystrophy (RSMD1), and the two disorders are considered allelic ( ). Both are caused by recessive mutations in the gene for selenoprotein N encoded by the gene now known as SELENON , and the spectrum also includes cases with Mallory bodies (Ferreiro et al 2004; see Chapter 12, Chapter 5 ).

Multiple minicores can also be seen in RYR1 -related myopathy with similar proximal and axial weakness and also showing partial or complete external ophthalmoplegia. Mutations (usually recessively inherited) in the RYR1 gene in these patients form part of the spectrum of changes associated with the RYR1 gene (see above; ), emphasizing the overlap in the core myopathies. Primary cardiac malfunction is not a typical feature of these groups of patients with multiple minicores, and many of the cases reported in the literature showing minicores with cardiac involvement are molecularly unsolved and probably heterogeneous. Some cases, however, have been shown to have a mutation in the MYH7 gene ( ) or the gene encoding titin ( ). Recently, two patients were identified with mutations in the ACTN2 gene encoding α-actinin 2 and biopsies showed unusual large multiple peripheral structured cores ( ); neither case, however, presented with hypotonia