5 Molecular Genetic Basis

The genetics of congenital myopathies with respect to the histopathological types is complex. One histopathologic type of CM can be caused by mutation(s) in any one of multiple different genes (genetic heterogeneity) and mutations in the same gene can lead to different types of CMs. Moreover, there can be significant intrafamilial and interfamilial variability in severity even for the same gene mutation (North, 2011; North et al. & International Standard of Care Committee for Congenital Myopathies, 2014). Genotype and phenotype correlations of congenital myopathies are hindered by the clinical variability of the phenotype but certain specific features may point to the involvement of a particular gene. Table 2 lists the various genes known to be associated with CMs and their patterns of inheritance.

6 Clinical Manifestations

Common clinical presentations of CMs include hypotonia, hyporeflexia and muscle weakness which can overlap with other neuromuscular disorders including congenital muscular dystrophies, spinal muscular atrophy, congenital myasthenic syndromes, congenital myotonic dystrophy and metabolic myopathies. Certain features of diagnostic importance include facial weakness associated with ptosis/ophthalmoplegia, bulbar and respiratory weakness and orthopedic complications like pectus carinatum and kyphoscoliosis. The severity may range from profound weakness in neonates with death in early infancy to mild weakness and survival to adulthood (North, 2011). Most of the congenital myopathies have generalized or proximal muscle weakness. Some have prominent axial or respiratory muscle weakness or weakness of ankle dorsiflexion. Cardiac involvement is unusual in congenital myopathies except in patients with variants in genes encoding titin (TTN) and myosin heavy chain 7 (MYH7). Table 3 lists some of the clinical features which are specific to certain types of congenital myopathies and can help in narrowing down the possible genetic diagnoses.

7 Clinical Approach

A systematic clinical approach helps in appropriate assessment and management of the patient and in accurate interpretation of the results of molecular genetic tests.

7.1 History/ Clinical examination

History of decreased fetal movements, early neonatal death due to respiratory insufficiency, floppiness and failure to thrive due to feeding difficulty during infancy, motor developmental delay, difficulty in walking and a waddling gait, and difficulty in climbing stairs and getting up from a sitting or squatting position, with or without history of similar features in other family members, suggest the possibility of a congenital myopathy. Clinical examination findings include myopathic facies, hypotonia, diminished deep tendon reflexes, reduction in muscle power more significantly in the axial and proximal limb muscles, and joint laxity. There is significant overlap between the clinical features of CMs with other genetic neuromuscular disorders. Clinical features of tongue fasciculations, facial dysmorphism other than myopathic faces, rapid progression and extreme joint laxity may be pointers for an alternative diagnosis.

7.2 Differential diagnosis

The following conditions can have significant phenotypic overlap with CMs and should be considered as possible differential diagnoses.

a. During infancy: Spinal muscular atrophy (SMA) type 1, congenital muscular dystrophies (CMD), congenital myotonic dystrophy, congenital myasthenic syndromes (CMS), metabolic myopathies, Prader–Willi syndrome and congenital hypomyelinating neuropathy can have overlapping phenotypic features. Table 4 lists the features that help to differentiate these conditions from congenital myopathies.

b. During childhood and in adults: Limb-girdle muscular dystrophies, SMA types 3 and 4, myotonic dystrophy, hereditary motor sensory neuropathy (HMSN) and acquired causes including autoimmune and inflammatory myopathies can mimic the milder forms of congenital myopathy which have survival until adulthood. Table 5 lists the features that help to differentiate these conditions from congenital myopathies.

7.3 Laboratory evaluation

The following laboratory evaluation is done in patients presenting with a CM phenotype:

a.

Serum CPK: is usually within normal limits or is only mildly elevated in CMs.

b.

Electromyography (EMG): shows myopathic changes or may be normal.

c.

Nerve conduction study (NCS): is usually normal but low-amplitude motor responses may be seen if there is marked loss of muscle bulk.

7.4 Muscle biopsy

Most congenital myopathies can be diagnosed using muscle biopsy followed by light microscopy and electron microscopy, unlike cases of muscular dystrophies where immunohistochemical studies are needed. Electron microscopy helps in histopathological diagnosis of subtypes of congenital myopathies. However, with the availability of next generation sequencing in recent years and the consequent ease of molecular diagnosis and accurate characterization, many clinicians prefer to defer muscle biopsy due to its invasive nature. But muscle biopsy can help to corroborate the diagnosis in cases where molecular genetic testing identifies novel variants or variants of unknown significance and also helps in characterization when genetic evaluation does not yield the diagnosis.

The typical histopathology findings in different types of CMs are as follows:

a. Nemaline myopathy: The biopsy shows characteristic nemaline rods (‘nema’ in Greek=thread) on modified Gomori’s trichrome stain (MGT) (Figure 2). These appear as red colored rods in clusters at the centre or periphery of the fibers and are seen in all fiber types. At later stages of disease, the biopsy can reveal changes of chronicity like fatty infiltration, fibrosis and fiber splitting. On electron microscopy (EM) rods have a lattice structure similar to the Z-line and can show continuity with the Z-line.

b. Core myopathy: The core myopathies are characterized by presence of central cores on staining with the oxidative stains succinic dehydrogenase (SDH), nicotinamide adenine dinucleotide (NADH) and cytochrome oxidase (COX) (Figure 3A). These are areas of absent oxidative enzyme stain on muscle biopsy that reflect the absence of mitochondria. These cores run along the longitudinal axis of the muscle fiber and may be central, peripheral, more than one per fiber and of variable size (Dubowitz et al., 2013). On electron microscopy, these cores may be structured (without any disruption of the intrinsic sarcomeric structure) or unstructured (with disruption of sarcomeric structure) with absence of mitochondria in the cores. Unlike central cores, minicores are identified as multiple focal areas devoid of oxidative enzyme activity which appears only as uneven stain on transverse section (Figures 3B&C).

 Figure 3: Muscle biopsy showing features of Core myopathy. A. Central area of absence of oxidative staining on NADH representing central cores (100X); B. & C. Multiple areas of absence of oxidative enzyme activity on NADH representing minicores.

c. Centronuclear myopathy: The characteristic feature is the presence of internalized and central nuclei (Figures 4 A & B). The biopsy in X-linked myotubular myopathy reveals fiber atrophy with many fibers showing centralized nuclei resembling myotubes; these are seen on longitudinal section as a row of nuclei. The central nuclei have reduced ATPase reaction and increased periodic acid-Schiff (PAS) and oxidative enzyme staining surrounded by a clear halo (Romero & Bitoun, 2011). Electron microscopy shows central nuclei with aggregates of mitochondria.

 Figure 4: Muscle biopsy with hematoxylin and eosin (H&E) staining showing features of centronuclear myopathy. A. Transverse section of muscle biopsy showing central nuclei (40X); B. Central nuclei are seen arranged in rows (40X).

d. Congenital fibre type disproportion: Patients of CFTD associated with known genetic abnormalities show type 1 fibers that are generally at least 40% to over 80% smaller than type 2 fibers (Figures 5 A & B).

It is important to emphasize that each of these structured and non-structured abnormalities described on biopsy can be seen in various other disorders including muscle degeneration, aging, metabolic changes, muscular dystrophies and even exercise induced changes. Therefore, it is essential that these changes are interpreted in the light of complete clinical examination and genetic results for a definite diagnosis of congenital myopathy.

 Figure 5: Muscle biopsy showing features of Congenital fibre type disproportion. A. Transverse section of muscle biopsy showing atrophic fibers (H&E 40X). B. Atrophic type I fibers seen with ATPase staining (40X).

7.5 Muscle imaging

Magnetic resonance imaging (MRI) of muscles can help to differentiate between different forms of congenital myopathies based on the pattern of selective muscle involvement. Imaging also helps in identifying the exact site from where the muscle biopsy has to be taken and has been used along with muscle biopsy to prioritize gene testing in the pre-NGS era.

7.6 Molecular genetic testing

Molecular genetic testing is now the preferred modality for confirmation of the clinical diagnosis of congenital myopathy and for exact characterization of the subtype, and is being used as the first line confirmatory test in place of muscle biopsy in many centres. Apart from accurate diagnosis for the proband, identification of the exact genetic etiology helps in accurate assessment of the risk of recurrence in other family members, in presymptomatic screening and carrier testing of other at-risk family members and in prenatal genetic testing to prevent recurrence in the family.

Availability of next generation sequencing (NGS) has significantly reduced the cost and time for molecular genetic testing for CMs. NGS has also vastly improved the mutation detection rate and helped in identification of many novel genes and gene variants related to CMs (Gonorazky et al., 2018). NGS-based congenital myopathy-associated multigene sequencing panel or Focused exome sequencing panel can be used as the first-tier test and if no significant variants are found, Whole exome sequencing and/or Whole genome sequencing can be done further. RNA sequencing (RNA-seq) is likely to result in improved diagnostic yield for congenital myopathies in unsolved cases (Ravenscroft et al., 2018).

8 Management

There are no definite curative therapies available at present for any of the congenital myopathies. Multidisciplinary management involving Neurology, Pulmonology, Gastroenterology, Clinical Genetics, Orthopedics and Physiotherapy is required for optimum care of affected individuals (Wang CH, et al). In the less severe cases, as the disorder is nonprogressive, good supportive care and physical therapy to maintain mobility and reduce joint contractures, and aggressive treatment of respiratory problems can help achieve a satisfactory outcome and better life expectancy. Management includes supportive and symptomatic care in the form of:

• regular physiotherapy for maintenance of muscle power and function and for improving cardiorespiratory capacity
• assessment of feeding difficulty to determine the need for gastrostomy insertion for early onset severe forms
• regular monitoring of respiratory capacity and signs of nocturnal hypoventilation and hypoxia every 6-12 monthly
• conservative treatment of contractures at the initial stages to avoid surgery
• care of scoliosis by bracing and surgery if required
• avoidance of anesthetic drugs associated with malignant hyperthermia (especially for individuals with RYR1 gene mutation)

9 Genetic Counseling

Following accurate diagnosis of the affected individual, appropriate genetic counseling can be provided to the patient and the family about the nature of the disorder, natural course and prognosis, available management options and the surveillance/ monitoring plan. Based on the identified underlying genetic etiology, the exact pattern of inheritance i.e. autosomal dominant, autosomal recessive or X-linked can be clearly ascertained and the risk of recurrence in subsequent pregnancies in the family can be determined. Prenatal diagnosis can be offered for at-risk pregnancies through targeted mutation analysis after identifying the exact pathogenic variant(s) in the affected proband and/ or carrier parents. Variable expressivity (ranging from mild to severe) even amongst affected members of the same family for certain autosomal dominant congenital myopathies such as RYR1 gene-associated central core disease can make counseling, especially about the prognosis and postnatal outcome, difficult.

10 Recent Advances

Certain therapeutic modalities for congenital myopathies are being investigated. Tyrosine has been reported as a beneficial supplement for nemaline myopathy mainly for sialorrhea (Ryan et al., 2008). N-acetylcysteine is being tried as an antioxidant therapy for RYR1-related congenital myopathies (Dowling et al., 2012). Pyridostigmine, an acetylcholinesterase inhibitor, has been reported in some studies to show significant clinical improvement in centronuclear myopathy. A single intravenous dose of AAV8 hMTM1 for X-linked myotubular myopathy is under Phase I/II clinical trials (ASPIRO trial, ClinicalTrials.gov).

11 Conclusion

Congenital myopathies are an important group of genetic neuromuscular disorders. Precise genetic diagnosis has important implications for disease management, monitoring for potential complications, avoidance of anesthetic complications, for genetic counseling and screening of other family members, and for prenatal diagnosis of subsequent pregnancies in the family to prevent recurrence.

References