Additional Investigation After the Muscle Biopsy Diagnosis

Next generation sequence panel showed 2 novel frameshift mutations of the nebulin (NEB) gene which were predicted to be pathogenic: maternally inherited c.5244_5254delCTACACTGAAAlnsG,p.Tyr1749Asnfs∗23 and paternally inherited c.24770_24771dupTT,p.Ser8258Leufs∗10.

Final Diagnosis

Autosomal recessive nemaline myopathy from NEB mutations (compound heterozygous)

Patient Follow-Up

At the time of last follow-up 1.5 years from the initial diagnosis, there is no progression of his muscle weakness. He had negative cardiology and pulmonary evaluations. He was getting regular physical therapy and did not require assistive devices for ambulation.

Discussion

The child in this vignette presented with proximal lower limb weakness since early childhood. In addition to proximal limb weakness he had neck flexor weakness, facial weakness, nasal dysarthria, mild scapular winging and reduced tendon reflexes at the knees. Pertinent negatives were absence of family history, diurnal variation of symptoms, ocular findings, calf hypertrophy, joint contractures , and normal sensory examination.

The differential diagnosis for this presentation was broad and involves different parts of the lower motor unit. Anterior horn cell disorders particularly spinal muscular atrophy (SMA) type 3 was a consideration in this case due to proximal weakness and reduced knee reflexes. The non-progressive course and lack of diffuse hypo/areflexia argued against the possibility of diffuse polyradiculopathy. Peripheral neuropathies seemed unlikely given lack of distal weakness and sensory findings. Presence of fatigue, facial weakness, and nasal dysarthria might suggest the possibility of a neuromuscular junction (NMJ) disorder . Early onset presentation and chronic course could suggest congenital myasthenic syndrome (CMS) rather than acquired autoimmune myasthenia gravis. However, lack of ocular manifestations and diurnal variation of symptoms were against NMJ disorder. Myopathy seemed the most likely possibility given symmetric limb girdle pattern of weakness. Chronic course and early onset of symptoms suggested a genetic myopathy. Negative family history either suggested autosomal recessive inheritance or de novo mutation. Myopathies with facial weakness in children can be seen in congenital myopathies, mitochondrial myopathies, facioscapulohumeral muscular dystrophy (FSHD), myotonic dystrophy and rarely other muscular dystrophies [1].

The investigative approach in a child with myopathy includes measurement of serum CK, EMG, muscle ultrasound or muscle MRI, muscle biopsy and finally genetic testing [2, 3]. Measurement of serum CK level is an inexpensive and excellent screening tool in patients with suspected myopathies. Among the myopathic disorders with normal CK, congenital myopathies are the most common causes [2, 3]. Though high CK is commonly encountered in most of the muscular dystrophies, certain muscular dystrophies like FSHD and myotonic dystrophy can have normal CK [1, 2]. However, the pattern of weakness in this case did not suggest FSHD. SMA type 3 closely mimics myopathies due to limb girdle pattern of weakness. EMG in this case helped to rule out SMA which shows chronic neurogenic changes. Absence of myotonic discharges in EMG in this case made myotonic dystrophy less likely. EMG in CM can be myopathic or normal. Repetitive nerve stimulation was not performed, so a NMJ disorder could not be completely ruled out. A muscle biopsy was considered as the next step in this case which showed features of NM. Next generation sequencing confirmed the final diagnosis of NM due to NEB mutation.

Congenital myopathies are a group of clinically and genetically heterogeneous conditions characterized by muscle weakness and distinctive structural abnormalities in muscle biopsies [3, 4]. Traditionally CM is classified histologically into 4 distinct types: central core disease, multi-minicore disease, centronuclear myopathy and nemaline myopathy based on the presence of central cores, multi-minicores, central nuclei and nemaline rods respectively [5–8]. The overall prevalence of CM is not clearly known but estimated to be around 1:20,000 children [9].

NM is the commonest form of CM [10]. The hallmark feature on muscle pathology is the accumulation of Z-disk and thin filament proteins into aggregates called nemaline bodies or rods, usually accompanied by disorganization of the muscle Z-disks [11]. Nemaline rods are protein aggregates which stain red with the modified Gomori trichrome stain [4]. They can appear within the sarcoplasm as isolated or diffuse structures, compact subsarcolemmal clusters, or both [12]. On electron microscopy, nemaline rods appear as electron dense structures [13]. Another common histologic hallmark of NM is type I fiber predominance [12, 13]. Our patient’s biopsy showed all those features.

NM can be classified into six clinical types based on the age of onset, severity of weakness and respiratory muscle involvement [14]. Severe neonatal form is characterized by severe hypotonia and respiratory failure at birth. Intermediate congenital NM patients fail to achieve motor milestones, or become wheelchair dependent and/or develop respiratory failure by 11 years. Typical congenital forms are characterized by delayed motor milestones, proximal limb, lower facial or bulbar weakness which is either static or progresses very slowly. The patient described in this vignette fits with this phenotype. Childhood-onset NM develop their symptoms in the late first or second decade of life and most remain ambulatory. Adult onset NM develops symptoms much later in life. NM is typically not associated with extraocular muscle weakness which differentiates it from other forms of CM like centronuclear myopathies [4]. Extramuscular manifestations are uncommon in NM [3, 4]. Patients are cognitively normal. Cardiac involvement is rare except for some patients with ACTA1 or MYPN mutations . Patients can develop respiratory failure, feeding difficulties or scoliosis which is usually secondary to the muscle weakness [15].

Genetics of CM is changing rapidly. To date, mutations in more than 30 different genes have been associated with CM, though it accounts for approximately 60% of the cases. There are 12 known genetic causes of NM: ACTA1, NEB, TPM2, TPM3, TNNT1, CFL2, KBTBD13, KLHL40, KLHL41, LMOD3, MYO18B, and MYPN [4]. ACTA1 mutations are the most common dominant/de novo mutations, and NEB mutations are the most common recessive mutations [4]. The nebulin gene in the chromosomal region 2q23 with its 183 exons, encodes one of the biggest proteins in vertebrates (600–900 kDa) [16]. This protein plays an important role in the skeletal muscle sarcomere by determining the minimum lengths of the actin filaments and regulating actin-myosin interactions and the calcium sensitivity of force generation [17]. A repetitive region in the middle of the nebulin gene complicates analytical testing [18]. All pathogenic variants known in NEB have been recessive, mostly compound heterozygous. The most common types of variants are splice-site mutations (34%), followed by frameshift mutations (32%) caused by small (<20 bp) deletions or insertions, nonsense mutations (23%), missense mutations (7%); large deletions and duplications (>1 kb) are rare (4%) [18].

The clinical and histological spectrum of disorders caused by NEB mutations is a continuum, ranging in severity from the severe form with perinatal onset to the mild forms. The distribution of weakness can vary from generalized muscle weakness, more pronounced in proximal limb muscles, to distal-only involvement in early-onset distal myopathy without nemaline bodies, a distal form of NM, although neck flexor weakness appears to be rather consistent [3, 4, 18]. Histological patterns range from a severe, but almost invariably non-dystrophic disturbance of the myofibrillar pattern to an almost normal picture on hematoxylin-eosin staining, with or without nemaline bodies, sometimes combined with cores, core-rod myopathy with generalized muscle weakness and a childhood-onset distal myopathy with rods and cores [18].

Useful laboratory investigations include measurement of serum creatine kinase levels, which are typically normal or slightly elevated. Neurophysiological studies , such as electromyography and nerve conduction studies, are useful mainly for excluding congenital neuropathies, myotonic disorders and congenital myasthenic syndromes [3]. Muscle imaging, in particular, muscle ultrasonography as a screening test and muscle MRI for a more detailed assessment, can reveal diagnostic patterns of selective muscle involvement [19]. Assessment of muscle biopsy samples with a standard panel of histological, histochemical and immunohistochemical stains will confirm the specific congenital myopathy and exclude distinct conditions with overlapping pathological features, such as the congenital muscular dystrophies, myofibrillar myopathies and autophagic vacuolar myopathies [3]. Electron microscopy helps to clarify the pathognomonic structural abnormalities that are seen with light microscopy [3]. Analysis of multiple congenital myopathy-associated genes through next generation sequencing is rapidly becoming the preferred diagnostic approach [3].

At present, there are no specific therapies for NM, in particular those due to NEB mutations [3]. Treatment is mainly supportive with regular physical therapy and follow-up to detect and manage respiratory failure, bulbar symptoms (dysphagia and dysarthria) and orthopedic complications.

Pearls