Clinical report:
The patient presented at 8-month-old boy, who was the fifth pregnancy of non-consanguineous Canadian (European) parents, with an uneventful pregnancy and delivery. He presented with a history of hypotonia, developmental delay, and failure to thrive. He was severely delayed in motor milestones but appropriate in social and language development.There was a significant family history of a paternal first cousin with a undifferentiated muscular dystrophy, the details or diagnosis of which could not be confirmed. There were no other familial neuromuscular, genetic or congenital diseases.
The patient had shown signs of hypotonia since birth and at three months he was admitted to hospital for failure to thrive. Upon presentation to the neuromuscular clinic, he continued to demonstrate difficulty with head movement, including holding up his head. He was however, able to spontaneously move all extremities against gravity.
His general exam was normal. On neurological examination, he was hypotonic with evidence of poor muscle bulk to his extremities, truncal hypotonia and head-lag. There were no obvious dysmorphic features or evidence of tongue fasciculations. Sensation appeared intact. He was a reflexic with downgoing toes. His mobility was limited to side-to-side rolling.
The patient underwent multiple admissions for respiratory difficulties and additional investigations. Given the significant hypoventilation and inability to increase respiratory effort, the decision was made for the patient to initiate nighttime BiPAP. At age 9 months he had a tracheostomy placed and has been stable on intermittent BiPap via tracheostomy.
The creatine kinase (CK) level was normal. Echocardiography was normal.Sequencing of the SMN1 gene showed no pathogenic mutations,andalpha-Glucosidase DBS(dried blood spot) enzyme test was normal.EMG and nerve conduction studies demonstrated spontaneous activity in proximal muscles, consistent with possible myopathy. An MRI of the thigh and pelvic girdle muscles showed no obvious abnormalities.
The patient underwent a quadriceps skeletal muscle biopsy.Histologically,the muscle demonstrated fairly dramatic fibre-size variability with both atrophic/hypotrophic (‘small’) and hypertrophic fibres seen (including scattered fibre-splitting). Fibrosis was increased among both the endomysium and perimysium. The Gomoritrichrome staining revealed numerous subsarcolemmal and sarcoplasmic ‘red’ granules and rods, consistent with nemaline rods, primarily in the small fibres but also noted in the histologically ‘normal’ fibres.
Enzyme histochemistry demonstrated that the small fibres were type 1 (ATPase 4.2 and MHC-s immunohistochemistry) and that the type 2 fibres (and scattered type 1 fibres) were of relatively normal size. Overall, there was type 2 fibre predominance with many fibres co-expressing both slow (MHC-s) and fast (MHC-f) myosin heavy chains.
Prominent sarcoplasmic inclusions were noted among the atrophic fibres on semi-thin Toluidine blue-stained sections. Ultrastructural analysis demonstrated abundant and robust Z-line-like electron densities with lattice morphology. These were seen in highest density among the small fibres, but also noted among the more ‘normal’ appearing fibres.
. Generally, it is thought that in alpha-tropomyosin NEM cases, the nemaline inclusions are largely restricted to Type-1 fibres and that the majority of these fibres are atrophic/hypotropic (small).
Molecular genetics :
Clinicopathological correlation via genetic testing (Nemaline myopathy panel, Invitae) showed three Variants of Uncertain Significance (VUS) identified in MEGF10 , NEB and TPM3 , and all of them were heterozygous. The first was a missense VUS, c.2974G>A (p.Glu992Lys) identified in MEGF10 .The MEGF10 gene is known to be associated with autosomal recessive early-onset myopathy, areflexia, respiratory distress and dysphagia (EMARDD), but not nemaline rod histology on muscle biopsy. The second was asynonymous VUS, c.17619C>T (p.Gly5873=) identified in NEB . TheNEB gene is only known to be associated with autosomal recessive nemaline myopathy 2 (NEM2). The third was a missense VUS, c.43G>C (p.Asp15His) identified in TPM3 . This variant was not present in population databases including ExAC and gnomAD. Algorithms developed to predict the effect of missense changes on protein structure and function (SIFT, PolyPhen-2,Align-GVGD andMutationTaster) all suggest that this variant is likely to be disruptive. The TPM3 gene is associated with autosomal dominant or recessive NEM1and congenital myopathy with fibre-type disproportion (CFTD). Absence of a second causative variant in the autosomal recessive MEGF10 and NEB genes, suggested it is unlikely that these are causative variants. In contrast, genetic analysis confirmed that neither of the parents were carriers for the pathogenic TPM3 mutation in peripheral blood, suggesting a de novo mutation in the affected child in a gene with known dominant inheritance.