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Nemaline myopathy is one of the major subtypes of congenital myopathy and is known to be caused by mutations in nine genes including cofilin-2 (CFL2).1 Typical nemaline myopathy presentations include proximal weakness, hypotonia, respiratory difficulties and facial weakness.1
This study was approved by The University of Western Australia Human Research Ethics Committee with written consent from patients.
After an uneventful birth the proband was admitted to the intensive care unit (ICU) with respiratory distress, although he did not require mechanical ventilation and was discharged after 5 days. A few weeks later he was readmitted following recurrent episodes of apnoea lasting seconds, associated with cyanosis. Most episodes presented after feeding and were aborted by stimulation. Upon admission to ICU, hypotension and respiratory acidosis were detected and orotracheal intubation and mechanical ventilation initiated. After correcting metabolic disturbances, the patient remained hypotonic, though fully awake and alert when taken off sedation. Extraocular movements were estimated to be normal and deep tendon reflexes were present. Head circumference was on the third centile. Extubation failed and the patient required 24-h continuous ventilation support before passing away at 12 months. Nerve conduction studies, MRI, EEG and echocardiography were normal. An electromyograph (EMG) showed apparent myotonic discharges and serum creatine kinase (CK) was mildly elevated.
The proband was Saudi Arabian and his parents were first cousins (figure 1A). Two cousins of the proband (figure 1A III:3 and III.4) had a similar disease, both requiring 24-h mechanical ventilation. One cousin (III:3) passed away at 14 months, while the 10-month-old brother (III:4) is still on 24-h ventilation. Due to the severity of the disease in the proband, diagnostic mutation analysis of SMN1 and ACTA1 was performed, but both were excluded.
Muscle biopsy of the quadriceps was carried out on the proband. This demonstrated an increased variability in fibre size ranging from 3 µm to 15 µm diameter. Nemaline bodies were frequently seen (figure 1B). A pathological diagnosis of nemaline myopathy was therefore made. There was significant increase of fatty and fibrous connective tissue, signalling replacement of contractile tissue by connective tissue elements and dropped out myofibres. No features consistent with myofibrillar myopathy were observed.
To identify the molecular cause of the disease, either in one of the eight known nemaline myopathy genes not yet sequenced in the proband, or in a novel gene for nemaline myopathy, whole exome sequencing and data analysis were performed on DNA from the proband as described previously, including filtering against variants present in the 1000 Genomes Project and NIH Heart, Lung and Blood Institute (NHLBI) Exome Sequencing Project datasets.1 After filtering, 36 homozygous variants matched the autosomal recessive and consanguineous pattern of inheritance of the family. One of the homozygous variants was a 4 bp deletion (c.100_103delAAAG, p.Lys34Glnfs*6) in the known nemaline myopathy gene, cofilin-2 (CFL2, OMIM 601443). This variant was therefore the most likely cause of the disease in the family. Sanger sequencing confirmed the homozygous deletion in the proband as well as heterozygosity for the mutation in both parents and the consanguineous aunt and uncle (figure 1A II:3 and II:4) who had had the two similarly affected children (figure 1C). DNA was not available from the two affected cousins III:3 and III:4.
To investigate the effect of the putative disease-causing 4 bp deletion in CFL2, immunostaining of the proband muscle biopsy for cofilin-2 was performed. The cofilin-2 immunostaining indicated virtual absence of cofilin-2 compared with an unaffected adult and an aged-matched patient with non-CFL2 nemaline myopathy (figure 1D–F).
We report the identification of a novel homozygous null mutation in the CFL2 gene encoding the muscle specific actin-binding protein cofilin-2 in a Saudi Arabian consanguineous family, through the use of whole exome sequencing. The identified 4 bp deletion (c.100_103delAAAG, p.Lys34Glnfs*6) is inferred to delete a highly conserved lysine residue and shift the reading frame, introducing a premature stop codon in exon 2 of CFL2. This premature stop codon would likely induce nonsense mediated decay of the CFL2 mRNA.2 This would be consistent with the absence of cofilin-2 protein observed in muscle sections.
The 4 bp deletion removed one of two (AAAG)n microsatellite repeats and therefore most likely originated through repeat instability, where misalignment of repetitive DNA strands during DNA synthesis results in contractions or expansions.
Previously, only two CFL2-related nemaline myopathy families have been described, both harboured homozygous missense mutations in CFL2.3 ,4 Agrawal et al 3 associated the disease with cofilin-2 deficiency but suggested the protein deficiency was caused by misfolding and degradation of the abnormal protein instead of the nonsense-mediated decay we propose as the mechanism in the family we have studied. Features additional to nemaline bodies have been described in muscle biopsies of the two previously described families including occasional fibres with minicores, actin aggregation3 and also features consistent with myofibrillar myopathy.4 This suggests that CFL2 mutations may result in a wide spectrum of pathological phenotypes.
A recent Cfl2 knockout mouse model (Cfl2−/− ) displays similar histopathological features to patients with CFL2 mutations, reinforcing the association between cofilin-2 abnormalities and nemaline myopathy.5 Genetically, the homozygous deletion in the family we describe is more similar to the Cfl2−/− knockout mice 5 than the missense mutations previously described in the two other families.3 ,4 Clinically, the patients in the family reported here were more severely affected than the patients in the two published families, closer in severity to the disease in the Cfl2−/− mice, none of which survived past postnatal day 8. The proband presented with typical nemaline myopathy at birth but deteriorated to require 24-h ventilation, as did both affected cousins. In contrast, the patients harbouring the missense mutations did not require mechanical ventilation, with the eldest in each family in either their 20s or their teens.3 ,4 This suggests that CFL2 null mutations result in more severe disease than missense mutations, although the precise mechanism through which cofilin-2 abnormality results in nemaline myopathy remains elusive.
Our results highlight the ability of next generation sequencing to rapidly ascertain the genetic cause in heterogeneous diseases and extend the phenotype of CFL2-associated nemaline myopathy such that patients with severe nemaline myopathy should be screened for mutations in CFL2.
Contributors RWO contributed to the drafting and revision of the manuscript and for its content, study concept and design, analysis and interpretation of data and performed the molecular analysis of the patient. AAlS was the clinician in charge of the patient, led the initial investigations and clinical analysis into the patient and oversaw the review of the clinical information on the patient. DS performed pathology analysis for the patient, contributed to writing the pathology section of the manuscript and interpretation of histological data. AA contributed to the drafting and revision of the manuscript, drafting/revising the manuscript for content and analysis or interpretation of data. KSY developed the next generation sequencing analysis pipeline and contributed to drafting/revising the manuscript for content. GR performed the molecular analysis of the patient, contributed to drafting the manuscript, drafting/revising the manuscript for content and analysis and/or interpretation of data. RMD assisted in conceptualising the study and contributed to drafting/revising the manuscript for content. VA was responsible for performing next generation sequencing of patient's DNA and contributed to drafting/revising the manuscript for content. RJA oversaw the process of next generation sequencing and contributed to drafting/revising the manuscript for content. NGL conceptualised the study, had overall responsibility for coordinating the study, assisted with drafting and revision of the manuscript and analysis of data, drafting/revising the manuscript for content, including medical writing for content, study concept or design, analysis or interpretation of data.
Funding This research was supported by the National Health and Medical Research Council of Australia (Fellowships APP1035955 to GR and APP1002147 to NGL, and project grant APP1022707 to NGL).
Competing interests None.
Ethics approval The University of Western Australia.
Provenance and peer review Not commissioned; externally peer reviewed.