In this report we describe how a novel missense mutation in CFL2
causes a congenital myopathy with features of both MFM and NM in two siblings from a consanguineous Iraqi Kurdish family. To our knowledge, this is the second family with a congenital myopathy caused by a CFL2
Congenital myopathies are generally characterized by symptoms of hypotonia and muscle weakness, usually present at birth or in early infancy. Classification is based on clinical symptoms in combination with typical structural abnormalities of muscle fibers, visible on electron microscopic images of the muscle biopsy. However, it can be difficult to distinguish between different types of congenital myopathies as there is extensive clinical variability and pathological heterogeneity, often with multiple histological features in a single biopsy and overlap in findings amongst the various pathologically defined entities.
NM is a rare form of congenital myopathy, with an estimated frequency of 0.02 per 1000 live births [2
]. In NM, there is primary proximal muscle weakness. Patients show a delayed motor development. Fine motor skills are intact. Joint hypermobility is common. The respiratory muscles are always involved, which can cause nocturnal hypoxia and hypercapnia. Cardiomyopathy is seen in a small percentage of patients [17
]. NM patients have normal intelligence [2
]. In contrast, MFM patients present with progressive muscle weakness which can be distal, limb-girdle, scapuloperoneal or generalized in distribution. Cardiomyopathy is somewhat more common and sometimes precedes signs of muscle weakness. Selcen et al. [18
] reported cardiomyopathy in 10 out of 63 MFM patients (16%). Respiratory insufficiency is also a common feature. The group of MFMs varies markedly in clinical presentation and age of onset. However, most forms of MFM have their onset in adulthood and they are usually autosomal dominant in inheritance. Several gene defects are known to cause MFM, including mutations in the genes encoding for desmin, α-B-crystallin, myotilin, ZASP, filamin C, plectin, FHL1 and BAG3. Specific phenotypic features like cataract or polyneuropathy may contribute to the identification of the underlying genetic defect. For example, scoliosis, rigid spine and contractures are classical features of MFM caused by BAG3
Within our family intrafamilial variability is seen, with the older sister having an earlier onset of symptoms and a more progressive course. Both patients had predominant axial and limb girdle weakness. Their phenotypic features fit with NM or a childhood onset form of MFM. Specifically, the severe scoliosis and childhood-onset of myopathy in patient 1 is compatible with some forms of MFM such as those caused by mutations in BAG3
]. The two affected siblings with a CFL2
mutation reported by Agrawal et al. [5
] presented with hypotonia at birth, delayed early motor milestones and the inability to run. Their symptoms were slowly progressive with a predominant proximal muscle weakness and poor head control.
Since cofilin-2 is known to be expressed in cardiac muscle, it is noteworthy that no cardiac abnormalities were identified in either of our patients, or in the two previously reported siblings [5
]. However, all four patients are still relatively young and continued cardiac follow-up is clearly warranted. Since cofilin-1 is present at higher levels in murine cardiac muscle than in skeletal muscle [23
], it is plausible that this isoform at least partially compensates for cofilin-2 abnormalities in the heart, explaining the absence of cardiac disease at a young age.
Muscle imaging at the age of 12 in patient 1 revealed a pattern of involvement that seems most compatible with the description of MRI abnormalities in some forms of MFM [20
] and not with that of NM [22
The presence of many nemaline rods, predominance of type 1 fibers or type 1 fiber atrophy, and marked variance in fiber size in muscle biopsies are indicative of NM [1
]. Furthermore, some of the NM biopsies show core-like structures [12
]. The biopsy of patient 2 with many fibers having clusters of rods and type 1 predominance is consistent with a NM. MFM on the other hand can be viewed as a dystrophy [8
], characterized by significant myofibrillar disorganization. The classical pathological features of MFM comprise subsarcolemmal and sarcoplasmic protein aggregates which may include some nemaline rods, cytoplasmic bodies, rubbed-out fibers, and core-like structures [7
]. Type 1 predominance is only seen in 15% of MFM biopsies [19
]. Dystrophic features such as fiber splitting, internal nuclei, fatty infiltration and fibrosis may be seen later in the evolution of a nemaline myopathy [24
], but it is unusual to see it in such an extent as in the biopsy of patient 1, at 13 years of age.
The two siblings provide insight into potential evolution of the pathological findings as the first, somewhat older patient’s muscle exhibited a more dystrophic pattern with a disturbed sarcoplasm and accumulation of more heterogenous proteins. Not only desmin, which can be seen at the border of nemaline rods, but also myotilin, ZASP, dystrophin, sarcoglycans, dysferlin, caveolin-3 and cytoplasmic bodies are suggestive of a MFM. The younger patient exhibited a less dystrophic pattern, with fewer central nuclei, but desmin accumulation was already present and there was already some slight interstitial fibrosis. The findings in the biopsy of patient 2 taken at the age of 3 years are similar to those in the two patients reported by Agrawal et al. [5
] (biopsies taken at 2 and 4 years of age). All showed accumulations of rods and unstructured cores. These data suggest that the pathology associated with CFL2
mutations may evolve from a less dystrophic, NM phenotype at younger ages, towards one more closely resembling MFM with increasing age.
So far, mutations in seven different genes have been identified as a cause of NM [6
]. With the exception of KBTBD13
], whose protein localization remains unknown, all the NM genes encode for proteins that are a component of sarcomeric thin filaments. In MFM there is also considerable genetic heterogeneity; mutations in eight different genes are responsible for different forms of MFM [7
]. The Z-disk may be the link between the two groups of diseases; the MFMs consisting of myofibrillar disorganization beginning at the Z-disk [8
], whereas the NMs consist of accumulation of Z-disk material along defective thin filaments. As cofilin is also a component of thin filaments, we would argue that the present disorder should be considered a form of NM.
encodes the protein cofilin-2, that is present predominantly in skeletal and cardiac muscle. Cofilin-2 belongs to the actin-cofilin (AC) group of proteins that include actin-depolymerizing factor (ADF), also called destrin (DSTN), and cofilin-1. AC proteins are actin binding molecules that are essential for actin dynamics, apoptosis cascades, phospholipid metabolism, and gene expression [13
]. Cofilin-2 is an important regulator of actin dynamics [14
] and has recently been shown to be essential for muscle maintenance [26
]. We identified a novel missense mutation in exon 2 of CFL2
(c.19G>A, p.Val7Met), which results in an aminoacid change from Valine to Methionine. Since this is a change of an evolutionary highly conserved aminoacid, it is likely to have a negative effect on cofilin- 2 protein formation. In all AC proteins, Ser3 phosphorylation and dephosphorylation mark the association and dissociation of cofilin with actin. The close proximity of the Val7Met substitution to the Ser3 phosphorylation site may have an effect on cofilin phosphorylation and activity. In addition, since a Methionine is introduced by the mutation, it may be speculated that the translation of cofilin-2 may be shifted to this codon, resulting in a truncation of the six most N-terminal amino acids, including Ser3. Unfortunately, the biopsy tissue of our patients was not of sufficient quantity to perform any additional stainings or functional studies to test this hypothesis. Agrawal et al. [5
] propose that the A35T-CFL2
mutation they identified results in a misfolding or destabilization of the protein’s tertiary structure, which leads to a reduction in the amount of cofilin-2 in muscle tissue in vivo
In conclusion, we identified the cause of a rare congenital myopathy with features of both nemaline and myofibrillar myopathy in two siblings by applying homozygosity mapping followed by targeted gene analysis in the homozygous regions. Homozygosity mapping is a valuable diagnostic tool in families with congenital myopathies with autosomal recessive inheritance. To our knowledge, this is the second report of such a myopathy caused by a CFL2 mutation. Given the clinical variability and the multitude of histological features of congenital myopathies, CFL2 mutation analysis should be considered in patients presenting with an autosomal recessive form of congenital myopathy.