Introduction
Myofibrillar myopathies (MFM) are a group of protein aggregate myopathies (PAM) sharing the histological features of Z-disk dissolution, myofibrillar degeneration, and accumulation of degradation products into protein aggregates [
1‐
4]. The clinical spectrum is wide and consists mainly in progressive muscle weakness of upper and/or lower limbs; limb-girdle and scapuloperoneal phenotypes can be observed as well as involvement of hand, facial, pharyngeal, and respiratory muscles [
1‐
4]. Cardiomyopathy, peripheral neuropathy, and cataract are frequent associated conditions [
1‐
4]. The diagnosis is established by muscle biopsy which shows as main morphological hallmark, abnormal fibers containing amorphous material of irregular shape and size positive to several proteins including αB-crystallin, desmin, and myotilin [
1‐
3]. MFM are usually transmitted as an autosomal dominant trait; however, X-linked, autosomal recessive and sporadic cases have been described [
1‐
4]. Causative mutations have been identified in a minority of patients in one of the following genes: desmin (
DES), αB-crystallin (
CRYAB), myotilin (
MYOT), Z-band alternatively spliced PDZ-containing protein (
LBD3/ZASP), Bcl2-associated athanogene-3 (
BAG3), and filamin C (
FLNC) [
1‐
5]. Recent mutations in
FHL1,
TTN,
DNAJB6,
PLEC,
ACTA1,
HSPB8,
LMNA,
KY,
PYROXD1, and
SQSTM1 have also been reported in patients featuring MFM pathology highlighting the variability and complexity of these muscular disorders [
5].
We performed whole-exome sequencing (WES) in four patients with PAM from three unrelated families in whom classical genetic approach had failed to identify a causative mutation in one of the known MFM causing genes.
Discussion
This study provides evidence that (1) p.(Arg89Cys) LMNA mutation causes a myopathy featuring PAM pathology, (2) RYR1 mutations may be an additional cause of autosomal dominant PAM with an unusual phenotype of tibial myopathy, and (3) the p.(Cys30071Arg) mutation in TTN gene responsible for hereditary myopathy with early respiratory features (HMERF) is associated with cardiac involvement.
Lamins are intermediate filament proteins that constitute the nuclear lamina, a structure that underlies and provides mechanical support to inner nuclear membrane [
25,
26]. Beside the structural role, nuclear lamins are involved in chromatin organization and transcription regulation as well as in physical connection between the nucleus and the cytoskeleton [
25,
26].
LMNA encodes for lamin A and lamin C that results from alternative splicing of exon 10; lamins A and C are expressed in most differentiated cells [
25,
26]. Despite mutations in
LMNA have been associated with a heterogeneous group of diseases, known as the “laminopathies,” four main clinical phenotypes affecting skeletal and cardiac muscle have been reported including limb-girdle muscular dystrophy type 1B (LGMD1B), the Emery-Dreifuss muscular dystrophy (EDMD), congenital muscular dystrophy (MDCL), and dilated cardiomyopathy [
25,
26]. Proband of Family 1 and her daughter harbored the same p.(Arg89Cys) mutation in
LMNA. The mutation was previously reported in a patient with EDMD who had a different clinical phenotype and without pathological description [
22]. Both members of our family clinically presented with arrhythmogenic cardiomyopathy and developed a proximal limb muscle weakness only in adulthood while in EDMD patient clinical disease onset was in early childhood with selective muscle involvement of the lower limbs and elbows contractures, cardiac conduction defects occurred at a later age. There was also no evidence of joint contractures in the mother and her daughter. MFM pathology has been reported in patients with
LMNA myopathy but the occurrence is extremely rare and never associated with the described mutation [
27,
28]. Interestingly, the brother of the proband who had a late-onset limb-girdle clinical phenotype was found to not carry the
LMNA variant suggesting a mutation in a different gene or a sporadic or acquired myopathy. In addition, the patient did not develop an early arrhythmia unlike the proband and her daughter.
The
RYR1 gene encodes the skeletal muscle ryanodine receptor, a calcium channel that resides in the junctional sarcoplasmic reticulum (SR) membrane and, by interacting with the dihydropyridine receptor located on the T-tubules, releases calcium from the SR to the sarcoplasm in response to an action potential, triggering muscle contraction [
29]. Autosomal dominant mutations in
RYR1 have been classically reported in patients with susceptibility to malignant hyperthermia (MH) and congenital central core disease (CCD) [
30,
31]. Other RYR1-related phenotypes with both autosomal dominant and recessive inheritance pattern include multiminicore disease (MnD), centronuclear myopathy (CNM), congenital fiber type disproportion (CFTD), and King-Denborough syndrome (KDS) [
30,
31]. Proband of family 2 was found to carry heterozygous missense mutation p.(Asn4807Phe) in
RYR1, a variation which maps to the C-terminal MH3 domain of the protein. Several lines of evidence suggest that the
RYR1 variant identified in our patient is disease-causing; the variant was previously reported in a patient with CCD [
32], it occurred at highly conserved positions, and it is predicted to have a damaging effect. Unfortunately, segregation analysis could not be performed because the proband’s father was dead and his old mother lived in a different region of the country. The onset and distribution of muscle weakness in our patient involving only distal lower limbs in the fourth decade of life is quite different from that observed in individual patients carrying the same
RYR1 variant [
32] or the adjacent p.(Phe4808Asn) mutation [
33‐
35] in which muscle weakness was proximal starting during early childhood [
32,
34,
35] or there was only a susceptibility to MH without clinical myopathy [
33]. In addition, he had a relevant cardiac history with atrial fibrillation which required ablation. Despite
RYR1 expression in cardiac muscle is limited, recently rare patients with cardiac involvement have been described [
30].
Finally, we studied an Italian family and found the previously reported p.(Cys30071Arg) mutation in the
TTN gene [
23,
24].
TTN encodes the giant protein titin which spans through half of the sarcomere, with N- and C-terminal regions located at the Z-disk and M-line, respectively, and which is involved in thick filaments assembling and stabilization [
36]. Titin sequence is characterized by fibronectin type III (FN3) and immunoglobulin-like modules that are repeated and organized in complex sequence arrangements [
36]. The missense p.(Cys30071Arg) mutation occurs in exon 343 encoding for the FN3 domain 119 of the titin A-band, a region of the protein that establishes strong interactions with the thick filaments providing a molecular template for their assembling and represents the most common mutation in patients with HMERF, reported in more than 20 families [
23,
24,
37‐
41]. The clinical manifestations and the histological features described in our family are consistent with HMERF phenotype, including autosomal dominant inheritance, adult onset, weakness in proximal, distal and respiratory muscles, and MFM pathology [
23,
24,
37‐
41]. The significant involvement of cardiac muscle in our patients represents a new aspect of the disease. The proband died suddenly and cardiac investigations documented severe sinus tachycardia; furthermore, his sister developed heart conduction defects including several episodes of supraventricular paroxysmal tachycardia. HMERF does not appear to be associated with major cardiac involvement [
42] but our observations suggest that cardiac surveillance should be recommended in patients carrying this mutation.
Additionally, we identified multiple VUS in other genes linked or not with myopathy besides the potentially causative mutations (Table
S1). Novel variants in
DCTN1,
MYH14, and
RBM20 were identified in Family 1 with cardiac and limb-girdle muscle involvement and in
TRIM63,
ACTC1, and
TTN in the proband of family 2 with a distal lower limb phenotype and atrial fibrillation. Clinical significance of these additional variants remains speculative and needs to be elucidated; they might contribute to the variability of the phenotype due to a synergistic or an antagonistic effect [
43]. Alternatively, it has been suggested that the cumulative burden of variants might affect the functioning of target tissue modulating the penetrance and/or the expression of clinical phenotype [
5].
In summary, whole-exome analysis in our PAM patients identified novel variants in known disease genes, a novel candidate disease gene, and documented phenotypic expansion for known causative genes.
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