1 INTRODUCTION

Hereditary myosin myopathies are a group of muscle disorders with variable age of onset and heterogeneous clinical features, caused by mutations in the skeletal muscle myosin heavy chain (MyHC) genes which are organized in a multigene cluster on chromosome 17 (Oldfors, 2007). In adult human skeletal muscle, there are three major MyHC isoforms: MyHC I (slow/ß-cardiac MyHC) encoded by MYH7 (OMIM *160760) and expressed in slow type 1 muscle fibers and in heart ventricles; MyHC IIa, encoded by MYH2 (OMIM *160740) and expressed in fast type 2A and 2B muscle fibers, and MyHC IIx, encoded by MYH1 (OMIM *160730) and expressed only in fast type 2B muscle fibers (Oldfors, 2007). MYH7 mutations have been associated with either hypertrophic or dilated cardiomyopathy, pure skeletal myopathies, and a combination of myopathy and cardiomyopathy (Oldfors, 2007). So far, MYH1 has not been associated to any genetic disease.

Mutations in MYH2 have been associated to myopathies with both dominant and recessive autosomal transmission (OMIM #605637). The autosomal dominant (AD) MyHC IIa myopathy is characterized by congenital joint contractures that resolve with time, late-onset and progressive proximal limb muscle weakness, ophthalmoplegia, and ptosis. The reduction in the number and size of type 2A fibers together with rimmed vacuoles are the main features of muscle biopsy (Cabrera-Serrano et al., 2015; D’Amico et al., 2013; Martinsson et al., 2000). The autosomal recessive (AR) MyHC IIa myopathy is characterized by mild, non-progressive, early onset muscle weakness, mild facial involvement, ophthalmoplegia and, occasionally, ptosis. Muscle biopsy shows a complete loss of 2A fibers along with unspecific myopathic changes such as fiber size variability, internalized nuclei, and interstitial fatty infiltration (Hernandez-Laın, Esteban-Perez, Domınguez -Gonzalez, & Cantero Montenegro, 2017; Lossos et al., 2005, 2013; Tajsharghi et al., 2010, 2014; Tsabari, Daum, Kerem, Fellig, & Dor, 2017; Willis et al., 2016).

Recently, a patient with homozygous recessive MYH2 mutation and phenotypic features consistent with the AD form was described (Findlay, Harms, Pestronk, & Weihl, 2018), opening the route to a possible new genotype-phenotype correlation.

The case we describe here shows overlapping features with the abovementioned case report (Findlay et al., 2018), thus contributing to widen the clinical and genetic spectrum of MYH2 myopathies, in particular of the AR form. It also broadens the knowledge of these very rare muscle diseases of which only few cases have been reported so far, globally scattered (Cabrera-Serrano et al., 2015; D’Amico et al., 2013; Hernandez-Laın et al., 2017; Lossos et al., 2005, 2013; Martinsson et al., 2000; Tajsharghi et al., 2010, 2014; Tsabari et al., 2017; Willis et al., 2016).

2 METHODS

3 RESULTS

3.2 Histopathological analysis and muscle MRI

The patient underwent two biopsies of the biceps brachii muscle. The first one, performed when he was 17 years old, showed scattered mildly hypotrophic fibers and many internal nuclei (Figure 1a). The second one, performed at the age of 39 years, showed rimmed vacuoles, severe fiber size variability, scattered necrotic fibers, fatty infiltration, and few 2B fibers (Figure 1b,d). ATPase staining and immunofluorescence for myosin isoforms showed marked predominance of type 1 fibers and few type 2 fibers. The latter were type 2B fibers, co-expressing both MyHCIIa and MyHCIIx (Figures 2b,d,f, and 1d). No type 2A fibers have been detected. Ultrastructural examination of the first biopsy showed scattered nuclei indentations (Figure 3a); the second biopsy showed rimmed vacuoles, chaotic myofibrillar organization, and myofilament nuclear inclusions (Figure 3b–d).

A muscle MRI was performed at the time of the second biopsy when the patient was 39. Axial TSE T1 and STIR T2 sequence targeted to pelvic girdle, lower limbs, and right arm were obtained. The study showed diffuse fibro-adipose substitution that was particularly overt in lumbar paraspinal muscles and in the gluteus maximus. In thighs, fibro-adipose replacement was prominent in the medial and posterior compartments (mostly in adductor longus and semitendinosus), while quadriceps were relatively spared. In calves, soleus and gastrocnemius were deeply substituted by fibrous tissue. Triceps brachii was the most involved muscle in the arm. No inflammation was detected in any of the examined muscles (Figure 4).

3.3 Genetic analysis

Genetic analysis for oculopharingeal muscular dystrophy (PABPN1), hereditary inclusion body myopathy (GNE), facio-scapulo-humeral dystrophy (D4Z4 fragment length screening), for late-onset Pompe disease (GAA), myotonic dystrophy type 1 and 2 (DMPK and ZNF9) resulted normal. NGS targeted gene panel was used to investigate the coding regions of 58 genes responsible for non-syndromic muscle disorders and led to the identification of two novel truncating mutations in MYH2 (ref. seq.: NM_017534) in compound heterozygosity: c.2377C>T/p.Arg793Ter in exon 21 and c.4381G>T/p.Glu1461Ter in exon 32. Mutations were confirmed by Sanger sequencing in the patient and his parents: the mother was heterozygous for c.2377C>T and the father was heterozygous for c.4381G>T (Figure 5). Clinical examination of both parents was normal: they did not show any kind of muscle involvement.

We analyzed the relative expression of MyHCI, MyHCIIa, and MyHCIIx transcripts in a muscle sample from the second brachial biceps biopsy as previously described (Tajsharghi et al., 2002). In the patient MyHCI mRNA was predominant (92.8%), whereas MyHCIIa and MyHCIIx mRNAs represented the 3% and the 4.2%, respectively (mean of three independent PCRs; cDNA was from two independent reverse transcriptions; Figure 2g). In the control the distribution of the three mRNAs was different: 22.4% of MyHCI mRNA, 38.3% of MyHCIIx Mrna, and 39.3% of MyHCIIa transcript (sample: brachial biceps). Sequencing of MyHCIIa cDNA was performed to evaluate the presence of nonsense-mediated mRNA decay (NMD) and revealed the presence of both mutated alleles.

4 DISCUSSION

Myosin heavy chain IIa myopathies are very rare diseases and exact data on prevalence and incidence are not well known: to date, 21 AD (Cabrera-Serrano et al., 2015; D’Amico et al., 2013; Martinsson et al., 2000) and 34 AR (Hernandez-Laın et al., 2017; Lossos et al., 2005, 2013; Tajsharghi et al., 2010, 2014; Tsabari et al., 2017; Willis et al., 2016) cases have been reported. Clinical and morphological phenotypes are continuously updated by new reports.

Recently, a case was reported by Findlay and colleagues (Findlay et al., 2018) describing a patient with homozygous p.Arg246His mutation in the MYH2 gene that affects the myosin motor domain and shows rimmed vacuoles and marked myopathic alterations at muscle biopsy. Residual MyHCIIa expression was demonstrated in hybrid fibers. The clinical phenotype was characterized by ophthalmoplegia and late-onset progressive muscle weakness.

Similar clinical and pathological findings were present also in our patient (see Table 1). Disease onset in adulthood, progressive muscle weakness, elevated CK levels, ophthalmoplegia, mild facial weakness, and histological features recall those described by Findlay and colleagues. Moreover, the absence of congenital contractures and the pattern of diffuse weakness are characteristics more similar to those described for AR patients. The first EMG revealed complex repetitive discharges and sporadic neuromyotonic discharges, no longer present in the second EMG: neuromyotonic discharges have never been described in myosin diseases and are characteristics of other neuromuscular diseases excluded in our patient; these findings were probably aspecific. Although muscle biopsy showed rimmed vacuoles, dystrophic changes, and fibro-fatty substitution that are typical of AD cases, we detected also the absence of type 2A fibers as in biopsies of AR patients so far described. However, our patient has two truncating mutations that led to the synthesis of a residual mutated protein that determines a histopathological picture similar to that of AD patients, as described up to now.

Our patient carried two new heterozygous truncating mutations: p.Arg793Ter, which is located in the myosin motor domain and interrupts the protein 1148 amino acids before the natural stop codon, and p.Glu1461Ter, which interrupts the coiled coil rod domain 480 amino acids before the stop codon. Surprisingly, despite double truncating mutations, our patient expressed minor amounts of MYH2 mRNA (Figure 2g) that led to the synthesis of residual myosin IIa in type 2B fibers, as shown by immunofluorescence (Figure 2b).

The NMD pathway is able to select and degrade mRNAs harboring premature termination codons (PTCs), so it was possible that both mutated alleles were degraded by NMD, but this is in contrast with the results of immunofluorescence assay. At first, we hypothesized that the allele carrying p.Arg793Ter was degraded by NMD and that the residual protein shown by the immunofluorescence was produced by the other mutated allele, which although mutated was able to produce a reduced amount of truncated and non-functional, or partially functional, protein that can be detected by the MyHCIIa antibody. Sequencing of skeletal muscle mRNA ruled out this hypothesis, showing the presence of both MYH2 mutated alleles, although in smaller amounts than the control (patient: 3% vs. control: 39%), as shown in Figure 2g. The low amount of both mutated MyHCIIa mRNAs in presence of two truncating mutation is probably due to an incomplete efficiency of NMD: some mRNAs escape degradation and are translated. Indeed, a different NMD efficiency is documented due to the distance between PTC and exon-junction complex, the number of exon-junction complexes downstream the PTC, and the exon sequence downstream PTC; furthermore, the NMD efficiency is variable in different tissues and in different cells (Hoek et al., 2019; Linde, Boelz, Neu-Yilik, Kulozik, & Kerem, 2007; Zetoune et al., 2008).

Tajsharghi and colleagues first hypothesized the pathogenic role of the mutated protein in AD patients, arguing that clinical symptoms are linked to the amount of protein present in muscle during development and growth: that is, joint contractures, typically present at birth, are due to the high expression of MyHCIIa in fetus, whereas the further downregulation of the protein in childhood explains the resolution of contractures (Tajsharghi et al., 2002). MyHCIIa expression increases in adulthood, particularly in extraocular muscles, explaining the adult-onset of ocular palsy and weakness of proximal muscles (Cabrera-Serrano et al., 2015; D’Amico et al., 2013). Rimmed vacuoles are typical of the AD form and selectively occur in muscle fibers expressing high levels of MyHCIIa (Tajsharghi et al., 2002).

Our case and the previous one by Findlay demonstrate that AR patients can show a clinical phenotype partially overlapping with AD patients (progressive myopathy, ptosis, severe ophthalmoparesis) and a muscle histopathology similar to that described in these patients (rimmed vacuoles and disruption of myofibrils organization in the sarcomere). This is probably due to the combination of the lack of WT MyHCIIa expression and the residual production of mutated proteins.

We had the outstanding opportunity to follow-up this patient for over two decades both from the clinical and the morphological point of view, recording a unique disease history. At the clinical examination the chronic progression was evident and was also confirmed by the two muscle biopsies: electronic microscopy performed in both samples showed how the slow accumulation of abnormal myosin protein had progressively subverted the sarcomeric arrangement up to a complete myofibrillar disorganization.

The lack of a specific morphological pattern and the absence of congenital joint contractures made this patient's early diagnosis difficult. Clinical-pathological reports are of pivotal importance to collect information that can guide and help physicians and researchers to broaden their knowledge of these rare diseases and to investigate in depth the pathogenetic and pathological mechanism.

In conclusion, the phenotype of MyHCIIa myopathy, that so far seemed exist in two well-established and rather different forms, could be more similar to a continuum of symptoms between two extremes that nowadays are classified as the AD and AR forms. This report wishes to reinforce physicians and geneticist attention on this disease whose presentation could vary from classical patterns.

CONFLICT OF INTEREST

Authors declare absence of any conflict of interest.

AUTHORS’ CONTRIBUTION

RT, SP, LP contributed to the conception and writing of the work; AL and LP contributed to the clinical evaluation of the patient; SP, DC, and FMS contributed to genetic analysis; PC, GF, and NG contributed to pathological processing of muscle biopsy; GC and CC contributed to imaging studies; GPC and MS contributed to the final revision of the work. All Authors have approved the submitted version and have agreed both to be personally accountable for the author's own contributions.