Novel SPEG Mutations in Congenital Myopathy without Centralized Nuclei

Congenital myopathies define a group of clinically and genetically heterogeneous rare muscle diseases. They cause neonatal or early onset hypotonia and/or muscle weakness, and the muscle biopsies of affected individuals exhibit characteristic structural defects [1, 2]. Recessive mutations in the SPEG gene, encoding the striated muscle preferentially expressed protein kinase (SPEG), were previously reported in five families with centronuclear myopathy (CNM) and dilated cardiomyopathy [3, 4]. SPEG interacts with myotubularin (MTM1), mutated in X-linked CNM (also called myotubular myopathy), but its cellular functions are barely understood. In this report, we describe a peculiar case with biallelic truncating SPEG mutations, but without histopathological CNM hallmarks and without cardiomyopathy, demonstrating that recessive SPEG mutations can give rise to myopathies with non-specific features on muscle biopsy.

The proband is a 10-year-old girl born to non-consanguineous healthy parents. The pregnancy was uneventful. At birth, the patient had hypotonia without respiratory distress, and poor sucking. Motor milestones were delayed with independent sitting at 11 months and walking at 30 months of age. Cognitive development was normal. The patient developed a scoliosis around age 4 treated since with a back brace. Reduced myocardial contraction without ventricular dilatation appeared at age 5 and progressively normalized with digoxin and captopril treatment. At the age of 6 years, non-invasive ventilation was started to treat obstructive sleep apnea and alveolar hypoventilation. No supraclavicular, intercostal, subcostal or intercostal retractions were observed. Forced vital capacity was above 70%. Clinical investigation at 9 years of age revealed proximal and distal muscle weakness, positive Gowers’ sign and facial weakness without ptosis or external ophthalmoplegia (Fig. 1A). The patient showed hypotonic pes planus and complained about difficulties climbing stairs. Maximal walking distance was 300 meters. Retractions were not noted. Swallowing was normal, but chewing difficulties required an enteral gastrostomy feeding from age 9 years and 10 months to prevent malnutrition. Serum creatine kinase levels were repeatedly normal. MRI scans of the thighs and lower legs was performed twice at age 3 and 5 years, and revealed a mild and non-progressive atrophy of the vastus lateralis, the gastrocnemius, and the soleus muscles (Fig. 1B). EMG at 1 year of age did not reveal any abnormalities. Motor examination was performed for the median, ulnar, common peroneal and tibial nerves, and sensory examination was performed for the median and ulnar nerves. Conduction velocities, amplitudes and distal latencies were normal, and intramuscular EMG from the tibialis anterior muscles showed an interferential pattern. A quadriceps muscle biopsy was performed at 16 months, and histological analyses of H&E, NADH-TR, SDH, PAS, Oil Red O, ATPase stains revealed hypotrophy of both fiber types and discrete fiber size heterogeneity (Fig. 1C). Typical CNM features such as centralized nuclei, necklace fibers, or type I fiber predominance were not observed.

Genetic counselling was provided, and informed consent was obtained for genetic analysis. The patient’s DNA was sequenced for a targeted panel of 136 genes implicated in neuromuscular disorders, but no causative mutation was identified. Thus, exome sequencing was performed for the affected child and her healthy parents. Variants were filtered based on their frequency in gnomAD and an in-house control database composed of 1550 exomes, and ranked with our in-house bioinformatics pipeline. Data analysis revealed compound heterozygous SPEG mutations segregating with the disease. We identified an out-of-frame duplication of 4 nucleotides in exon 4 (c.1071_1074dup; p.Lys359Valfs*35), predicted to induce a premature stop codon on the maternal allele, and a nonsense mutation in exon 20 (c.4399C>T; p.Arg1467*) on the paternal allele (Fig. 1D). None of these variants was listed in the gnomAD and ClinVar databases.

To date, SPEG mutations were reported in five families with severe neonatal hypotonia and muscle weakness [3, 4]. A single patient deceased shortly after birth, while all others exhibited delayed motor milestones. Facial weakness and eye involvement were noted in the majority of patients, and abnormal respiration requiring ventilation was reported in 2 families [3, 4]. A dilated cardiomyopathy was diagnosed in the surviving patients with exception of the youngest patient of 3 years of age. Histologically, all biopsies of the published cases revealed prominent central nuclei and some showed necklace fibers. Overall, both clinical and histological findings were similar to X-linked centronuclear myopathy resulting from MTM1 mutations [5, 6], with the exception of the cardiac involvement.

Our patient is the oldest and mildest reported patient with SPEG mutations. Although presenting with neonatal hypotonia and delayed motor milestones, she reached independent walking before the age of three, did not show eye involvement and did not develop a dilated cardiomyopathy. Noteworthy, her muscle biopsy did not reveal internal or central nuclei nor necklace fibers. Similarly, one previously reported MTM1 patient displayed no central nuclei on muscle biopsy, highlighting that mutations in CNM genes might cause an atypical muscle morphology [7].

MRI revealed a similar pattern for the published and new SPEG patients with predominant involvement of the anterior compartment of the thigh and of the posterior compartment of the lower legs [4]. Our patient shows a mild picture compared to previously described SPEG cases, correlating with the mild phenotypic presentation.

All SPEG mutations previously associated with the severe CNM-like muscle disorder are evenly distributed nonsense or frameshift mutations predicted to induce a premature stop codon resulting in nonsense-mediated mRNA decay or alternatively the translation of a truncated protein. Both mutations found in our patient have not yet been described and involve a premature stop codon, predicted to remove the myotubularin binding domain located between the SPEG residues 2530 and 2674 [3].

The SPEG locus encodes at least four differentially expressed isoforms [8]. Among the murine SPEG protein isoforms, SPEGα and SPEGβ were shown to be the main isoforms present in skeletal and cardiac muscles. The only reported patient without cardiomyopathy carries a homozygous truncating mutation in exon 4, predicted to affect SPEGβ but not SPEGα thereby suggesting that the presence of SPEGα might be sufficient to protect from the development of a cardiomyopathy. Accordingly, our patient carries one of the truncating mutations in exon 4 and did not develop a cardiomyopathy. However, murine and human isoforms might be different, and this is substantiated by the human gene expression databases (www.gtexportal.org/). To better understand the pathomechanisms of SPEG mutations, further characterization of the human SPEG isoforms in human muscle and heart is necessary. It is nevertheless possible that patients with the milder phenotype will develop a dilated cardiomyopathy at later stages.

Strikingly, the muscle biopsy of our patient is the only one without central nuclei, potentially correlating with the disease severity. Of note, the mutation in exon 20 (c.4399C>T; p.Arg1467*) is one of the most distal mutation reported and might impact differently on protein stability and thereby explain the comparably milder phenotype in our patient.

Overall, our study expands the phenotype of congenital myopathies associated with recessive SPEG mutations. Specific SPEG mutations can induce a severe CNM-like or, as described in this study, a rather moderate congenital myopathy without central nuclei. This is of major diagnostic importance, as SPEG mutations should be considered in patients with congenital hypotonia even in the absence of central nuclei.

The authors thank the family who participated in this study, Nicolas Dondaine who analyzed the results of the MYOdiagHTS panel and Gratiela Mac-Caby who performed MRI studies. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), University of Strasbourg, the France Génomique National infrastructure, funded as part of the Investissements d’Avenir program managed by the Agence Nationale pour la Recherche (ANR-10-INBS-09) and by Fondation Maladies Rares whithin the frame of the “Myocapture” sequencing project, ANR-10-LABX-0030-INRT under the frame program Investissements d’Avenir ANR-10-IDEX-0002-02, Fondation pour la Recherche Médicale, and AFM-16992 and CREGEMES for the MYOdiagHTS project.