We present the clinical, morphological and molecular data of an Italian family with centronuclear myopathy, carrying a novel pathogenic mutation of BIN1 gene in heterozygous state, consistent with autosomal dominant inheritance. The proband, a 56-years-old man suffered of lower limbs myalgia and slight CK elevation. Clinical examination revealed no muscle weakness, short stature, mild symmetric eyelid ptosis, scapular winging, ankle retraction and well-developed muscles. Muscle biopsy showed nuclear centralization and clustering, deep sarcolemmal invaginations and type 1 fibers hypotrophy. Muscle MRI revealed fatty infiltration of posterior legs compartments, lumbar paraspinal and serratus muscles. By sequencing BIN1, we identified a heterozygous pathogenic mutation [c.107C>A (p.A36E)], and we demonstrate that the mutation strongly impairs the membrane tubulation property of the protein. One affected sister with similar phenotype carried the same mutation. Our findings expand the clinical, morphological and genetic spectrum of the autosomal dominant CNM associated with BIN1 mutations.
Centronuclear myopathies (CNM) are a group of congenital myopathies characterized by prominent nuclear centralization in muscle fibers [1]. Distinctive clinical and histopathological features have been associated to the different genes so far: X-linked severe neonatal ‘myotubular myopathy’ and mild late onset ‘necklace fibers myopathy’ are due to myotubularin gene mutations (MTM1) [2, 3], autosomal dominant early onset CNM with frequent and distinctive ‘radiating sarcoplasmic strands’ (RSS) (“spoke-of-wheels”) is linked to dynamin-2 (DNM2) [4], and autosomal recessive and dominant CNM with particular ‘central nuclei clustering’ is caused by mutations in the gene coding for amphiphysin-2 (BIN1) [5, 6].
The proband is a 56-years-old male. He has three siblings, and his parents were not related. The mother died at 60 years from hepatic failure and the father, who was reported having gait disturbances since his sixties, died at 80 years. Pregnancy and delivery were uneventful. Motor development was normal. He started to complain about lower limbs myalgia and discomfort at the age of 52. Clinical examination revealed mild bilateral eyelid ptosis, scoliosis, scapular winging, Achilles tendons retraction and pes cavus, short stature and well-developed muscles. Muscle weakness was not detected. Cardiac and respiratory assessments were normal. CK level was 2–5 fold normal value. EMG showed myopathic changes and pseudo-myotonic discharges. Whole body MRI revealed selective fatty replacement of posterior lower limbs compartment, which was predominant in the distal part of the hamstring muscles with sparing of the semitendinosus (Fig. 1A). The only mildly affected muscle in the upper limbs was the serratus.
The deltoid muscle biopsy revealed increased centralization and internalization of myonuclei, which were occasionally found in central clusters (Fig. 1C). Several fibers showed vacuolization around central nuclei (Fig. 1G) which were often strongly stained by PAS (Fig. 1F), and some fibers presented deep sarcolemmal invaginations (Fig. 1D). Oxidative staining disclosed moderate type I fiber atrophy, while RSS were not observed (Fig. 1E).
Myotonic Dystrophy type1 and type2, Pompe Disease, and MTM1 and DNM2-related CNMs were ruled out. BIN1 sequencing (NM_139343.2) by targeted NGS panel using the MiSeq platform (Illumina, San Diego, California, USA) revealed a heterozygous c.107C>A (p.A36E) missense mutation in exon 1 not listed in the human variation databases. The array-Comparative Genomic Hybridization (aCGH) study did not show any duplications/deletions in the whole genome. Regarding the investigated BIN1 gene it was covered by five oligos. The Log Ratio average in that region was in accordance to a normal profile.
DNA samples from the parents were unavailable, but segregation analysis of siblings detected the same BIN1 c.107C>A (p.A36E) mutation in one of the three sisters who is now aged 65 years. She reported lower limbs discomfort since the age of 60 and difficulties in climbing stairs. Clinical examination revealed short stature, calf hypertrophy, Achilles and hand finger flexors retractions. Mild weakness of posterior thigh compartment (MRC4/5) was detected. Patient was unable to rise up from the floor. CK levels were normal. EMG was not performed. Muscle MRI revealed a similar but more severe involvement of the lower limb muscles compared to the brother (Fig. 1B). Patient refused to undergo muscle biopsy.
The c.107C>A (p.A36E) mutation affects a highly conserved amino acid in the N-terminal amphipathic helix, and is in close proximity to previously reported recessive (K35N) and dominant (DelK21, R24C) BIN1 mutations (Fig. 1K) [5, 6]. In order to assess the impact of the BIN1 c.107C>A (p.A36E) mutation on the protein function, we generated and transfected wild type and mutant GFP-BIN1 constructs into COS-1 cells. In cultured cells, exogenously expressed wild type BIN1 promotes membrane tubulation in 86.3% of the transfected cells (Fig. 1H) [5, 6]. In contrast, the BIN1 construct harboring the p.A36E mutation did not induce tubulation in any transfected cell (Fig. 1J). This is in accordance with previously reported heterozygous BIN1 mutations as R24C (Fig. 1I) [6], and represents a strong functional proof for the pathogenicity of the p.A36E mutation in BIN1.
In contrast to the previously reported dominant BIN1-related CNM cases [6], our patients had a later disease onset and milder clinical phenotype revealed by lower limbs myalgias and mild CK elevation. Previously reported patient had a disease onset between 22 and 50 years. Lower limb muscle weakness was prominent in all reported patients (3 had waddling gait, 3 required a cane and 3 were wheelchair dependent) and muscle MRI revealed a predominant lower limb involvement without a specific pattern. By contrast disease onset in our patients was at the age of 52 and 60 in the proband and the sister respectively. None of them had gait difficulties and only the sister had a mild muscle weakness in lower limbs. Nevertheless, disease predominantly involves the lower limbs also in our patients, as previously observed [6]. Indeed, even if subclinically in the proband, the muscle MRI detected a lower limb involvement in both patients. The first and most affected muscles were biceps femoris (long head) and semimenbranosus in the thighs, and the gastrocnemius (medial head) in the legs. The more severe involvement in the sister might be age-related. By contrast, both our patients complained about myalgias and lower limb discomfort, but only one patient of the previously described families had the same. Concerning patients’ father, even if he had gait disturbances since his sixties and the family mutation cause a very mild disease that could be started late in the life course, we cannot prove that his gait difficulties arose from BIN1 mutation.
Muscle biopsy revealed prominent nuclear centralization with clusters of myonuclei, perinuclear vacuolization, deep sarcolemmal invaginations, and type1 hypotrophy. All this findings are typical of BIN1 related CNM, as previously reported both in dominant and recessive forms. However, unlike DNM2 or certain case of dominant BIN1–related CNM, RSS in our patient were not detected [1, 6].
The BIN1 c.107C>A (p.A36E) mutation segregates with the disease and affects a highly conserved aminoacid in the N-terminal amphipathic helix. Amphiphysin 2, encoded by BIN1, is implicated in T-tubule biogenesis in skeletal muscle and thereby plays a central role in excitation-contraction coupling [7]. The amphipathic helix induces membrane curvature and specific missense mutations repress the amphiphysin 2 membrane tubulating properties [5, 6]. Accordingly, we observed a strong impact of the BIN1 A36E mutation on membrane tubulation, supporting the hypothesis that the disease arises at least in part from membrane remodeling defects in muscle. Our findings confirm that specific BIN1 mutations cause either dominant or recessive CNM, and expand the clinical and genetic spectrum of BIN1-related CNM.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
