Genetic results in our MFM cohort (n = 43): known and novel pathogenic mutations
In 14 index patients, we identified a heterozygous pathogenic mutation in one of the nine genes causing MFM (Table
1), corresponding to a diagnostic yield of 37% in our MFM cohort. In ten patients we found the underlying gene defect using the initial Sanger sequencing study and in four patients by directly employing NGS panel diagnostics. Nine out of the 14 index patients had a positive family history corresponding with an autosomal dominant inheritance (for pedigrees see Figure
1), whereas the others occurred sporadically (Patient F6.1 with a
DES mutation, Patient F13.1 with a mutation in
BAG3 and all three patients, F9.1, F10.1 and F11.1, with a
MYOT mutation, Table
1). We identified six heterozygous mutations in
DES, four of these were the Arg350Pro mutation, two different mutations in
FLNC, and three
MYOT mutations, including two times the Ser60Phe mutation in exon 2. One mutation each was found in
ZASP,
CRYAB and in
BAG3 (Table
1, Figure
1). The mutation in
BAG3 represents a novel mutation, whereas the other mutations have been described previously.
Table 1
Clinical characteristics of myofibrillar myopathy patients with an identified mutation
F1.III.1
| M | + |
DES
| 6 | Arg350Pro | 39 | 47 | weakness LL | + | - | + | + | - | - | - | - | - | - | - | - | - | 1031 |
F2.III.4
| M | + |
DES
| 6 | Arg350Pro | 42 | 74 | weakness LL | - | - | + | + | - | + | - | - | - | - | - | - | - | 2690 |
F3.II.3
| M | + |
DES
| 6 | Arg350Pro | 48 | 59 | dyspnoea | + | + | + | + | + | - | - | - | - | VC 45%,NV | TAA intermittent, LV hypertrophy | - | + | 2044 |
F4.II.2
| M | + |
DES
| 6 | Arg350Pro | 36 | 42 | weakness LL | + | - | + | + | - | - | - | + | - | - | - | - | + | 1159 |
F5.II.2
| M | + |
DES
| 1 | Ser2Ile | 60 | 72 | syncopes | + | - | + | + | - | + | + | - | - | - | 3°AVB, PM | - | - | nl |
F6.1
| F | - |
DES
| 3 | Glu245Asp | 51 | 62 | weakness LL | - | - | + | + | - | - | - | - | - | - | DCM, AF, 3 °AVB, PM, TI | - | - | nl |
F7.IV.2
| M | + |
FLNC
| 18 | Val930_Thr 933del | 28 | 40 | weakness LL | - | - | + | - | - | - | + | - | - | - | - | - | - | 629 |
F7.III.4
| F | + |
FLNC
| 18 | Val930_Thr 933del | 53 | 60 | weakness LL | - | - | + | + | - | - | - | - | - | - | - | - | + | 388 |
F8.III.3
| F | + |
FLNC
| 48 | Trp2710X | 53 | 59 | dyspnoea | + | - | + | + | - | - | + | - | - | VC 35%,NV | - | - | - | 500 |
F9.1
| M | - |
MYOT
| 2 | Ser60Phe | 67 | 71 | dyspnoea | - | - | - | + | + | - | - | - | - | - | LBBB, HF, AF, 3°AVB intermittent, PM | ad/sm | - | 206 |
F10.1
| M | - |
MYOT
| 2 | Ser60Phe | 63 | 67 | myalgia LL | - | - | + | - | - | - | - | - | - | - | - | - | - | 589 |
F11.1
| M | - |
MYOT
| 2 | Ser55Phe | 55 | 60 | weakness LL | + | - | + | + | - | - | - | - | - | mild | AF, PM, ICD, DCM, HTx, | - | - | 608 |
F12.III.3
| F | + |
ZASP
| 6 | Ala165Val | 46 | 57 | walking difficulties | - | + | - | - | - | - | - | - | - | - | - | - | - | 182 |
F13.1
| M | - |
BAG3
| 3 | Pro209Gln | 34 | 43 | weakness LL | + | - | + | + | - | - | + | - | - | - | - | a/sm | - | 1050 |
F14.1
| M | + |
CRYAB
| 3 | Gly154Ser | 69 | 69 | rhabdomyolysis | - | - | - | - | - | - | - | - | - | VC 65% | - | - | - | 2000 |
The c.626C > A, p.Pro209Gln mutation in exon 3 of
BAG3 (Patient F13.1, Table
1) is so far not known in the databases of the NHLBI exome sequencing project (ESP) or the 1000 genome project and was predicted to be disease causing by all used prediction programmes. Further arguments for pathogenicity came from familial segregation analysis, in which both healthy parents did not harbour the mutation, indicating a
de novo mutation.
Genetic results in our MFM cohort (n = 43): polymorphisms and unclassified variants
With means of NGS panel diagnostics we found a large number of variants, especially in
FLNC. The suspicious variants with a minor allele frequency beneath 1% are listed in Additional file
1. We assessed these variants with various databases and prediction programmes. Especially the pathogenicity of the c.6595G > A, p.Gly2199Arg mutation in exon 40 of
FLNC (patient F20.1, Additional file
1) finally remained unclear. This genetic variant has only been counted once by the NHLBI exome sequencing project (ESP) and was predicted to be benign by PolyPhen-2, but disease causing by Mutation Taster. The glycin residue at position c.6595 is highly conserved, as well as the nucleotide at this position. Unfixed muscle tissue of this patient was not available anymore and no new muscle biopsy could be obtained, so that additional proteomic analyses for possible further clarification could not be performed. Moreover, family members were not available for segregation analysis to further clarify the pathogenicity.
Clinical findings in MFM patients with an identified gene defect (n = 14)
The 14 index patients with an identified mutation included eleven males and three females (Table
1). The age at onset ranged from 28 to 69 years (mean 49 years). The most common initial symptom was weakness in the legs (64%). In three patients the first symptom was dyspnoea (21%) and in one syncopes (7%), indicating a respiratory or cardiac onset of the disease. At the time of examination, the disease persisted already up to 32 years (mean disease duration 9 years). Muscle weakness was distributed in the distal and proximal lower limbs, combined with weakness in the proximal upper limbs in six of the 14 patients. Patients F3.II.3 and F6.1 were wheelchair bound since the age of 59 and 60 years respectively, both after 11 years of disease progression. Scapular winging was evident in 29% of the patients, 14% showed additional axial weakness and 14% a ptosis without ophthalmoparesis or facial weakness (Table
1). In 64% of the patients, atrophy of the distal lower limbs was present, among them the two patients with an additional PNP. EMG revealed a myopathic pattern in 58% and a mixed pattern in 42%, in three of the patients with a mixed pattern also pseudomyotonic discharges were present. The serum CK levels varied from normal to 16 fold elevated (mean: 5.5 N) (Table
1). Only patient F14.1 did not show permanent skeletal muscle weakness.
Multisystemic symptoms in the whole MFM cohort (n = 43)
In general, 16% of the patients included in the study (n = 43) had respiratory involvement and 60% presented one or more multisystemic symptom(s) (Table
2). The most striking finding was the large number of MFM patients with mainly a sensorimotor axonal-demyelinating polyneuropathy (12/43 or 28%), which occurred as frequently as cardiac disease (Table
2). In 50% (n = 6) of the MFM patients with a polyneuropathy, no other possible cause was present. However, another 33% (n = 4) suffered from diabetes mellitus and 17% (n = 2) had taken neurotoxic medications, as possible (additional) causes of a polyneuropathy. The PNP was diagnosed after 0–6 years of disease duration (mean: 1.7 years). 14% of the MFM patients suffered from hearing impairment, with a symptom onset between 16 and 80 years of age. Half of these patients used hearing devices. We did not detect diarrhoea, intestinal malabsorption or pseudoobstruction in our cohort.
Table 2
Multisystemic symptoms of all included MFM patients (n = 43)
Respiratory symptoms (6)
| restricted vital capacity | 6** | (1x DES, 1x FLNC, 1x MYOT, 1x CRYAB) |
ventilation assistance | 1 | |
nocturnal ventilation | 2 | (1x DES, 1x FLNC) |
Cardiac symptoms (12)
| atrial fibrillation | 8 | (1x DES, 2x MYOT) |
tachyarrhythmia absoluta | 1 | (1x DES) |
ventricular tachycardia | 1 | |
bradycardia | 1 | |
ventricular extrasystoles | 1 | |
3° AV block | 3* | (2x DES, 1x MYOT) |
LBBB | 2 | (1x MYOT) |
RBBB | 1 | |
bifascicular block | 1 | |
tricuspid insufficiency | 2 | (1x DES) |
aortic/mitral/tricuspid insufficiency | 1 | |
heart failure | 3 | (1x MYOT) |
LV hypertrophy | 3 | |
DCM | 2 | (1x DES, 1x MYOT) |
pace maker | 6 | (2x DES, 2xMYOT) |
ICD | 1 | (1x MYOT) |
heart transplantation | 1 | (1x MYOT) |
sudden cardiac death | 0 | |
Polyneuropathy (12)
| axonal | 3 | (1x BAG3) |
demyelinating | 0 | |
axonal-demyelinating | 9 | (1x MYOT) |
sensory | 0 | |
sensory + autonomic | 1 | |
motor | 1 | |
sensorimotor | 10 | (1x MYOT, 1x BAG3) |
Bulbar symptoms (8)
| dysphagia | 5 | (1x DES) |
dysphonia | 2 | (1x DES) |
Hearing impairment
| | 6 | (2x DES, 1x FLNC) |
Gynaecomastia
| | 1 | |
Multisystemic symptoms in MFM patients with an identified mutation (n = 14 and affected relatives)
In the group of index patients with a pathogenic mutation (n = 14) 64% showed at least one multisystemic symptom (Figure
1, Tables
1 and
2). Patient F3.II.3 harbours the Arg350Pro mutation in
DES and presented with exertional dyspnoea as initial symptom at 48 years of age. Subsequently, he developed dyspnoea at rest, had a vital capacity (VC) of 45% of the theoretical value and needed non-invasive continuous positive airway pressure (CPAP) ventilation at night. Furthermore, he presented an intermittent tachyarrhythmia absoluta and a hypertrophic left ventricle at echocardiography. He also suffered from bilateral hypacusis and used a hearing aid on the right side since the age of 52 years.
Patient F4.II.2 carried the same Arg350Pro mutation in DES and recognised swallowing problems after six years of disease progression. He also presented a bilateral hypacusis since the age of 38 years.
Only the affected mothers of two more patients with the Arg350Pro mutation in DES (patient F1.III.1; F2.III.4) showed multisystemic symptoms (respiratory insufficiency, cardiac involvement with a pace maker implantation, polyneuropathy).
Patient F6.1 carried the Glu245Asp mutation in DES and exhibited cardiac symptoms: a dilated cardiomyopathy was diagnosed approximately ten years after disease onset and a chronic atrial fibrillation and complete atrioventricular block occurred, necessitating pace maker implantation.
The patient carrying the Ser2Ile mutation in DES (patient F5.II.2) presented with syncopes as the first symptom at the age of 60 years, due to a complete atrioventricular conduction block leading to pace maker implantation. At the age of 65 years, he developed mild distal lower leg weakness and dysphonia. Other causes of dysphonia were excluded. Interestingly, the patient’s father also had dysphonia and a pacemaker implantation at the age of 58 years. The patient’s brother, carrying the same mutation, received a pacemaker at the age of 45 years and had mild distal involvement of the lower legs, but no dysphonia.
Patient F8.III.3 with the Trp2710X mutation in FLNC developed respiratory problems three years before weakness in the extremities occurred. After five years of disease progression, the patient was dependent on non-invasive nocturnal ventilation. VC decreased to 35%.
The affected mother of the index patient harbouring the Val930_Thr933del mutation in FLNC (patient F7.III.4) presented with bilateral hearing impairment and hearing aids at the age of 60 years.
Patient F9.1 carrying the Ser60Phe mutation in MYOT developed exertional dyspnoea due to heart failure at the age of 64 years as the first symptom. A left bundle branch block, an intermittent atrioventricular conduction block and atrial fibrillation were diagnosed, and a pace maker was implanted. The patient also suffered from coronary disease. Four years later, a sensorimotor axonal-demyelinating polyneuropathy was diagnosed.
In Patient F11.1 with a Ser55Phe mutation in MYOT, cardiac symptoms began shortly after skeletal weakness became apparent at the age of 55 years. He had a permanent atrial fibrillation with tachyarrhythmia absoluta and received an implantable cardioverter defibrillator (ICD). In addition, he had several myocardial infarctions due to coronary disease. One year later, a dilated cardiomyopathy was diagnosed and heart transplantation was performed.
Patient F14.1 carries the p.Gly154Ser mutation in CRYAB and presented at the age of 69 years with several episodes of rhabdomyolysis. The patient also complained about exercise intolerance and fatigue. The neurological examination was normal, but a respiratory involvement with a VC of 65% in lying position was diagnosed. His 42-year-old son, carrying the same mutation, was asymptomatic and only showed a moderate serum CK elevation.
Histological findings of the MFM cohort
In the 14 index patients with an identified mutation we consistently found an increased variability of fibre diameter (100%), frequent nuclear bags (64%), internal nuclei (86%), vacuoles (64%), rimmed vacuoles (50%) and increased endomysial connective tissue (79%). In 43% of the biopsies, cytoplasmic bodies and core-like lesions on the NADH-TR staining were revealed. Myofibrillar disorganisation was seen in all cases, but characteristic protein aggregations in the cytoplasm of mGT stained fibres were found in only 10 of the 14 cases (71%) (Patient F11.1: in the vastus medialis muscle biopsy protein aggregations were seen, but not in the simultaneously obtained tibialis anterior muscle biopsy). So 29% only showed typical MFM findings at the ultrastructural level (EM was performed in 11 of the 14 cases). Z-disk streaming and myofibrillar disorganisation were seen in 100%, granulofilamentous material in 64%. Abnormal mitochondria (64%), tubulofilamentous accumulations (29%) and cytoplasmic bodies (36%) were other frequent findings. Immunhistochemistry with antibodies directed against desmin showed immunoreactivity in all 8 performed cases.
In the group of 24 patients, in whom the underlying genetic defect had not been identified, we made similar observations. Characteristic protein aggregations in the cytoplasm of mGT stained fibres were found in 76% of the cases, myofibrillar disorganisation in 82%.
In ten of the 12 MFM patients additionally presenting a polyneuropathy, we observed neurogenic muscle atrophy, which found expression in angular shaped muscle fibres and fibre type grouping.
Clinicopathological phenotype of the patient with a novel BAG3 mutation
Patient F13.1 harbours the novel Pro209Gln mutation in
BAG3 (Table
1)
. He presented first symptoms of distal lower limb weakness and symmetrical calve atrophy at the age of 34 years. The skeletal muscle weakness spread to the proximal lower limbs and finally to the proximal upper limbs with scapular winging after 2 more years of disease progression. Moreover, he developed an axonal sensorimotor polyneuropathy, as multisystemic symptom of MFM. No other obvious cause for the PNP could be found. The PNP was first diagnosed at the age of 39 years and finds expression in decreased vibration sense and ataxic, clumsy gait as well as an increased sensitivity for cold. EMG showed a mixed pattern and the maximum CK level was 1050 U/L (Table
1). The muscle biopsy showed desmin positive protein deposits, vacuoles and core-like lesions as well as some necrotic fibres. At the ultrastructural level, tubulofilamentous accumulations, lobulated nuclei and glycogen accumulations were seen, in addition to typical MFM findings like Z-disk streaming and the accumulation of granulofilamentous material. We did not observe regenerating fibres or apoptotic nuclei. The ultrastructural study of nerve tissue revealed no giant axons.