Abstract
Most myotilinopathy patients present with a dominant late onset distal phenotype and myofibrillar pathology, although the first MYOT mutation in a family reported to have LGMD phenotype. We report here a French family affected with a late onset proximal and distal muscle weakness and myofibrillar myopathy on muscle pathology, in which the siblings known to be clinically affected were homozygous for the c.179C>T (p.Ser60Phe) myotilin gene mutation. One subjectively asymptomatic member of the family was heterozygous for this mutation. This is the first report of a family with patients being homozygous for a known dominant MYOT mutation. Dominant negative mutations are generally considered not to cause a more severe disease in homozygosity, but our data clearly demonstrate the existence of dominant MYOT mutations with a possible dose effect causing a more severe disease phenotype in homozygosity in the spectrum of myofibrillar myopathies (MFM).
INTRODUCTION
Myotilin is a sarcomeric protein localized at the myofibrillar Z-discs, and exclusively expressed in skeletal and cardiac muscle. It is a 498 amino acid peptide encoded by a single copy gene (MYOT) with 10 exons located on chromosome 5q31 [1]. Myotilin plays a significant role in sarcomere assembly, acting together with α-actinin and filamin C to cross-link actin into tightly packed bundles. Dominant mutations in MYOT were originally described in limb-girdle muscular dystrophy type 1A (LGMD1A) [2] and subsequently identified more frequently in patients with late onset myofibrillar and distal myopathy [3, 4], as well as in a family reported with spheroid body myopathy [5]. Eight different dominant MYOT mutations have been identified [6], most of them in exon 2 (Ser60Cys, Ser60Phe, Ser55Phe, Thr57Ile, Lys36Glu, Ser39Phe, Ser95Ile), and one in exon 9 (Arg405Lys) [7]. These known dominant mutations give rise to some degree of variation of the clinical and pathological phenotype [8], regarding proximal or distal muscle involvement, muscle atrophy, ankle contracture, late cardiomyopathy, or peripheral neuropathy [9]. One case with generalized pseudohypertrophy and stiffness has been described, which is very much outside the general spectrum [10]. A novel mutation in the extreme N-terminus p.Arg6Gly was reported as a recessive mutation with a late onset LGMD phenotype and myofibrillar pathology [11].
To our knowledge, only 14 patients with the c.179C>T (p.Ser60Phe) mutation have been reported [4, 13]. We describe here a French family with a late onset proximal and distal muscle weakness in which the affected siblings clinically characterized with a limb-girdle phenotype were homozygous for the c.179C>T (p.Ser60Phe) mutation, and one sibling heterozygous for this mutation was subjectively asymptomatic with a subclinical myopathy onexamination.
MATERIALS AND METHODS
We have studied a French family with five siblings by neurological examination (III-2, III-3, II-3, III-4 and III-5), serum CK level assessment, electrophysiological studies (III-2 and III-5), and muscle Magnetic Resonance Imaging (MRI) in four patients (III-1, III-3, III-4 and III-5). All individuals gave their informed consent prior to their inclusion in the study.
Muscle biopsy was performed in patients III-2 and III-5. Patient III-2 had a muscle biopsy at 51 years of age while in patient III-5 two muscle biopsies were performed at ages 53 and 56. Muscle histology was assessed by routine histochemistry H&E, modified Gomori trichrome and PAS, enzyme histochemistry NADH-TR and SDH as well as immunohistochemistry using antibodies directed against myotilin and desmin. Semi-thin sections were applied in all three biopsies, and electron microscopy was performed in the latest biopsy in patient III-5.
Mutation screening and genotyping
Genomic DNA was extracted using standard procedures. Exon 2 of MYOT gene (NM_006790) was amplified by PCR (PCR Master Mix, Fermentas, St. Leon-Rot, Germany) and directly sequenced using the Big-Dye Terminator v3.1 kit on ABI3130xl automatic DNA sequencer system (Applied Biosystems, Foster City, CA, USA). The primers used were: forward, 5’-CAAGGAAGAGCAGACCAAGG-3’ and reverse, 5’- ATTGCCAGTACCCTGCTT-3’. Sequences were analyzed with Sequencer 4.10.1 software (Gene Codes Corporation, Ann Arbor, MI, USA). Haplotype analysis was performed with microsatellite markers D5S1995, D5S396, D5S816, D5S479, D5S414, D5S2009, spanning some 5.7 Mb area around the MYOT gene. Fluorescently labeled PCR products were analyzed using ABI3730xl DNA Analyzer and GeneMapper v4.0 software (Applied Biosystems).
Targeted next-generation sequencing was performed to patients III-1 and III-5 with SeqCap EZ Choice Library (Roche NimbleGen) which was designed to target exons of 236 myopathy related genes [14]. The targeted genes are listed in Supplementary data Table 1. DNA capture and paired-end sequencing with read length of 150 bp was performed at the Functional Genomics unit (FuGU) in Biomedicum Helsinki using Illumina MiSeq. Reads were processed following the Genome Analysis Toolkit best practices.
RESULTS
The pedigree of the family is shown in Fig. 1. Four of the siblings were referred to neurological examinations because of lower limb muscle weakness. Their sister III-1 was primarily reported to be healthy. Both parents were deceased and they were not related. Reportedly the father (II-1) had walking difficulties at age 60, but in absence of examination data the underlying cause remains elusive. Their mother (II-2) died at age 70 with cancer. No gait difficulties were reported.
Clinical, EMG and MRI data
The proband (III-5) developed gait difficulties with weakness in lower limbs at the age of 40. When first examined at age 51 he had a clear proximal and a mild distal weakness. At age 56 his walking distance was 500 meters, and at age 59 he needed a stick for walking. Deep tendon reflexes were present. Speech was not affected. CK level was mildly elevated (415 IU/l, normal range 40–250). EMG showed a myopathic pattern with small polyphasic motor unit potentials and early recruitment; there were no signs of neuropathy or neurogenic abnormality. Electrocardiogram and echocardiography examinations were normal. Muscle MRI demonstrated severe fatty degenerative changes in all gluteal muscles, vasti, soleus and tibial anterior, with relatively preserved rectus femoris, semitendinosus, gracilis and sartorius and gastrocnemius lateralis.
His brother (III-2) developed progressive gait difficulties at the same age as the proband. At age 52, neurological examination showed weakness in proximal upper and lower limbs, and distally in upper limbs. CK level was slightly elevated (289 IU/l). He became more severely disabled and was wheelchair-dependent at age 66. Both brothers were clinically diagnosed with limb-girdle musculardystrophy.
Two of their sisters (III-3 and III-4) developed gait difficulties later. Patient III-3 reported onset of proximal weakness at age 67 with difficulty to climb stairs. At age 72, she used a stick for walking, and had lower limb proximal weakness and very mild proximal upper limb weakness. CK level was normal (117 IU/l). Muscle MRI at age 72 showed almost total dystrophic fatty replacement in all hamstring, adductor magnus and longus muscles in the thigh and in soleus, medial gastrocnemius and anterior compartment muscles in the lower leg, with a proximo-distal gradient (Fig. 2a and b).
Patient III-4 reported gait difficulties from age 60 and examinations 7 years later showed a waddling and steppage gait, with hip extension (3-/5), knee extension-flexion (4/5), ankle extensors deficit (1/5), and a milder upper limb deficit (proximal and distal 4/5). She had difficulties getting up from the chair without using her arms and needed a walker also for short distance. CK level was 325 IU/l. Muscle MRI performed at age 67 demonstrated subtotal dystrophic fatty replacement in all hamstring and adductor magnus muscles on the thigh, total replacement in almost all lower leg muscles sparing only the lateral compartment peroneal muscles and right lateral gastrocnemius muscle (Fig. 3a and b).
Patient III-1 was subjectively asymptomatic. However, MRI performed for this study at age 75 demonstrated clear mild-moderate myopathic changes with fatty degeneration in all three gluteal muscles, biceps femoris, semimembranosus and adductor magnus muscles (Fig. 4) (lower legs were not included in the study). CK level was normal. She could walk on tiptoes and on heels and ankle function testing was 5/5.
None of the affected members exhibited dysphonia.
Muscle biopsy findings
Deltoid muscle biopsy performed at 51 years of age in III-2 showed rare small groups of atrophic mostly angulated fibres. Deltoid muscle biopsy performed at age 53 in III-5 showed a few fibres containing small subsarcolemal inclusions, eosinophilic on H&E and dark blue on Gomori trichrome. These inclusions proved to be spheroid bodies on semithin sections. Another biopsy was performed at age 56 on the left vastus lateralis muscle, showing more clear signs of myofibrillar myopathy with more fibers containing spheroid bodies (Fig. 5a) and irregular oxidative enzyme stains (Fig. 5b). Immunohistochemistry with anti-myotilin antibodies revealed the typical focal accumulations and irregular distribution of myotilin in the affected myofibers (Fig. 5c).
Ultrastructural analysis revealed areas of myofibrillar destruction, accumulation of myofibrillar material with Z line streaming together with disorganized material containing autophagic vesicles and spheroid bodies (Fig. 6).
Molecular genetics
Candidate gene studies by direct Sanger sequencing in patient III-5 based on the pathology diagnosis of myofibrillar myopathy (MFM) showed no mutation in DES and ZASP genes. However, sequencing of MYOT exon 2 showed homozygosity of the previously known mutation c.179C>T (p.Ser60Phe) (Fig. 7).
Further genetic assessment of all siblings showed that subjects III-2, III-3 and III-4 were all homozygous for the MYOT c.179C>T(p.Ser60Phe) mutation. The subjectively asymptomatic sister (III-1) was heterozygous for the same mutation. No genetic study could be performed in the parents.
Haplotype analysis of a 5.7 Mb area around the MYOT gene showed only one allele present in the 4 homozygotes whereas two alleles were seen in patient III-1 (Table 2). To ensure the homozygosity, targeted next generation sequencing (NGS) was performed on patients III-5 and III-1 to exclude the possibility that only one allele was amplified in Sanger sequencing because of poor primer design. In accordance with haplotype analysis, c.179C>T (p.Ser60Phe) mutation was homozygous in NGS data and no heterozygous SNPs were seen in the area covered by the microsatellite markers. No other variants that could be considered as a modifying mutation or that could explain the differences in the phenotypes of homozygous patient III-5 and heterozygous patient III-1 were detected. Target regions covered <20X are listed in the Supplementary data Table 2.
DISCUSSION
We report the first family with patients being homozygous for a known dominant MYOT mutation, c.179C>T (p.Ser60Phe). Three main phenotypes have previously been described with dominant myotilin mutations: LGMD1A with dysphonia in two published families [13, 15], spheroid body myopathy with LGMD phenotype, dysphonia and respiratory failure [5], and late onset MFM presenting clinically in most patients with a distal myopathy phenotype [4]. Various clinical phenotypes have been described in patients with c.179C>T (p.Ser60Phe) mutation: distal myopathy, predominant proximal weakness and mixed proximal and distal weakness patterns. Some variability was also reported regarding age of onset although always late: after age 40 years or very late, after age 70 years. The pathology was always identified as MFM with rimmed vacuoles.
In our family there was a mixed pattern of proximal and distal muscles weakness in the homozygous patients. In all these patients the onset of weakness was after 40 years of age. In the homozygous sisters III-3 and III-4, the onset of weakness was reported to be later than in the brothers and after age 60, but the extensive dystrophic fatty replacement found in muscle MRIs suggests the onset must have been decades before the actual MRI study. Notably, the heterozygous sister III-1 was subjectively healthy at the age of 75 years as asymptomtic patients reported in the literature [4] and her muscle MRI changes were definitely much milder compared to the muscle MRI findings in the homozygous patients although she was older. CK level was normal in the heterozygous patient and mildly elevated in the homozygotes.
The haplotyping and NGS results strongly indicate that the detected homozygosity is real, because the other alternative, deletion of at least 5.7 Mb, would most likely have more severe consequences than the phenotype seen in these patients. No possibly modifying or other pathogenic mutations were detected in NGS analysis. The interest of this family with homozygous patients, relates to the understanding of the pathogenic molecular mechanism of MYOT mutations in exon 2. The disease in the homozygotes is not more severe than what has been reported earlier in heterozygosity with c.179C>T (p.Ser60Phe), but the clearly more severe outcome of the homozygosity compared to heterozygosity in this family suggests there is a dose effect of the mutant protein, and that the dominant effect in heterozygosity cannot be just inhibition of the functions of the normal allele. Similar dose effect was reported in the literature [16-18] These patients would constitute a category of double dominant mutation effects in the spectrum of MFM.