MYO-MRI diagnostic protocols in genetic myopathies

Whole-body magnetic resonance imaging has emerged as a useful imaging tool in diagnosing and characterizing the progression of myopathies and muscular dystrophies. Whole-body MRI indications and diagnostic efficacy are becoming better defined with the increasing number of cases, publications and discussions within multidisciplinary working groups. Advanced Whole-body MRI protocols are rapid, lower cost, and well-tolerated by patients. Accurate interpretation of muscle Whole-body MRI requires a detailed knowledge of muscle anatomy and differential pattern of involvement in muscle diseases. With the surge in recently identified novel genetic myopathies, Whole-body MRI will become increasingly useful for phenotypic validation of genetic variants of unknown significance. In addition, Whole-body MRI will be progressively used as a biomarker for disease progression and quantify response to therapy with the emergence of novel disease modifying treatments. This review outlines Whole-body MRI indications and updates refined protocols and provides a comprehensive overview of the diagnostic utility and suggested methodology of Whole-body MRI for pediatric and adult patients with muscle diseases.     

Sub-cellular localisation of fukutin related protein in different cell lines and in the muscle of patients with MDC1C and LGMD2I

MDC1C and LGMD2I are two allelic forms of muscular dystrophies caused by mutations in the gene encoding for fukutin related protein (FKRP). FKRP encodes for a putative glycosyltransferase, the precise function of which is unknown. However, the marked reduction of -dystroglycan glycosylation in the muscle of MDC1C and LGMD2I patients suggests a role for FKRP in dystroglycan processing. Using a polyclonal antibody raised against FKRP we now show that endogenous FKRP locates to the Golgi apparatus of neuronal, oligodendroglial, and the cardiac muscle cell line H9c2. In differentiated C2C12 myotubes and in transverse sections of normal skeletal and cardiac muscle, endogenous FKRP surrounded the myonuclei. This localisation was unaffected in the skeletal muscle of patients with MDC1C and LGMD2I carrying various FKRP mutations. These observations imply a specific role for FKRP during striated muscle, neuronal and glial development and suggest that protein mis-localisation is not a common mechanism of disease in FKRP-related dystrophies.     

A novel missense mutation in POMT1 modulates the severe congenital muscular dystrophy phenotype associated with POMT1 nonsense mutations

Mutations in POMT1 lead to a group of neuromuscular conditions ranging in severity from Walker–Warburg syndrome to limb girdle muscular dystrophy. We report two male siblings, ages 19 and 14, and an unrelated 6-year old female with early onset muscular dystrophy and intellectual disability with minimal structural brain anomalies and no ocular abnormalities. Compound heterozygous mutations in POMT1 were identified including a previously reported nonsense mutation (c.2167dupG; p.Asp723Glyfs*8) associated with Walker–Warburg syndrome and a novel missense mutation in a highly conserved region of the protein O-mannosyltransferase 1 protein (c.1958C>T; p.Pro653Leu). This novel variant reduces the phenotypic severity compared to patients with homozygous c.2167dupG mutations or compound heterozygous patients with a c.2167dupG mutation and a wide range of other mutant POMT1 alleles.     

Long-term clinical and MRI follow-up in two POMT2-related limb girdle muscular dystrophy (LGMDR14) patients

IntroductionPOMT2-related limb girdle muscular dystrophy (LGMDR14) is a rare muscular dystrophy caused by mutations in the POMT2 gene. Thus far only 26 LGMDR14 subjects have been reported and no longitudinal natural history data are available.Case reportWe describe two LGMDR14 patients followed for 20 years since infancy. Both patients presented a childhood-onset, slowly progressive pelvic girdle muscular weakness leading to loss of ambulation in the second decade in one patient, and cognitive impairment without detectable brain structural abnormalities. Glutei, paraspinal, and adductor muscles were the primarily involved muscles at MRI.DiscussionThis report provides natural history data on LGMDR14 subjects, with a focus on longitudinal muscle MRI. We also reviewed the LGMDR14 literature data, providing information on the LGMDR14 disease progression. Considering the high prevalence of cognitive impairment in LGMDR14 patients, a reliable application of functional outcome measures can be challenging, therefore a muscle MRI follow-up to assess disease evolution is recommended.     

Myopathology in times of modern imaging

Over the last two decades, muscle (magnetic resonance) imaging has become an important complementary tool in the diagnosis and differential diagnosis of inherited neuromuscular disorders, particularly in conditions where the pattern of selective muscle involvement is often more predictive of the underlying genetic background than associated clinical and histopathological features. Following an overview of different imaging modalities, the present review will give a concise introduction to systematic image analysis and interpretation in genetic neuromuscular disorders. The pattern of selective muscle involvement will be presented in detail in conditions such as the congenital or myofibrillar myopathies where muscle imaging is particularly useful to inform the (differential) diagnosis, and in disorders such as Duchenne or fascioscapulohumeral muscular dystrophy where the diagnosis is usually made on clinical grounds but where detailed knowledge of disease progression on the muscle imaging level may inform better understanding of the natural history. Utilizing the group of the congenital myopathies as an example, selected case studies will illustrate how muscle MRI can be used to inform the diagnostic process in the clinico‐pathological context. Future developments, in particular, concerning the increasing use of whole‐body MRI protocols and novel quantitative fat assessments techniques potentially relevant as an outcome measure, will be briefly outlined.     

A novel MYH7 mutation links congenital fiber type disproportion and myosin storage myopathy

This study aimed to identify the genetic defect in a multigenerational family presenting an autosomal dominant myopathy with histological features of congenital fiber type disproportion. Linkage analysis and genetic sequencing identified, in all affected members of the family, the c.5807A > G heterozygous mutation in MYH7, which encodes the slow/β-cardiac myosin heavy chain. This mutation causes skeletal but not cardiac involvement. Myosin heavy chain expression pattern was also characterized by immunohistochemistry, western blot and q-PCR in muscle biopsies from two patients aged 25 and 62, respectively. While only congenital fiber type disproportion was observed in the younger patient, older patient’s biopsy presented aggregates of slow myosin heavy chains, in fiber sub-sarcolemmal region. These clinico-pathologic findings suggest a novel phenotype within the emerging group of hereditary myosin myopathies, which in this family presents typical characteristics of congenital fiber type disproportion in early stages and later evolves to myosin storage myopathy.     

Fukutin mutations in non-Japanese patients with congenital muscular dystrophy: less severe mutations predominate in patients with a non-Walker-Warburg phenotype

Six genes including POMT1, POMT2, POMGNT1, FKRP, Fukutin (FKTN) and LARGE encode proteins involved in the glycosylation of α-dystroglycan (α-DG). Abnormal glycosylation of α-DG is a common finding in Walker-Warburg syndrome (WWS), muscle-eye-brain disease (MEB), Fukuyama congenital muscular dystrophy (FCMD), congenital muscular dystrophy types 1C and 1D and some forms of autosomal recessive limb-girdle muscular dystrophy (LGMD2I, LGMD2K, LGMD2M), and is associated with mutations in the above genes. FCMD, caused by mutations in Fukutin (FKTN), is most frequent in Japan, but an increasing number of FKTN mutations are being reported outside of Japan. We describe four new patients with FKTN mutations and phenotypes ranging from: severe WWS in a Greek-Croatian patient, to congenital muscular dystrophy and cobblestone lissencephaly resembling MEB-FCMD in two Turkish patients, and limb-girdle muscular dystrophy and no mental retardation in a German patient. Four of the five different FKTN mutations have not been previously described.     

Characteristic form of standing up from squatting in Miyoshi's distal muscular dystrophy

A 27-year-old woman with Miyoshi’s distal muscular dystrophy devised a unique form of standing up from a squatting position; She held her ankles with her hands to support the weight transfer, fixed the heels, extended the knees to elevate the hips, raised the upper half of the body, and finally stood up. This strategy illustrates the characteristic and specific distribution of the wasted muscle in this disease.     

Serial MRI changes in a patient with infantile Alexander disease and prolonged survival

Alexander disease is a major entity of leukodystrophy; magnetic resonance imaging (MRI) studies of the brain typically show extensive changes in the cerebral white matter with frontal predominance. Heterozygous missense mutations of GFAP are thought to be sufficient for the molecular diagnosis, which has widened the Alexander disease entity beyond the classical one. We report the patient, a 16-year-old Japanese boy, with infantile-onset Alexander disease, showing striking MRI findings; extreme white matter loss of cerebrum through cerebellum, severe atrophy of basal ganglia, cerebellum, brain stem, and cervical spinal cord. Molecular analysis showed a heterozygous mutation R239L (c.730G > T) in GFAP. A relative long disease course, over 15 years, with the help of mechanical ventilation revealed the striking MRI progression.     

Mutation and haplotype analysis of oculopharyngeal muscular dystrophy in Thai patients

Oculopharyngeal muscular dystrophy (OPMD) is an inherited neuromuscular disease associated with a short trinucleotide repeat expansion in Exon 1 of the PABPN1 gene. OPMD is uncommon in East Asian populations, and there have been no previous reports of Thai patients. We studied clinical and molecular genetic features of six unrelated Thai patients with autosomal dominant OPMD. All patients had expansions of the guanine-cytosine-guanine (GCG) repeat ranging from three to seven additional repeats in the PABPN1 gene. Haplotype analysis showed that these mutations might have originated independently. Analysis of the size of the GCG repeat in the PABPN1 gene in 200 Thai control patients showed that 0.5% of the control subjects possessed (GCG)₇, thereby suggesting that the prevalence of autosomal recessive OPMD in the Thai population was approximately 1 in 160,000. In conclusion, our data suggest that OPMD in Thailand may be more common than previously thought.     

Improvement of the diagnostic procedure in proximal myotonic myopathy/myotonic dystrophy type 2

Proximal myotonic myopathy/myotonic dystrophy type 2 (PROMM/DM 2) is caused by an expansion of the (TG)_n(TCTG)_n(CCTG)_n repeat tract in intron 1 of the ZNF9 gene located on chromosome 3q21. Because these expansions show a marked mitotic instability, expanded alleles are often difficult to detect. In order to improve the diagnostic procedure, we applied a combination of pulsed-field gel electrophoresis and semi-quantitative Southern blot analysis with a novel hybridization probe. The combination of these methods led to unequivocal results in about 98% of cases with a clinical diagnosis of PROMM/DM 2. Furthermore, we report the genotype/phenotype correlation in a patient lacking a normal ZNF9 allele and a further proband with a grey zone allele.     

Ultrastructure of the skeletal muscle in the X chromosome-linked dystrophic (mdx) mouse

Summary Ultrastructurally there are some clear differences in the pathology of muscle in X chromosomelinked muscular dystrophy of the mouse (mdx) and Duchenne muscular dystrophy (DMD). In particular the mouse muscle does not become infiltrated by large aggregations of connective tissue. It has been proposed that the differences are due to secondary biochemical changes consequent on the absence of dystrophin in both conditions. If this is the case, attention should be directed to the earliest ultrastructural changes held in common by both disorders. The most conspicuous of these, preceding myofibril breakdown, is dilation of the sarcoplasmic reticulum. Any physiological link between this and the absence of dystrophin remains to be determined. We suggest that in themdx mouse, the widespread myofibre necrosis occurring at 3–4 weeks is triggered by increased mechanical demands causing the lack of dystrophin to become critical at this time. Subsequent regeneration of the myofibres appears to be almost completely successful. The ultimate failure of regeneration in DMD, in contrast, may be due to an additional factors acting in DMD exacerbating the lack of dystrophin. This additional factor may be associated with the plasma membrane lesions (not seen inmdx). Alternatively there may be factors present in the mouse that compensate for the lack of dystrophin. It is pointed out that to understand better the different processes occurring inmdx and DMD we need to learn more about the factors which control the balance between the growth of muscle and the growth of connective tissue in both normal and pathological human and mouse muscle.Supported by the Muscular Dystrophy Group of Great Britain and the Medical Research Council     

Skeletal muscle of mice with a mutation in slow α-tropomyosin is weaker at lower lengths

Skeletal muscle function was measured in anaesthetised transgenic mice having a mutation in the TPM3 gene (slow alpha-tropomyosin), a similar mutation as found in some patients with nemaline myopathy, and was compared with control muscles. Measurements of isometric and dynamic muscle performance were done with electrical nerve stimulation at physiological temperatures. No muscle weakness was found in the transgenic muscles when performance was measured at muscle optimum length. This was true not only with full activation but also at lower activation levels, indicating that calcium sensitivity was not affected at this length. Also, fatigability was not affected in these conditions. However, isometric force of the muscles with the mutation in TPM3 was lower at lengths below optimum, with more impairment at decreasing length. As the muscles are active over a large range of different muscle lengths during daily activities, this finding may explain, at least in part, the muscle weakness experienced by patients with nemaline myopathy.     

A novel FKRP gene mutation in a Taiwanese patient with limb-girdle muscular dystrophy 2I

Limb-girdle muscular dystrophy (LGMD) is a group of hereditary muscle diseases with preferential involvement of the shoulder and pelvic girdle muscles, but with no pathognomonic features as in facioscapulohumeral and congenital muscular dystrophies. We report 18-year-old female with progressive shoulder and pelvic muscle weakness. She had marked restrictive pulmonary dysfunction. Echocardiogram showed mild decrease in ejection fraction of 52% (normal: >55%). She was first seen in our hospital at age 2 years with progressive proximal muscle weakness and elevated creatine kinase (CK) level to 15,290 IU/L, with what clinically and pathologically appeared to be steroid-responsive inflammatory myopathy. She responded dramatically to steroid therapy. Progressive proximal muscle weakness began again at age 8 years. Serum CK was 14,910 IU/L. She was wheelchair-bound by age 12. Muscle biopsy showed dystrophic changes without inflammation with reduced immunoreactivity to an antibody against sugar chain (VIA4-1) of alpha-dystroglycan. On laminin overlay assay, there was a nearly complete loss of laminin-binding activity to alpha-dystroglycan. Genetic analysis of fukutin-related protein (FKRP) gene revealed a novel compound heterozygous mutation of c.823C>T (p.R275C) and c.948delC, confirming the diagnosis of LGMD2I, the first reported case in East Asia.     

Intragenic mutations in SMN1 may contribute more significantly to clinical severity than SMN2 copy numbers in some spinal muscular atrophy (SMA) patients

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by deletion or intragenic mutation of SMN1. SMA is classified into several subtypes based on clinical severity. It has been reported that the copy number of SMN2, a highly homologous gene to SMN1, is associated with clinical severity among SMA patients with homozygous deletion of SMN1. The purpose of this study was to clarify the genotype-phenotype relationship among the patients without homozygous deletion of SMN1. Methods: We performed molecular genetic analyses of SMN1 and SMN2 in 112 Japanese patients diagnosed as having SMA based on the clinical findings. For the patients retaining SMN1, the PCR or RT-PCR products of SMN1 were sequenced to identify the mutation. Results: Out of the 112 patients, 106 patients were homozygous for deletion of SMN1, and six patients were compound heterozygous for deletion of one SMN1 allele and intragenic mutation in the retained SMN1 allele. Four intragenic mutations were identified in the six patients: p.Ala2Val, p.Trp92Ser, p.Thr274TyrfsX32 and p.Tyr277Cys. To the best of our knowledge, all mutations except p.Trp92Ser were novel mutations which had never been previously reported. According to our observation, clinical severity of the six patients was determined by the type and location of the mutation rather than SMN2 copy number. Conclusion: SMN2 copy number is not always associated with clinical severity of SMA patients, especially SMA patients retaining one SMN1 allele.     

Breathing patterns and HbSaO2 changes during nocturnal sleep in patients with Duchenne muscular dystrophy

Summary A night-time polygraphic sleep recording with continuous HbSaO₂ monitoring was performed in 11 chair-bound Duchenne muscular dystrophy patients with severe restrictive lung disease but with blood gas values within normal limits when awake. No abnormalities of sleep pattern were detected. Nocturnal sleep did not have significant adverse effects on respiration. However, in 6 patients, infrequent central apnoeas or hypopnoeas occurred which were associated with falls in HbSaO₂ greater than those that have been reported to be in normal subjects. The magnitude of HbSaO₂ falls appeared to be significantly correlated with functional residual capacity values. Overall, the findings revealed a relatively preserved, although unstable, blood O₂ balance during nocturnal NREM and REM sleep in patients with Duchenne muscular dystrophy, even in an advanced stage of their illness.     

Pre-clinical study of 21 approved drugs in the mdx mouse

Duchenne muscular dystrophy, a genetic disease caused by the absence of functional dystrophin, remains without adequate treatment. Although great hopes are attached to gene and cell therapies, identification of active small molecules remains a valid option for new treatments.We have studied the effect of 20 approved pharmaceutical compounds on the muscles of dystrophin-deficient mdx5Cv mice. These compounds were selected as the result of a prior screen of 800 approved molecules on a dystrophin mutant of the invertebrate animal model Cænorhabditis elegans. Drugs were administered to the mice through maternal feeding since 2 weeks of life and mixed in their food after the 3rd week of life. The effects of the drugs on mice were evaluated both at 6 weeks and 16 weeks. Each drug was tested at two concentrations. Prednisone was added to the molecule list as a positive control. To investigate treatment efficiency, more than 30 histological, biochemical and functional parameters were recorded. This extensive study reveals that tricyclics (Imipramine and Amitriptyline) are beneficial to the fast muscles of mdx mice. It also highlights a great variability of responses according to time, muscles and assays.