A new dysferlin gene mutation in a Portuguese family with Miyoshi myopathy

Abstract

Dysferlinopathies are autosomal recessive muscular dystrophies caused by mutations in the dysferlin gene (DYSF). A 33-year-old man was born to a non-consanguineous couple. At the age of 25 he stared to feel weakness of the distal lower limbs and also experienced episodes of rhabdomyolysis. Electromyography showed a myopathic pattern, and muscle biopsy revealed dystrophic changes with absence of dysferlin. Genetic analysis was positive for a mutation in the c3367_3368del DYSF gene (p.Lys1123GLUFS*2). After 8 years of disease evolution the symptomatology worsened. This is the first report of this mutation of the DYSF gene identified in a non-consanguineous Portuguese family, studied over 8 years. We believe the mutation is responsible for the Miyoshi myopathy. Disease progression cannot be predicted in either the patient or carrier family because there are no similar cases previously described in the literature.

Background

Muscular dystrophies constitute a genetically heterogeneous group of disorders.¹ They are characterised by progressive muscle weakness, atrophy and degeneration.² The dystrophies differ depending on the location and function of the mutated protein in the muscle cells, and consequently differ by which muscles are primarily affected, age of onset, severity and symptom progression.^{3 4} Dysferlinopathies are autosomal recessive muscular dystrophies caused by mutations in the dysferlin gene (DYSF), a 150 kb gene on chromosome 2p13 that contains 55 exons encoding for 2080 amino acids.⁵

Dysferlin is a transmembrane protein highly expressed in the striated muscle vesicles, sarcolemma and to a lesser extent in other tissues such as monocytes, syncytiotrophoblast, endothelium, brain, pancreas and kidney.^{6 7} Dysferlin has an essential role in membrane repair through regulation of calcium dependent vesicle patch fusion on the sarcolemma and to a lesser extent of secretory vesicles of the Golgi network.^{6 8 9} In the absence of dysferlin, membrane damage is not adequately repaired and the myofibre will undergo necrosis.⁹ Dysferlinopathies can manifest either as the phenotypes Miyoshi myopathy (distal myopathy) or limb girdle muscular dystrophy type 2B (LGMD2B).^{4 5}

We report a new dysferlin gene mutation in a Portuguese family. This mutation identified on the DYSF gene was responsible for Miyoshi myopathy in a homozygous individuals which could lead to a progressive myopathy and probably to a mild form in a heterozygous person. This is significant because Miyoshi myopathy should be a part of the differential diagnosis of symptomatic patients with distal muscle weakness, mistakenly classified as another pathology, such as polymyositis.

Case presentation

The patient was a 33-year-old man born to a non-consanguineous couple. At the age 25 he manifested weakness and fatigue of the lower limbs, namely of the gastrocnemius, which worsened on exercise, walking or cycling, and he was unable to run. On clinical evaluation the patient was alert, without signs of cognitive deterioration. The neurological examination was positive for lower left limb strength weakness compared with the right limb, affecting mostly the distal muscles. The patient could not walk on his heels, and on the left limb could not walk on tip-toes. He experienced recurrent episodes of rhabdomyolysis with increased concentrations of creatine kinase (CK, 20–60 times normal values), increased levels of myoglobin (approximately 10 times normal values) and mildly raised lactate dehydrogenase levels.

Family history

The patient’s parents were in their sixties and he had a 37-year-old brother, all of whom were asymptomatic of the disease all their lives which was confirmed during the 8 year study. The 82-year-old maternal great aunt denied any symptomatology as a young adult. A few years ago, she had muscle weakness of the lower limbs with difficulty in walking fast, running or climbing stairs, with no increase in CK levels. Neurological examination showed that the patient was alert, with no signs of cognitive deterioration and no alterations in the neurological examination.

Investigations

The patient underwent muscle biopsy of the right leg vastus lateralis which showed marked variability of the size of the fibres with many hypertrophied fibres and atrophied fibres dispersed by the fascicles. Increased interstitial connective tissue, with fibrosis of the endomysium and perimysium, and slight adipose infiltration of the perimysium, were found. We observed some fibres in necrosis and myofagocytosis. There was no evidence of ragged red fibres, inflammation or fibres with vacuoles or inclusions. In the immunocytochemical study, absence of the protein dysferlin in the sarcolemma was found.

Electromyography of the musculature of the upper limbs showed changes in the characteristics of the motor unit potentials, namely polyphasic and a marked reduction in amplitude and duration (myopathic pattern). There were no changes in peripheral nerve conduction.

Genetic analysis by polymerase chain reaction and dysferlin gene sequencing was positive for the mutation c3367_3368del in the DYSF gene (p.Lys1123GLUFS * 2) in homozygosity. The parents had normal or slightly elevated CK levels, although the 37-year-old brother had no changes in CK levels. They were carriers of the mutation p.Lys1123GLUFS*2, apparently in heterozygosity. The great aunt also showed the c3367_3368del mutation (p.Lys1123GLUFS*2) on dysferlin gene sequencing, probably in heterozygosity. During the study, the great aunt died from a different pathology. Therefore, it was not possible to quantify dysferlin mRNA to further characterise the patient's disease.

Differential diagnosis

The two main phenotypes of dysferlinopathies which arise from the same gene mutation, with a prevalence of 1-9/1 000 000,⁹ are Miyoshi myopathy with predominantly distal weakness or limb girdle muscular dystrophy type 2B (LGMD2B) with primarily proximal weakness.^{1 10} This suggests that some additional factors apart from dysferlin might be involved, so a thorough differential diagnosis is needed.^{1 10}

Treatment

Available therapeutic options include supportive treatment to prolong survival and improve quality of life, and management techniques such as emotion support, weight control to avoid obesity, physical therapy and stretching exercises to promote mobility.^{3 9} Most patients with Miyoshi myopathy need to use support devices to walk 10–15 years after the onset of symptoms, and after 20–25 years of the illness, they often need to use a wheelchair.^{9 11}

Outcome and follow-up

We followed this family for 8 years. After 8 years of evolution of the disease, the patitent had severe worsening of the symptomatology despite physical therapy and stretching exercises to promote mobility. Therefore, the patient felt the symptoms worsening with progressive loss of muscular strength and the need for support devices to walk. Currently, the patient is alert, without signs of cognitive deterioration and the neurological examination shows myopathic gait, steppage, positive Gowers’ sign, unable to rise from the floor, and muscle atrophy of the shoulder girdle, vastus medialis, and anterior and posterior compartment of the legs. We found no cardiac or ventilatory mechanic changes. During the study, the patient died from a different pathology, therefore, it was not possible to quantify dysferlin mRNA to further characterise the patient's disease.

Discussion

In this study, we identified a frameshift variation in DYSF (p.Lys1123GLUFS*2), namely a deletion of two nucleotides on exon 31 (gene with 55 exons) that induces a premature stop codon creation that leads to a predictably truncated protein and probably results in complete loss of protein function, causing dysferlinopathy. According to published data, this is the first report of clinical implications of this mutation, which has been described as pathogenic in the databases dbSNP(rs74854262672)¹² and ClinVar (ID:284027).¹³

The patients in this family, with symptoms and signs of myopathy, showed primarily distal weakness of the leg distal muscles with difficulty in walking and climbing stairs. The patient showed characteristic clinical findings of Miyoshi myopathy, such as autosomal recessive heredity; onset of symptoms between 15 and 30 years of age; involvement of the distal muscles of the lower limbs; elevated serum CK levels; pathologic evidence of dystrophic features with a necrotic regeneration pattern without vacuoles on muscle biopsy; and absent dysferlin on immunohistochemistry.4 9 14 15 The dysferlin deficit on the muscle biopsy was confirmed in the molecular study that associated with the patient's symptoms and identified a new pathogenic mutation in the dysferlin gene. Therefore, it can be inferred that the new mutation identified induces Miyoshi myopathy characterised by initially affecting the distal muscles of the lower limbs, the gastrocnemius and soleus muscles.^{1 10 14} Over several years, the symptoms spread to the thighs and gluteal muscles. But the arms, hands, anterior tibial and peroneal muscles remain relatively spared early in the disease, as observed in our patient.^{10 14 16}

The rate of progression of Miyoshi myopathy can be variable, with some patients remaining fairly stable with distal weakness, while other progress rapidly over a few years with a more aggressive pattern involving both proximal and distal muscles, as in our patient.^{9 17} The great aunt was an elderly woman with symptoms of distal myopathy, with the mutation likely in heterozygosity, and therefore, she was a symptomatic heterozygote with the Miyoshi myopathy phenotype. This suggests that the phenotype is directly related to the accumulation of lesions in the sarcolemma over time due to the reduction or dysfunction of dysferlin. Corroborating this hypothesis, the patient's parents had levels of CK in the upper limit of normal or a slight increase.

Definitive confirmation of dysferlinopathy could not be performed on the great aunt in this study by DNA testing. The question arises whether the identified mutation is the single cause for the patient’s symptomatology or whether there were other factors modifying gene expression, such as a second mutation. Meznaric et al described symptomatic cases with only one mutated allele, confirmed by reduction in expression of dysferlin mRNA.¹⁸ This suggests that the heterozygous parents and brother, who had normal or slightly elevated CK levels but were asymptomatic, may develop a mild phenotypic disease in the future.

There is currently no definitive treatment for Miyoshi myopathy and therefore mutation carriers should receive genetic counselling. Miyoshi myopathy and specifically this mutation may be underdiagnosed in elderly heterozygous patients with similar symptomatology, mistakenly classified as another pathology such as polymyositis. Miyoshi myopathy should be part of the differential diagnosis of symptomatic patients with distal muscle weakness.

The 8 year study of a non-consanguine Portuguese family where two patients had distal muscle weakness identified a new frameshift in the DYSF gene (p.Lys1123GLUFS * 2), which was shown to be responsible for Miyoshi myopathy. The mutation identified in homozygotes leads to a rapidly progressive myopathy, and in heterozygotes is probably responsible for a mild form of the disease in old age due to the accumulation of lesions in the sarcolemma by the likely truncated dysferlin dysfunction resulting from this mutation. There is no definitive treatment for this pathology so supportive treatment aims to improve quality of life. It is appropriate and necessary to offer genetic counselling to at risk couples (both carriers of disease causing mutation) informing them of the 25% risk of having a child affected by the rapidly progressive illness.

Since this is the first report of this new mutation responsible for Miyoshi myopathy, the evolution and prognosis of the disease through time is not known, and follow-up of the family is essential. The longitudinal study of this family will clarify whether heterozygous patients will present with Miyoshi myopathy.

Learning points.

• New dysferlin gene mutation (p.Lys1123GLUFS*2) is responsible for Miyoshi myopathy.

• In homozygous individuals, the mutation leads to a progressive myopathy.

• In heterozygous persons, this mutation probably leads to a mild form of the disease.

• Miyoshi myopathy should be part of the differential diagnosis for patients with distal muscle weakness.

• The longitudinal study of this family will clarify if heterozygous patients will present with Miyoshi myopathy symptoms.

Ethics statements

Patient consent for publication

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