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Year : 2011  |  Volume : 54  |  Issue : 2  |  Page : 350-354
Dysferlinopathy: Spectrum of pathological changes in skeletal muscle tissue

1 Department of Neuropathology, National Institute of Mental Health & Neurosciences, Bangalore, Karnataka, India
2 Department of Neurology, National Institute of Mental Health & Neurosciences, Bangalore, Karnataka, India

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Date of Web Publication27-May-2011


Background: Dysferlinopathy is an autosomal recessive-limb girdle muscular dystrophy (AR-LGMD) caused due to the defect in gene encoding dysferlin, a sarcolemmal protein. Awareness of the variants and their relative frequency is essential for accurate diagnosis. Aim: To study the spectrum of morphologic changes in immunohistochemically proven cases of dysferlinopathies, to correlate the findings with clinical phenotype and durations of illness and determine the frequency. Materials and Methods: Dysferlinopathies seen over a period of 2 years at a tertiary neurological center were analyzed. Results: Clinically, majority had Miyoshi phenotype (46.6%) with distal involvement and LGMD phenotype (40%) with proximal muscle involvement. In addition, a proximo-distal and tibial muscle phenotype was encountered. Morphologically, rimmed vacuoles were noted in the Miyoshi phenotype. The presence of ragged red fibers, lobulated fibers and inflammation had no preference to a particular phenotype. Significant atrophy and lobulated fibers were noted in patients with longer duration of illness. Conclusions: Dysferlinopathy was the second most common identifiable cause (21%) of LGMD next to sarcoglycanopathies (27%).

Keywords: Dysferlinopathy, limb girdle muscular dystrophy, Miyoshi myopathy, ragged red fibers, rimmed vacuoles

How to cite this article:
Gayathri N, Alefia R, Nalini A, Yasha T C, Anita M, Santosh V, Shankar S K. Dysferlinopathy: Spectrum of pathological changes in skeletal muscle tissue. Indian J Pathol Microbiol 2011;54:350-4

How to cite this URL:
Gayathri N, Alefia R, Nalini A, Yasha T C, Anita M, Santosh V, Shankar S K. Dysferlinopathy: Spectrum of pathological changes in skeletal muscle tissue. Indian J Pathol Microbiol [serial online] 2011 [cited 2020 Jul 6];54:350-4. Available from: http://www.ijpmonline.org/text.asp?2011/54/2/350/81636

   Introduction Top

Limb girdle muscular dystrophies are a heterogeneous group of inherited muscle diseases characterized by wasting and weakness of skeletal muscle. [1] Mutations in dysferlin gene (DYSF) causes either limb girdle muscular dystrophy (LGMD 2B) or a Miyoshi myopathy (MM) phenotype [2] and muscle biopsy reveals dystrophic features. The defective gene is localized to chromosome 2p13. [3],[4] Awareness of the variants and their relative frequency is essential for accurate diagnosis. We report a spectrum of pathological changes in skeletal muscle biopsies from 30 patients with dysferlinopathy, seen over a period of 2 years (Jan 2005-Dec 2007). The findings are clinicopathologically correlated.

   Materials and Methods Top

Skeletal muscle biopsies from 30 patients, clinically diagnosed as limb girdle muscular and/or MM, were subjected to a battery of immunostains. Patients with total absence of dysferlin were included in the study. Most of the muscle biopsied were from biceps (n = 17). The remaining biopsies were from quadriceps (9), gastrocnemius (1), peroneous brevis (1), hamstring (1) and deltoid (1). Detailed clinical history of all the patients was noted. Laboratory evaluation included complete hemogram, serum creatine kinase (CK), nerve conduction and electromyography (EMG). Serial cryosections were stained for routine battery of stains Haematoxilin Eosin (HE), modified Gomori trichrome (MGT), succinic Dehydrogenase(SDH), Nicotinamide adenine dinucleotide Tetrazolium Reductase(NADH -TR),adenosine tri phosphatase (ATPase) pH 9.5, 4.6 and acid phosphatase]. Immunostaining to monoclonal antibodies against Dystrophin (1, 2, 3), Sarcoglycans (α, β, γ, δ), Dysferlin, Merosin and β-dystroglycan as primary and HRP tagged goat-antimouse as secondary was carried out on cryosections. To characterize the inflammatory cell infiltrates, monoclonal antibodies to T-cells (CD 3) and B-cells (CD 20) were used on selected cases. Tiny pieces of tissue fixed in 3% glutaraldehyde from eight cases were post fixed in osmium tetroxide and embedded in araldite for electron microscopy. Ultrathin sections contrasted with uranyl acetate and lead citrate were observed under FEI Technai Spirit (120 kV) transmission electron microscope.

To distinguish between MM and LGMD phenotypes with respect to morphologic changes, an attempt was made to classify biopsy findings as (1) myopathic (m) (mild fiber size variation, increase in number of internal nuclei, splitting) (2) dystrophic (d) (marked variation in fiber size, necrosis, myophagocytosis, regeneration, fibrosis) and (3) dystrophic with additional neurogenic changes (d-NA) (dystrophic features plus the presence of atrophic angulated fibers with pyknotic nuclei and/or fiber grouping). The frequency of occurrence of structural abnormalities such as moth-eaten fibers, lobulated fibers, ragged red fibers (RRF) and ring fibers in different phenotypes was noted. The findings were correlated with duration of illness and clinical phenotype [Table 1], [Table 2].
Table 1: Muscle biopsy findings categorized based on different phenotypes

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Table 2: Frequency of histopathologic changes in different phenotypes

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   Results Top


Case histories from all 30 cases were critically analyzed. There were 20 males and 10 females with mean age of onset at 21.4 ± 6.0 years (range 14-48 years) and the mean duration of illness was 6.4 ± 4.2 years (range 1-15 years). MM with distal involvement and inability to stand on toes and marked calf muscle atrophy [Figure 1] was present in 14 cases (46.6%), while LGMD phenotype with proximal muscle weakness of lower limbs was seen in 12 cases (40%). A proximo-distal involvement, with onset of weakness in proximal followed by distal, was noted in two cases. Interestingly, in two patients, the first symptom was progressive weakness in tibialis anterior muscle with foot drop, which later progressed to involve the proximal muscle as described in tibialis muscular dystrophy (TMD phenotype). Consanguinity (uncle-niece and first cousin) was noted in 13 (43.3%) cases. There was family history of similar illness in eight with siblings affected in six families. Cardiac and respiratory system was normal. Mean CK was 10,033.8 ± 9283 (range 435-27,460 u/l) (normal 20-170 u/l). Electromyography was myopathic in all.
Figure 1: (a, b) Clinical photograph showing marked wasting of the distal muscles in Miyoshi phenotype

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Light microscopy [Figure 2]a - f

Routine histology revealed myopathic pattern in 9 (30%), dystrophic changes in 11 (36.3%) and dystrophy with additional neurogenic features in 10 (33.3%) cases [Table 1]. Dystrophy with neurogenic changes was seen in patients with long standing disease. Correlation of structural abnormalities with different phenotypes and duration of illness is depicted in [Table 2]. Lobulated fibers [Figure 2]b were noted in patients with longer duration of illness. Rimmed vacuoles [Figure 2]c were observed only in the distal forms [4/14 (28%) cases with MM phenotype and in one case with TMD]. The other findings include RRF [Figure 2]d, e in six cases and ring fibers in seven cases. The duration of illness in cases with RRF ranged from 3 to 12 years. Perivascular inflammation [Figure 2]f detected in 10 cases comprised predominantly T-cells. Two of 10 cases showed dense inflammation and hence a differential diagnosis of polymyositis was made. Creatine phosphokinase (CPK) in one of the cases with dense inflammation was markedly elevated (12,600 u/l). Type-II fiber predominance was noted in 9/30 cases, while type-I predominance was noted in 2 cases. Immunostaining to dysferlin showed total absence of labeling along the membrane in all the cases [Figure 3]. Normal immunolabeling was noted to the rest of the antibodies in all, except in two cases which showed reduced intensity to β-sarcoglycan.
Figure 2: Photomicrographs showing (a) transversely cut skeletal muscle with variation in fiber diameter, internal nuclei and myophagocytosis (Hematoxylin and eosin, 100), (b) lobulated fibers (SDH), (c) rimmed vacuoles (MGT), (d, e) ragged blue fibers (SDH) and RRF (MGT) and (f) perimysial perivascular cuffing (b, c, d, f ) (Hematoxylin and eosin ×200

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Figure 3: Immunohistochemical staining shows total absence of labeling to dysferlin, while being positive to rest of the panel (IHC, ×200) (dys, Dystrophin; sarc, sarcoglycan)

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Electron microscopy

Electron microscopy carried out in eight cases (LGMD-1, MM-5, TMD-2) revealed the following changes.

Plasma membrane discontinuity was noted along the length of the muscle fiber in majority of the fibers [Figure 4]a. The membrane discontinuity ranged from 78 to 219 nm in the non-necrotic fibers, while that in the necrotic fiber it was as large as 1412 nm. The gaps were plugged by numerous vesicles [Figure 4]b. The necrotic fibers showed increased numbers of membrane vesicles as compared to the non-necrotic fibers. Deep invagination of the sarcolemma was noted.
Figure 4: Electron micrographs showing (a) plasma membrane discontinuity (ƒq) and (b) gaps plugged with numerous vesicles (ƒq) [bar: (a) 1 nm; (b) 200 nm]. (c) Electron dense granular material within the sleeves of the duplicated basal lamina (ƒq) and increased golgi cisterns in the subsarcolemmal region (ƒq) (d) [bar: (c) 1 ìm; (d) 500 nm]. (e) Abnormal mitochondria with type-I (ƒq) and type-II (ƒq) paracrystalline inclusions in the subsarcolemmal region [bar: (e) 2 ìm; (f) 500 nm]

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Basement membrane was thickened and/or duplicated in both necrotic and non-necrotic fibers. Electron dense granular material was seen within the sleeves of the duplicated basal lamina in an occasional fiber [Figure 4]c.

Subsarcolemmal region with increased granular sarcoplasm comprising mitochondria, glycogen, free ribosomes, randomly oriented filaments, markedly dilated T-tubules and smooth surfaced tubules was noted in majority of the non-necrotic fibers [Figure 4]d. Increased Golgi cisterns and an occasional T-tubule network were seen. In addition, myofibrillar disorganization, streaming and clumping of Z-band and increased subsarcolemmal mitochondria was evident in some of the fibers. In cases with rimmed vacuoles, the vacuoles contained multi-laminated membrane structures, glycogen granules, dense bodies, granular and fibrillar material. One case with RRF showed abnormal mitochondria with paracrystalline inclusions (type-I and type-II) [Figure 4]e, f.

   Discussion Top

Thirty cases with total absence of dysferlin on immunohistochemistry, on analysis, revealed the presence of Miyoshi's phenotype with distal involvement clinically in 14 cases and LGMD with proximal muscle involvement in 12 cases. Of the remaining four patients, two had proximo-distal involvement and two had onset like tibial muscular dystrophy. This is the largest series from the Indian subcontinent. A detailed account on clinical findings has been reported earlier from our center. [5] Histologically, a spectrum of pathological changes was encountered. 36% of the cases showed dystrophic features, while subtle myopathic features were noted in 30% of the cases. Ten cases revealed the presence of atrophic fibers in addition to myogenic features. Amongst these, large group atrophy noted in four cases mimicked a neurogenic lesion and an initial histological diagnosis of spinal muscular atrophy was suspected even though EMG showed myopathic pattern. The neurogenic pattern with group atrophy and hypertrophy was noted in patients with longer duration of illness. The presence of atrophic fibers is probably due to splitting and inadequate regeneration of the fibers. [6]

Lobulated fiber, a feature predominantly noted in LGMD 2A (calpainopathy) and facio scapulo humeral dystrophy (FSHD), has been suggested to be frequent and numerous in patients with long standing and slowly progressive course of LGMD. [7] In the present study, all five patients with lobulated fibers had longer duration of illness (6-8 years). A 2-year follow up in these patients revealed that the disease was stationary. Long-term follow up will determine the progression of the disease in these cases with lobulated fibers. In the present series, lobulated fibers were seen in all clinical phenotypes.

The presence of rimmed vacuoles was noted in the Miyoshi phenotype and in one case with TMD phenotype. None of the biopsies from the LGMD phenotype or proximo-distal had rimmed vacuoles. There was no correlation between the duration of illness and the presence of rimmed vacuoles. In TMD, vacuolar degeneration has been described in a few cases in a study from 66 Finnish patients. [8] However, defective protein responsible for TMD has not been mentioned in their series.

Inflammatory infiltrates have been previously described in patients with dysferlin deficiency [9] and in FSHD. Inflammatory response in FSHD is linked to chromosome 4. [10] In dysferlinopathies, splice site mutations at nucleotide 5711, which disrupt dysferlin, produce the phenotype associated with inflammation. [11] Immunophenotyping of inflammatory infiltrates in our study revealed predominantly T-cells, similar to the observation of Gallardo et al. [9] Although the origin and role of inflammatory cells is unclear, a T-cell mediated effector response directed against either connective tissue and/or vascular elements and/or altered muscle fiber component(s) produced by dystrophic muscle fiber degeneration has been suggested. [10] There was no correlation between duration of illness and the presence of inflammation. Molecular genetic study was not performed in our cases to determine the type of mutation involved in patients with inflammation. RRF, a feature considered diagnostic of mitochondrial myopathy, have been reported in several myopathies including polymyositis, inclusion body myositis and muscular dystrophies. Six cases in the present series showed evidence of RRF. Three out of six cases had longer duration of illness. The presence of RRF is a probably reparative response to muscle fiber damage.

According to "patch hypothesis", [12] membrane repair requires accumulation and fusion of vesicles with each other and with the plasma membrane at/near the site of disruption, wherein dysferlin acts as a mediator in vesicle fusion and membrane repair process. [13] Ultrastructural evidence of plasma membrane disruption plugged with vesicles noted in the necrotic and non-necrotic fibers and increased tubules and tubular sacs, suggests that even though vesicle formation is noted, membrane repair mechanism is affected due to dysferlin deficiency, resulting in contraction involved muscle fiber damage, further resulting in motor weakness in these patients.

In conclusion, our study reveals dysferlinopathies to be the common (21%) identifiable cause of LGMD next to sarcoglycanopathies (27%). In the absence of a comprehensive international patients' registry, the overall prevalence of dysferlinopathies is still hard to estimate. However, it accounts for 30% in certain geographical areas. [14] Immunohistochemistry helped in delineating it from other forms of LGMD. Presentation was heterogeneous with majority having MM and LGMD phenotypes. Morphologically, rimmed vacuoles were noted in the distal phenotype while presence of RRF, LF and inflammation had no preference to a particular phenotype. Significant atrophy and LF were noted in patients with longer duration of illness. Molecular genetics will help to identify the type of mutation prevalent in Indian subcontinent.

   Acknowledgment Top

The authors thank Mrs. Nagina, Mrs. Hemavathy and Mr. Ramesh for their untiring technical help.

   References Top

1.Kaplan JC, Beckmann JS, Fardeau M. Limb girdle muscular dystrophies. In: Karpati G, Hilton-Jones D, Griggs RC, editors. Disorders of Voluntary muscle, United Kingdom: Cambridge University Press; 2000; p. 433-63.  Back to cited text no. 1
2.Liu J, Aoki M, Illa I, Wu C, Fardeau M, Angelini C, et al. Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 1998;20:31-6.  Back to cited text no. 2
3.Bejaoui K, Hirabayashi K, Hentati F, Haines JL, Weissenbach BJ, Rowland LP. Linkage to Miyoshi myopathy (distal autosomal recessive muscular dystrophy) locus to chromosome 2p12-14. Neurology 1995;45:768-72.  Back to cited text no. 3
4.Bashir R, Strachan T, Keers S, Stephenson A, Mahajneh I, Marconi G, et al. A gene for autosomal recessive limb-girdle muscular dystrophy maps to chromosomes 2p. Hum Mol Genet 1994;3:455-7.   Back to cited text no. 4
5.Nalini A, Gayathri N. Dysferlinopathy: a clinical and histopathological study of 28 patients from India. Neurol India 2008;56:379-85.  Back to cited text no. 5
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6.Yamanouchi Y, Arikawa E, Arahata K, Ozawa E, Nonaka I. Limb girdle muscular dystrophy: Clinical and pathological evaluation. J Neurol Sci 1995;129:891-5.  Back to cited text no. 6
7.Van der kooi AJ, Ginjaar HB, Busch HF, Wokke JH, Barth PG, De Visser M. Limb girdle muscular dystrophy: A pathological and immunohistochemical reevaluation. Muscle Nerve 1998;21:584-90.  Back to cited text no. 7
8.Udd B, Partanen J, Halonen P, Falck B, Hakamies L, Heikkila H, et al. Tibial Muscular dystrophy- Late adult-onset Distal myopathy in 66 finnish patients. Arch Neurol 1993;50:604-8.  Back to cited text no. 8
9.Gallardo E, Rojas-Garcia R, de Luna N, Pou A, Brown RH, Illa I. Inflammation in dysferlin myopathy: Immunohistochemical characterisation of 13 patients. Neurology 2001;57:2136-8.  Back to cited text no. 9
10.Arahata K, Ishihara T, Fukunaga H, Orimo S, Lee JH, Goto K, et al. Inflammatory response in facioscapulohumeral muscular dystrophy (FSHD): Immunohistochemical and genetic analysis. Muscle Nerve 1995;2:S56-66.  Back to cited text no. 10
11.Mc Nally EM, Ly CT, Rosenmann H, Rosenbaum S, Jiang W, Anderson LV. Splicing mutation in dysferlin produces limb-girdle muscular dystrophy with inflammation. Am J Med Genet 2000;91:305-12.   Back to cited text no. 11
12.Miyake K, Mc Neil PL. Vesicle accumulation and exocytosis at sites of plasma membrane disruption. J Cell Biol 1995;131:1747-58.  Back to cited text no. 12
13.Bansal D, Miyake K, Vogel SS, Gral S, Chen C, Williamson R, et al. Defective membrane repair in dysferlin - deficient muscular dystrophy. Nature 2003;423:168-72.  Back to cited text no. 13
14.Andoni UJ, Guillaume B, France L, Karine N, Martin K, Nicolas L. Dysferlinopathies. Neurol India 2008;56:289-97.  Back to cited text no. 14

Correspondence Address:
N Gayathri
Department of Neuropathology, National Institute of Mental Health & Neurosciences, Bangalore - 560 029, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.81636

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]

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