Congenital myopathies: Clinical and immunohistochemical study
Congenital myopathies (CMs), a group of relatively non-progressive disorders presents with weakness and hypotonia of varying severity, morphologically recognized by specific structural abnormalities within the myofiber. This report presents the clinical and Histopathological features of 40 patients with CMs. Centronuclear myopathy was the commonest (40%) followed by congenital fiber type disproportion (37.5%). Other less common CMs included: myotubular myopathy (5%), nemaline myopathy (5%), central core disease (5%), multicore disease (2.5%) and congenital myopathy with tubular aggregate (5%). Immunolabeling to desmin corresponded to morphological changes within the myofibers while vimentin was negative in all the patients. There is no combined role of these proteins in the disease process.
Keywords: Central core disease, centronuclear myopathy, congenital myopathy, desmin, fiber type disproportion, myotubular myopathy, nemaline myopathy, tubular aggregate
Congenital myopathies (CMs) are a separate category of muscle disorders with characteristic histopathological features  and include several types. , These disorders usually have onset in infancy/childhood and occasionally in adulthood, can be either familial or sporadic, and are often mild, non-progressive or slowly progressive. Since the first description of central core disease,  several types of CMs have been described. . Recent studies suggest that protein aggregate myopathy is related not only to excess myofibrillar protein and "inclusion-like bodies" (reducing bodies) but also structural abnormalities in muscle (cores, nemaline rods).  Desmin and vimentin are the most widely studied proteins and the role of both vimentin and desmin in the disease process has been suggested.  The present study was undertaken to analyze the clinical spectrum, pathologic characteristics and expression pattern of desmin and vimentin, and the combined role of these proteins if any in the disease process of a cohort of CMs at our institute.
A retrospective analysis of the case records of patients with CMs was done with emphasis on details of perinatal history, onset and progression, associated symptoms, and inheritance pattern. Serum creatine kinase (CK) values and electromyography (EMG) findings were also noted. Biopsy slides of all the patients were reassessed. Immunostaining using monoclonal antibodies against desmin and vimentin as primary, and peroxidase-labeled streptavidin biotin as secondary was carried out. Descriptive statistics (mean and standard deviation for continuous variables, and frequency percentage for categorical variables) were used while analyzing clinical data using SPSS Version 11.0. Follow-up information was obtained by postal correspondence. A simple questionnaire was posted to patients to assess the progression of weakness and present disability. In some patients follow-up information was also obtained by telephonic interview.
Analysis revealed 137 case records with diagnostic code CM. Of the 137 patients with the diagnosis of CM, 77 patients had undergone muscle biopsy and a definitive diagnosis of CM was established in 25 (32%) patients based on morphological and clinical findings. Another 15 patients with morphological diagnosis of CM were also included in the analysis, thus the total cohort was 40 patients. The types of CM included centronuclear myopathy (CNM=16, 40%), congenital fiber type disproportion (CFTD=15, 37.5%), myotubular myopathy (MTM =2), nemaline myopathy (NM=2), central core disease (CCD=2), congenital myopathy with tubular aggregate (TA=2) and multicore disease (MCD=1).
Demographic profile of the total cohort
Of the 40 patients, males were 23 (57.5%) and mean age at presentation was 13.5 ± 9.6 years (range 1 to 37 years; median 10 years) and mean duration of illness was 9.4 ± 7.2 years (range 9 months to 30.5 years; median 8.7 years). Onset was variable: infantile-18; first decade-16; second decade- 2; and third decade-4. Clinical characteristics are presented in [Table 1].
Muscle biopsy revealed central nucleus (25-100%) with perinuclear halo. On oxidative stains, central intense reactivity, peripheral halo and/or radiating pattern giving spoke-like appearance (12 cases), and evidence of Type 1 fiber hypoplasia (5 cases) was noted. Ultrastructurally, central nucleus with perinuclear non-contractile areas rich in glycogen and/or mitochondria and an occasional fiber with lipofuscin at the nuclear poles were noted. The spoke-like appearance seen on light microscope corresponds to radial organization of the myofibrils and mitochondria. Desmin labeling corresponded to oxidative staining with central dense reaction, intense perinuclear labeling and a radiating spoke-like pattern. In cases with Type 1 fiber hypoplasia, the small-diameter fibers showed diffuse intense reaction. Vimentin showed intensely labeled endothelial cells of blood vessels while myofibers were negative in all the cases [Figure 1] a-d.
Histochemically, biopsies showed presence of fetal myotubes with central condensation of oxidative reaction. These fibers revealed strong desmin labeling in the center of the majority of small fibers. Vimentin was negative in all the fibers and labeled only the endothelial cells [Figure 2] a-c.
Congenital fiber type disproportion
The cases diagnosed as CFTD were categorized basing on Brooke's criteria,  fiber Type 1/Type 2 predominance showed diffuse faint labeling in the majority of the fibers while in fiber Type 1/fiber Type 2 hypoplasia punctate positivity was noted in hypoplastic fibers. Vimentin was negative in all the fibers in all the cases.
Nemaline rod myopathy
Red-stained rod bodies were identified on modified Gomori's trichrome in all the myofibers. Ultrastructurally, rods were square to rectangular bodies of varying sizes seen in clusters in the subsarcolemmal region and in singles in the intermyofibrillar region oriented to the long axes of the myofibrils. Thin filaments were attached to the rods. Intranuclear rods were not observed. Desmin failed to label the rod bodies. However, intense immunlabeling was noted around the rod bodies, with the rest of the sarcoplasm showing diffuse staining. Vimentin neither labeled the rods nor the myofibers.
Central core disease
Type I fiber predominance and absence of oxidative enzyme reaction (SDH and NADH-TR) in the center/eccentric (central cores) was observed in all fibers. The core region ultrastructurally showed absence of mitochondria, distortion of myofilamentous pattern, streaming of Z-band and displaced triads. In the non-core region, the alignment of sarcomeres and mitochondria was preserved . Desmin showed intense labeling around the core in most of the fibers causing a demarcation, and a strong staining in the core region in a few fibers, while normal staining pattern was noted in rest of the fiber. Vimentin was negative in all the fibers [Figure 3] a-d.
Tubular aggregate myopathy
Two siblings with myopathy and tubular aggregates have been reported. 
Muscle biopsy showed predominance of Type 1 fibers and multiple small areas (multicores) with absence of oxidative reaction and focal disruption of myofibrils affecting a few sarcomeres, Z-band streaming and absence of mitochondria. Immunostaining to desmin was characterized by a wrinkled paper appearance. Staining to vimentin showed intense labeling of endomysial and perimysial endothelial cells of blood vessels while negative in all the fibers [Figure 4] a-d.
The study describes clinical, morphological features and expression pattern of desmin and vimentin in a large cohort of congenital myopathies from India. In this cohort CNM (40%) was the commonest CM followed by CFTD (37.5%). In a study of 25 patients by Jain et al., CCD (24%) was the commonest CM followed by NM and MCD (20%), while CFTD and CNM accounted for 16% and 12% respectively. However, the overall incidence of CM among the spectrum of neuromuscular diseases is unknown and so is the frequency of individual CMs. ,,,,,,,,, This lack of epidemiological data is probably related to variation in the patient population studied, requirement for muscle biopsy and availability of special techniques for characterization.
There are several proteins in the skeletal muscle and normal function depends on the correct organization of these proteins.  Pathogenesis and morphogenesis of these protein aggregates within the muscle fibers are incompletely understood. Myosin and actin form 70% of the myofibrillar proteins. Desmin and vimentin are dispersed intermediate filaments which aggregate as massive perinuclear bundles during the early stages of development. As differentiation proceeds, desmin gets associated to the lateral edges of the sarcomeres while vimentin gradually disappears as the cell matures.  In the recent years, protein aggregate myopathy is related not only to excess myofibrillar protein and "inclusion-like bodies" (reducing bodies) but also to structural abnormalities in muscle (cores, nemaline rods). Desmin and vimentin are the most widely studied proteins and a combined causative role of these proteins in the disease process has been suggested. , The present study reveals disease-specific disorganization of desmin as a pathogenic mechanism in CMs. In CNM, desmin labeling corresponded to oxidative staining with central dense reaction, intense perinuclear labeling and a radiating spoke-like pattern. While, diffuse reaction, and intense perinuclear and central labeling in MTM probably suggests dysmaturation of these fibers. None of the fibers showed labeling to vimentin. In CFTD, there was no particular pattern of labeling seen in cases with fiber predominance. However, punctuate positivity seen in cases with hypoplasia was similar to the staining pattern seen in the atrophic fibers of infantile spinal muscular atrophy (unpublished data) pointing to a neurogenic origin.  Desmin failed to label the rod bodies in NM, nevertheless diffuse labeling of the fibers was noted. Our findings differ from van der Van et al,  who demonstrated desmin as a component of rod bodies. Immnuolabeling to desmin in CCD paralled morphological alteration seen on oxidative stains. The wrinkled paper appearance seen in MCD is noteworthy. Vimentin labeling in all cases was restricted to endothelial cells of endo and perimyseal blood vessels. It has been reported that abnormalities in the distribution of vimentin parallel those of desmin.  However, in the present study, there is no combined role of vimentin and desmin in the disease process as seen by the absence of vimentin in the myofibers in all the cases.
In conclusion, this is the largest series of CM reported from India.CNM and CFTD constitute the major groups. Since most of the disorders are caused by the proteins that form the structural constituent or are involved during the development of the sarcomere, it is likely that the mutant proteins and/or abnormal synthesis, structural or functional alterations lead to disease process and pathogenesis. Proteomic studies and evaluation of altered expression of desmin would provide better insights into the pathobiology of CMs.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]