Dystrophin Immunohistochemistry Research Articles

An Ultra-Rare Manifestation of an X-Linked Recessive Disorder: Duchenne Muscular Dystrophy in a Female Patient

Duchenne muscular dystrophy (DMD) is the most common inherited muscle dystrophy. Patients are characterized by muscle weakness, gross motor delay, and elevated serum creatinine kinase (CK) levels. The disease is caused by mutations in the DMD gene located on the X chromosome. Due to the X-linked recessive inheritance pattern, DMD most commonly affects males, who are generally diagnosed between the age of 3-5 years. Here we present an ultra-rare manifestation of DMD in a female patient. Cytogenetic examination showed that she has a t(X;10)(p21.1;p12.1) translocation, which turned out to affect the DMD gene with one of the breakpoints located in exon 54 (detected by genome sequencing). The X-inactivation test revealed skewed X-inactivation (ratio 99:1). Muscle histology and dystrophin immunohistochemistry showed severe dystrophic changes and highly reduced dystrophin expression, respectively. These results, in accordance with the clinical picture and a highly elevated serum CK, led to the diagnosis of DMD. In conclusion, although in very rare cases, DMD can manifest in female patients as well. In this case, a balanced X-autosome reciprocal translocation disrupts the DMD gene and skewed X-inactivation leads to the manifestation of the DMD phenotype.

Severe cardiac involvement with preserved truncated dystrophin expression in Becker muscular dystrophy by +1G>A DMD splice-site mutation: a case report

Becker muscular dystrophy (BMD) is caused by specific mutations in the DMD gene that causes progressive muscle weakness and primarily affects skeletal and cardiac muscle. Although cardiac involvement is a significant cause of mortality in BMD, the genetic–phenotype correlation for skeletal and cardiac muscles has not been elucidated. Here, we described a 39-year-old man with BMD, who presented with subtle skeletal muscle weakness in the right leg in his 20s and underwent left ventricular restoration for severe dilated cardiomyopathy at the age of 29. He had difficulty climbing stairs after the age of 35. Neither duplication nor deletion of exons was detected by multiplex ligation-dependent probe amplification. A hemizygous c.264 + 1G>A mutation in intron 4 of the DMD was identified by next-generation sequencing. Furthermore, exon 4 skipping of the DMD was confirmed in both skeletal and cardiac muscles evaluated by reverse transcriptase PCR. Endomyocardial and skeletal muscle biopsies revealed dystrophic pathology characterized by muscle fiber atrophy and hypertrophy with a mild degree of interstitial fibrosis. Interestingly, dystrophin immunohistochemistry demonstrated patchy and faint staining of the skeletal muscle membranes but almost normal staining of the cardiac muscle membranes. Western blot analysis revealed a decreased amount of truncated dystrophin in skeletal muscle but surprisingly almost normal amount in cardiac muscle. This case indicates that BMD patients may have severe cardiac dysfunction despite preserved cardiac truncated dystrophin expression.

Non-professional phagocytosis: a general feature of normal tissue cells

Non-professional phagocytosis by cancer cells has been described for decades. Recently, non-professional phagocytosis by normal tissue cells has been reported, which prompted us to take a closer look at this phenomenon. Non-professional phagocytosis was studied by staining cultured cells with live-cell staining dyes or by staining paraffin-embedded tissues by immunohistochemistry. Here, we report that each of 21 normal tissue cell lines from seven different organs was capable of phagocytosis, including ex vivo cell cultures examined before the 3rd passage as well as the primary and virus-transformed cell lines. We extended our analysis to an in vivo setting, and we found the occurrence of non-professional phagocytosis in healthy skin biopsies immediately after resection. Using dystrophin immunohistochemistry for membrane staining, human post-infarction myocardial tissue was assessed. We found prominent signs of non-professional phagocytosis at the transition zone of healthy and infarcted myocardia. Taken together, our findings suggest that non-professional phagocytosis is a general feature of normal tissue cells.

Abstract B16: Dystrophin is a tumor suppressor in peripheral nerve sheath tumors

Abstract Introduction: We previously demonstrated that dystrophin inactivation results from genomic DMD 5-end deletions in advanced GIST and leiomyosarcoma, fostering migration, invasion, anchorage independence, and invadopodia formation. Hence, dystrophin inactivation, the most common cause of muscular dystrophy, is also a tumorigenic mechanism in sarcoma. CDKN2A/CDKN2B inactivation is an early event in peripheral nerve sheath tumor (PNST) progression, associated with progression from neurofibroma (NF) to atypical NF. PRC2 inactivation is a later event during progression of some MPNST. Additional biologic markers are needed to distinguish NF from low-grade MPNST. In the present study, we identify and characterize dystrophin dysregulation at an early phase in PNST progression. Methods: Dystrophin immunohistochemistry (IHC) was performed in FFPE sections using the DYS1 monoclonal antibody. DYS1 and other dystrophin monoclonals were used in immunoblotting (IB) studies of snap-frozen PNST. Genomic analyses were performed by targeted gene capture and sequencing (Haloplex custom panel of 812 cancer-associated genes) and Cytoscan HD SNPs. Dystrophin restorations were performed in MPNST cultures using Dp116 (puromycin resistance) and Dp427 (neomycin resistance), either singly or in combination. Results: Dystrophin IHC against TMAs containing common subtypes of benign and malignant soft-tissue tumors showed that most soft-tissue tumor subtypes lacked detectable dystrophin expression whereas 18 of 19 schwannomas expressed dystrophin strongly and diffusely, at levels comparable to that in non-neoplastic skeletal muscle. Dystrophin was also expressed strongly in the neoplastic Schwann-cell component of neurofibromas (NF), whereas dystrophin expression was absent or weak in each of 17 MPNST. IB studies in a separate series of snap-frozen specimens showed that the dystrophin expressed strongly in schwannomas (classic histology = 4; cellular = 6) was coexpression of 427kDa (so-called myogenic) and 116kDa Schwann-cell specific isoforms. IB studies in snap-frozen MPNST showed loss of dystrophin 116kDa and 427kDa expression in 16 cases, retained expression in 1 case, and retained expression of 427kDa only, in 1 case. Genomic surveys demonstrated intragenic DMD deletions or inactivating mutations in 4 of 11 MPNST. Each of 3 NF transitioning to MPNST had strong dystrophin expression in the NF component but not in the associated MPNST. Conjoined restoration of 116kDa and 427kDa dystrophin isoforms (but not restoration of the 116kDa isoform, on its own) inhibited MPNST migration. IHC and IB studies (in progress) suggest that PNST dystrophin inactivation occurs concurrent with or subsequent to CDKN2A/CDKN2B inactivation during transition from NF to low-grade MPNST, and prior to PRC2 inactivation. Conclusions: We show that Dp116 and Dp427 dystrophin isoforms have tumor-suppressor properties in PNST. Extinction of these isoforms occurs in most MPNST, and in laboratory models is associated with increased cell migration. Dystrophin inactivation coincides with transition from NF to low-grade MPNST; therefore, dystrophin IHC might be useful in diagnostic distinction between atypical NF and MPNST. Citation Format: Inga-Marie Schaefer*, Armelle Dufresne*, Nacef Bahri, Marije A. J. de Rooij, Stacy M. Yanofsky, Yuexiang Wang, Chandrajit P. Raut, Laurence H. Baker, Adrian Marino-Enriquez, Matt van de Rijn, Jonathan A. Fletcher. Dystrophin is a tumor suppressor in peripheral nerve sheath tumors [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B16.

A novel point mutation affecting Asn76 of dystrophin protein leads to dystrophinopathy

Mutations in the DMD gene lead to Duchenne and Becker muscular dystrophy (DMD/BMD). Missense mutations are rare cause of DMD/BMD. A six-month-old male patient presented with mild generalized muscle weakness, hypotonia, and delayed motor development. Dystrophinopathy was suspected because of highly elevated serum creatine kinase level (1497 U/L) and tiered DMD gene analysis was performed. Multiplex ligation-dependent probe amplification (MLPA) assay showed deletion of exon 4, which could not be confirmed by another method. Sequencing of exon 4 revealed a novel de novo point mutation (c.227A>T, p.Asn76Ile) in the N-terminal actin-binding domain (N-ABD) of dystrophin protein. The false positive MLPA result was explained by the fact that the affected nucleotide lies directly at the 3' ligation site of the MLPA probe. Sequencing of the whole coding region of DMD gene proved c.227A>T to be the sole variant being potentially pathogenic. According to in silico analyses the mutation was predicted to be highly destabilizing on N-ABD structure possibly leading to protein malfunction. Muscle biopsy was performed and dystrophin immunohistochemistry results were suggestive of BMD. Our results highlight the importance of confirmatory testing of single-exon deletions detected by MLPA and we describe a novel, destabilizing missense mutation in the DMD gene.

Vacuolar myopathy in an adult Warmblood horse

Histopathological interpretation of semimembranosus muscle samples from an adult Warmblood mare with clinical signs suggestive of exertional rhabdomyolysis and intermittent mild elevations in muscle enzyme activities revealed abundant sarcoplasmic vacuoles in all fibre-types containing fine, apparently proteinaceous debris. Vacuolar contents stained lightly with PAS, but did not appear to contain amylopectate, lipid or acid phosphatase and their periphery was unstained with dystrophin immunohistochemistry. Electron microscopy revealed that vacuoles were not membrane bound. No vacuoles were detected in muscle samples evaluated at post mortem following 4 months of rest. To our knowledge, this is the first report of a presumed primary vacuolar myopathy in a horse.

Congenital myopathy in Braunvieh × Brown Swiss calves

A hitherto unknown skeletal muscle disorder is described in six Braunvieh x Brown Swiss calves. The animals showed rapidly progressing muscular weakness and became recumbent within 2 weeks of birth. Histological examination of skeletal muscle revealed a marked variation in muscle fibre size, internally placed nuclei, segmental loss of cross-striation with disorganization of myofibrils, and accumulation of nemaline rods. The most distinctive histological finding was intracytoplasmic, homogeneous, mostly crescent-shaped areas at the periphery of numerous muscle fibres. Electron microscopically, accumulations of tightly packed, parallel filamentous structures, about 20 nm in diameter, were detected in these areas. Enzyme histochemistry showed that all muscle fibre types were affected. Vimentin and dystrophin immunohistochemistry revealed normal antigen distribution within connective tissue components and at the periphery of each muscle fibre, respectively. The lesions could be readily distinguished from other neurological and neuromuscular disorders previously described in Braunvieh x Brown Swiss or American Brown Swiss Cattle. The disease appears to be a novel congenital myopathy in this breed, and a hereditary aetiology is suspected.

Duplication of dystrophin gene and dissimilar clinical phenotype in the same family

We report here three related patients with a duplication of exons 19–41 of the dystrophin gene, having dissimilar clinical phenotype and dystrophin immunohistochemistry. Two brothers aged six and three years had myalgia, proximal muscular weakness and hypertrophic calves, with 10- to 20-fold increase of serum creatine kinase. Muscle biopsy showed dystrophic changes and reduced, patchy binding of dystrophin. The clinical and laboratory findings were consistent with a diagnosis of Becker muscular dystrophy with early onset. Their 14-year-old cousin had only mild hyperCKemia. His muscle biopsy was normal with only mild reduction of dystrophin immunostaining. At follow-up, he is still without symptoms and signs at age 19. All three patients had the same gene duplication and an increased dystrophin size of 507 kDa. Expression of the dystrophin-associated glycoproteins adhalin, α-dystroglycan, and, β-dystroglycan were normal in the three patients. An intrafamilial variability in patients carrying a partial duplication of the dystrophin gene may be related to a quantitative difference in mRNA.

Absence of dystrophin and spectrin in regenerating muscle fibers from Becker dystrophy patients

We studied muscle biopsies from 36 Becker muscular dystrophy patients, and correlated dystrophin negative fibers with regenerating and degenerating myofibers. Dystrophin immunohistochemistry was used to identify dystrophin-negative and dystrophin-positive fibers. Immunohistochemical staining for fetal myosin and acid ATPase identified regenerating fibers, and calcium glioxalate and beta-spectrin staining identified necrotic fibers. All Becker biopsies contained detectable dystrophin in the majority of muscle fibers. 13 cases (36%) showed no dystrophin negative fibers, 9 cases (25%) showed a generalized, markedly decreased immunostaining pattern, and 14 cases (39%) showed a subset of dystrophin negative fibers (0.3–8% of total). Most dystrophin-negative fibers in Becker muscle were judged to be in the process of regeneration, and not in degeneration. No correlation was observed between the age of the patients and number of dystrophin negative fibers. We conclude that the absence of dystrophin and spectrin labeling in some BMD myofibers is associated with regeneration, probably due to incoplete expression of dystrophin secondary to myofibers immaturity. Our results might be explained by a developmental delayed expression of these two proteins, or by abnormal assembling in membrane's components during regeneration in dystrophy. Furthermore, our results rationalize the recently reported finding of some dystrophin-negative fibers in polymyositis.

Dystrophin analysis in the diagnosis of childhood muscular dystrophy: an immunohistochemical study of 75 cases.

Seventy-five consecutive pediatric muscular dystrophy (MD) cases were analyzed by dystrophin immunohistochemistry (75/75), Western blot analysis (26/75), DNA analysis (30/75), and immune electron microscopy (8/75). The patients included 64 males and 11 females and the clinical diagnoses were Duchenne MD (DMD) (41), Becker MD (BMD) (8), intermediate/outlier MD (4), female DMD (3), limb girdle or Becker (1), congenital MD (CMD) (10), Fukuyama CMD (1), facioscapulohumeral MD (FSH) (3), limb girdle MD (2), and other uncharacterized dystrophies (2). Dystrophin analysis was performed on all cases using the N- and C-terminal antidystrophin antibodies. Dystrophin analysis helped to exclude an Xp21 dystrophy in four patients. Except for two patients who showed normal staining with the N-terminal and abnormal staining with the C-terminal antibody, all DMD cases showed absent staining except for the immunoreactive revertant fibers, which were generally under 5%. A variety of staining patterns was seen in BMD, ranging from normal to abnormal (variable intensity of staining, partially stained/unstained fibers). Abnormalities were observed with the C-terminal antibody in one case of CMD and Fukuyama CMD, and normal staining was present in the other dystrophies. Immune electron microscopy confirmed absence of staining in DMD and normal membrane staining in other dystrophies. Our study underscores the importance of using antibodies with specificities to different regions of the dystrophin molecule for accurate diagnosis. As abnormal staining may be encountered in non-Xp21 dystrophies such as CMD, dystrophin staining should not be used in isolation to make a diagnosis of a dystrophinopathy.

Dystrophin immunostaining in muscles from patients with different types of muscular dystrophy: a Brazilian study

The localization of the protein dystrophin was studied using the immunofluorescence method, in muscle biopsies from 74 patients affected by different types of muscular dystrophy and 4 normal controls. In 15 patients with limb-girdle muscular dystrophy (LGMD) the pattern was indistinguishable from normal. Among 42 Duchenne patients (DMD), 3 were totally negative and 39 showed a variable proportion (4–30%) of partially labelled fibers. With one exception 17 Becker dystrophy patients (BMD), showed a positive sarcolemmal reaction. A diffuse reaction inside the fibers, which was not observed in normal controls, was seen in the majority of DMD and also in some of the BMD patients. Based on these observations it is suggested that in DMD, a small quantity of protein is still present or there is a cross-reaction with other proteins which share some homology with dystrophin. The present results suggest that it is possible to make a differential diagnosis between DMD and BMD through dystrophin immunohistochemistry. However, to distinguish between patients with BMD and LGMD phenotypes, or DMD and outliers, complementary immunoblot studies and quantitative determination of dystrophin are necessary.