Muscular dystrophy refers to a group of genetically determined, progressive, degenerative disorders of the muscle (Shukla et al., 2004). Duchenne muscle dystrophy (DMD) represents the most common and severest form of all known muscular dystrophies. The disorder is an X-linked recessive disease with development of muscle weakness in the early childhood, progressing to a disabling state by early adulthood and usually resulting in early age of death by the third decade of life because of the involvement of cardiac and respiratory muscles (Emery, 1983). About 70% of the DMD and Becker muscle dystrophy (BMD) are inherited as X-linked recessive disorders and, therefore, the disease affects mainly the male sex. About two-thirds of the mothers of the affected boys are believed to be carriers (Arahata, 2002). DMD and BMD are called dystrophinopathies as they are caused by the mutations of the dystrophin gene (Emery, 1991). The dystrophin gene in the human genome spans 2.4 Mb of DNA. It is composed of 79 exons and maps to Xp21.2; hence, it is difficult to perform full gene screening by sequencing. It is transcribed into 16 kb mRNA that is translated into the dystrophin protein, which consists of a 427 kDa protein (Blake et al., 2002; Bellayou et al., 2009). The dystrophin is a sarcolemmal membrane spanning protein connecting the muscle cell cytoskeleton to the extracellular environment. It functions by supporting the integrity of muscle cell membrane from being squeezed during forcible muscle contractions (Ozawa, 2010). Patients with dystrophinopathies lack this protective mechanism of their muscle fibers. Consequently, muscle cells are damaged by the successive muscle contractions, and degenerate and become replaced by fibrofatty tissue. This results in a pseudohypertrophic appearance that particularly affects the calf muscles, and progressively, patients lose their muscular power and become wheelchair bound in their early life (Corrado and Jhon, 1994).
The diagnosis of muscular dystrophy usually begins with a clinical suspicion that has proven to be very sensitive and to have a high positive predictive value (Shukla et al., 2004). The diagnosis can be confirmed biochemically, histopathologically (Emery, 1983), and by using dystrophin immunohistochemical staining (Pereira et al., 2006), which can easily reflect any mutation in the dystrophin gene.
The aim of the present study is to clinicopathologically characterize an Egyptian family of a nonconsanguineous couple, with five (three males and two females) out of seven siblings clinically suspected to have muscular dystrophy.
Materials and methods
The current study was approved by the Alexandria Faculty of Medicine, Research Ethics Committee, and the family members who participated in the study were provided appropriate genetic counseling and signed an informed written consent to participate in the study.
The index case was a woman, 21 years old, complaining of frequent falls, inability to climb stairs, and hold objects, starting at the age of 12 years, with a gradual onset and progressive course. The clinical examination indicated proximal bilateral symmetrical muscle weakness, muscle hypotonia, hyporeflexia, pseudohypertrophy of the calves, waddling gait, and positive Gower’s sign.
The patient had four siblings who were clinically affected and had the same complaints. The elder sister was bed ridden, whereas the other three brothers presented with a wide range of clinical severity. The eldest (26 years) and the youngest (10 years) brothers were clinically normal at the time of their visit to the clinic.
The index case was referred to the clinical genetics outpatient clinic for pedigree construction and for verification of the mode of inheritance in that family. The two severely affected siblings did not participate in our study as they were bed ridden, and were residents of a remote rural area.
The index case and the other two male siblings who were clinically suspected to have primary muscular dystrophies were subjected to further investigations as detailed below. The youngest apparently normal male sibling agreed to participate in the study as well.
Biochemical and electrophysiological studies
Serum creatinine phosphokinase (CPK) was assessed for all participants. Electrophysiological study was performed as follows:
- Standard nerve conduction study for both lower limbs including a sensory conduction study for the sural nerve and a motor conduction study for both posterior tibial and common peroneal nerves. In addition, F-wave of the posterior tibial nerve and H-reflex were also performed according to the standard techniques.
- Needle electromyography for the right tibialis anterior, left vastus medialis, and right biceps brachii according to the standard techniques.
Pathological examination of muscle biopsies of affected participants
Quadriceps muscle biopsy was obtained from three of the affected members – the index case and two of her brothers – using an open method. The tissue biopsy was immediately placed on a dry filter paper for immobilization and was then fixed in 10% neutral-buffered formalin, to be processed to formalin-fixed paraffin-embedded tissue blocks. Five micrometer tissue sections were cut and stained with conventional hematoxylin and eosin stain for routine histopathologic examination.
Immunostaining was performed on 5-μm-thick sections cut from the formalin-fixed paraffin-embedded tissue blocks mounted on polylysine-coated microslides. Tissue sections were dewaxed, rehydrated, and then incubated for 15 min in 3% hydrogen peroxide to block endogenous tissue peroxidase. Heat-induced antigen retrieval was performed by microwaving tissue sections in 10 μl citrate buffer, pH 6.0, for 10–20 min, followed by cooling at room temperature for 20 min. Then, tissue sections were incubated with the dystrophin monoclonal antibody (DS9910-20; US Biological; 0.2 mg/ml; Fremont, California, USA) at a dilution of 1 : 200 for 30 min at room temperature. The antigen–antibody reaction was visualized using the UltraVision LP Detection System (Thermo Scientific). Immunohistochemical reactions were developed with diaminobenzidine and sections were counterstained with Harris hematoxylin. Normal muscle biopsies were used as a positive control and showed normal dystrophin membranous immunostaining (Preston and Shapiro, 2007).
The three clinically suspected primary muscular dystrophies patients and the apparently normal youngest male sibling were included in this descriptive study. The clinical and the electrophysiological findings of the family members are described in Table 1. The echocardiographic study was unremarkable for all affected siblings. The pedigree is shown in Fig. 1. The family pedigree was not conclusive of an X-linked recessive inheritance pattern because of the lack of other family members affected from the maternal side.
The biochemical marker CPK was markedly elevated to more than 1000 IU in the three affected participants. The histopathological examination of the hematoxylin and eosin-stained sections prepared from the quadriceps muscle biopsy obtained from three of the affected members showed muscle fiber size variability, hypertrophic muscle fibers, and increased internalization of nuclei, increased endomysial fibrosis, and fat metaplasia. Inflammation around the necrotic fibers, fiber splitting, and myophagocytosis, in addition to regenerating fibers with large vesicular sarcolemmal nuclei with prominent nucleoli and basic cytoplasm, were also noted (Figs 2 and 3). These findings could be noted in either limb girdle muscle dystrophy or DMD/BMD; however, the clinical picture in the affected males in the family was more in favor of DMD/BMD. Therefore, dystrophin immunohistochemical staining was considered mandatory. Immunohistochemical staining of the paraffin-embedded tissue sections with dystrophin monoclonal antibodies was carried out on the muscle biopsies obtained from the three affected siblings. Dystrophin immunostaining indicated a heterogeneous pattern of protein expression, ranging from almost complete absence of membranous staining (Fig. 4), with the exception of few and tiny foci showing some muscle cells with residual membranous (sarcolemmal) staining in the female patient, to weak, faint, and interrupted staining in the biopsies of the two male siblings (mosaic pattern) (Fig. 5).
The DMD gene (MIM 300377) is one of the largest human genes so far, known to span 2.4 MB of DNA on Xp21.2. It consists of 79 exons encoding dystrophin sarcolemmal protein; hence, immunohistochemistry (IHC) was considered a convenient cost-effective method for screening of the defective gene product. The majority of DMD/BMD cases (∼65%) show partial gene deletions (60%) or duplications (5%) that are preferentially clustered in two major hotspots, spanning exons 3–7 and 44–55. Point mutations and small deletion/insertion alterations account for the remaining cases (35%). The difference between DMD and BMD is in the degree of phenotype severity and is explained by reading frame hypothesis, mutations that disrupt the reading frame causing a premature termination, and loss of dystrophin that can lead to the severe phenotype of DMD. Mutations that retain the reading frame generate a shortened protein; the dystrophin may still have been limited to altered or subnormal function, leading to a milder phenotype of BMD.
Both the DMD and the BMD are genetic disorders in which the defective gene is localized on the X chromosome and the disease is transmitted in an X-linked recessive manner, thereby primarily affecting the males, whereas females who are the carriers of the disease are only rarely affected (Sajid et al., 2011).
Usually, 90% of females with dystrophinopathies are asymptomatic carriers; however, they still can manifest the disease ((Sajid et al., 2011)). This can result from skewed X inactivation and X autosome translocation; females with Turner’s syndrome (45,X) can also develop this. In addition, a case report showed uniparental disomy of the entire X chromosome to be the pathogenetic mechanism in some affected females (Boyd et al., 1986; Boileau and Junien, 1989; Bakel et al., 1995; Sajid et al., 2011; Zürich, 2011).
In this study, the diagnosis of the patients was established after correlating the clinical, biochemical, morphological, and immunohistochemical findings in each affected family member. Electrophysiology suggested DMD/BMD.
In the present study, the index case (female patient) showed typical features of muscular dystrophies including progressive proximal muscle weakness, frequent falls, and muscle wasting similar to the manifestations observed in her affected male siblings. Similarly, serum CPK level was also elevated in all affected siblings. Muscle biopsies indicated typical myopathic features and dystrophin immunohistochemical staining of the three affected siblings showed abnormal expression supporting the diagnosis of DMD/BMD.
However, the lack of other affected maternal family members and the lack of consanguinity between the parents that sometimes causes a female to express diseases with an X-linked recessive inheritance pattern may suggest one of two explanations: the occurrence of the maternal Xp21.2 mutation at the dystrophin gene is probably because of a maternal new mutation resulting in an asymptomatic carrier status of the mother of the affected cases or mutation is confined to the maternal gonads (gonadal mosaicism), and affected females in the family may be attributed to skewed X inactivation.
The increased use of dystrophin analysis on muscle biopsies for molecular diagnosis has aid the diagnosis of many female patients with no previous family history of any neuromuscular disease through having a mosaic dystrophin immunostaining pattern of their muscle biopsies (Zürich, 2011). The staining pattern of dystrophin in the affected females with a mosaic appearance of dystrophinopathies was further reported in several studies (Arikawa et al., 1991; Sajid et al., 2011).
This supports the utility and relevance of using dystrophin IHC for confirmation of the diagnosis of various muscular dystrophies.
Because of similarities in the clinical presentation and molecular pathology underlying both DMD and BMD, the differentiation of both syndromes is largely dependent on clinical severity and the type of mutation in the dystrophin gene. The gene-level diagnosis is mandatory in these patients; yet, it is difficult to make a molecular diagnosis in every suspected case. This is because of financial and technical limitations resulting from the huge size of the gene and the presence of variable mutations encompassing the whole gene. This can therefore be substituted by study of the gene expression using IHC, which is considered to be an essential preliminary step before pursuing molecular diagnosis. Dystrophin immunohistochemical analysis of muscle biopsy is a powerful tool in distinguishing various muscular dystrophies and it has proven to be better than genetic analysis using the multiplex PCR technique because genetic analysis may sometimes fail to detect some mutations in the dystrophin gene because of technique-dependent limitations, unless multiple techniques are used (Cohen and Muntoni, 2004).
Genetic testing of the dystrophin gene in DMD/BMD necessitates complete genetic screening of the whole dystrophin gene. However, this testing is currently not available in all centers on a routine clinical basis. The immunohistochemical procedure is rapid, easy, and accurate. Moreover, the increasing availability of dystrophin antibodies and its associated proteins for paraffin IHC makes it an applicable tool that can be introduced routinely in certain clinical settings, particularly in developing countries, because of the huge expenses of full gene sequencing in addition to other techniques for the detection of large deletions.
Conflicts of interest
There are no conflicts of interest.
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