The August, 1970 issue of Indian Pediatrics reported an article describing the histopathological and enzyme histochemical features in the muscle biopsy of children with Duchenne muscular dystrophy (DMD) [1]. This article provides an opportunity to introspect on the evolution of diagnosis of children with Duchenne muscular dystrophy. In the present era of molecular genetics, the article provides an insight into the importance of muscle biopsy, which has gradually lost its way in this tale of last 50 years in diagnosis of children with DMD.

Naryananan, et al. [1] reported a descriptive study on clinical, histopathological and enzyme histochemical features of 23 children with Duchenne muscular dystrophy. Among the 23 patients, 5 children had an onset before 3 years of age while the remaining 18 children had onset between 4 to 10 years of age. Two girls were included in those 23 children with rest being boys. Histopathological features described by authors include marked variation in muscle fibre size with hyaline degeneration of muscle, alteration in sarcoplasmic nuclei with central nuclei and clumps of atrophic nuclei, and increased endomysial connective tissue. Enzyme histochemical analysis revealed increased lactate dehydrogenase activity in the dystrophic muscle fibre suggestive of increased anaerobic metabolism along with decrease in aerobic metabolism in terms of decrease in succinic dehydrogenase. Increased ATPase activity observed in the muscle was postulated by authors to decrease ATP content with consequent impairment of muscular activity. Authors have emphasized on correlation of clinical symptoms to enzyme histochemical features like muscle weakness to increase in ATPase activity in the muscle fiber and increase in LDH activity in connective tissue to increased fat deposition.

In late 1860s, Duchenne performed muscle biopsy on a patient with a myopathy. Till 1970s, pathologists largely relied on histopathological features based on routine hematoxylin and eosin staining. Enzyme histo-chemistry was introduced in the early 1970s. The present article is one of the first few articles that describe the enzyme histochemical features in children with Duchenne muscular dystrophy. Electron microscopy had limited application for muscular dystrophies, but played a major role in the pathological diagnosis of congenital myopathies in the early 1980s [2]. Immunohistochemistry was introduced in 1980s that described absence of dystrophin protein in children with DMD. Western blot also provided qualitative information on expression of proteins in muscular dystrophy [3]. In late 1980s, with the advent of molecular diagnostics, polymerase chain reaction (PCR) was available world-wide and gradually replaced muscle biopsy as the first line investigation for diagnosis of DMD. This technique detects large deletions in 60-65% of patients with DMD. Subsequently, by the year 2003, multiplex ligation probe analysis (MLPA) was introduced for detecting mutations in DMD gene. MLPA is a quantitative method to detect deletion and duplication in all the 79 exons of dystrophin gene, and is also useful in carrier testing.

DMD is an X-linked recessive disorder resulting from deletion or duplication in the DMD gene. Indian studies have revealed a yield of 73% for detecting mutations (deletion and duplications) in DMD gene by MLPA [4]. Conventionally, patients with suspected DMD/Becker muscular dystrophy (BMD) who test negative for DMD mutation by MLPA were subjected to immuno-histochemistry on muscle biopsy. Studies have revealed that 36% of MLPA negative patients were detected to have DMD by immunohistochemistry [5]. With the advent of next generation sequencing (NGS), single nucleotide variations, small deletions, insertions and splice site changes in the DMD gene could be further screened among MLPA negative patients.

In the study by Tallapaka, et al. [4], 10 of the 14 MLPA negative patients were detected to have sequence variants in DMD gene. Kohli, et al. [6] have reported 97% yield of NGS among patients with DMD. As NGS is good at detecting large deletion and duplications, it might become the investigation of choice in years to come. Although, there has been gradual decline in the cost of NGS, still, for many in India, cost remains a major constraint in adopting the same as the first line investigation.

In this genetic era, when we look back at the study by Narayanan,et al. [1] the diagnosis of DMD was made purely on the basis of clinical phenotype, high creatine phosphokinase (CPK) levels and muscle histopathology. There is an overlap in the clinical and histological features of DMD with limb girdle muscular dystrophy (LGMD 2I) resulting from mutation in Fukutin related protein (FKRP) [7].Dubowitz, et al. [8], way back in the year 1965, have considered LGMD and late onset spinal muscular atrophy to mimic DMD and have beautifully outlined how their histological features differ.

There is a paradigm shift from histopathological diagnosis to genetic diagnosis that has far more implications beyond establishing a diagnosis. Accurate genetic diagnosis is an essential tool for designing personalized treatment of DMD [9]. Detection of point mutation in exon 53 and exon 51 provides an avenue for recently approved treatment strategies for DMD including exon 51 skipping (Eteplirsen) and exon 53 skipping therapies [10,11]. Similarly, non-sense mutation in DMD provides opportunity to enrol the patient in the research for drug Ataluren (Stop codon read through). There is emerging interest in CRISPR-Cas9 mediated genome editing as a potential therapy for DMD, which obviously will require identification of point mutation [12]. Apart from clinical benefits, improvement in the dystrophin expression as determined by semiquantitative immunohistochemistry and Western blot are often considered as an outcome measure in clinical trials among children with DMD [10]. Apart from treatment implications, genetic diagnosis will have implications for estimating the reproductive risk, enabling carrier testing and prenatal diagnosis.

The rapid advances in genetic diagnosis have bypassed burdensome workup including invasive muscle biopsy, and prevent uncertainty in the diagnosis. However, parents must be always involved in decision- making, and the cost, utility, validity, and limitations of genetic testing must be clearly explained. Muscle biopsy remains the investigation of choice among MLPA negative patients who are either unable to afford NGS or whose NGS have revealed variants of unknown significance (VUS). In this era of molecular diagnosis, histopathological features and enzyme histochemistry might have limited historical role.

Despite rapid genetic advances, few patients remain undiagnosed. Recent studies have demonstrated that non-coding mutations could be an important source of unresolved genetic disease that could be detected by analyzing DMD transcripts in muscle biopsy using mRNA [13]. Hence, muscle biopsy is useful not only for establishing the diagnosis but may be useful for genetic counselling of patients who remain undiagnosed on MLPA and NGS. There is a lot of emerging interest in proteomics of DMD for better understanding of pathogenesis and possible avenues for treatment [14]. Hence, a tale that started 50 years back on advances in improving the diagnosis of DMD by muscle biopsy, has implications even in this advanced molecular era.

1. Narayanan I, Das S, Vaishnava S, Sriramachari. Duchenne muscular dystrophy. Indian Pediatr. 1970;7:429-41.

2. Hudgson P. The value of electron microscopy in muscle biopsies. Proc R Soc Med. 1970;63:470-4.

3. Barresi R. From proteins to genes: Immunoanalysis in the diagnosis of muscular dystrophies. Skeletal Muscle. 2011; 1:24.

4. Tallapaka K, Ranganath P, Ramachandran A, Uppin MS, Perala S, Aggarwal S, et al. Molecular and histopathological characterization of patients presenting with the Duchenne muscular dystrophy phenotype in a tertiary care center in Southern India. Indian Pediatr. 2019;56:556-9.

5. Manjunath M, Kiran P, Preethish-Kumar V, Nalini A, Singh RJ, Gayathri N. A comparative study of mPCR, MLPA, and muscle biopsy results in a cohort of children with Duchenne muscular dystrophy: a first study. Neurol India. 2015;63:58-62.

6. Kohli S, Saxena R, Thomas E, Singh K, Bijarnia Mahay S, Puri RD, et al. Mutation spectrum of dystrophinopathies in India: Implications for therapy. Indian J Pediatr. 2020 May 2. [E-pub ahead of print]

7. Rocha CT, Hoffman EP. Limb–girdle and congenital muscular dystrophies: Current diagnostics, management, and emerging technologies. Curr Neurol Neurosci Rep. 2010;10:267-76.

8. Dubowitz V. Muscular dystrophy and related disorders. Postgrad Med J. 1965;41:332-46.

9. Bello L, Pegoraro E. Genetic diagnosis as a tool for personalized treatment of Duchenne muscular dystrophy. Acta Myol. 2016;35:122-7.

10. Lim KRQ, Maruyama R, Yokota T. Eteplirsen in the treatment of Duchenne muscular dystrophy. Drug Des Devel Ther. 2017;11:533-45.

11. Clemens PR, Rao VK, Connolly AM, Harper AD, MahJK, Smith EC, et al. Safety, tolerability, and efficacy of viltolarsen in boys with Duchenne muscular dystrophy amenable to exon 53 skipping: A phase 2 randomized clinical trial. JAMA Neurol. 2020 May 26. [E-pub ahead of print].

12. Wong TWY, Cohn RD. Therapeutic applications of CRISPR/Cas for Duchenne muscular dystrophy. Curr Gene Ther. 2017;17:301-8.

13. Gonorazky H, Liang M, Cummings B, Lek M, Micallef J, Hawkins C, et al.RNAseq analysis for the diagnosis of muscular dystrophy. Ann ClinTransl Neurol. 2015;3:55-60.

14. Capitanio D, Moriggi M, Torretta E, Barbacini P, Palma SD, Viganò A, et al. Comparative proteomic analyses of Duchenne muscular dystrophy and Becker muscular dystrophy muscles: Changes contributing to preserve muscle function in Becker muscular dystrophy patients. J Cachexia Sarcopenia Muscle. 2020;11:547-63.