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Year : 2022  |  Volume : 65  |  Issue : 5  |  Page : 259-270
Genetics and muscle pathology in the diagnosis of muscular dystrophies: An update

1 Department of Pathology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India
2 Department of Pathology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad, Telangana, India
3 Department of Medical Genetics, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad, Telangana, India

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Date of Submission02-Nov-2021
Date of Decision19-Nov-2021
Date of Acceptance28-Nov-2021
Date of Web Publication11-May-2022


Muscular dystrophies are a clinically and genetically heterogeneous group of disorders involving the skeletal muscles. They have a progressive clinical course and are characterized by muscle fiber degeneration. Congenital muscular dystrophies (CMD) include dystroglycanopathies, merosin-deficient CMD, collagen VI-deficient CMD, SELENON-related rigid spine muscular dystrophy, and LMNA-related CMD. Childhood and adult-onset muscular dystrophies include dystrophinopathies, limb-girdle muscular dystrophies, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic dystrophy. Traditionally, muscle biopsy and histopathology along with special pathology techniques such as immunohistochemistry or immunoblotting were used for the diagnosis of muscular dystrophies. However, recent advances in molecular genetic testing, especially the next-generation sequencing technology, have revolutionized the diagnosis of muscular dystrophies. Identification of the underlying genetic basis helps in appropriate management and prognostication of the affected individual and genetic counseling of the family. In addition, identification of the exact disease-causing mutations is necessary for accurate prenatal genetic testing and carrier testing, to prevent recurrence in the family. Mutation identification is also essential for initiating mutation-specific therapies (which have been developed recently, especially for Duchenne muscular dystrophy) and for enrolment of patients into ongoing therapeutic clinical trials. The 'genetic testing first' approach has now become the norm in most centers. Nonetheless, muscle biopsy-based testing still has an important role to play, especially for cases where genetic testing is negative or inconclusive for the etiology.

Keywords: Molecular genetic testing, muscle biopsy, muscular dystrophy, next-generation sequencing technology

How to cite this article:
Narasimhaiah D, Uppin MS, Ranganath P. Genetics and muscle pathology in the diagnosis of muscular dystrophies: An update. Indian J Pathol Microbiol 2022;65, Suppl S1:259-70

How to cite this URL:
Narasimhaiah D, Uppin MS, Ranganath P. Genetics and muscle pathology in the diagnosis of muscular dystrophies: An update. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 May 24];65, Suppl S1:259-70. Available from: https://www.ijpmonline.org/text.asp?2022/65/5/259/345037

   Introduction Top

Muscular dystrophies are a clinically and genetically heterogeneous group of inherited disorders of the skeletal muscle, which are characterized by progressive muscle fiber degeneration.[1] The age of onset of manifestations can be variable, ranging from onset at birth (e.g., congenital muscular dystrophies), to childhood (e.g., Duchenne muscular dystrophy) to adulthood (e.g., facioscapulohumeral muscular dystrophy). The features common to most muscular dystrophies include progressive muscle weakness (with predominant involvement of proximal muscles) and elevated serum creatine kinase (CPK), but the severity and rate of progression can be variable for the different types. Additional associated findings in many muscular dystrophies include cardiomyopathy and respiratory muscle weakness, especially towards later stages of the disease.[2]

Genetic basis and classification of muscular dystrophies

Muscular dystrophies are caused by mutations in genes coding for important structural and functional components of the skeletal muscle, especially the extracellular matrix, sarcolemma, cytoskeleton, and cytosolic and nuclear membrane proteins.[3] Many genes associated with muscular dystrophy encode components of the dystrophin-glycoprotein complex (DGC). The DGC helps to link the intracellular cytoskeleton to the extracellular matrix, and mutations in genes that code for components of this complex result in loss of sarcolemmal integrity, making the muscle fibers more susceptible to damage.[3]

Muscular dystrophies can be broadly categorized into congenital muscular dystrophies and the childhood and adult-onset types. Congenital muscular dystrophies (CMDs), as outlined in [Table 1], include dystroglycanopathies, merosin-deficient CMD, collagen VI-deficient CMD, SELENON-related rigid spine muscular dystrophy, and LMNA-related CMD.[4],[5] The childhood and adult-onset muscular dystrophies include dystrophinopathies, limb-girdle muscular dystrophies (LGMDs), Emery-Dreifuss muscular dystrophy (EMD), facioscapulohumeral muscular dystrophy (FSHD), and myotonic dystrophy. The subtypes, associated genes and patterns of inheritance of these childhood and adult-onset disorders are listed in [Table 2].[1],[6] In addition to these, there are a group of disorders called myofibrillar myopathies and distal myopathies, some of which were formerly classified as LGMDs, such as DES gene-related LGMD1E and LGMD2R and MYOT gene-related LGMD1A.[6] These are now considered to be a distinct group characterized by disintegration of the myofibrils and intracellular accumulation of the degradation products as inclusions.[7],[8]
Table 1: Classification of congenital muscular dystrophies[4],[5]

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Table 2: Types of childhood and adult-onset muscular dystrophies[1],[6]

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Evaluation of muscle pathology in muscular dystrophies

Muscle biopsy is usually taken from the moderately involved muscle (power 3/5). The tests done in the muscle biopsy sample include routine histopathology, enzyme histochemistry, immunohistochemistry, and Western blot (Immunoblot).

  • Histopathology findings: Muscular dystrophies are characterized by features of chronic muscle damage. There is usually partial or complete effacement of the muscle architecture with diffuse variation in size of both type 1 and 2 myofibers.[9] A combination of hypertrophic, atrophic and rounded fibers is seen with the hypertrophied fibers demonstrating the internalization of myonuclei. Internalized nuclei in more than 10% of myofibers indicate myopathy, and if seen in more than 30% of myofibers suggest chronic dystrophy. Myotonic dystrophy is one condition with numerous myofibers (>60%) with internalized nuclei appearing as nuclear chains in longitudinal sections.[9]

    The atrophic fibers can be small angulated fibers or appear as multinuclear clumps. Other non-specific features observed are fiber splitting, whorled fibers and ring fibers. Necrosis and fibrosis are the two hallmarks of muscular dystrophy.[10] Fibrosis is generally accompanied by adipose tissue infiltration. Myonecrosis tends to involve individual or clusters of myofibers followed by myophagocytosis. As any injury to the muscle is followed by regeneration of myoblasts by satellite cells, the biopsy may feature variable numbers of regenerating fibers. These fibers have basophilic cytoplasm and vesicular nuclei with prominent eosinophilic nucleoli.

    Inflammation can be present in some muscular dystrophies such as FSHD, DMD, dysferlinopathy and calpainopathy.[11],[12] The endomysial inflammatory infiltrate is generally composed of lymphocytes, with predominance of T-cells over B-cells. Muscle biopsies of a third of patients with FSHD show lymphocytic infiltrates comprising CD4+ and CD8+ T-cells. Similarly, dysferlinopathies can show non-specific myopathic features and dense inflammatory infiltrate comprising lymphocytes and macrophages. Few biopsies from calpainopathy can show eosinophils.[9],[11] Inflammation is also reported in congenital muscular dystrophy.[13] The presence of inflammation, particularly when dystrophic features are not well-developed, may lead to misdiagnosis as myositis. Careful consideration of clinical features, laboratory parameters and judicious application of immunohistochemistry can help in correct diagnosis.
  • Enzyme Histochemistry: Unlike in other myopathies like congenital and metabolic myopathies, where enzyme histochemistry demonstrates specific diagnostic features, in dystrophies, the enzymes can be non-specific. Modified Gomori trichrome (MGT) can show rimmed vacuoles in certain limb girdle muscular dystrophies (LGMD2G, LGMD2J)[14],[15] and oculopharyngeal muscular dystrophy.[16] Lobulated fibers in NADH-TR can be a prominent feature in calpainopathy, but can also be seen in FSHD and other myopathies.[17],[18] Lobulated fibers show increased reaction product at the periphery due to accumulation of mitochondria. Increased expression of actin-binding proteins may contribute to the architectural changes seen in lobulated fibers.[19] Type 1 fiber predominance is found in various myopathies such as DMD, congenital myopathies and CMDs.[20] In quadriceps biopsies, type 1 predominance is present if type 1 fibers comprise >55% of the total.[21]
  • Immunohistochemistry: Immunohistochemistry (IHC) plays a major role in identifying the type of muscular dystrophy since this is not possible based on the morphological features and enzyme histochemistry. The deficiency of the protein product in muscular dystrophies can be either primary or secondary. In addition, certain developmentally regulated proteins not expressed in normal mature muscle may be re-expressed in muscular dystrophies.[9]

    The primary protein product deficiency is due to mutation in the causative gene. The ability to detect the primary protein abnormality by IHC in muscular dystrophies depends on the genetic alteration and its effect on the protein product.[9] In autosomal recessive disorders, the protein is not detectable by IHC if the mutation leads to a stop codon. On the other hand, if the mutation results in production of some protein product, then IHC may not be helpful, but other quantitative methods such as Western blot may be of help in demonstrating the decreased or abnormal protein.[9] Likewise, in muscular dystrophies with autosomal dominant pattern of inheritance, IHC is generally not of much help since protein production is ensured by the normal allele. However, in rare autosomal dominant MDs, IHC may be of use, one example being LGMD1C.[9] This is because, in LGMD1C, the mutant caveolin-3 interferes with the function of wild-type caveolin-3 by inducing proteolysis.[22]

    The secondary deficiency in protein product is due to mutations in other gene (s). This is to be expected since some of the major skeletal muscle proteins are part of an inter-linked complex. For example, dystrophin protein links the extracellular matrix with the actin cytoskeleton, forming a complex comprising laminin-α2, alpha-dystroglycan and sarcoglycans, amongst others. One should remember the possibility of secondary deficiency of proteins while ordering and assessing the immunohistochemical panels for muscular dystrophies.

    The localization of proteins in skeletal muscle varies from sarcolemma (dystrophins, sarcoglycans, laminin-α2), nuclear membrane (emerin) to myofibrils (telethonin, titin).[9]

    IHC is a qualitative assay performed on cryosections. As many of the proteins are localized to the sarcolemma, it is necessary to ensure the integrity of the plasma membrane. Usually, β-spectrin is used to assess that the sarcolemma is intact. As is the routine practice in IHC, it is essential to run positive and negative controls.

    In routine myopathology practice, IHC is commonly performed for dystrophins, sarcoglycans (α, β, γ, δ), dysferlin, laminin-α2 (merosin), collagen VI, emerin, α-dystroglycan and β-dystroglycan. For large proteins like dystrophin, antibodies that target different regions of the protein are used to avoid false-negative results. For dystrophin IHC, it is common practice to apply three antibodies that target the C-terminal, N-terminal and rod domain of the protein. For some proteins, Western blot is a more suitable method of assessment. LGMDR2 is due to mutation in the DYSF gene, resulting in the deficiency of dysferlin. However, secondary deficiency of dysferlin can occur in calpainopathy. In this situation, a quantitative method such as Western blot can be useful in differentiating between primary and secondary protein deficiency.[9] Another useful antibody for IHC is α-dystroglycan (DG), one of the proteins of the dystrophin-associated complex. Alpha-dystroglycan is essential to maintain stability of the skeletal muscle and is heavily glycosylated. Glycosylation of proteins is essential to maintain various biological processes, and hypoglycosylation disrupts the protein function.[23] Mutations in various genes implicated in LGMDs and CMDs lead to either primary or secondary deficiency of α-DG, the latter more common than the former.[24],[25] Antibodies against the glycosylated epitope of α-DG are helpful in demonstrating the decrease in the protein. However, suitable commercial antibodies are not available for the core protein of α-DG. In dystrophinopathies, both core protein and glycosylated epitope of α-DG, as well as β-DG are decreased. Whereas, in CMDs and LGMDs, β-DG is normal and the glycosylated epitope of α-DG is decreased.[9],[26],[27] Here again, Western blot can be a useful adjunct for IHC.

    The classic biopsy features of dystrophies with corresponding IHC are depicted in [Figure 1]. [Table 3] provides the primary and secondary protein deficiencies in muscular dystrophies.
  • Western blot: Western blot (immunoblot) is a qualitative method useful for evaluation of molecular weight and abundance of a protein. Multiplex blots can simultaneously interrogate multiple proteins with different molecular weights in the muscle homogenate. It is a useful adjunct for IHC, particularly when suitable antibodies are not available like for calpain-3 and multiplexing helps in assessment of secondary reduction of the protein. However, Western blot is labor-intensive and costlier than IHC.[28]
Figure 1: Muscle histopathology images: (a) 6 years-old male patient with Duchenne muscular dystrophy showing fibre size variation [hematoxylin and eosin (H and E), X40], fibrosis (Masson trichrome) and complete absence of D1, D2 and D3 along the sarcolemma with dystrophin IHC. (b) 14 years-old male patient with Becker muscular dystrophy showing marked fibre size variation and fibrosis (H and E, X40), and irregular staining patterns with dystrophin IHC. (c) 12 years-old female patient with gamma sarcoglycanopathy showing dystrophic picture (H and E, X40), loss of checkerboard pattern (ATPase pH 9.4), and absence of gamma sarcoglycan staining along the sarcolemma with sarcoglycan IHC. (d) 33 years-old female patient with dysferlinopathy showing endomysial inflammation (H and E, X40) and absence of dysferlin immunostaining along the sarcolemma. (e) One-and-half-year-old floppy baby with dystrophic picture (H and E, X40), fibrosis and effacement of architecture (Masson trichrome) and absence of immunostaining for merosin in LAMA2-associated congenital muscular dystrophy

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Table 3: Immunohistochemistry features in muscular dystrophies

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Genetic testing modalities for muscular dystrophies

Majority of the muscular dystrophies are monogenic disorders i.e., each of the disorders is caused by mutation (s) in a single gene. Therefore, molecular genetic tests are used for their diagnosis. Of the various technologies used for molecular genetic testing, the most commonly used ones are Sanger sequencing, next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA). These techniques are described in brief, hereunder.

Molecular genetic testing techniques

  • Sanger sequencing: Sanger sequencing, the 'first-generation' sequencing technique, was developed in the 1970s and until about a decade ago, was almost exclusively the method used for DNA sequencing applications.[52] This method requires prior amplification of the specific gene or genomic region of interest through the polymerase chain reaction (PCR) process. One standard PCR reaction usually amplifies a DNA fragment of 500 to 1000 nucleotides-size. As majority (95-98%) of the disease-causing mutations lie in the coding portions (exons) of the gene or in the intron-exon splice junctions, PCR amplification is usually done for the exonic and exon-flanking intronic sequences of the gene, without including the deep intronic regions. The PCR products or 'amplicons' are then subjected to Sanger sequencing. Sanger sequencing again involves a special type of PCR called chain-termination PCR, which is similar to standard PCR, but involves the addition of modified nucleotides called dideoxyribonucleotides (ddNTPs) in place of deoxyribonucleotides (dNTPs). The sequence data is then analysed manually through visual assessment of the sequence chromatogram as well as through in silico alignment with the reference sequences, to detect the variants. Though Sanger sequencing is accurate and suitable for sequencing individual small genes and for targeted sequencing of specific individual gene variants, it has a number of limitations when dealing with larger genes or genomic regions, in terms of being labour-intensive, expensive and time-consuming.[52]
  • Multiplex ligation-dependent probe amplification (MLPA): MLPA is a modified multiplex PCR assay. In each assay up to 40 probes can be used, to evaluate the relative copy number of different target genomic sequences (which could be either individual exons of genes, or specific chromosomal regions). Each probe consists of two half-probes (5' and 3' half-probes) that are complementary to 2 contiguous parts of the target genomic sequence. After hybridization of the probes with the patient's DNA, each pair of two half-probes get ligated only if their contiguous sequences perfectly hybridize with the target genome sequence. During the subsequent PCR reaction, only the ligated probes get amplified, and the amplified ligation products are then separated through capillary electrophoresis. The number of probe ligation products is analysed and this gives a measure of the number of target sequences in the patient's DNA. MLPA is used for gene dosage analysis as well as for testing chromosomal aneuploidies and chromosomal microdeletions and microduplications.[53]
  • Next-generation sequencing (NGS): The term 'next-generation sequencing' is used for any one of different 'massively parallel' DNA sequencing methodologies. A number of NGS technologies have been developed which use different sequencing chemistries and principles, but most of them involve the following basic steps: i. fragmentation of the entire genomic DNA into multiple pieces, either through physical shearing using mechanical methods such as sonication and nebulization, or through enzymatic digestion using nucleic acid endonucleases; ii. addition of specific adaptors at both ends of each fragment which serve as primer-binding sites for amplification; iii. size-selection of adaptor-ligated DNA fragments through agarose gel electrophoresis or with paramagnetic beads; iv. simultaneous PCR amplification of all the multiple fragments and creation of fragment libraries (unlike for Sanger sequencing where each target fragment requires a unique set of primers and an individual PCR reaction for amplification, for NGS a common set of primers is used which binds to all the adaptors ligated to the fragments, thereby ensuring universal simultaneous PCR amplification of all the millions of fragments); v. sequencing the libraries multiple times to ensure high depth of coverage (the chemistry used for sequencing varies for different NGS technologies); and vi. in silico reassembling of all the individual fragment sequences to form complete genomic sequences, and alignment of the sequence reads with the reference genome to detect the variants.[52],[54],[55],[56] For specific genomic regions of interest, 'targeted capture' or 'selective enrichment' of the desired regions is done through any one of different methods such as hybridization-based strategy, transposon-mediated fragmentation, molecular inversion probes method, or PCR-based target enrichment, and only those regions are subjected to sequencing.[57] With the help of these selective enrichment techniques, different customized platforms can be generated such as whole-exome sequencing (WES), clinical exome sequencing (CES), or multigene panels. The CES panel includes exons (with flanking intron-exon junctions) of only the genes established to be associated with human genetic disorders, whereas the WES platform includes exons (with flanking intron-exon junctions) of all the known genes in the genome. For whole genome sequencing (WGS), prior targeted capture is not required.[54],[55] NGS overcomes many of the limitations of Sanger sequencing. It is much more cost-effective and much less time consuming than Sanger sequencing, when larger genomic regions and multiple genes have to be sequenced. It also ensures a much higher depth of coverage and can detect even variants present at a low level of mosaicism.[54],[55]

Choice of genetic test in the clinical setting

Detailed delineation of the phenotype, through thorough clinical evaluation and ancillary laboratory testing, is essential for choosing the apt genetic test, as well as for accurate interpretation and analysis of the test results. Some of the important aspects of molecular genetic testing with reference to muscular dystrophies are highlighted here:

  • Next generation sequencing (NGS) is the preferred technique for genetic diagnosis of most of the muscular dystrophies including CMDs, LGMDs and Emery-Dreifuss muscular dystrophy. This is because these conditions have multiple subtypes with very similar phenotypes that are difficult to differentiate clinically. These subtypes are caused by mutation (s) in any one of many different genes, and within each gene, the mutation can involve any nucleotide position. Moreover, most of these genes are large (more than 10 kilobases in size); therefore, NGS would be more cost-effective than Sanger sequencing for individual genes.[58],[59]
  • Amongst the NGS-based platforms, multigene panels or clinical exome sequencing (CES) can be used if the features are suggestive of a specific entity e.g., dystroglycanopathy, autosomal recessive LGMD, etc.
  • Whole exome-sequencing (WES) helps to identify causal variants in genes already known to be associated with the phenotype (the ones included in a multigene panel and/or CES), as well as in other genes not hitherto reported in association with the phenotype (and hence not included in the multigene panel/CES). Trio-WES i.e., simultaneous whole exome sequencing of the proband and of both parents is the ideal approach, as it allows for simultaneous comparison of the variant data of the proband with that of the parents.[60]
  • Whole-genome sequencing (WGS) covers the entire genome. Therefore, not just the exons, but also the introns and promoter regions of genes as well as the intergenic regions are covered in WGS. It can therefore detect sequence variants in the deep intronic regions and promoter regions of genes, which are not covered in CES or WES. WGS data can also be analysed to look for copy number variations across the genome.[61] Until recently, WGS was being used only in research settings, but over the past few years it is being increasingly used as a second-tier or third-tier diagnostic test for evaluation of patients in whom first-level genetic tests such as CES and/or WES fail to identify the causative gene mutations.
  • When a large number of genes and/or genomic regions are covered with high throughput techniques like NGS, a large number of genetic variants are detected. The American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) have formulated guidelines for the classification of gene variants, using criteria based on population data, computational data, functional data, and segregation data. Based on these guidelines, variants are categorized as 'pathogenic', 'likely pathogenic', 'variant of uncertain significance' (VOUS), 'likely benign', and 'benign'.[62] Usually, in the clinical setting, identification of a pathogenic or likely pathogenic gene variant (s) which matches the clinical phenotype and follows the expected familial segregation pattern, is considered to be confirmatory for the diagnosis of the particular genetic disorder associated with that gene.
  • Interpretation of VOUS is tricky, difficult and often inconclusive. Additional evidences can help to corroborate the pathogenicity of a VOUS. For example, if CES identifies a VOUS in the SGCA gene in a patient with clinical features of LGMD, muscle biopsy and IHC with alpha sarcoglycan can be done for corroboration of the diagnosis of SGCA-associated LGMD (LGMDR2). If sufficient evidence regarding the pathogenicity of a VOUS is not obtained, it should not be assumed to be the disease-causing variant, and definitive actions such as prenatal or preimplantation genetic testing should not be performed based on such a presumptive, unconfirmed diagnosis.
  • Sanger sequencing is the preferred technique for targeted testing of SNVs and small insertions/deletions (involving a few nucleotides), and for individual small genes. Targeted variant testing is done to look for a specific, known/identified genetic variant. Sanger sequencing-based targeted variant testing helps to validate the variant identified through NGS-based testing in the proband [Figure 2]. Targeted variant testing of family members (for the variant identified in the proband) is done to study the segregation pattern of the variant in the parents, affected and unaffected siblings, and other relatives (as required), to check whether the pattern is consistent with what is expected for the mode of inheritance. Targeted testing of family members is also done for carrier testing, presymptomatic testing, and prenatal genetic testing.
  • MLPA is the test of choice for detection of large intragenic deletions and duplications that involve one or more exons. As large deletions and duplications are the most common types of mutations that occur in the DMD gene, accounting for around 65-70% of Duchenne muscular dystrophy and around 80% of Becker muscular dystrophy, MLPA is the preferred first-line testing modality when dystrophinopathies are suspected clinically [Figure 3].[63] For MLPA-negative cases of DMD, further NGS-based CES or WES is done, to look for sequence variants (including SNVs, small insertion-deletions, splice variants etc.) which constitute the remaining 30-35% of mutations. CES or WES also helps detect mutations in genes associated with other muscular dystrophies that mimic DMD, especially the childhood-onset autosomal recessive LGMDs such as sarcoglycanopathies.[64]
  • Myotonic dystrophy type 1 is caused by expansion of the trinucleotide repeat CTG in the 3' untranslated region of the DMPK gene and myotonic dystrophy type 2 is caused by expansion of the tetranucleotide repeat CCTG in the CNBP (ZNF9) gene. The tests of choice for detecting such repeat expansions are repeat-primed PCR assay [Figure 4] and Southern blot testing.[65] Recently developed software and improvements in the analysis pipeline, have made it possible to detect repeat expansions in even short-read sequencing datasets generated through NGS.[66]
  • The molecular mechanisms of FSHD are distinct from those of other muscular dystrophies and therefore a different testing strategy is used. FSHD1, which accounts for around 95% of FSHD cases, is caused by a heterozygous pathogenic contraction of the D4Z4 repeat array in the subtelomeric region of chromosome 4q35. In normal alleles, there more than 11 repeat units (each unit consisting of around 3300 bases), whereas alleles with less than 11 units cause disease. The diagnosis of FSHD1 is established by haplotyping of the 4q alleles and documenting the reduced number of D4Z4 repeats through the Southern blot test. FSHD2, which accounts for the remaining around 5% of FSHD cases, is caused by hypomethylation of the D4Z4 repeat array, which can be detected with the help of Southern blot testing using a methylation-sensitive restriction enzyme.[67] Recently, a unique method using the novel single-molecule optical mapping approach has been developed to detect the D4Z4 repeat sizes.[68]
Figure 2: Targeted variant testing through Sanger sequencing showing the homozygous pathogenic variant c.1376A>G; p.Gln459Arg in the CAPN3 gene (reference sequence ENST00000397163.3), thereby validating presence of the variant detected through clinical exome sequencing in a patient with autosomal recessive limb-girdle muscular dystrophy (LGMDR1)

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Figure 3: Multiplex ligation-dependent probe amplification (MLPA) of the DMD gene showing hemizygous deletion of 5 exons (exon 46 to exon 50) in a male child with Duchenne muscular dystrophy

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Figure 4: Repeat-primed PCR assay showing evidence of trinucleotide CTG repeat expansion in the DMPK gene in a patient with myotonic dystrophy type 1

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Role of muscle biopsy versus genetic testing

The advent of NGS-based molecular genetic testing has led to a paradigm shift in the diagnostic strategy for genetic neuromuscular disorders, with the 'non-invasive genetic testing first' approach being followed in most centres, nowadays.[69] Genetic testing helps to overcome some of the limitations faced with muscle biopsy, in the diagnostic evaluation of muscular dystrophies. Though muscle biopsy is a minimally invasive procedure, it may be difficult to perform in young children. The expertise for performing and interpreting techniques such as IHC, immunoblotting etc., which are required for precise typing of the muscular dystrophy, is available in only a limited number of centers. As there is considerable overlap between various muscular dystrophies, many different IHC stains may be required for accurate characterization. For some types, the antibody may not be readily accessible or may not be available, and hence IHC may not be feasible. There is also a possibility of false-positives with IHC, due to secondary deficiency of sarcolemmal proteins. With reduction in the costs and availability of higher throughput NGS technologies in recent times, genetic testing has become far more accessible and affordable.

Identification of the exact disease-causing mutation (s) through genetic testing of patients with muscular dystrophies, helps in molecular confirmation of the diagnosis. This is important because different muscular dystrophies have overlapping phenotypes, and accurate characterization through genetic testing helps in accurate prognostication, appropriate genetic counselling and suitable management of the disease. Furthermore, identification of the exact pathogenic gene variants in the index case is essential for accurate carrier testing and prenatal/preimplantation genetic testing, to prevent recurrence of the disorder in the family.

In recent times, mutation-specific therapies have also become available or are being developed for some of the muscular dystrophies, especially DMD.[70] Mutation identification is essential for initiating these therapies and for enrolment of patients into ongoing therapeutic clinical trials. Some of the approved and investigational mutation-specific therapies for DMD include exon-skipping, stop codon readthrough and gene editing using CRISPR/Cas9.[71]

Based on the accumulating evidence, one can say that genetic testing takes precedence over muscle biopsy as the first-tier diagnostic test for muscular dystrophies. However, genetic testing does have its limitations and the diagnostic yield of even NGS-based tests is limited at present. The diagnostic yield of WES in LGMDs has been found to range from around 40% to 50% in many studies.[58],[60],[72],[73],[74] A recent study from India has reported a diagnostic yield of 54% with WES in a cohort of 22 patients with CMDs.[75]

It is also important to remember that if the differential diagnosis includes acquired conditions such as inflammatory myopathy, a muscle biopsy can be very helpful to ascertain the etiology and guide further management decisions. In cases where genetic testing detects VOUS, as discussed above, muscle biopsy helps to corroborate the diagnosis. Muscle biopsy also provides valuable material for RNA sequencing, functional studies and basic research to understand disease biology.[76]

   Conclusion Top

With the availability of next-generation sequencing technology and other recent advances, molecular genetic testing has emerged as the preferred first-line diagnostic test for patients with muscular dystrophies. However, there is still a role for muscle biopsy in the evaluation of muscular dystrophies, especially where genetic testing does not yield the diagnosis or is inconclusive. Therefore, myopathologists need not put away their microscopes yet!

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Conflicts of interest

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Correspondence Address:
Prajnya Ranganath
Additional Professor and Head, Department of Medical Genetics, Nizam's Institute of Medical Sciences, Panjagutta, Hyderabad - 500 082, Telangana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijpm.ijpm_1074_21

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