Challenges and Advances in Molecular Diagnosis of Myopathies and Dystrophies in Perspective of Their Use in Developing Countries: Past, Present, and Future
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.325313
Source of Support: None, Conflict of Interest: None
Keywords: Diagnosis, dystrophy, genetics, GNE, myopathyKey Message: Accumulating knowledge clearly shows a higher frequency of certain genetic mutations inducing neuromuscular disorders in specific populations. Identifying and screening such markers using molecular technologies may better manage patients prone to such diseases in developing countries.
Myopathies are a collection of genetic alterations that ultimately leads to abnormal structure or functioning of skeletal muscles., Apart from managing treatment after symptomatic detection, their timely screening and diagnosis can help in better management., Further, late diagnosis and delay in proper management of such patients can also affect appropriate treatment.,,,,, With the rapid advancement in myopathy research in the last few decades, we can now establish region- and subcontinent-wise prevalence and molecular variations in myopathies and dystrophies.,,,,,,,,,,,,,, For example, more than 200 mutations have been reported in GNE myopathy; however, some of these SNPs are more prevalent in Japan, China, Middle East, and the Roma population in Bulgari and UK., Consequently, accumulating knowledge clearly shows the frequency of many mutations inducing other myopathies and dystrophies, showing a trend toward the population in certain regions. Thus, such mutations can be used for timely detection of myopathies and dystrophies in families that have a history of such disorders or could be carriers for the next generation.,,
Molecular alterations associated with myopathy
Various key genetic alterations responsible for myopathies have been identified in the last few decades.,,,,,,,, To identify these mutation patterns specific to myopathies and dystrophies, we first looked for a publicly available “ClinVar” database for reported genetic alterations. We used the search terms “OMIM” [submitter] and “dystrophy” [disease/phenotype] or “myopathy” [disease/phenotype] for our analysis. This led to the identification of 499 gene variants across various genes previously correlated with myopathy and 1028 gene variants associated with dystrophy [Table 1]. Interestingly, single nucleotide variation was seen in majority of studies that represented 83.8% (418/499 cases) of myopathies and 77.5% (797/1028) dystrophy cases. Findings from this cohort suggest the prevalence of single nucleotide variations across these disorders and suggest the importance of screening diseased and carrier populations in any susceptible population cohort. As covering methods to detect all these mutations are beyond the scope of this review article, we focused on known genetic mutations, especially in context but not limited to hereditary myopathies, whose large types are well reported in the literature,,,,,,, [Table 2]. We also discuss muscular dystrophies, which are generally characterized by pathological evidence of ongoing muscle degeneration and regeneration.,, This review explores some of the key nonsequencing molecular methods used in screening in past decades, some of which still hold a strong potential, especially for use under low-cost clinical setups in developing countries for timely screening of hotspot mutations prevalent in a population. In addition, this study discusses the next-generation sequencing (NGS) methods that hold great potential in the coming future.
Amplification refractory mutation system
Amplification refractory mutation system (ARMS-PCR) is a general technique used for detection of any point mutation or small deletion, which are some of the commonly found genetic alterations. This technique may detect normal, heterozygous, and homozygous states in the genotype of a sample using two complementary reactions, including specific primers for the amplification of typical DNA sequence at a given locus and other primers containing a mutant specific primer for the amplification of mutant DNA [Table 3]. In recent decades, these techniques have been used to detect both myopathy and dystrophy cases. In one such study by Fanim M et al. (2004), ARMS-PCR in combination with other techniques (including SSCP, DHPLC, and direct sequencing) was used to detect mutation in the LGMD2A mutation cohort. In recent years, this technique has also been used to evaluate sequence variants in the patient, carrier, and fetus for detecting a nonsense variant (c.C799T: p.R267X) in the CHM gene. Bai RK (2004) and Attali R et al. (2013) used the ARMS-PCR method for detection of mitochondrial and congenital myopathy, respectively. Bai RK specifically analyzed for A3243G mitochondrial tRNAleu (UUR) point mutation in 36 patients. Interestingly, this study demonstrated consistent results between PCR-restriction fragment length polymorphism (RFLP) analysis and real-time ARMS-qPCR. However, the findings of this study also showed ARMS-qPCR method was much more sensitive for detecting low percentages of mutant heteroplasmy. Attali R used the ARM-PCR approach along with Genome-wide linkage and sequencing techniques to screen congenital myopathy-specific mutations in a family using healthy individuals as controls. Collectively, these studies indicate that the ARMS-PCR technique can be used as a potential molecular tool in diagnosing genetic diseases in prenatal, newborns, and adults.
Nested PCR uses two or more successive PCRs, where the product of the initial PCR reaction is used as a template for the next PCR. This type of PCR is employed to amplify templates in low copy numbers in specimens. It has the benefits of increased sensitivity and specificity [Table 3]. In the past few years, this technique has also been used to screen various forms of myopathies. Huang W et al. (2004) and Malcov (2005) et al. used nested PCR to analyze dystrophin and SRY gene as well as to evaluate the possibility of using this technique for pre-implantation genetic diagnosis (PGD) of deleted Duchene muscular dystrophy (DMD) with family history. Huang W et al. reported that this technique of single lymphocytes 3-plex nested PCR for dystrophin and SRY gene was highly sensitive and reliable for PGD of deleted DMD with family history. Similarly, Malcov et al. analyzed biopsies of 156 embryos and was found them to be reliable in DMD diagnosis. Nested PCR was also used by Fardeau M et al. (2005) to check transgene expression and safety of intramuscular administration of a plasmid containing a full-length dystrophin cDNA. Gergely P and Joshi PR also used nested PCR for idiopathic inflammatory and myofibrillar myopathies. Interestingly, Gergely P et al. assessed the prevalence of TTV infection in IIM compared to patients with rheumatoid arthritis and healthy blood donors. However, Joshi PR used it for microdissected fibers analysis for multiple mtDNA deletions. These studies demonstrated the effective use of nested PCR to diagnose and screen myopathy and dystrophy-associated patients.
Restriction fragment length polymorphism
RFLP technique was invented in 1984 by Alec Jeffreys and was later widely used for the analysis of Variable Number of Tandem Repeats to genetically differentiate between organisms. This technique has also been used to study genetic variation in various myopathies and dystrophies due to its advantages [Table 3]. In the last few decades, this technique, either alone or in combination with other techniques, has been used widely. In one such study by Scuderi C et al., RFLP and DNA sequence analyses revealed pathogenic mutations m. 3243A > G in muscle and blood and a new heterozygous insertion at nt697 in doublecortin gene (DCX). In work done by Shojasaffar B et al., a healthy population in Iran was analyzed for CTG expansion and haplotype analysis in DM1 gene in a healthy Iranian population using PCR/RFLP from control healthy individuals. In another study on McArdle disease (a metabolic myopathy) by Rubio JC et al., RFLP along with other techniques was used to study 55 unrelated Spanish patients with McArdle disease for screening for three more frequent mutations in the PYGM gene in the Spanish population. Interestingly, the findings of this study suggest that molecular diagnosis of disease based on blood DNA would avoid muscle biopsy in 75.8% [95% confidence interval (95% CI): 62.1%–78.6%] of patients with McArdle disease. In another study by Cardaioli E et al. on mitochondrial myopathy, RFLP analysis confirmed that 63% of muscle mtDNA harbored mutation while it was absent in all other tissues. In another study by Malayeri FA et al. on DMD and BMD dystrophies, six RFLP markers were used for carrier detection and prenatal diagnosis in patients and their family members in the Iranian population. Interestingly, the group reported that linkage analysis using these six RFLP markers for carrier detection and the prenatal diagnosis was a rapid, easy, reliable, and inexpensive method, suitable for most routine diagnostic services. In another study by Wojcik KA et al. in keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD) that included 279 patients with KC, 225 patients with FECD and 322 control individuals were studied. The group reported an association between two SNPs, c.-441G > A (rs174538) and g. 61564299G > T (rs4246215), in the FEN1 gene using PCR and RFLP techniques. Interestingly, this group reported that SNP g. 61564299G > T and c.-441G > A polymorphisms in the FEN1 gene can modulate the risk of keratoconus and FECD. Further, in a recent study by Foja S et al., RFLP was used to study novel missense mutations of cornea-specific TGFBI gene in patients and two generations of a family diagnosed with unique corneal dystrophy phenotypes. This group reported two novel mutations: c. 1640T > G (p.Phe574Cys) in exon 12 and c. 393G > T (p.Glu131Asp) in exon 4 of TGFBI gene. Thus, RFLP seems to be a very relevant molecular method that has also been used in recent years to study the molecular cause of various critical myopathies and dystrophies.
Multiplex ligation-dependent probe amplification
Multiplex ligation-dependent probe amplification (MLPA) is a multiplex polymerase chain reaction-based method that can simultaneously detect changes in gene copy number status, DNA methylation, and point mutations. MLPA can be performed using a standard thermocycler and capillary electrophoresis equipment, thus making it a robust, reliable, and cost-effective method [Table 3]. Moreover, the procedure requires a short working time and results can be obtained in one day. MLPA can also be used on highly fragmented DNA as probes used in this procedure bind to a sequence of only 50–100 nucleotides in length. Hence, this technique is good for the detection of small deletions encompassing only a single exon. As nearly 50 different genomic locations can be tested in a single reaction, it is a reliable method for detecting genetic alterations of diagnostic and prognostic significance in various myopathies and dystrophies. In the past few decades, MLPA has been used in numerous studies that specially focused on dystrophies.,,,, In two such studies by Tallapaka K et al. and Polavarapu K et al. in India, 510 and 606 patients were analyzed, respectively, for dystrophies using MLPA and sequencing methods. Interestingly, a study by Polavarapu K et al. reported MLPA to be identifying significantly larger mutations in sporadic (88.2%) than in familial cases (75.3%). A study by Tallapaka et al. showed that MLPA detected mutation in the DMD gene in 372/510 (72.9%), of whom 342 (67.1%) had exonic deletions and 30 (5.9%) had exonic duplications. Similar studies in China by Zhao HH et al. and Kong X et al. showed the importance of MLPA based diagnosis in detecting muscular disorders. Zhao HH reported MLPA to have better productivity and sensitivity than multiplex PCR, whereas studies by Kong X showed that a combination of MLPA, NGS, and Sanger sequencing is of great significance for family analysis, gene diagnosis, and gene therapy. Thakur N et al. and Mohammed F et al. also used MLPA to report DMD mutation patterns in Sri Lankan and Kuwait populations, respectively. Overall, there are a large set of studies that have shown the importance of MLPA in the diagnosis and screening of neuromuscular disorders.
Multiplex PCR is a highly sensitive and specific test for detection and proper diagnosis in patients with Duchenne and Becker muscular dystrophy (DMD and BMD). This method is effective in DMD because a majority of DMD-associated mutations are deletions. Thus, rapid polymerase chain reaction (PCR) assays can be easily developed for deletion detection in patients in a day. This method provides multiple advantages in diagnosis [Table 3]. Literature suggests a high-level use of this method for DMD/BMD detection. This method has widely been used in combination with MLPA for effective patient diagnosis. However, many groups have found it effective as an individual detection method. In one such study in India, Kumari P et al. studied 225 samples for deletion from 25 exons of DMD gene using multiplex PCR. They found that deletions were present in 169 (75.1%) patients of DMD/BMD, with the most frequent deletions observed in exon 50 (14.9%) and exon 49 (10.8%). Another study by Li Y et al. on 239 DMD patients reported that multiplex PCR is an effective tool for identifying alterations in 19 exons. In a study by Sedghi M et al., the efficiency of MLPA and multiplex PCR was analyzed. This study reported that MLPA analysis can determine all deletions detected by multiplex PCR and suggested that multiplex PCR might be used for initial analysis of boys affected with DMD in the Iranian population as it can detect 95% of rearrangements in patients with DMD. However, few studies have modified multiplex PCR for rapid and accurate diagnosis of DMD. In one such study by Syu JR et al., a short-end injection CE (short-end CE) was used after PCR that speeded up genotyping of the DMD gene. This method involved no extra purification and was completed within 9 min, with results showing good agreement with those of MLPA. Overall, multiplex PCR holds a promising approach for the diagnosis of DMD/BMD in patients that has promising diagnostic importance.
Future challenges in using nonsequencing methods
Nonsequencing-based molecular techniques have faced some drawbacks that limit their use in many labs [Table 3]. One of the major limitations with most of these is the variation of standardization from lab to lab. Further, as each known mutation needs different sets of primer sets, their processing needs to be standardized. This results in such techniques requiring a comparatively long time for standardization. Nonsequencing molecular methods have another limitation of not detecting multiple mutations in a short region. Thus, any gene having multiple mutations needs an individual procedure for detecting mutations, making this process very complex and time-consuming. This process requires a large number of samples as well as appropriate positive and negative controls to confirm that the assays are working well. Moreover, some of the nonsequencing-based methods (e.g., ARMS-PCR) may end up in nonspecific signals due to the high DNA concentration. Thus, such samples may need to be diluted and restandardized for samples specific results analysis. Overall, the nonsequencing-based methods can be helpful in detecting limited genetic mutations that have been well correlated with a specific disease or phenotype. However, unlike NGS techniques, it does not support a personalized healthcare approach for individual patients, where all mutations specific to a disease can be simultaneously analyzed.
The most prominent breakthrough of DNA sequencing came in 1977 which was Sanger Dideoxy synthesis. In 1980, the Maxam–Gilbert chemical cleavage method was also introduced by Maxam and Gilbert, which is based on the chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides. The Sanger sequencing method has been the most preferred method of identifying alterations in small regions of the genes. Further, with the innovation of a better laser detection system and progress in computational hardware and software, more efficient methods have been developed. In 2005, Next-Generation Sequencing (NGS) technique was developed; since then, it has been used for various studies. In 2009, this technique was used for studying Human genome-specific mutations. In 2011, this technique was approved by FDA to be used for diagnosis in clinical setups. Till now, a large number of sequencing methods have been developed that provide extensive options as per user requirements [Table 4]. Some of the advanced NGS platforms can generate billions of reads per run and may analyze a large set of samples per run. Thus, NGS has evolved as one of the most advanced techniques for identifying genetic disorders in patients. Further, due to the association of many genetic alterations in myopathies and dystrophies, Sanger-Sequencing and NGS also seem to be suitable techniques in identifying such neuromuscular disorders [Figure 1]. Moreover, with the advancement in sequencing from single cells and from circulating nucleic acids, better methods for many neuromuscular disorders can be achieved in the future.
Early detection of myopathies and dystrophies can be an essential step that may lead to better health outcomes than treating them at a later stage. Moreover, screening such patients at an early stage, before any symptoms become noticeable, can also be a very effective technique. Developing novel methods using nonsequencing-based methods, including Tetra ARMS-PCR, Nested PCR, and RFLP, can be very helpful in the timely screening of such mutations across regions that are more susceptible to such mutilation. This study also values the importance of NGS and coincide with its central role in identifying and better-classifying myopathies and dystrophies in the future. However, we also highlight the importance and role of various screening methods that hold great potential in economically poor and developing countries. Although earlier screening holds a promising role in myopathies, the reliability of screening methods should be confirmed by scientists and medical investigators. Further, the importance of screening methods can benefit patients from starting treatment earlier rather than later. As many myopathies and dystrophies are associated with multiple genetic alterations, cases found positive during any single genetic alteration during initial screening should also be validated using a more reliable method for more accurate diagnosis. Alongside known benefits, screening may also notably cause harm to many patients who are given either a false positive test result or in such disorders where no effective treatment is available. Thus, potential participants involved in any such study should be provided all the necessary information to decide to participate in such screenings. Overall, we promote nonsequencing-based molecular methods for early-stage diagnosis and management of myopathies in developing countries and suggest the importance of emerging NGS-based methods in identifying new genetic players in neuromuscular disorders.
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Conflicts of interest
There are no conflicts of interest.
[Table 1], [Table 2], [Table 3], [Table 4]