Open access peer-reviewed chapter - ONLINE FIRST

Duchenne Muscular Dystrophy (DMD) Diagnosis: Past and Present Perspectives

By Nahla O. Mousa, Ahmed Osman, Nagia Fahmy, Ahmed Abdellatif, Suher Zada and Hassan El-Fawal

Submitted: August 17th 2019Reviewed: December 14th 2019Published: January 8th 2020

DOI: 10.5772/intechopen.90862

Downloaded: 33

Abstract

Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder, characterized by progressive skeletal muscle wasting. The disease is caused by various types of mutations in the dystrophin gene (DMD). The disease occurs at a frequency of about 1 in 5000 male births, making it the most common severe neuro-muscular disease. In addition to clinical examinations of muscle strength and function, diagnosis of DMD usually involves a combination of immunological assays using muscle biopsies, typically immunohistochemistry and western blotting, and molecular techniques such as DMD gene sequencing or Multiplex Ligation Dependent Probe Amplification (MLPA) using blood samples. In fact, precise molecular diagnosis is a prerequisite for determining the appropriate personalized therapeutic approach such as exon-skipping, gene therapy or stem cell-based therapies in conjunction with gene editing techniques like CRISPR-Cas9. However, the quest for reliable biomarkers with high sensitivity and specificity for DMD from liquid biopsy is still a hotspot of research, as such non-invasive biomarker(s) would not only facilitate disease diagnosis but would also help in carrier detection, which will eventually result in better disease management. In this chapter, we will illustrate the detailed current and prospect strategies for disease.

Keywords

  • DMD
  • diagnosis
  • biomarkers

1. Introduction

Dystrophin protein is present in myocytes in skeletal, cardiac, and smooth muscles, acting to connect the actin microfilaments, via N-terminus of the protein, to the extracellular matrix by binding membrane—bound (sarcolemma) glycoprotein complex (dystrophin associated glycoprotein complex; DGC) to the C-terminal end of the protein, and thus, plays an important role in normal muscle function [1]. Inactivating mutations occurring in DMD gene causes immature termination of protein translation, giving rise to C-terminally truncated protein product that fails to transmit muscle impulses, which causes increasing intracellular Ca2+ influx and thus, activating apoptotic machineries and eventually causes cell death and muscle atrophy/necrosis [2]. Death usually occurs in the third decade of life as a result of respiratory or heart failure [3].

2. Methods for DMD diagnosis

2.1 Clinical picture

Affected DMD boys are usually normal at birth but in early childhood they suffer from inability to get up from floor or climb stairs or run and they fell very often. Also, enlarged calf muscles (pseudo hypertrophy) are always noticed [4]. From the age of 7–12, the cases become more deteriorated, and the patients start to suffer from scoliosis [5], and joint contracture [6]. Also, patients will have an apparent reduction in bone-mineral density and will have hypocalciuria and osteoporosis [7].

Because the disease affects proximal as well as distal muscles, thus, in early teenage, DMD boys usually get respiratory infections and sleep apnea [8], and later, the patient will develop cardiomyopathy and eventually heart failure [9].

2.2 Circulating blood biomarkers

2.2.1 CK levels and other proteins/enzymes

One of the dystrophin protein main functions is to stabilize the muscle tissue, since it exists and binds to sarcolemma. The absence of dystrophin will eventually lead to the increased permeability of the muscular tissue and consequently the release of the muscle proteins [10], of which the creatine kinase (CK) enzyme that is responsible for the production of phosphocreatine and ADP from creatine and ATP as part of energy homeostasis. In normal condition, normal myocytes turnover, serum levels of CK ranges from 20 to 200 U/L, however, it can be slightly increased in some neurological disorders. On the other hand, in case of DMD boys, due to the accelerated muscular destruction, it may reach higher levels reaching several thousands of units/L, and in severe muscle damage it can reach 200,000 U/L [11, 12, 13]. However, CK levels sometimes can be misleading because in advanced stages of DMD, CK levels may come within normal range due to progressive muscular atrophy [14].

CK is considered one of the most used serum biomarkers in DMD diagnosis, however, many studies were performed to detect alterations in other muscle related proteins using immunoassay and MS-based detection to screen for other potential diagnostic biomarkers (Table 1).

Tested markerLevels (high or low) in DMD patients and/or other MDsLocation (serum/muscle)Detection methodRef.
Alkaline phosphatase (AP)-AElevated in Grade 1 and Grade 2 patientsSerumMeasuring enzyme activities[15]
AP-BNo change
Gly-APElevated in Grade 1 and Grade 2
Ala-APElevated in Grade 1
Ser-APElevated in Grade 1,2,3
Leu-APElevated in Grade 1
Met-APNo Change
Phe-APElevated in Grade 1,2,3
Trp-APElevated h in Grade 1,2,3
Gly-pro-APElevated in Grade1
Reduced in Grade 3
Gly-Pro-Leu-APReduced in Grade1 and Grade 2
TrypsinReduced in Grade 1
Cathepsin CReduced in Grade 1 and Grade 2
SulphataseNo change
PhosphataseNo change
Acetyl-choline esteraseReduced in Grade 2
EsteraseElevated in Grade 1,2,3
RNaseReduced in Grade 1 and Grade 2
Angiotensin Converting enzymeReduced in Grade 3
Myostatin (Growth and differentiation factor 8; GDF8)Elevated in DMD patientsSerumELISA[16]
Interleukin 17Elevated in Emery-Dreifuss MD and Limb-Girdle MD 1BSerumELISA[17, 18]
TGF-β2Elevated in Emery-Dreifuss MD and Limb-Girdle MD 1B
IL6Variable
Skeletal troponin I (sTnI),Elevated in DMD, BMD, LGMD2BSerumELISA
Myosin light chain 3 (Myl3),Elevated in DMD, BMD, LGMD2B
Fatty acid binding protein 3 (FABP3)Elevated in DMD, BMD, LGMD2B
Muscle-type creatine kinase (CKM)Elevated in DMD, BMD, LGMD2B
N-terminal α Dystroglycan (αDG-N)Reduced in DMD patientsSerumELISA[19]
FibronectinElevated in DMD
Normal in BMD
SerumELISA[20]
Basic fibroblast growth factorElevated in DMD patientsSerumELISA[21]
cardiac myosin light chain IElevated in DMD patients (correlated with CK levels)SerumImmunoradiometric assay[22]
Troponin I, fast skeletal muscleElevated in DMDSerumSOMAscan assay
“Aptamer-based proteomic technology”
[23]
Carbonic anhydrase 3Elevated in DMD
Fatty acid-binding protein, heartElevated in DMD
Troponin I, cardiac muscleElevated in DMD
Creatine kinase M-typeElevated in DMD
Mitogen-activated protein kinase 12Elevated in DMD
Alanine aminotransferase 1Elevated in DMD
MyoglobinElevated in DMD
FibrinogenElevated in DMD
Phospholipase A2, membrane associatedElevated in DMD
Acidic leucine-rich nuclear phosphoprotein 32 family member BElevated in DMD
Hepatoma-derived growth factor-related protein 2Elevated in DMD
40S Glucose-6-phosphate isomerase ribosomal protein S7Elevated in DMD
Heparin cofactor 2Elevated in DMD
PersephinElevated in DMD
Calcium/calmodulin-dependent protein kinase II αElevated in DMD
Malate dehydrogenase, cytoplasmicElevated in DMD
l-lactate dehydrogenase B chainElevated in DMD
Aminoacylase-1Elevated in DMD
Proteosome subunit α type-2Elevated in DMD
C-X-C motif chemokine 10Elevated in DMD
cAMP-dependent protein kinase catalytic subunit αElevated in DMD
Heat-shock 70 kDa protein 1A/1BElevated in DMD
Proto-oncogene tyrosine-protein kinase receptor RetReduced in DMD
Growth/differentiation factor 11Reduced in DMD
Complement decay-accelerating factorReduced in DMD
Cadherin-5Reduced in DMD
Tumor necrosis factor receptor superfamily member 19 LReduced in DMD
GelsolinReduced in DMD
Wnt inhibitory factor 1Reduced in DMD
Contactin-5Reduced in DMD
Prolyl endopeptidase FAPReduced in DMD
Jagged-1Reduced in DMD
Netrin receptor UNC5CReduced in DMD
Kunitz-type protease inhibitor 1Reduced in DMD
Protein SETReduced in DMD
Disintegrin metalloproteinase domain-containing protein 9Reduced in DMD
Cell adhesion molecule L1-likeReduced in DMD
OsteomodulinReduced in DMD
WAP, Kazal, Ig, Kunitz and NTR domain-containing protein 1Reduced in DMD
Bone sialoprotein 2Reduced in DMD
Interleukin-34Reduced in DMD
Neurogenic locus notch homolog protein 3Reduced in DMD
Cytoplasmic aspartate aminotransferaseElevated in DMDSerumMeasuring enzyme activity[24]
mitochondrial aspartate aminotransferaseElevated in DMD
Alanine transaminase (ALT)Elevated in DMDSerumELISA[25]
Aspartate transaminase (AST)Elevated in DMD
Muscle-specific enolase (MSE, beta beta and alpha beta enolases)Elevated in DMD and another progressive muscular dystrophiesSerumEnzyme immunoassay[26]
Serum carbonic anhydrase III (CA-III)Elevated in DMD, limb-girdle dystrophy, facioscapulohumeral dystrophy and congenital dystrophySerumEnzyme immunoassay[27]
Creatine kinase (CK) isoenzymes (MM, MB, and BB)Elevated in DMDSerumSensitive enzyme immunoassay[28]
Matrix metalloproteinase-9 (MMP-9)Elevated in DMDSerumELISA[29]
Tissue inhibitors of metalloproteinase-1 (TIMP-1)Elevated in DMD
Osteopontin (OPN)Normal
MT-1-MMPElevated in autosomal dominant EDMDSerumELISA and zymography[30]
MMP2Elevated in autosomal dominant EDMD and in X-linked EDMD
MMP9Non-significant elevation
TIMP-1Normal in AD-EDMD
Elevated in X-linked EDMD
SerumELISA sandwich immunoassay[31]
TIMP-2Non-significant decrease AD-EDMD/X-EDMD cases
TIMP-3Reduced in AD-EDMD/X-EDMD
Carbonic anhydrase III (CA-III, EC 4.2.1.1)Elevated in DMD, congenital (Fukuyama-type), limb-girdle, also elevated in: polymyositis myotonic dystrophy amyotrophic lateral sclerosis spinal progressive muscular atrophy or Kugelberg-Welander disease and in carriers of DMDSerumRadioimmunoassay[32]
Vitamin D binding protein (GC)Reduced in DMDSerum2D-HPLC off-line coupled to LC-MALDI-TOF-MS verified with ELISA[33]
Fibulin-1 (FBLN1)Elevated in DMD
Gelsolin (GSN)Reduced in DMD
Carbonic anhydrase 1 (CA1)Elevated in DMD
Apolipoprotein B100Reduced in DMD
ALT, AST, LDH, and ALPElevated in DMDSerumEnzymatic assay[34]
ALT, AST, and LDHElevated in BMD and LGMD
FSHD and EDMDlack of abnormal serum enzyme levels
ALPHighly elevated in LGMD2B
Elevated in non-LGMD2B
Vascular endothelial growth factorHighly elevated in BMD
Elevated in Bedridden DMD, spinal muscular atrophy, myotonic dystrophy
SerumELISA[35]
Creatine kinase MB fractionElevated in DMDSerumMultiplex, microsphere-based immune-fluorescent assay[36]
Tissue-type plasminogen activator PLATSlightly elevated in DMD
MyoglobinSlightly elevated in DMD
Epidermal growth factorSlightly elevated in DMD
Chemokine (C-C motif) ligand 2Slightly elevated in DMD
CD 40 ligandSlightly elevated in DMD
VitronectinSlightly elevated in DMD
Carboxyterminal propeptide of type I procollagenNo significant alterationSerumRadioimmunoassay[37]
Aminoterminal propeptide of type III procollagenNo significant alteration
Laminin P1No significant alteration
Creatine kinaseElevated in DMD and BMDSerumMeasuring enzyme activity[38]
Pyruvate kinaseElevated in DMD and BMD
Myosin light chain—3Elevated in DMDSerumaffinity proteomics-based screening approach using an antibody suspension bead array[39]
Carbonic anhydrase IIIElevated in DMD
Electron transfer flavoprotein AElevated in DMD
Mitochondrial malate dehydrogenase 2Elevated in DMD
Electron transfer flavoprotein BReduced in DMD
Fast skeletal muscle troponin TElevated in DMD
Matrix metalloproteinase 9Elevated in DMDSerumImmunoassay[40]
Matrix metalloproteinase 2Reduced in BMD
Myostatin (GDF-8)Reduced in DMD
Follistatin (FSTN)Elevated in DMD and BMD
N-terminal fragment of titinElevated in DMD patientsUrineELISA[41]

Table 1.

List of potential protein biomarkers that could be utilized in the diagnosis of Duchenne muscular dystrophy.

2.2.2 MicroRNA

MicroRNAs (miRNAs) are a tissue—specific class of small, non-coding RNA molecules that function as gene regulators/silencers and consequently they are considered sensitive indicators for different cellular contexts. MiRNAs act through binding to a specific region in the 3′-UTR in the target mRNA molecules, thus, inducing mRNA degradation and inhibiting the translation process [42]. The circulating levels of miRNAs in serum reflect the intracellular status and hence, they are excellent biomarkers for many pathological conditions as they can be detected from liquid biopsies and/or tissue specimens [43]. Many studies attempted to study the modulation in the levels of different miRNAs (Table 2).

microRNAStatusDiseaseLocationRef.
miR-133aUpregulatedDMD, BMD, LGMD, FSHDSerum and skeletal muscles[44, 45, 46, 47]
miR-206UpregulatedDMD, BMD, LGMD, FSHDSerum and skeletal muscles
miR-1UpregulatedDMD, BMD, LGMD, FSHDSerum and skeletal muscles
miR-499UpregulatedDMDSerum[45]
miR-208aUpregulatedDMDSerum
miR-208bUpregulatedDMDSerum
miR-95UpregulatedDMDSerum[48]
miR-539DownregulatedDMDSerum
miR-30cUpregulatedDMDSerum[49]
miR-181aUpregulatedDMDSerum
miR-21DownregulatedDMDUrine[50]
miR-29DownregulatedDMDUrine
miR-23DownregulatedDMDUrine
miR-181aUpregulatedDMDSerum[51]
miR-4538UpregulatedDMDSerum
miR-4539UpregulatedDMDSerum
miR-606UpregulatedDMDSerum
miR-454DownregulatedDMDSerum
miR-483UpregulatedDMDSerum[52]
hsa_miR_146b, hsa_miR_368, hsa_miR_381, hsa_miR_487b, hsa_miR_495, hsa_miR_376a, hsa_miR_299_5p, hsa_miR_155, hsa_miR_382, hsa_miR_199a, hsa_miR_379, hsa_miR_335, ambi_miR_5021, hsa_miR_432, hsa_miR_199b, hsa_miR_369_5p, hsa_miR_21, hsa_miR_34a, hsa_miR_199a*, hsa_miR_154, hsa_miR_221, hsa_miR_214, hsa_miR_518a_2*, hsa_miR_409_3p, hsa_miR_452, ambi_miR_2537, hsa_miR_127, hsa_miR_493_3p, hsa_miR_130a, ambi_miR_4983, ambi_miR_13145, hsa_miR_148a, hsa_miR_210, hsa_miR_485_5p, hsa_miR_299_3p, hsa_miR_134, hsa_miR_222, hsa_miR_181d, ambi_miR_13258UpregulatedDMDSerum[53]
hsa_miR_423, hsa_miR_361, hsa_miR_197, hsa_miR_92, hsa_miR_26a, ambi_miR_7075, hsa_miR_30b, hsa_miR_30e_5p, hsa_miR_29a, ambi_miR_13156, hsa_miR_30a_5p, hsa_miR_193b, hsa_miR_331, hsa_miR_486, hsa_miR_30d, hsa_miR_29b, hsa_miR_101, hsa_miR_30c, hsa_miR_22Downregulated

Table 2.

List of different microRNAs that could be used as potential biomarkers in the diagnosis of DMD.

2.2.3 Lipids, metabolites, amino acid, and organic acid

In addition to the previously mentioned biomarkers, lipid profile and metabolites in the blood or urine are also very important parameters that reflect the status of the muscles and thus, they could be measured to indicate the extent of muscular dystrophy and can serve as good candidates for diagnostic purposes (Table 3).

Tested markerLevels (high or low)Location (serum/muscle)Ref.
24,25(OH)2D3Reduced in DMDSerum[54]
1,25(OH)2D3No change
25(OH)D3No change
CreatinineReduced in DMD, BMD, LGMD2A and LGMD2BSerum[55]
Imidazole acetic acidReduced in DMD and LGMD2B
5α Dihydrotestosterone glucuronide // androsterone glucuronide // Etiocholan-3alpha-ol-17-one 3-glucuronideReduced in DMD
DL-p-Hydroxyphenyllactic acid // Isohomovanillic acidReduced in DMD
CreatineElevated in DMD, DM1, LGMD2Aand LGMD2B
Guanidinoacetic acidReduced in DMD, BMD, DM1 and LGMD2A
p-Coumaric acidReduced in DMD
CitrullineReduced in DMD
5-Methoxyindoleacetate // Indoleacetic acidReduced in DMD
L-Aspartic acidReduced in DMD
OrnithineReduced in DMD
2-Hydroxycaproic acidReduced in DMD
L-SerineReduced in DMD
Dehydroisoandrosterone 3-sulfateReduced in DMD
ErythroseReduced in DMD, BMD, FSHD
GlutamineReduced in DMD, BMD, LGMD-2B, FSHD and elevated in DM-1Serum[56]
AcetateElevated in DMD, BMD, FSHD, LGMD-2B and DM-1
TyrosineElevated in BMD
LysineReduced in FSHD, LGMD-2B and DM-1
CitrateReduced in FSHD
Elevated in LGMD-2B
LactateReduced in LGMD-2B
HistidineReduced in FSHD
Serum creatinineElevated in BMD
Decreased in DMD
Serum[57]
3-MethylhistidineDeduced in DMD and LGMDUrine[58]
N epsilon,N epsilon-dimethyllysineNo alteration
N epsilon, N epsilon, N epsilon-trimethyllysineNo alteration
NG,NG-dimethylarginineElevated in DMD and LGMD
NG,N’G-dimethylarginineNo alteration
Tetranor PGDM (PGD2 metabolite)Elevated in DMDUrine[59]
Nitric oxideReduced in DMDSerum[60]

Table 3.

List of metabolites that can be used as potential biomarkers in DMD diagnosis.

2.3 Muscle imaging

Magnetic resonance imaging (MRI) is now used to visualize the composition of skeletal muscles and detect structural abnormalities in the of DMD patients [61]. The produced images can reveal the presence of fat infiltration of muscle tissue, a characteristic consequence of DMD, and thus, can be used for monitoring disease progression and response to treatment [62].

2.4 Genetic diagnosis

2.4.1 RFLP

Detecting the mutation, especially non-sense point mutations, in the 2.4 Mb gene represents a challenging task. In this context, restriction fragment length polymorphism (RFLP) analysis could be used by digesting the genomic DNA using specific restriction endonucleases followed by Southern blotting using DMD-specific DNA probes (genomic or cDNA probes). At 1985, Bamkan et al. developed 11 RELP markers that are present in the X chromosome and can be used for diagnosis. However, RFLP can detect only small percentage of the mutation and hence it cannot be used as gold standard technique in the diagnosis process [63, 64, 65].

2.4.2 Multiplex PCR

Multiplex PCR is one of the modified PCR protocols that allows the co-amplification of multiple products using different primer pairs that specially bind complementary regions in the target segment. This method showed a great potential to diagnose DMD since the multiple primers covered commonly mutated locations across the entire DMD gene, hotspot regions [66, 67, 68]. This technique was first developed by Chamberlain et al. [69] through utilization of 6 primer sets that were modified to 9 sets and later to 10 by Beggs et al. [70] (to amplify exons 45, 48, 19, 17, 51, 8, 12, 44, 4). If no amplification take place, this will confirm deletion of this exon. The developed primer sets were successfully able to detect deletion mutations in the hot spot regions. One of the limitations of such technique was its inability to diagnose all cases with other deletion mutation in other regions, or patients with SNPs or deep intronic mutation.

2.4.3 Multiplex ligation dependent probe amplification (MLPA)

In order to simultaneously investigate the status of the 79 exons of the DMD gene, a PCR-based technique was developed to diagnose DMD in a multiplex PCR reaction. The assay uses multiple probes to target different exons in the DMD gene. Each probe consists of two oligonucleotides; one consists of a 5′-adapter and a 3′-exon-specific region, and vice versa for the second oligonucleotide, where the 3′-end of the first primer and the 5′-end of the second hybridize to two adjacent nucleotides in the target exon. Hybridized probes are subjected to ligation reaction, thus, only hybridized probes get ligated, amplified by PCR using adapter-specific primers and separated by capillary electrophoresis. Positive PCR product indicates the presence of the target exon, while deleted exon(s) will not produce corresponding product(s). In this assay, it is also possible to detect exon duplication, which will be detected as larger peak [71, 72]. However, this assay cannot detect non-sense nor in/del point mutations.

2.4.4 Microarray

High-throughput methods such as DNA microarrays were adopted using specific oligonucleotide probes that cover the entire 2.4 mbp DMD gene (targeted high density comparative genomic hybridization (CGH) microarray). Such method could effectively be used to detect known as well as novel intronic mutations [73, 74, 75].

2.4.5 Next generation sequencing (NGS)

The development of NGS and the massively parallel sequencing allowed the sequencing of 100 s of millions of independent short reads (100–300 bp) at the same time. Such approaches generate huge amount of data that uses bioinformatic analysis for annotations and alignments of the generated sequences to produce sequence information for large genes such as DMD and titin and delineate the exact locations of mutations [76]. One major advantage of resorting to NGS for DMD diagnosis is that it could be used for the analysis of MLPA-negative samples that could have small deletions/duplications or single nucleotides variants [77].

Also, RNA sequencing by NGS (RNA-seq) is very useful in detecting the splicing pattern that occur in the DMD transcripts in the muscles through different developmental stages, muscle breakdown or muscle regeneration [78, 79, 80].

2.4.6 Muscle biopsy

In some cases, muscle biopsy is required to fully characterize the phenotypic effect of the mutation. The muscle tissue is used in immunoassays, using different antibodies targeting different regions of dystrophin protein (C-terminal, Rod and N-terminal domains), such as western blotting [81, 82, 83] or immunohistochemistry [83]. Uchino et al. [83] developed a multiplex western blotting assay to analyze the expression of other muscle proteins like dysferlin, merosin, different forms of sarcoglycan (alpha, beta, gamma, delta), and calpain in addition to dystrophin protein, due to the frequent epigenetic changes incited in these proteins as a consequence to the alteration in dystrophin expression.

3. Conclusion

In this chapter, we have presented a comprehensive review for the methods that have been used in the diagnosis of DMD. Because of the nature of the disease, an X-linked disorder, DMD symptoms of the first affected male births of asymptomatic carrier mothers are usually go unnoticed until the age of 5, where the progressive muscle weakness becomes obvious and fibrotic fatty tissue infiltration is prominent. However, it is well known that early diagnosis and treatment results in better disease management and improve the clinical outcomes. In fact, some studies have pointed out to the fact that initiating corticosteroids therapy early enough has delayed the loss of ambulation in most cases by about 2 years [84]. In addition, with the fast-paced progress in molecular/personalized therapies such as exon-skipping and gene-editing based approaches, precise diagnosis and mutation detection becomes a necessity. Moreover, the genetic testing has been extensively used in prenatal diagnosis and has assisted in decreasing disease burden by aborting affected male pregnancies. In a retrospective study conducted in the Netherland, the authors reported 145 abortions of male fetuses over 26 years that had been found to carry inactivating mutations of the DMD gene [85]. Furthermore, identifying female carriers, is gaining momentum to decrease the possibility of giving birth to affected males and consequently contributes to the overall disease management.

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Nahla O. Mousa, Ahmed Osman, Nagia Fahmy, Ahmed Abdellatif, Suher Zada and Hassan El-Fawal (January 8th 2020). Duchenne Muscular Dystrophy (DMD) Diagnosis: Past and Present Perspectives [Online First], IntechOpen, DOI: 10.5772/intechopen.90862. Available from:

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