2.3 Muscle biopsy

The assessment of skeletal muscle biopsy is an essential procedure for an accurate diagnosis when a muscle disease is suspected, providing evidence of pathological changes in muscle and guides for appropriate tests.

#### 2.3.1 Muscle histopathological analysis

Even though the muscle biopsy is a highly invasive procedure, the data gained from it has the utmost importance for histopathological diagnosis and is an essential component in the diagnosis of muscle disorders that could identify the cause of the disease process and distinguish between different types of muscular dystrophy. The overall structure of the tissue as well as all specific histological features of dystrophic muscle can be observed by hematoxylin and eosin (H&E) staining performed on frozen sections sampled from the quadriceps or deltoid muscle. Generally, the features observed in all dystrophic muscle biopsy include fibers'size variation, round shape muscle fibers, the presence of atrophy, regenerating and degenerating fibers, splitting of fibers, proliferation of the connective tissue, and increased number of internal nuclei. In the end phases of the disease, the fibers are replaced by adipose tissue [36]. Histopathological changes differ widely in severity among the types of muscular dystrophy, as well as among allelic variants of the same genotype. Also, some features are specific for each type of muscular dystrophy. For example, lobulated fibers are characteristic for LGMD 2A; a high variability in fibers'size is specific for LGMD 1C, and increased internally nuclei are specific for myotonic dystrophy; the presence of rimmed vacuoles suggests a myotilinopathy, while prominent vacuoles are found in LGMD [36]. With all signs, none of the specific forms of muscular dystrophy can be diagnosed just based only on histological analysis.

immunohistochemical protocols based on the use of specific antibodies for antigen

A large array of antibodies directed against different muscle protein is now available for current diagnosis and allows the use of these techniques for the diag-

about integrity of muscle complexes, secondary reduction of proteins, and also gene

overexpression of closely protein levels [49]. For example, utrophin, an autosomal protein which presents around 80% sequence homology to dystrophin [50, 51] and localized in normal skeletal muscle at the neuromuscular junction (NMJ) [52], is overexpressed in dystrophinopathies (see Figure 2) [53, 54]. Mutations in one sarcoglycan often lead to reduced expression of other sarcoglycans [49], and reductions of dysferlin (see Figure 3) are observed in other forms of muscular dystrophy such as calpainopathy, caveolinopathy, and anoctaminopathy [55].

However, IF is not always helpful in diagnosis of all forms of MD. For example, in LGMD 2A some available antibodies have no immunoreactions on the sections. Few studies tried to analyze calpain 3 on cryosections but, because of rapid degradation of calpain 3 after harvesting, concluded that immunostaining analysis alone does not predict the presence of CAPN3 mutations [40]. However, the staining of other proteins such us dystrophin and sarcoglycans which appear normal on

Also, in dominant forms LGMD1B caused by changes in the LMNA gene which encode for lamin A/C, immunostaining of cryosection reveals normal expression

cryosection

Dilution for Western blot

function by compared normal and affected patients (see Figure 1) [48]. In addition, IF method identifies also the secondary reduction and

cryosections by IF could be informative for further analysis.

Details of the most used antibodies in muscular dystrophy diagnosis.

Antibody Manufacturer Clone Dilution for

Dystrophin C-terminus Novocstra Dy8/6C5 1:40 1:200 Dystrophin N-terminus Novocstra Dy10/12B2 1:50 1:50 Dystrophin rod domain Novocstra Dy4/6D3 1:20 1:200 Utrophin Novocstra DRP3/20C 1:50 1:200 Calpain 3 Novocstra 2C4 — 1:100 Calpain 3 Novocstra 12A2 — 1:50 Caveolin 3 Santa Cruz A-3 1:50 1:200 Dysferlin Novocstra Ham1/7B6 1:50 1:100 α-Sarcoglycan Novocstra Ad1/20A6 1:50 1:100 β-Sarcoglycan Novocstra BSarc/5B1 1:50 1:100 γ-Sarcoglycan Novocstra 35DAG/21B5 1:50 1:100 Merosin Novocstra Mer3/22B2 1:50 1:50

even in the presence of a mutation [56].

Table 1.

15

The use of antibodies directed against muscle protein is useful to gain information

IF or fluorescent antibody staining is a helpful routinely technique widely used to determine the localization of fluorescent-tagged protein and changes in protein expression on a cryosection (presence, reduced, and absence). It is also used to

localization through antigen-antibody interaction.

Clinical and Molecular Diagnosis in Muscular Dystrophies

DOI: http://dx.doi.org/10.5772/intechopen.85339

nosis of many muscle disorders (see Table 1).

investigate muscle architecture.

Also, the muscle biopsy analysis can not only denote the specific genetic cause of the disease but can also provide clues for further investigation. In combination with protein analysis, the genetic investigations can provide an accurate diagnosis.

#### 2.3.2 Protein analysis

The study of muscle protein expression is important for diagnosis, for genotypephenotype correlations, and to identify possible genetic defect [37–39]. There are many methods used for the study of muscle protein expression [40], but the most used are immunostaining methods (immunohistochemistry or similar methods immunofluorescence and immunoblotting/Western blotting (WB)). Both methods use labeled antibodies for a specific muscle protein involved in a type of muscular dystrophy. While the immunohistochemistry/immunofluorescence method is used to identify the localization and relative abundance of the proteins, in tissue cryosections, the WB method is useful to detect the total amount of proteins as well as the normal or reduced size of the proteins in homogenized sample.

#### 2.3.2.1 Immunofluorescence (IF)

In the past, the diagnosis of muscular dystrophy consisted only on clinical assessment, serum CK levels, and histological investigations of muscle biopsy [41, 42].

The discovery in 1986 of the first muscle protein involved in a type of muscular dystrophy, dystrophin [43], has later led to the identification of the dystrophinassociated protein complex (DAPC) [44] and other additional proteins from the muscle cytosol (calpain 3, TRIM32) [45], from extracellular matrix (a2-laminin, collagen VI) from the sarcomere (telethonin, myotilin, titin, nebulin) [46]. Each of these proteins is involved in a type of muscular dystrophy; so far over 40 types of muscular dystrophy are known [47].

The development of specific antibodies (Abs) for affected proteins has improved the diagnosis for these diseases, over time. Now, there are many

#### Clinical and Molecular Diagnosis in Muscular Dystrophies DOI: http://dx.doi.org/10.5772/intechopen.85339

2.3.1 Muscle histopathological analysis

Muscular Dystrophies

2.3.2 Protein analysis

2.3.2.1 Immunofluorescence (IF)

muscular dystrophy are known [47].

[41, 42].

14

Even though the muscle biopsy is a highly invasive procedure, the data gained from it has the utmost importance for histopathological diagnosis and is an essential component in the diagnosis of muscle disorders that could identify the cause of the disease process and distinguish between different types of muscular dystrophy. The overall structure of the tissue as well as all specific histological features of dystrophic muscle can be observed by hematoxylin and eosin (H&E) staining performed on frozen sections sampled from the quadriceps or deltoid muscle. Generally, the features observed in all dystrophic muscle biopsy include fibers'size variation, round shape muscle fibers, the presence of atrophy, regenerating and degenerating fibers, splitting of fibers, proliferation of the connective tissue, and increased number of internal nuclei. In the end phases of the disease, the fibers are replaced by adipose tissue [36]. Histopathological changes differ widely in severity among the types of muscular dystrophy, as well as among allelic variants of the same genotype. Also, some features are specific for each type of muscular dystrophy. For example, lobulated fibers are characteristic for LGMD 2A; a high variability in fibers'size is specific for LGMD 1C, and increased internally nuclei are specific for myotonic dystrophy; the presence of rimmed vacuoles suggests a myotilinopathy, while prominent vacuoles are found in LGMD [36]. With all signs, none of the specific forms of muscular dystrophy can be diagnosed just based only on histological analysis.

Also, the muscle biopsy analysis can not only denote the specific genetic cause of the disease but can also provide clues for further investigation. In combination with protein analysis, the genetic investigations can provide an accurate diagnosis.

The study of muscle protein expression is important for diagnosis, for genotypephenotype correlations, and to identify possible genetic defect [37–39]. There are many methods used for the study of muscle protein expression [40], but the most used are immunostaining methods (immunohistochemistry or similar methods immunofluorescence and immunoblotting/Western blotting (WB)). Both methods use labeled antibodies for a specific muscle protein involved in a type of muscular dystrophy. While the immunohistochemistry/immunofluorescence method is used

to identify the localization and relative abundance of the proteins, in tissue

as the normal or reduced size of the proteins in homogenized sample.

cryosections, the WB method is useful to detect the total amount of proteins as well

In the past, the diagnosis of muscular dystrophy consisted only on clinical assessment, serum CK levels, and histological investigations of muscle biopsy

The development of specific antibodies (Abs) for affected proteins has improved the diagnosis for these diseases, over time. Now, there are many

The discovery in 1986 of the first muscle protein involved in a type of muscular dystrophy, dystrophin [43], has later led to the identification of the dystrophinassociated protein complex (DAPC) [44] and other additional proteins from the muscle cytosol (calpain 3, TRIM32) [45], from extracellular matrix (a2-laminin, collagen VI) from the sarcomere (telethonin, myotilin, titin, nebulin) [46]. Each of these proteins is involved in a type of muscular dystrophy; so far over 40 types of

immunohistochemical protocols based on the use of specific antibodies for antigen localization through antigen-antibody interaction.

IF or fluorescent antibody staining is a helpful routinely technique widely used to determine the localization of fluorescent-tagged protein and changes in protein expression on a cryosection (presence, reduced, and absence). It is also used to investigate muscle architecture.

A large array of antibodies directed against different muscle protein is now available for current diagnosis and allows the use of these techniques for the diagnosis of many muscle disorders (see Table 1).

The use of antibodies directed against muscle protein is useful to gain information about integrity of muscle complexes, secondary reduction of proteins, and also gene function by compared normal and affected patients (see Figure 1) [48].

In addition, IF method identifies also the secondary reduction and overexpression of closely protein levels [49]. For example, utrophin, an autosomal protein which presents around 80% sequence homology to dystrophin [50, 51] and localized in normal skeletal muscle at the neuromuscular junction (NMJ) [52], is overexpressed in dystrophinopathies (see Figure 2) [53, 54]. Mutations in one sarcoglycan often lead to reduced expression of other sarcoglycans [49], and reductions of dysferlin (see Figure 3) are observed in other forms of muscular dystrophy such as calpainopathy, caveolinopathy, and anoctaminopathy [55].

However, IF is not always helpful in diagnosis of all forms of MD. For example, in LGMD 2A some available antibodies have no immunoreactions on the sections. Few studies tried to analyze calpain 3 on cryosections but, because of rapid degradation of calpain 3 after harvesting, concluded that immunostaining analysis alone does not predict the presence of CAPN3 mutations [40]. However, the staining of other proteins such us dystrophin and sarcoglycans which appear normal on cryosections by IF could be informative for further analysis.

Also, in dominant forms LGMD1B caused by changes in the LMNA gene which encode for lamin A/C, immunostaining of cryosection reveals normal expression even in the presence of a mutation [56].


#### Table 1.

Details of the most used antibodies in muscular dystrophy diagnosis.

2.3.2.2 Multiplex Western blotting

Clinical and Molecular Diagnosis in Muscular Dystrophies

DOI: http://dx.doi.org/10.5772/intechopen.85339

abnormal molecular weight [59, 60].

cryosection (8 μm thick, 10 mm<sup>2</sup>

proteins involved in a specific pathology.

specific antibodies [58].

an enzyme.

Figure 3.

regenerating fiber.

(see Figure 4).

17

Western blot (WB), also called immunoblot, is a semiquantitative method used to detect specific proteins from cell lysate samples or tissue homogenates using

Immunofluorescent staining of dysferlin in normal control muscle and patient with LGMD 2B: (a) expression of dysferlin from normal patients; (b) reduced expression of dysferlin around some muscle fiber; the presence of

Due to higher sensitivity and specificity of the method, WB brings more information about proteins that cannot be offered by other immunoassay techniques. The WB technique is an extremely powerful technique which gives information about the presence, absence, and size of the proteins, identifying the proteins with

Usually, this analytical method involved three major processes [61]: (i) separation of proteins into a SDS-polyacrylamide gel based on their molecular weight; (ii) protein blotting on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane; and (iii) visualization of the protein of interest by incubation of membrane with specific primary antibody and then labeled by secondary antibody conjugate with

For the preparation of tissue lysates, around 20–80 mg of muscle tissue are required which may be a significant part of a severely affected muscle. Cooper et al. [62] reported in 2003 obtaining of the lysate from 16 mg muscle tissue using a single

Given the complexity of muscular dystrophies, in recent years, has become a necessity in the analysis and comparison of the expression of multiple target

Also, the technology has improved over time going from the detection of a single protein to identification of multiple proteins in complex samples using a biphasic polyacrylamide gel systems and a cocktail of primary antibodies [63]. In our laboratory, polyacrylamide gel system is performed as previously described [63] with some modifications [64, 65] which permitted separation of the large proteins more than 200 kDa (e.g., dystrophin) in the top part of the gel while and smaller proteins under 150 kDa (e.g., calpain 3), in the bottom. The intensity and thickness of the specific protein bands correspond to the relative abundance of protein of interest. The amount of target protein is determined by comparing stained band of control with the patients. Quantification of protein based on densitometry of bands using ImageJ software provides information about the relative level of protein in muscle. In DMD patients, the total absence of the bands for dystrophin at 427 kDa is observed, while BMD patients show a reduced intensity of the bands for dystrophin

).

Figure 1. Workflow chart for muscle protein analysis in muscular dystrophy diagnostic process.

#### Figure 2.

Representative immunofluorescent staining from dystrophin and utrophin in normal human (control) and DMD and BMD skeletal muscle.

It is important to note that to obtain good results in protein evaluation by IF is a necessary investigation of the integrity of muscle fiber membrane by analysis of spectrin [54, 57].

All these facts suggest the difficulty of identification with accuracy of a type of MD based only on histochemical findings and immunofluorescence analysis and indicate a further investigation of proteins using immunoblot analysis.

#### Figure 3.

Immunofluorescent staining of dysferlin in normal control muscle and patient with LGMD 2B: (a) expression of dysferlin from normal patients; (b) reduced expression of dysferlin around some muscle fiber; the presence of regenerating fiber.

#### 2.3.2.2 Multiplex Western blotting

Western blot (WB), also called immunoblot, is a semiquantitative method used to detect specific proteins from cell lysate samples or tissue homogenates using specific antibodies [58].

Due to higher sensitivity and specificity of the method, WB brings more information about proteins that cannot be offered by other immunoassay techniques. The WB technique is an extremely powerful technique which gives information about the presence, absence, and size of the proteins, identifying the proteins with abnormal molecular weight [59, 60].

Usually, this analytical method involved three major processes [61]: (i) separation of proteins into a SDS-polyacrylamide gel based on their molecular weight; (ii) protein blotting on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane; and (iii) visualization of the protein of interest by incubation of membrane with specific primary antibody and then labeled by secondary antibody conjugate with an enzyme.

For the preparation of tissue lysates, around 20–80 mg of muscle tissue are required which may be a significant part of a severely affected muscle. Cooper et al. [62] reported in 2003 obtaining of the lysate from 16 mg muscle tissue using a single cryosection (8 μm thick, 10 mm<sup>2</sup> ).

Given the complexity of muscular dystrophies, in recent years, has become a necessity in the analysis and comparison of the expression of multiple target proteins involved in a specific pathology.

Also, the technology has improved over time going from the detection of a single protein to identification of multiple proteins in complex samples using a biphasic polyacrylamide gel systems and a cocktail of primary antibodies [63]. In our laboratory, polyacrylamide gel system is performed as previously described [63] with some modifications [64, 65] which permitted separation of the large proteins more than 200 kDa (e.g., dystrophin) in the top part of the gel while and smaller proteins under 150 kDa (e.g., calpain 3), in the bottom. The intensity and thickness of the specific protein bands correspond to the relative abundance of protein of interest. The amount of target protein is determined by comparing stained band of control with the patients. Quantification of protein based on densitometry of bands using ImageJ software provides information about the relative level of protein in muscle.

In DMD patients, the total absence of the bands for dystrophin at 427 kDa is observed, while BMD patients show a reduced intensity of the bands for dystrophin (see Figure 4).

It is important to note that to obtain good results in protein evaluation by IF is a necessary investigation of the integrity of muscle fiber membrane by analysis of

Representative immunofluorescent staining from dystrophin and utrophin in normal human (control) and

All these facts suggest the difficulty of identification with accuracy of a type of MD based only on histochemical findings and immunofluorescence analysis and

indicate a further investigation of proteins using immunoblot analysis.

Workflow chart for muscle protein analysis in muscular dystrophy diagnostic process.

spectrin [54, 57].

DMD and BMD skeletal muscle.

Figure 2.

16

Figure 1.

Muscular Dystrophies

disease prognosis. Additionally, diagnostic accuracy leads to more specific genetic

The great technological advances in molecular assays over the last 20 years have led to the identification of the molecular genetic cause for many forms of muscular dystrophy. This advance enabled, thus, the diagnosis of muscular dystrophy to evolve from the analysis of 20 exons out of 79 exons within hotspot region of DMD gene by PCR Multiplex [68–70] to multiple gene analysis using next-generation

Different forms of muscular dystrophy are caused by a variety of mutations that occurred in many human genes. Mutations that occurred in gene that encode for proteins from DAPC are responsible for many types of muscular dystrophy. The most common types of mutations involve large rearrangement (deletion and duplication) and point mutations. A correct characterization gene mutation for each type of muscular dystrophy represents the key for genotype-phenotype correlation. Here, we describe some of the more used molecular techniques in the muscular

Among the different methods used for detection of gene deletions and duplication, the MLPA assay is the most used due to rapid analysis up to 45 different DNA fragments in a single PCR amplification with only a single primer pair [71] and low amounts of genomic DNA. In addition to large deletions and duplications, MLPA may also identify single exon deletion. This apparent result should be always checked by an alternative method avoiding false-positive results that can occur in the presence of a single-nucleotide variation in a gene. This method proves its usefulness for female's carrier screening as well as for prenatal testing [72].

Because require equipments that exist in most molecular diagnosis laboratory (a thermocycler and a capillary electrophoresis), is a very cost-effective method

Data analysis can be done by using the free MLPA data analysis software Coffalyser (MRC-Holland, Amsterdam, the Netherlands) or additional software

The advantages of MLPA method for diagnosis are (i) low input of DNA required; (ii) the high specificity and sensitivity, the method being able to distinguish sequences differing in only one nucleotide; and (iii) plenty of MLPA kits available for different genes involved in muscle pathology such as CAPN3, DYSF,

High-resolution melting curve analysis (hrMCA) is a highly sensitive molecular post PCR method introduced in 1997 by Wittwer et al. [75, 76], to identify pathogenic variants in nucleic acid sequences. The improvement of real-time equipment regarding highly controlled temperature transitions, data acquisition software to monitor and analyze the melting, as well as the development of a new functional

HRM analysis starts with PCR amplification of the region of interest with specific primers in the presence of a specialized double-stranded DNA (dsDNA) fluo-

The method is based on the DNA property of dissociating (or melting) from double-stranded DNA into single-stranded DNA (ssDNA) when exposed to a gradual temperature [77, 78]. The melting process can be real time monitored by

counseling for families and possible preimplantation prenatal diagnosis.

Clinical and Molecular Diagnosis in Muscular Dystrophies

DOI: http://dx.doi.org/10.5772/intechopen.85339

2.4.1 Multiplex ligation-dependent probe amplification (MLPA)

widely used for both diagnostic and research.

SGCA, SGCB, SGCD, SGCG, and FKRP.

2.4.2 High-resolution melting curve analysis (hrMCA)

class of dyes have made this technology possible.

rescent binding dye.

19

such as MLPAinter [73] or GeneMapper v4.0 software [74].

sequencing (NGS) technique.

dystrophy diagnosis.

#### Figure 4.

Representative Western blotting analysis of muscle homogenates from four patients with muscular dystrophy: (A) nitrocellulose membrane labeled with antibody against rod domain of dystrophin (NCL-Dys1). (B) Blot labeled with antibody against C-terminus domain of dystrophin (NCL-Dys2); M-normal control; lane 1-MD with normal expression of dystrophin; lane 2-BMD; lane 3-DMD, absence of dystrophin band; lane 4-DMD; lane 5 MD with normal expression of dystrophin. MHC—Corresponding myosin heavy chain bands on the post-blotted gel, stained with Coomassie blue.

WB plays an important role in distinguishing between DMD and BMD patients especially for patients with discordant phenotype (do no respect reading frame rule).

Analysis by WB of calpain 3, protein found to be involved in LGMD 2A, could show a total or a reduced intensity of bands at 94 kDa. There are cases with LGMD 2A which displayed normal or almost normal bands for calpain 3 compared with control (see Figure 5).

This fact suggests a poor specificity of WB analysis for this protein [66]. Also, false-negative results provided by WB can be found in analysis of dysferlin when this protein is accumulated in the cytoplasm, and deficiency of lamin A/C could not be identified in all patients with LMNA mutations [67].

However, for these proteins, genetic analyses are required to confirm the exact diagnosis.

Western blot has the advantage of simultaneous analysis of several proteins which reduced cost and time for analysis. This method is useful in differential diagnosis of muscular dystrophies providing information on the relative location of mutation.

#### 2.4 DNA diagnosis methods

The identification and characterization of genetic defect involved in pathology is often essential both for diagnosis and treatment options as well as in predicting

#### Figure 5.

Calpain 3 band patterns on WB. Representative Western blotting analysis of muscle homogenates from three patients analyzed with antibody against calpain 3. (a) Nitrocellulose membrane labeled with antibody against calpain 3 NCL-CALP-2C4 detects bands at 90 and 30 kDa in normal patients. (b) Nitrocellulose membrane labeled with antibody against calpain 3 NCL-CALP-12A2 detects bands at 90 kDa and two bands at 60 and 30 kDa in normal patients.

#### Clinical and Molecular Diagnosis in Muscular Dystrophies DOI: http://dx.doi.org/10.5772/intechopen.85339

disease prognosis. Additionally, diagnostic accuracy leads to more specific genetic counseling for families and possible preimplantation prenatal diagnosis.

The great technological advances in molecular assays over the last 20 years have led to the identification of the molecular genetic cause for many forms of muscular dystrophy. This advance enabled, thus, the diagnosis of muscular dystrophy to evolve from the analysis of 20 exons out of 79 exons within hotspot region of DMD gene by PCR Multiplex [68–70] to multiple gene analysis using next-generation sequencing (NGS) technique.

Different forms of muscular dystrophy are caused by a variety of mutations that occurred in many human genes. Mutations that occurred in gene that encode for proteins from DAPC are responsible for many types of muscular dystrophy. The most common types of mutations involve large rearrangement (deletion and duplication) and point mutations. A correct characterization gene mutation for each type of muscular dystrophy represents the key for genotype-phenotype correlation.

Here, we describe some of the more used molecular techniques in the muscular dystrophy diagnosis.

#### 2.4.1 Multiplex ligation-dependent probe amplification (MLPA)

Among the different methods used for detection of gene deletions and duplication, the MLPA assay is the most used due to rapid analysis up to 45 different DNA fragments in a single PCR amplification with only a single primer pair [71] and low amounts of genomic DNA. In addition to large deletions and duplications, MLPA may also identify single exon deletion. This apparent result should be always checked by an alternative method avoiding false-positive results that can occur in the presence of a single-nucleotide variation in a gene. This method proves its usefulness for female's carrier screening as well as for prenatal testing [72].

Because require equipments that exist in most molecular diagnosis laboratory (a thermocycler and a capillary electrophoresis), is a very cost-effective method widely used for both diagnostic and research.

Data analysis can be done by using the free MLPA data analysis software Coffalyser (MRC-Holland, Amsterdam, the Netherlands) or additional software such as MLPAinter [73] or GeneMapper v4.0 software [74].

The advantages of MLPA method for diagnosis are (i) low input of DNA required; (ii) the high specificity and sensitivity, the method being able to distinguish sequences differing in only one nucleotide; and (iii) plenty of MLPA kits available for different genes involved in muscle pathology such as CAPN3, DYSF, SGCA, SGCB, SGCD, SGCG, and FKRP.

#### 2.4.2 High-resolution melting curve analysis (hrMCA)

High-resolution melting curve analysis (hrMCA) is a highly sensitive molecular post PCR method introduced in 1997 by Wittwer et al. [75, 76], to identify pathogenic variants in nucleic acid sequences. The improvement of real-time equipment regarding highly controlled temperature transitions, data acquisition software to monitor and analyze the melting, as well as the development of a new functional class of dyes have made this technology possible.

HRM analysis starts with PCR amplification of the region of interest with specific primers in the presence of a specialized double-stranded DNA (dsDNA) fluorescent binding dye.

The method is based on the DNA property of dissociating (or melting) from double-stranded DNA into single-stranded DNA (ssDNA) when exposed to a gradual temperature [77, 78]. The melting process can be real time monitored by

WB plays an important role in distinguishing between DMD and BMD patients especially for patients with discordant phenotype (do no respect reading frame rule). Analysis by WB of calpain 3, protein found to be involved in LGMD 2A, could show a total or a reduced intensity of bands at 94 kDa. There are cases with LGMD 2A which displayed normal or almost normal bands for calpain 3 compared with

Representative Western blotting analysis of muscle homogenates from four patients with muscular dystrophy: (A) nitrocellulose membrane labeled with antibody against rod domain of dystrophin (NCL-Dys1). (B) Blot labeled with antibody against C-terminus domain of dystrophin (NCL-Dys2); M-normal control; lane 1-MD with normal expression of dystrophin; lane 2-BMD; lane 3-DMD, absence of dystrophin band; lane 4-DMD; lane 5 MD with normal expression of dystrophin. MHC—Corresponding myosin heavy chain bands on the

This fact suggests a poor specificity of WB analysis for this protein [66]. Also, false-negative results provided by WB can be found in analysis of dysferlin when this protein is accumulated in the cytoplasm, and deficiency of lamin A/C could not

However, for these proteins, genetic analyses are required to confirm the exact

Western blot has the advantage of simultaneous analysis of several proteins which reduced cost and time for analysis. This method is useful in differential diagnosis of muscular dystrophies providing information on the relative location of mutation.

The identification and characterization of genetic defect involved in pathology is

often essential both for diagnosis and treatment options as well as in predicting

Calpain 3 band patterns on WB. Representative Western blotting analysis of muscle homogenates from three patients analyzed with antibody against calpain 3. (a) Nitrocellulose membrane labeled with antibody against calpain 3 NCL-CALP-2C4 detects bands at 90 and 30 kDa in normal patients. (b) Nitrocellulose membrane labeled with antibody against calpain 3 NCL-CALP-12A2 detects bands at 90 kDa and two bands at 60 and

be identified in all patients with LMNA mutations [67].

control (see Figure 5).

post-blotted gel, stained with Coomassie blue.

2.4 DNA diagnosis methods

diagnosis.

Figure 5.

18

30 kDa in normal patients.

Figure 4.

Muscular Dystrophies

measuring the gradually diminishing amount of fluorescence during DNA strand dissociation. The presence of a mutation in PCR products determines a modification in the shape of DNA melting curves comparative with melting profile of the wild type (normal) DNA (see Figure 6).

and for dysferlin (DYSF 58 exons) [81] that are associated with LGMDs, is more

Due to overlapping clinical symptoms and many possible genetic causes for LGMDs, obtaining a diagnosis is often difficult. Next-generation sequencing becomes a valuable option for an accurate diagnosis due to ability to analyze a large number

In DMD gene (79 exons), a high frequency of point mutations around 35% was reported [82]. Screening of this huge gene for point mutation is difficult to perform; therefore it is necessary to select only genomic regions that contain the variants [83]. However, the combined technology MLPA for identification of large deletions and duplication followed by HR-MCA and sequencing is a robust algorithm for diagnosis for male muscular dystrophy patients as well as for female carrier and also

The presence of clinical symptoms in a male child presented in Cap 2.1 along with increased serum levels of CK, transaminase enzymes or aldolase should trigger the diagnostic investigation for dystrophinopathies [11]. When DMD or BMD is suspected, diagnostic recommendation as first investigation is the screening of DMD gene for deletions and duplications by MLPA. The presence of a mutation in

A large number of research studies highlight the utility of MLPA as detection methods for DMD gene [84–86] which is the largest gene (2.2 Mb) of the human

Many different types of mutations that occurred in the DMD gene, encoding for

Previously reported studies have shown that all over the world, deletions of one

Because the mutations have been observed across all exons of the gene with a higher incidence of mutation in two "hotspots" regions between exons 2–20 and 45–

or more exons are most common mutation in dystrophinopathies (60–65%), followed by duplications (5–8%) and point mutations (30–35%) [88]. Around 90% from all dystrophinopathies cases present worldwide respect reading frame rule. For the remaining 10% as well as for patients with discordant phenotype, additional

50 [85, 89], MLPA method proves its utility for the molecular diagnosis of dystrophinopathies, by simultaneous screening of all 79 exons of DMD gene for large intragenic rearrangements [90]. All deletion and duplications identified should be checked for the validity of the reading frame rule on http://www.dmd.nl. If no deletions and duplication are identified, the DMD gene should be investigated for point mutations [91] by hrMCA followed by sequencing of exons with a modification of melting curve only. Full characterization of the mutation (type, size, and position) is important in identification of patients that are eligible for

the cytoskeletal protein, dystrophin, are responsible for both severe disease Duchenne muscular dystrophy (DMD) and the milder form of the disease Becker muscular dystrophy (BMD). The difference in disease severity between the two phenotypes, DMD and BMD, can be explained by the "reading frame rule" proposed by Monaco in 1988 [87]. According to this theory, DMD is caused by mutations which disrupt the reading frame, and no protein will be synthesized, while for BMD phenotype the mutations do not affect the reading frame leading to the

synthesis of a smaller and semi-functional dystrophin protein.

2.5 Diagnosis algorithm in the most common forms of MD

Clinical and Molecular Diagnosis in Muscular Dystrophies

DOI: http://dx.doi.org/10.5772/intechopen.85339

gene confirms the dystrophinopathies diagnosis.

analyses are required for an accurate diagnosis.

specific mutation gene therapy [89].

21

suitable for sequencing.

for prenatal diagnosis.

2.5.1 Dystrophinopathies

of targets.

genome.

HR-MCA is a rapid and accurate method for detection of genetic variation in population. Although HR-MCA is not locus specific, it can become a tool of choice for point mutations screening after identification of large mutation (deletion and duplications) by MLPA. The sequencing of the fragments with abnormal melting profile only, identified by HR-MCA, reduces costs and waiting time per archived results.

#### 2.4.3 Sanger sequencing

All point mutations identified by HR-MCA method need to be confirmed by sequencing. Sequencing, the most widely used approach for DNA analysis, remains the "gold standard" for mutation analysis.

The Sanger DNA sequencing method is applied to determine the sequence of a DNA molecule and to identify the subtle mutations in samples compared with a reference sequence [79, 80].

Because a lot of muscle proteins associated with different forms of muscular dystrophy are extremely large, full gene analysis using Sanger sequencing can lead to higher costs and is time-consuming for analysis. The method finds its usefulness in the analysis of small gene composed of only few exons in which the frequency of point mutation is higher. For instance gene that encode for sarcoglycans (e.g., SGCA 10 exons, SGCB 6 exons, SGCG 10 exons), for calpain 3 (CAPN3 26 exons),

#### Figure 6.

Representative high-resolution melting curve analysis which reveals difference in melting shape between wild type and variant sequence.

and for dysferlin (DYSF 58 exons) [81] that are associated with LGMDs, is more suitable for sequencing.

Due to overlapping clinical symptoms and many possible genetic causes for LGMDs, obtaining a diagnosis is often difficult. Next-generation sequencing becomes a valuable option for an accurate diagnosis due to ability to analyze a large number of targets.

In DMD gene (79 exons), a high frequency of point mutations around 35% was reported [82]. Screening of this huge gene for point mutation is difficult to perform; therefore it is necessary to select only genomic regions that contain the variants [83].

However, the combined technology MLPA for identification of large deletions and duplication followed by HR-MCA and sequencing is a robust algorithm for diagnosis for male muscular dystrophy patients as well as for female carrier and also for prenatal diagnosis.

### 2.5 Diagnosis algorithm in the most common forms of MD

#### 2.5.1 Dystrophinopathies

measuring the gradually diminishing amount of fluorescence during DNA strand dissociation. The presence of a mutation in PCR products determines a modification in the shape of DNA melting curves comparative with melting profile of the wild

HR-MCA is a rapid and accurate method for detection of genetic variation in population. Although HR-MCA is not locus specific, it can become a tool of choice for point mutations screening after identification of large mutation (deletion and duplications) by MLPA. The sequencing of the fragments with abnormal melting profile only, identified by HR-MCA, reduces costs and waiting time per archived results.

All point mutations identified by HR-MCA method need to be confirmed by sequencing. Sequencing, the most widely used approach for DNA analysis, remains

The Sanger DNA sequencing method is applied to determine the sequence of a DNA molecule and to identify the subtle mutations in samples compared with a

Because a lot of muscle proteins associated with different forms of muscular dystrophy are extremely large, full gene analysis using Sanger sequencing can lead to higher costs and is time-consuming for analysis. The method finds its usefulness in the analysis of small gene composed of only few exons in which the frequency of point mutation is higher. For instance gene that encode for sarcoglycans (e.g., SGCA 10 exons, SGCB 6 exons, SGCG 10 exons), for calpain 3 (CAPN3 26 exons),

Representative high-resolution melting curve analysis which reveals difference in melting shape between wild

type (normal) DNA (see Figure 6).

the "gold standard" for mutation analysis.

2.4.3 Sanger sequencing

Muscular Dystrophies

reference sequence [79, 80].

Figure 6.

20

type and variant sequence.

The presence of clinical symptoms in a male child presented in Cap 2.1 along with increased serum levels of CK, transaminase enzymes or aldolase should trigger the diagnostic investigation for dystrophinopathies [11]. When DMD or BMD is suspected, diagnostic recommendation as first investigation is the screening of DMD gene for deletions and duplications by MLPA. The presence of a mutation in gene confirms the dystrophinopathies diagnosis.

A large number of research studies highlight the utility of MLPA as detection methods for DMD gene [84–86] which is the largest gene (2.2 Mb) of the human genome.

Many different types of mutations that occurred in the DMD gene, encoding for the cytoskeletal protein, dystrophin, are responsible for both severe disease Duchenne muscular dystrophy (DMD) and the milder form of the disease Becker muscular dystrophy (BMD). The difference in disease severity between the two phenotypes, DMD and BMD, can be explained by the "reading frame rule" proposed by Monaco in 1988 [87]. According to this theory, DMD is caused by mutations which disrupt the reading frame, and no protein will be synthesized, while for BMD phenotype the mutations do not affect the reading frame leading to the synthesis of a smaller and semi-functional dystrophin protein.

Previously reported studies have shown that all over the world, deletions of one or more exons are most common mutation in dystrophinopathies (60–65%), followed by duplications (5–8%) and point mutations (30–35%) [88]. Around 90% from all dystrophinopathies cases present worldwide respect reading frame rule. For the remaining 10% as well as for patients with discordant phenotype, additional analyses are required for an accurate diagnosis.

Because the mutations have been observed across all exons of the gene with a higher incidence of mutation in two "hotspots" regions between exons 2–20 and 45– 50 [85, 89], MLPA method proves its utility for the molecular diagnosis of dystrophinopathies, by simultaneous screening of all 79 exons of DMD gene for large intragenic rearrangements [90]. All deletion and duplications identified should be checked for the validity of the reading frame rule on http://www.dmd.nl.

If no deletions and duplication are identified, the DMD gene should be investigated for point mutations [91] by hrMCA followed by sequencing of exons with a modification of melting curve only. Full characterization of the mutation (type, size, and position) is important in identification of patients that are eligible for specific mutation gene therapy [89].

If a muscle biopsy is the tool of choice as first step in analysis routine, histochemical staining and dystrophin analysis by immunohistochemistry/immunofluorescence and Western blot confirm or not the dystrophinopathies diagnosis based on the difference in the expression of dystrophin.

changes in protein expression. Muscle biopsy is not always informative taken into account the secondary protein reduction in addition to the primary protein deficiency but can guide to targeted genetic tests. Although genetic testing can be expensive, it will identify the exact defect of the disease. Being composed only few exons and considering that LGMDs could have many possible genetic causes, the

Autosomal dominant LGMDs' form is quiet rare in population. Recessive forms are more common and studied. The most commonly recessive form of LGMDs and the most studied are LGMD 2A (calpainopathy) and LGMD 2B (dysferlinopathy).

Besides the clinical manifestation and laboratory investigation of serum enzymes presented above, for characterization of this condition, searching for mutations in CAPN3 gene in correlation with protein calpain 3 investigations by Western blot represents the "gold standard" in LGMD2A. The identification of the mutations in CAPN3 gene is most often difficult due to many genetic variations which appear in

The improvement of next-generation sequencing technology (NGS), which screens genomic DNA for a large number of genes involved in neuromuscular disease, makes LGMD diagnosis easier to achieve. However, in most laboratories, the diagnosis starts with screening for mutation by direct Sanger sequencing analysis of the 24 exons of the CAPN3 gene. With this method more than 95% cases are diagnosed. Even if this analysis is successful, in most cases, there are not always identify large deletions and duplications as well as intronic splice mutations [98]. This fact shows importance of screening for large genomic rearrangements by MLPA, the most cost-effective techniques. Sanger sequencing combined with MLPA leads to an increase in the mutation detection rate and remains one of the most valuable diagnostic tools. Therefore, when DNA analysis is not conclusive, a

Until this moment, only few studies reported the success of immunohistochem-

In general, for analysis, two antibodies which produce characteristic patterns of bands are used: NCL-CALP-2C4 directed against exon 1 which recognizes the fullsized protein at 94 kDa and additional band at 30 kDa and NCL-CALP-12A2 against exon 8 that recognizes also to the full-sized protein at 94 kDa and additionally bands doublet at 60 and 54 kDa. An example of normal calpain on blot is shown in

Identification and interpretation of the pattern of bands obtained on WB by using the two antibodies for calpain 3 provide useful information about protein expression in muscle. Generally, it is widely accepted that the complete absence of all specific calpain 3 bands on blot is specific for LGMD2A diagnosis and is due to a primary defect in CAPN3 [100]. The presence of a normal amount of calpain-3 protein on blot does not exclude the LGMD 2A diagnosis. The normal amount of calpain-3 protein on blot in patients with LGMD 2A was reported [101] by several studies. The possible explanation of normal expression but functionally inactive

ical technique application for the diagnosis of LGMD using calp3d/12A2 and calp2C4 antibodies [40, 99] and demonstrated the localization of calpain 3 in

Nowadays, it is widely accepted that WB, even if it does not have a high

sequencing of genes is the most suitable method of choices for diagnosis.

LGMD 2A is the most common form of limb-girdle muscular dystrophy accounting for about 30% of all LGMDs, caused by mutation in CAPN3 gene which

encodes for calcium sensitive dependent protease-calpain 3 protein.

2.5.2.1 Limb-girdle muscular dystrophy 2A (LGMD2A)

Clinical and Molecular Diagnosis in Muscular Dystrophies

DOI: http://dx.doi.org/10.5772/intechopen.85339

this gene and the position they have in the gene.

muscle biopsy is required for protein analyses.

accuracy, is the most suitable method for calpain 3 analysis.

myofibrils and myonuclei.

Figure 5.

23

Analysis by IF of dystrophin in muscle samples using three monoclonal mouse antibodies against three domains of protein (C-terminal, rod domain, and Nterminal) revealed the localization of protein at the sarcolemmal level of skeletal muscle fibers and displays a normal expression of intensity signal around each muscle fiber. In DMD patients, dystrophin is absent or severely reduced, while BMD patients displayed a variable expression of signal for dystrophin. Also, labeling of dystrophin on sections plays a critical role in identification process of the DMD female carriers which display a mosaic pattern of dystrophin expression.

Western blot as additional method confirms the diagnostic.

It's important to note that the protein result should be confirmed by genetic analysis.

#### 2.5.2 Limb-girdle muscular dystrophies

Limb-girdle muscular dystrophies (LGMD) are a highly clinically and genetically heterogeneous group of muscle disorders that affect in both males' and females' voluntary muscles of the pelvic and shoulder areas [92].

Major advances in last decades, both in neuromuscular disorders field and diagnostic assays, made that many genes associated with LGMDs to be found such us: CAPN3 (encode for calpain 3), DYSF (encode for dysferlin), FKRP (encode for fukutin), SGCA, SGCB, SGCG, and SGCD (that encode for the α-, β-, γ-, and δsarcoglycan), and more than 30 forms of LGMDs to be characterized [11, 37]. However, a significant number of patients clinically diagnosed with LGMDs remain molecularly uncharacterized [93].

Based on their inheritance pattern were classified in dominant (type 1 LGMD) and recessive forms (type 2 LGMD) [94].

Giving the complexity of the clinical symptoms and different genes involved, a diagnosis algorithm for these pathologies is still waiting, but comprehensive guidelines for identifying these disorders have been published. The initial evaluation of a patient involves clinical examination, followed by laboratory test such as serum creatine kinase measurements, genetic tests, and muscle biopsy analysis. CK level can vary from lower level in dominantly inherited LGMD forms to very high level in recessively inherited forms [95].

All these diagnostic approaches should be sufficient to accurately predict the correct form of LGMDs. In the last years, the increasing use of next-generation sequencing technology for simultaneous analysis of known LGMD-related genes improved diagnostic rate and offered opportunity to identified new disease-related genes [96]. However, this technology is extremely expensive and is not yet available in all the molecular diagnostic laboratories.

If the clinical features, laboratory tests, and other investigations such as electromyography, magnetic resonance, or ultrasound imaging suggest a LGMD, muscle biopsy [97] may be considered an appropriate test to start the investigation.

Routine histochemical stains will display variable degree of typically dystrophic feature characteristic for LGMD: variation in fiber size, degeneration and regeneration fiber, presence of internal nuclei, and increased endomysial fibrosis [37, 40].

Disease prediction based on clinical and histochemical tests alone is difficult to achieve, so immunohistochemical stains (IHC or IF) of specific muscle proteins involved in LGMDs (calpain 3, dysferlin, caveolin3, the sarcoglycans, myotilin, lamin A/C, etc.) provides useful information about the presence, absence and

Clinical and Molecular Diagnosis in Muscular Dystrophies DOI: http://dx.doi.org/10.5772/intechopen.85339

If a muscle biopsy is the tool of choice as first step in analysis routine, histochemical staining and dystrophin analysis by immunohistochemistry/immunofluorescence and Western blot confirm or not the dystrophinopathies diagnosis based

Analysis by IF of dystrophin in muscle samples using three monoclonal mouse

antibodies against three domains of protein (C-terminal, rod domain, and Nterminal) revealed the localization of protein at the sarcolemmal level of skeletal muscle fibers and displays a normal expression of intensity signal around each muscle fiber. In DMD patients, dystrophin is absent or severely reduced, while BMD patients displayed a variable expression of signal for dystrophin. Also, labeling of dystrophin on sections plays a critical role in identification process of the DMD

female carriers which display a mosaic pattern of dystrophin expression. Western blot as additional method confirms the diagnostic.

It's important to note that the protein result should be confirmed by genetic

Limb-girdle muscular dystrophies (LGMD) are a highly clinically and geneti-

Major advances in last decades, both in neuromuscular disorders field and diagnostic assays, made that many genes associated with LGMDs to be found such us: CAPN3 (encode for calpain 3), DYSF (encode for dysferlin), FKRP (encode for fukutin), SGCA, SGCB, SGCG, and SGCD (that encode for the α-, β-, γ-, and δsarcoglycan), and more than 30 forms of LGMDs to be characterized [11, 37]. However, a significant number of patients clinically diagnosed with LGMDs remain

Based on their inheritance pattern were classified in dominant (type 1 LGMD)

Giving the complexity of the clinical symptoms and different genes involved, a diagnosis algorithm for these pathologies is still waiting, but comprehensive guidelines for identifying these disorders have been published. The initial evaluation of a patient involves clinical examination, followed by laboratory test such as serum creatine kinase measurements, genetic tests, and muscle biopsy analysis. CK level can vary from lower level in dominantly inherited LGMD forms to very high level

All these diagnostic approaches should be sufficient to accurately predict the correct form of LGMDs. In the last years, the increasing use of next-generation sequencing technology for simultaneous analysis of known LGMD-related genes improved diagnostic rate and offered opportunity to identified new disease-related genes [96]. However, this technology is extremely expensive and is not yet available

If the clinical features, laboratory tests, and other investigations such as electromyography, magnetic resonance, or ultrasound imaging suggest a LGMD, muscle biopsy [97] may be considered an appropriate test to start the investigation.

Routine histochemical stains will display variable degree of typically dystrophic feature characteristic for LGMD: variation in fiber size, degeneration and regeneration fiber, presence of internal nuclei, and increased endomysial fibrosis [37, 40]. Disease prediction based on clinical and histochemical tests alone is difficult to achieve, so immunohistochemical stains (IHC or IF) of specific muscle proteins involved in LGMDs (calpain 3, dysferlin, caveolin3, the sarcoglycans, myotilin, lamin A/C, etc.) provides useful information about the presence, absence and

cally heterogeneous group of muscle disorders that affect in both males' and

females' voluntary muscles of the pelvic and shoulder areas [92].

on the difference in the expression of dystrophin.

2.5.2 Limb-girdle muscular dystrophies

molecularly uncharacterized [93].

in recessively inherited forms [95].

in all the molecular diagnostic laboratories.

and recessive forms (type 2 LGMD) [94].

analysis.

Muscular Dystrophies

22

changes in protein expression. Muscle biopsy is not always informative taken into account the secondary protein reduction in addition to the primary protein deficiency but can guide to targeted genetic tests. Although genetic testing can be expensive, it will identify the exact defect of the disease. Being composed only few exons and considering that LGMDs could have many possible genetic causes, the sequencing of genes is the most suitable method of choices for diagnosis.

Autosomal dominant LGMDs' form is quiet rare in population. Recessive forms are more common and studied. The most commonly recessive form of LGMDs and the most studied are LGMD 2A (calpainopathy) and LGMD 2B (dysferlinopathy).

### 2.5.2.1 Limb-girdle muscular dystrophy 2A (LGMD2A)

LGMD 2A is the most common form of limb-girdle muscular dystrophy accounting for about 30% of all LGMDs, caused by mutation in CAPN3 gene which encodes for calcium sensitive dependent protease-calpain 3 protein.

Besides the clinical manifestation and laboratory investigation of serum enzymes presented above, for characterization of this condition, searching for mutations in CAPN3 gene in correlation with protein calpain 3 investigations by Western blot represents the "gold standard" in LGMD2A. The identification of the mutations in CAPN3 gene is most often difficult due to many genetic variations which appear in this gene and the position they have in the gene.

The improvement of next-generation sequencing technology (NGS), which screens genomic DNA for a large number of genes involved in neuromuscular disease, makes LGMD diagnosis easier to achieve. However, in most laboratories, the diagnosis starts with screening for mutation by direct Sanger sequencing analysis of the 24 exons of the CAPN3 gene. With this method more than 95% cases are diagnosed. Even if this analysis is successful, in most cases, there are not always identify large deletions and duplications as well as intronic splice mutations [98]. This fact shows importance of screening for large genomic rearrangements by MLPA, the most cost-effective techniques. Sanger sequencing combined with MLPA leads to an increase in the mutation detection rate and remains one of the most valuable diagnostic tools. Therefore, when DNA analysis is not conclusive, a muscle biopsy is required for protein analyses.

Until this moment, only few studies reported the success of immunohistochemical technique application for the diagnosis of LGMD using calp3d/12A2 and calp2C4 antibodies [40, 99] and demonstrated the localization of calpain 3 in myofibrils and myonuclei.

Nowadays, it is widely accepted that WB, even if it does not have a high accuracy, is the most suitable method for calpain 3 analysis.

In general, for analysis, two antibodies which produce characteristic patterns of bands are used: NCL-CALP-2C4 directed against exon 1 which recognizes the fullsized protein at 94 kDa and additional band at 30 kDa and NCL-CALP-12A2 against exon 8 that recognizes also to the full-sized protein at 94 kDa and additionally bands doublet at 60 and 54 kDa. An example of normal calpain on blot is shown in Figure 5.

Identification and interpretation of the pattern of bands obtained on WB by using the two antibodies for calpain 3 provide useful information about protein expression in muscle. Generally, it is widely accepted that the complete absence of all specific calpain 3 bands on blot is specific for LGMD2A diagnosis and is due to a primary defect in CAPN3 [100]. The presence of a normal amount of calpain-3 protein on blot does not exclude the LGMD 2A diagnosis. The normal amount of calpain-3 protein on blot in patients with LGMD 2A was reported [101] by several studies. The possible explanation of normal expression but functionally inactive

protein is due to a functional enzyme defect that impairs the autolytic or proteolytic activity of protein without elimination of protein from the muscle [102, 103].

hrMCA high-resolution melting curve analysis

Clinical and Molecular Diagnosis in Muscular Dystrophies

MLPA multiplex ligation-dependent probe amplification

LGMD limb-girdle muscular dystrophy

DOI: http://dx.doi.org/10.5772/intechopen.85339

IF immunofluorescence IHC immunohistochemistry

MD muscular dystrophy MEB muscle-eye-brain disease

WB Western blot

Author details

Bucharest, Romania

25

Gisela Gaina1,2\*, Magdalena Budisteanu1

provided the original work is properly cited.

1 Victor Babes National Institute of Pathology, Bucharest, Romania

2 Department of Biochemistry and Molecular Biology, University of Bucharest,

\*Address all correspondence to: giselagaina@yahoo.com; giss\_g@yahoo.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Emilia Manole<sup>1</sup> and Elena Ionica<sup>2</sup>

The reduction is more difficult to interpret because calpain-3 appears to reduce in amount as secondary effect in other forms of muscular dystrophies such as dysferlinopathies [40] and titinopathies [81, 92].

Even if muscle calpain-3 results on blot should always be confirmed by mutation analysis, Western blot remains one of the most valuable diagnostic tools in LGMDs allowing for the simultaneous analysis of multiple proteins, identifying both the primary defect and the secondary reductions.
