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membrane. Two distinct phenotypes are associated with mutations in this gene: Miyoshi myopathy, which is a predominantly distal muscle wasting (37), and LGMD2B, with a proximal weakness (36). Only a few mutations have been identified, due to the large size of the dysferlin gene (55 exons), and no apparent hot spot for mutations. Therefore, muscle protein analysis is very helpful.

Protein analyses in LGMD2B have shown a total deficiency of dysferlin, both through immunofluorescence and western blot (Fig. 6). Although a partial deficiency has been reported in LGMD2B patients (65), dysferlin deficiency seems to be specific to LGMD2B in our patients, and has not been seen as a secondary effect in other forms of MD (66). Dysferlin is an ubiquitously expressed protein, and can be detected also in the skin and in corionic villus biopsy (Fig. 6).

In DMD and sarcoglycanopathies, a normal localization and molecular weight (MW) for this protein was found, suggesting no interaction between dysferlin and the DGC.

3.3.5. Telethoninopathy

The sarcomere is the unit of skeletal and cardiac muscle contraction. In the past few years, there have been many studies focusing the role of skeletal and cardiac muscle proteins (67,68). Mutations in several sarcomeric proteins such as telethonin (33), myotilin (9), actin (69), tropomiosin 3 and 2 (70,71), nebulin (72), troponin T1 (73), have been associated to human muscle diseases. We have recently detected one nemaline myopathy affected patient, with a deficiency of only the SH3 domain of nebulin, through Western blot analysis (74).

Fig. 6. (A) Dysferlin through IF analysis showing the normal sarcolemmal pattern in the control muscle, the positive labeling in the normal villus and in skin, the negative pattern in the muscle from one LGMD-2B affected patient. (B) Multiplex Western blot analysis for dysferlin (with dystrophin, at 427 kDa), showing the presence of the dysferlin 230 kDa band in the normal villus sample, in the control, and in the LGMD-2A patient, and no dysferlin band in the LGMD-2B patient. (See color plate 11 appearing in the insert following p. 82)

Fig. 6. (A) Dysferlin through IF analysis showing the normal sarcolemmal pattern in the control muscle, the positive labeling in the normal villus and in skin, the negative pattern in the muscle from one LGMD-2B affected patient. (B) Multiplex Western blot analysis for dysferlin (with dystrophin, at 427 kDa), showing the presence of the dysferlin 230 kDa band in the normal villus sample, in the control, and in the LGMD-2A patient, and no dysferlin band in the LGMD-2B patient. (See color plate 11 appearing in the insert following p. 82)

The role of the majority of these proteins is still unknown. However, their presence in affected patients with the aforementioned conditions suggests an essential role in the constitution of the sarcomere, since total deficiencies are probably incompatible with life. New methodologies to detect possible alterations in sarcomeric proteins have to be developed to elucidate their role.

Telethonin is a sarcomeric protein of 19 kD, present in the Z disk of the sarcomere of the striated and cardiac muscle (38). Mutations in the telethonin gene at 17q cause LGMD2G (33). Telethonin was found to be one of the substrates of the serine kinase domain of titin, that acts as a molecular ruler for the assembly of the sarcomere by providing spatially defined binding sites for other sarcomeric proteins. The specific function of telethonin and its interaction with the other proteins from the muscle is still unknown.

Protein analysis in 6 LGMD2G Brazilian patients, from four unrelated families, showed deficiency of telethonin in all of them (Fig. 7), associated with frameshifted mutations in the LGMD2G gene. The possibility of other mutational mechanisms in this sarcomeric gene associated with the presence of the protein in the muscle cannot be ruled out yet.

Additional protein studies in these patients have shown normal expression for the proteins dystrophin, sarcoglycans, dysferlin, calpain, and titin. Immunofluorescence analysis for a-actinin-2 and myotilin showed a cross-striation pattern, suggesting that at least part of the Z-line of the sarcomere is preserved. Ultra-structural analysis con firmed the maintenance of the integrity of the sarcomeric architecture. Therefore, mutations in the telethonin gene do not seem to alter the sarcomere integrity (63).

Telethonin was clearly present in the rods, in muscle fibers from patients with nemaline myopathy, confirming its localization in the Z-line of the sarcomere. The analysis of telethonin on muscle biopsies from patients with LGMD2A, LGMD2B, SGpathies, and DMD showed normal localization, suggesting that the deficiency of calpain, dysferlin, sarcoglycans, and dystrophin does not seem to alter telethonin expression (63).

3.3.6. Caveolin 3 Deficiency

Caveolin3 is the protein present in the Caveolae, small invaginations in the plasma membrane that are present in most types of cells, and is probably involved in signal transduction.

Mutations in the caveolin-3 gene (CAV-3) with a negative dominant effect and reduction of the protein expression cause autosomal dominant LGMD1C muscular dystrophy (11). It has been suggested that CAV-3 mutations might also cause the AR-LGMD form (12). However, recent screening for mutations in the CAV-3 gene in 61 Brazilian LGMD patients and 100 normal controls has not confirmed the existence of the AR caveolin deficiency (75).

3.3.7. Congenital MD with Merosin Deficiency

Laminin 2 is a constituent of the basal lamina, which links to dystroglycan and which provides structural support in the extracellular matrix. It is composed by three chains: a-2, P-1 and y-1. Laminin a-2 deficiency due to mutations in the LAMA2 gene at 6q2 is the cause of the autosomal recessive Congenital MD (76,77). Laminin a2 is totally deficient in muscle biopsies from patients with the severe typical congenital dystrophy phenotype. However partial deficiencies have been described in patients with heterogeneity in the clinical picture (Fig. 8) (78,79). The protein is ubiquitously expressed, and may be detected in skin biopsy (80) as well as in chorionic villus, which is a very useful test for prenatal diagnosis (81,82) (Fig. 9).

We have studied 20 patients affected by the typical form of congenital MD, and detected a total deficiency of laminin a2, using both the 80 kDa and 300 kDa antibodies. In patients with partial deficiency, usually the 300 kDa antibody shows a more deficient pattern (83). We also have recently detected a partial deficiency of only the 300 kDa a2-laminin antibody in 5 patients with the classical LGMD clinical course (84-86). Screening for mutations in the LAMA2 gene will elucidate the primary or secondary etiology of these deficiencies.

3.3.8. Protein Study for Differential Diagnosis

Testing for defective protein expression is a powerful tool for deciding where to start the search for gene mutations (Fig. 9)

In adult MD forms, multiplex Western blot analysis for dystrophin, calpain, and dysferlin has shown to be very useful for preliminary screening of muscular dystrophy. With the exception of calpain 3, which may occur as a secondary effect of a dys-ferlinopathy, dysferlin and telethonin deficiencies seem to be the consequence of their respectively primary gene defect. Therefore the absence of dysferlin or telethonin on muscle biopsy strongly suggests a diagnosis of LGMD2B or LGMD2G, respectively.

Fig. 7. Double IF analysis for a-actinin 2 (a positive marker of Z-band of the sarcomere), and telethonin in one control and one LGMD-2G patient. Note the sarcolemmal deficiency of telethonin in the patient. Some unspecific reaction are commonly seen in the nucleus, which requires further studies. (See color plate 11 appearing in the insert following p. 82)

Fig. 7. Double IF analysis for a-actinin 2 (a positive marker of Z-band of the sarcomere), and telethonin in one control and one LGMD-2G patient. Note the sarcolemmal deficiency of telethonin in the patient. Some unspecific reaction are commonly seen in the nucleus, which requires further studies. (See color plate 11 appearing in the insert following p. 82)

Fig. 8. IF analysis for a2-Laminin with antibodies against the 80 kDa and 300 kDa fragments showing the pattern in a normal control, the positive pattern in a normal villus, the total deficiency, through the two antibodies in one CMD severely affected patient and one patient with partial deficiency. (See color plate 11 appearing in the insert following p. 82)

Fig. 8. IF analysis for a2-Laminin with antibodies against the 80 kDa and 300 kDa fragments showing the pattern in a normal control, the positive pattern in a normal villus, the total deficiency, through the two antibodies in one CMD severely affected patient and one patient with partial deficiency. (See color plate 11 appearing in the insert following p. 82)

If no protein or DNA alterations are found in patients with clinical diagnosis of LGMD, the possibility of spinal muscular atrophy (SMA) should be considered, due to the clinical overlap of these diseases.

Patients with suspected Xp21 dystrophy are first tested for deletions in the dystrophin gene, a less invasive test. The identification of a molecular deletion will confirm the diagnosis of DMD/BMD. If no deletion is detected (about 40% of the cases), muscle proteins are analyzed in an attempt to elucidate the possible diagnosis.

Fig. 9. Schematic representation of the procedures and methodology used for the diagnosis of NMD in our Center.

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