Rita C.M. Pavanello

Serum creatine-kinase (CK) and pyruvate-kinase (PK) activities in Duchenne (DMD) as compared with Becker (BMD) muscular dystrophy

Serum creatine-kinase (CK) activities were determined in 536 patients affected with X-linked muscular dystrophy (456 with Duchenne or DMD and 80 with Becker or BMD) and serum pyruvate-kinase (PK) in 360 among them (309 DMD and 51 BMD). The aim of this investigation was to assess the variability and rate of decrease in serum activity in DMD as compared with BMD as a function of age and in DMD as a function of Vignos scale as well. In DMD, maximum CK and PK activities were found around 1-6 years old and the average rate of decline according to age was estimated as 0.18 per year and 0.27-0.29 for both enzymes as a function of Vignos scale (assessed in 291 cases). For BMD, maximum serum enzyme levels were found around 10-15 years old and the rate of decline of serum activity per year was 0.06 for CK and 0.07 for PK. If maximum levels of serum enzyme reflect active muscle degeneration and the rate of decline per year to progressive loss of muscle mass (responsible for the release of muscle enzymes to the blood stream) our observations suggest: (a) active muscle degeneration occurs, on average, 5 years later in the group of outliers and 10 years later in BMD as compared with severe DMD; (b) the rate in which muscle mass is lost is significantly greater in DMD than in BMD and therefore serum enzyme determinations may represent an important test for evaluation of therapeutic trials; (c) serum enzymes determination may represent an important preliminary test to discriminate in a proportion of young patients if they will develop a severe or milder phenotype.

The facioscapulohumeral muscular dystrophy (FSHD1) gene affects males more severely and more frequently than females

We investigated 52 families of patients with facioscapulohumeral muscular dystrophy (FSHD1), including 172 patients (104 males and 68 females). Among 273 DNA samples which were analyzed with probe p13E-11, 131 (67 males and 64 females) were shown to carry an EcoRI fragment smaller than 35 kb; 114 among them were examined clinically and neurologically. Results of the present investigation showed that: a) there is no molecular evidence for autosomal or X-linked recessive inheritance of FSHD1; b) an excess of affected males, which is explained by a significantly greater proportion of females than males among asymptomatic cases and a significantly greater proportion of affected sons than daughters observed in the offspring of asymptomatic mothers; c) the penetrance of the FSHD1 gene until age 30 was estimated as 83% for both sexes but was significantly greater for males (95%) than for females (69%); d) new mutations occur significantly more frequently in females than males among somatic/germinal mosaic cases; and e) severely affected cases originated more often through new mutations or were transmitted through maternal than through paternal lines including somatic/germinal mothers. These observations have important implications for understanding the molecular mechanisms responsible for FSHD1 and for genetic and prognostic counseling according to the gender of the affected patient.

Clinical variability in calpainopathy: What makes the difference?

Limb girdle muscular dystrophies (LGMD) are a heterogeneous group of genetic disorders characterised by progressive weakness of the pelvic and shoulder girdle muscles and a great variability in clinical course. LGMD2A, the most prevalent form of LGMD, is caused by mutations in the calpain-3 gene (CAPN-3). More than 100 pathogenic mutations have been identified to date, however few genotype : phenotype correlation studies, including both DNA and protein analysis, have been reported. In this study we screened 26 unrelated LGMD2A Brazilian families (75 patients) through Single-Stranded Conformation Polymorphism (SSCP), Denaturing high-performance liquid chromatography (DHPLC) and sequencing of abnormal fragments which allowed the identification of 47 mutated alleles (approximately 90%). We identified two recurrent mutations (R110X and 2362-2363AG>TCATCT) and seven novel pathogenic mutations. Interestingly, 41 of the identified mutations (approximately 80%) were concentrated in only 6 exons (1, 2, 4, 5, 11 and 22), which has important implications for diagnostic purposes. Protein analysis, performed in 28 patients from 25 unrelated families showed that with exception of one patient (with normal/slight borderline reduction of calpain) all others had total or partial calpain deficiency. The effects of type of mutation, amount of calpain in the muscle, gender and ethnicity of affected patients on clinical course (age of onset and ascertainment) were analysed. Interestingly, it was observed that, on average, African–Brazilian calpainopathy patients are more severely affected than Caucasians.

Sarcoglycanopathies are responsible for 68% of severe autosomal recessive limb-girdle muscular dystrophy in the Brazilian population.

Sarcoglycanopathies (SGPs) constitute a subgroup of limb-girdle muscular dystrophies (LGMD), where the primary defect in one sarcoglycan (SG) glycoprotein (alpha-SG, beta-SG, gamma-SG or delta-SG) results in a deficiency of the whole complex. Four genes, at 17q, 4q, 13q and 5q, encode the four glycoproteins, and mutations in these genes cause diseases called LGMD2D, 2E, 2C and 2F. To estimate the prevalence, relative proportions and clinical features of SGPs, we have studied the SG proteins in muscle biopsies of 140 patients (from 115 unrelated Brazilian families) with a clinical diagnosis of LGMD. Alpha-SG immunofluorescence analysis showed a positive staining pattern in 70% (80/115) of the families, a patchy pattern in 14% (16/115) and a negative pattern in 16% (19/115) of the families. All the 19 alpha-SG negative, and four of the 16 alpha-SG patchy patients were also deficient for the other three SG proteins, confirming the diagnosis of SGP in 20% of the LGMD families. None of the positive alpha-SG patients were deficient for any of the other three SG proteins, supporting the view that the SG complex functions as a unit. DNA analysis for the four sarcoglycan genes showed that alpha-SG mutations accounted for 47%, beta-SG for 16%, gamma-SG for 16% and delta-SG for 21% of the cases. SG abnormalities were observed in only 8.5% of patients with milder LGMD forms, but were present in 68% of patients with a severe Duchenne-like course. The relatively high frequency of SGP among Brazilian people with LGMD may be due to the disproportionally high frequency of African Brazilian SGP patients with the same mutation (particularly among LGMD2C and 2F patients), suggesting a founder effect. Consanguinity is also common in our SGP families.

Dysferlin protein analysis in limb-girdle muscular dystrophies

Dysferlin is the protein product of the DYSF gene mapped at 2p31, which mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. To date, nine autosomal recessive forms (AR-LGMD) have been identified: four genes, which code for the sarcoglycan glycoproteins, are associated with both mild and severe forms, the sarcoglycanopathies (LGMD2C, 2D, 2E and 2F). The other five forms, usually causing a milder phenotype are LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2G (telethonin), LGMD2H (9q31-11), and LGMD2I (19q13.3).We studied dysferlin expression in a total of 176 patients, from 166 LGMD families: 12 LGMD2B patients, 70 with other known forms of muscular dystrophies (LGMD2A, sarcoglycanopathies, LGMD2G), in an attempt to assess the effect of the primary gene-product deficiency on dysferlin. In addition, 94 still unclassified LGMD families were screened for dysferlin deficiency.In eight LGMD2B patients from five families, no dysferlin was observed in muscle biopsies, both through immunofluorescence (IF) and Western blot methodologies, while in two families, a very faint band was detected. Both patterns, negative or very faint bands, were concordant in patients belonging to the same families, suggesting that dysferlin deficiency is specific to LGMD2B.Myoferlin, the newly identified homologue of dysferlin was studied for the first time in LGMD2B patients. Since no difference was observed between patients mildly and severely affected, this protein do not seem to modify the phenotype in the present dysferlin-deficient patients.Dystrophin, sarcoglycans, and telethonin were normal in all LGMD2B patients, while patients with sarcoglycanopathies (2C, 2D, and 2E), LGMD2A, LGMD2G, and DMD showed the presence of a normal dysferlin band by Western blot and a positive pattern on IF. These data suggest that there is no interaction between dysferlin and these proteins. However, calpain analysis showed a weaker band in four patients from two families with intra-familial concordance. Therefore, this secondary deficiency of calpain in LGMD2B families, may indicate an interaction between dysferlin and calpain in muscle.Dysferlin was also present in cultured myotubes, in chorionic villus, and in the skin.Dysferlin deficiency was found in 24 out of a total of 166 Brazilian AR-LGMD families screened for muscle proteins (∼14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population.

Immunofluorescence dystrophin study in Duchenne dystrophy through the concomitant use of two antibodies directed against the carboxy-terminal and the amino-terminal region of the protein.

Dystrophin immunohistochemical studies in muscle from Duchenne patients (DMD) have shown a population of fibers with partial labelling. In order to determine whether this is related to a cross reaction or to the presence of dystrophin. 22 DMD patients were studied immunohistochemically, through the concomitant use of antibodies from the N-terminal and the C-terminal regions of the protein. In 2, the reaction was negative while in 2 others 17 and 25% of fibers were positive with both antibodies. In the remainder, a population of partially stained fibers was seen: 11 were positive with both antibodies and in 7 only with the N-terminal one. Apparently, there is no correlation between the proportion of positive fibers and clinical progression, or the presence and pattern of DNA deletions in the central part of the gene. These observations led us to suggest that some truncated protein, intermediate synthesis or degradation products of dystrophin are present in muscle from Duchenne patients.

A defect in the RNA-processing protein HNRPDL causes limb-girdle muscular dystrophy 1G (LGMD1G)

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of genetically determined muscle disorders with a primary or predominant involvement of the pelvic or shoulder girdle musculature. More than 20 genes with autosomal recessive (LGMD2A to LGMD2Q) and autosomal dominant inheritance (LGMD1A to LGMD1H) have been mapped/identified to date. Mutations are known for six among the eight mapped autosomal dominant forms: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin-3), LGMD1D (desmin), LGMD1E (DNAJB6), and more recently for LGMD1F (transportin-3). Our group previously mapped the LGMD1G gene at 4q21 in a Caucasian-Brazilian family. We now mapped a Uruguayan family with patients displaying a similar LGMD1G phenotype at the same locus. Whole genome sequencing identified, in both families, mutations in the HNRPDL gene. HNRPDL is a heterogeneous ribonucleoprotein family member, which participates in mRNA biogenesis and metabolism. Functional studies performed in S. cerevisiae showed that the loss of HRP1 (yeast orthologue) had pronounced effects on both protein levels and cell localizations, and yeast proteome revealed dramatic reorganization of proteins involved in RNA-processing pathways. In vivo analysis showed that hnrpdl is important for muscle development in zebrafish, causing a myopathic phenotype when knocked down. The present study presents a novel association between a muscular disorder and a RNA-related gene and reinforces the importance of RNA binding/processing proteins in muscle development and muscle disease. Understanding the role of these proteins in muscle might open new therapeutic approaches for muscular dystrophies.

Transcriptional regulation differs in affected facioscapulohumeral muscular dystrophy patients compared to asymptomatic related carriers.

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder that has been associated with a contraction of 3.3-kb repeats on chromosome 4q35. FSHD is characterized by a wide clinical inter- and intrafamilial variability, ranging from wheelchair-bound patients to asymptomatic carriers. Our study is unique in comparing the gene expression profiles from related affected, asymptomatic carrier, and control individuals. Our results suggest that the expression of genes on chromosome 4q is altered in affected and asymptomatic individuals. Remarkably, the changes seen in asymptomatic samples are largely in products of genes encoding several chemokines, whereas the changes seen in affected samples are largely in genes governing the synthesis of GPI-linked proteins and histone acetylation. Besides this, the affected patient and related asymptomatic carrier share the 4qA161 haplotype. Thus, these polymorphisms by themselves do not explain the pathogenicity of the contracted allele. Interestingly, our results also suggest that the miRNAs might mediate the regulatory network in FSHD. Together, our results support the previous evidence that FSHD may be caused by transcriptional dysregulation of multiple genes, in cis and in trans, and suggest some factors potentially important for FSHD pathogenesis. The study of the gene expression profiles from asymptomatic carriers and related affected patients is a unique approach to try to enhance our understanding of the missing link between the contraction in D4Z4 repeats and muscle disease, while minimizing the effects of differences resulting from genetic background.

Dystrophin immunostaining in muscles from patients with different types of muscular dystrophy : a Brazilian study

The localization of the protein dystrophin was studied using the immunofluorescence method, in muscle biopsies from 74 patients affected by different types of muscular dystrophy and 4 normal controls. In 15 patients with limb-girdle muscular dystrophy (LGMD) the pattern was indistinguishable from normal. Among 42 Duchenne patients (DMD), 3 were totally negative and 39 showed a variable proportion (4-30%) of partially labelled fibers. With one exception 17 Becker dystrophy patients (BMD), showed a positive sarcolemmal reaction. A diffuse reaction inside the fibers, which was not observed in normal controls, was seen in the majority of DMD and also in some of the BMD patients. Based on these observations it is suggested that in DMD, a small quantity of protein is still present or there is a cross-reaction with other proteins which share some homology with dystrophin. The present results suggest that it is possible to make a differential diagnosis between DMD and BMD through dystrophin immunohistochemistry. However, to distinguish between patients with BMD and LGMD phenotypes, or DMD and outliers, complementary immunoblot studies and quantitative determination of dystrophin are necessary.

Central core disease due to recessive mutations in RYR1 gene : Is it more common than described?

Central core disease (CCD) is an autosomal‐dominant congenital myopathy, with muscle weakness and malignant hyperthermia (MH) susceptibility. We identified two of nine Brazilian CCD families carrying two mutations in the RYR1 gene. The heterozygous parents were clinically asymptomatic, and patients were mildly affected, differing from the few autosomal‐recessive cases described previously. Recessive inheritance in CCD may therefore be more common than previously appreciated, which has important implications for genetic counseling and MH prevention in affected families. Muscle Nerve, 2007