L.U. Yamamoto

Animal Models for Genetic Neuromuscular Diseases

The neuromuscular disorders are a heterogeneous group of genetic diseases, caused by mutations in genes coding sarcolemmal, sarcomeric, and citosolic muscle proteins. Deficiencies or loss of function of these proteins leads to variable degree of progressive loss of motor ability. Several animal models, manifesting phenotypes observed in neuromuscular diseases, have been identified in nature or generated in laboratory. These models generally present physiological alterations observed in human patients and can be used as important tools for genetic, clinic, and histopathological studies. The mdx mouse is the most widely used animal model for Duchenne muscular dystrophy (DMD). Although it is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle, this mouse is not useful for clinical trials because of its very mild phenotype. The canine golden retriever MD model represents a more clinically similar model of DMD due to its larger size and significant muscle weakness. Autosomal recessive limb-girdle MD forms models include the SJL/J mice, which develop a spontaneous myopathy resulting from a mutation in the Dysferlin gene, being a model for LGMD2B. For the human sarcoglycanopahties (SG), the BIO14.6 hamster is the spontaneous animal model for δ-SG deficiency, whereas some canine models with deficiency of SG proteins have also been identified. More recently, using the homologous recombination technique in embryonic stem cell, several mouse models have been developed with null mutations in each one of the four SG genes. All sarcoglycan-null animals display a progressive muscular dystrophy of variable severity and share the property of a significant secondary reduction in the expression of the other members of the sarcoglycan subcomplex and other components of the Dystrophin-glycoprotein complex. Mouse models for congenital MD include the dy/dy (dystrophia-muscularis) mouse and the allelic mutant dy2J/dy2J mouse, both presenting significant reduction of α2-laminin in the muscle and a severe phenotype. The myodystrophy mouse (Largemyd) harbors a mutation in the glycosyltransferase Large, which leads to altered glycosylation of α-DG, and also a severe phenotype. Other informative models for muscle proteins include the knockout mouse for myostatin, which demonstrated that this protein is a negative regulator of muscle growth. Additionally, the stress syndrome in pigs, caused by mutations in the porcine RYR1 gene, helped to localize the gene causing malignant hypertermia and Central Core myopathy in humans. The study of animal models for genetic diseases, in spite of the existence of differences in some phenotypes, can provide important clues to the understanding of the pathogenesis of these disorders and are also very valuable for testing strategies for therapeutic approaches.

Asymptomatic carriers for homozygous novel mutations in the FKRP gene: the other end of the spectrum

Autosomal recessive limb-girdle muscular dystrophy linked to 19q13.3 (LGMD2I) was recently related to mutations in the fukutin-related protein gene (FKRP) gene. Pathogenic changes in the same gene were detected in congenital muscular dystrophy patients (MDC1C), a severe disorder. We have screened 86 LGMD genealogies to assess the frequency and distribution of mutations in the FKRP gene in Brazilian LGMD patients. We found 13 Brazilian genealogies, including 20 individuals with mutations in the FKRP gene, and identified nine novel pathogenic changes. The commonest C826A European mutation was found in 30% (9/26) of the mutated LGMD2I alleles. One affected patient homozygous for the FKRP (C826A) mutation also carries a missense R125H change in one allele of the caveolin-3 gene (responsible for LGMD1C muscular dystrophy). Two of her normal sibs were found to be double heterozygotes. In two unrelated LGMD2I families, homozygous for novel missense mutations, we identified four asymptomatic carriers, all older than 20 years. Genotype–phenotype correlation studies in the present study as well as in patients from different populations suggests that the spectrum of variability associated with mutations in the FKRP gene seems to be wider than in other forms of LGMD. It also reinforces the observations that pathogenic mutations are not always determinant of an abnormal phenotype, suggesting the possibility of other mechanisms modulating the severity of the phenotype that opens new avenues for therapeutic approaches.

Muscle Protein Alterations in LGMD2I Patients With Different Mutations in the Fukutin-related Protein Gene

Fukutin-related protein (FKRP) is a protein involved in the glycosylation of cell surface molecules. Pathogenic mutations in the FKRP gene cause both the more severe congenital muscular dystrophy Type 1C and the milder Limb-Girdle Type 2I form (LGMD2I). Here we report muscle histological alterations and the analysis of 11 muscle proteins: dystrophin, four sarcoglycans, calpain 3, dysferlin, telethonin, collagen VI, α-DG, and α2-laminin, in muscle biopsies from 13 unrelated LGMD2I patients with 10 different FKRP mutations. In all, a typical dystrophic pattern was observed. In eight patients, a high frequency of rimmed vacuoles was also found. A variable degree of α2-laminin deficiency was detected in 12 patients through immunofluorescence analysis, and 10 patients presented α-DG deficiency on sarcolemmal membranes. Additionally, through Western blot analysis, deficiency of calpain 3 and dystrophin bands was found in four and two patients, respectively. All the remaining proteins showed a similar pattern to normal controls. These results suggest that, in our population of LGMD2I patients, different mutations in the FKRP gene are associated with several secondary muscle protein reductions, and the deficiencies of α2-laminin and α-DG on sections are prevalent, independently of mutation type or clinical severity.

Thomsen or Becker myotonia? A novel autosomal recessive nonsense mutation in the CLCN1 gene associated with a mild phenotype

We describe a large Brazilian consanguineous kindred with 3 clinically affected patients with a Thomsen myotonia phenotype. They carry a novel homozygous nonsense mutation in the CLCN1 gene (K248X). None of the 6 heterozygote carriers show any sign of myotonia on clinical evaluation or electromyography. These findings confirm the autosomal recessive inheritance of the novel mutation in this family, as well as the occurrence of phenotypic variability in the autosomal recessive forms of myotonia. Muscle Nerve, 2012

Protein and DNA analysis for the prenatal diagnosis of alpha2-laminin-deficient congenital muscular dystrophy.

Congenital muscular dystrophies (CMD) are characterized by neonatal hypotonia and/or artrogriposis associated with a dystrophic muscle biopsy. The CMD1A form is caused by a deficiency of the α2 chain of laminin 2 (LAMA2 gene at 6q2), a protein present in the basal lamina of muscle fibers, in Schwann cells, epidermis, and in fetal trophoblastic tissue. This allows its study for prenatal diagnosis in the chorionic villous (CV), which was performed in a family with one deceased affected CMD1A child. Immunohistochemical analysis of the CV using antibodies against the C- and N-terminal domains of the α2-laminin protein showed a normal positive labeling for both antibodies in the “at-risk” CV, which did not differ from the normal control CV. The integrity of the CV membrane was confirmed through the analysis with antibodies against α1, β1, and γ1 laminins. DNA study using markers flanking the 6q2 region showed that the affected patient and the “at-risk” fetus did not share the same haplotype. Therefore, the fetus was considered normal through both methodologies, which was confirmed after the birth of a clinically normal male baby. As the LAMA2 gene is very large and the spectrum of mutations causing disease is wide, the analysis of the protein in muscle biopsy has been largely used for the diagnosis. Besides, the possibility to detect it in the chorionic villous, mainly using positive markers, also offers a powerful tool for prenatal diagnosis.

A.P.14: A new in/del in the critical splicing region of the VMA21 gene causing X-linked myopathy with excessive autophagy (XMEA)

X-linked myopathy with excessive autophagy (XMEA) is an inherited, slowly progressive myopathy, characterized by membrane-bound sarcoplasmic vacuoles in muscle fibers. Proximal muscle weakness in early childhood is observed, but with no cardiac, nor cognitive impairment. Recent findings identified mutations in the vacuolar membrane ATPase activity 21 (VMA21), as causative of XMEA. Among Six different single-nucleotide substitutions in VMA21 (in 14 XMEA families), four were intronic, and in two of them, the IVS1–27A base is involved. These mutations result in a reduction in VMA21 mRNA, and protein, and a consequent elevated lysosomal pH with partial block the final degradation stage of autophagy. Only a few XMEA families have been worldwide identified. Here we describe the first XMEA Brazilian family carrying a small in/del in the VMA21 gene. The 5-year-old propositus presented a characteristic dystrophic phenotype. He walked at the age of 2 and showed difficulties for running, climbing stairs, and raising from the floor. No calf hypertrophy nor joint contractures were observed. CK level was 1330 U/l, and ECG showed altered conduction in the right branch. Muscle biopsy showed a dystrophic pattern and autophagic vacuoles. Emerin was normal. Family history revealed a recessive X-linked inheritance, with 5 affected males linked through asymptomatic females. The affected maternal grandfather, aged 48, was wheelchair bound since the age of 30, presenting also cardiac alterations and joint contractures in the upper limbs. Exome sequencing identified a small insertion-deletion, including the IVS1-27A base previously described. This new family/mutation reinforces the importance of this splice site branchpoint for the appropriate transcription/translation of VMA21, and normal lysosome function. Additionally, it expands the clinical variability, including cardiac involvement and joint contractures to the XMEA phenotype.

Genetic variability in the myostatin gene does not explain the muscle hypertrophy and clinical penetrance in myotonia congenita

Myostatin (MSTN), a member of the transforming growth factor b (TGF-b) superfamily, is expressed mainly in developing and adult skeletal muscle. Mutations in the myostatin gene cause the ‘‘double-muscling’’ phenotype observed in the Belgian Blue and Piedmontese cattle breeds. Mice that lack myostatin show marked increase in muscle mass, and, in humans, a missplicing mutation in the myostatin gene was identified in a child with gross muscle hypertrophy. Based on these observations, myostatin was defined as a negative regulator of muscle growth. However, several polymorphisms in the myostatin gene with no known phenotypic sequelae have been identified. Muscle hypertrophy is also observed in some myotonic disorders, including Thomsen (autosomal dominant) and Becker (autosomal recessive) congenital myotonias. Both are caused by mutations in the chloride channel 1 (CLCN1) gene that codes for the muscle chloride channel. Myotonia congenita is characterized by delayed relaxation of skeletal muscle (myotonia), and the phenotypic spectrum ranges from mild myotonia disclosed only by clinical examination to severe and disabling myotonia with transient weakness and myopathy. There is a variable association with muscle hypertrophy. To date, mutations in the CLCN1 gene have been identified as the primary cause of Thomsen and Becker myotonias. However, it is generally not possible to predict if certain point mutations will cause a dominant or a recessive phenotype, since severity varies greatly between heterozygous family members and may even vary with time in individual patients. Additionally, reduced penetrance has also been reported in several dominant pedigrees of Thomsen myotonia where the phenotypic spectrum of heterozygotes varies from pronounced myotonia to normality. Based on these observations, an influence of other genetic factors has been suggested but not identified so far. One such possibility could be interaction between the CLCN1 gene and common variants of the MSTN gene, which might also explain the variable muscle hypertrophy observed in these myotonias. We looked for genetic variation in both MSTN and CNCL1 in 15 individuals belonging to 2 Brazilian families with genetic myotonias, including 5 affected individuals, 7 unaffected carriers, and 3 normal individuals. For the CLCN1 analysis, family 1 carried a novel splice-site mutation g.IVS18þ1G>T, and in family 2 there was a novel stop codon K248X mutation in exon 6 of the CLCN1 gene. Among the 5 affected patients, 4 were homozygous, and 1 was heterozygous for the CLCN1 mutation. All presented with muscle hypertrophy and weakness (Fig. 1). Analysis of the MSTN gene included screening for variants in all 3 exons through polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism analysis, and direct sequencing. We were unable to identify any variants in any of the studied individuals. Therefore, our results show that the hypermusculature associated with myotonia in these families could not be attributed to modulation of muscle growth by variation in myostatin expression. These results are in accordance with the finding of no differences of mRNA myostatin expression in children with primary muscle diseases, and prominent muscle atrophy or hypertrophy. We conclude that either mutations in the CLCN1 gene alone are sufficient to induce muscle hypertrophy in addition to myotonia, or an alternative gene interaction with CLCN1 other than MSTN has to be sought to explain the hypermusculature observed in the Thomsen and Becker myotonias.

P.4.13 Central core disease (CCD): Improving the screening for mutations in RYR1 gene

Central core disease (CCD) is a congenital myopathy, characterized by the presence of central core-like areas in muscle fibers. Patients with RYR-related CCD usually have mild or moderate axial and proximal weakness, hypotonia and motor developmental delay. CCD is associated with susceptibility to malignant hyperthermia (MH), and both conditions have been linked to mutations in human RYR1 gene, which encodes a calcium release channel known as ryanodine receptor (RyR1). RYR1 mutational spectrum linked with CCD includes more than 200 described mutations mostly heterozygous dominant missense mutations and minor deletions or duplications. Definite diagnosis of CCD is done by clinical history and evaluation, histopathological analyses of muscle biopsy and, recently, genetic testing of gene’s “hot spots”, because RYR1 is a large gene and direct sequencing of each exon is laborious. Here, we tested Multiplex Ligation-dependent Probe Amplification (MLPA) as an alternative and simple method for screening patients with histological diagnosis or family history of CCD. This technique uses high-performance PCR to quantify up to 45 transcripts in small human DNA samples. It is based on annealing of two hemi-probes into its complementary sequence and was designed to detect the 33 most common mutations in RYR1 gene. From 41 patients diagnosed with CCD in three different institutions, 24 (58,5%) had mutations found in direct sequencing and 11 (26,8%) in MLPA. Seventeen patients (41,5%) had consistent results: 6 (14,6%) had their mutation diagnosed with sequencing and MLPA and neither method was able to find mutations in 11 patients (26,8%). Mutations in exon 101 were the most prevalent in sequencing (11 patients) and MLPA (4 patients). MLPA had shown to be a relevant tool to help diagnosing CCD; moreover, it is especially useful in diagnosing family members of patients with known mutations detectable by MLPA. Financial support: FAPESP-CEPID, CNPQ-INCT, FINEP.

T.P.38 Effects of steroid hormones on myostatin expression and on genes of muscle regeneration pathway

Abstract Myostatin is an important negative regulator of skeletal muscle growth, while decanoato de nadrolone, an anabolic steroid, is a strong positive effector. Inhibition of myostatin has been tested as an approach for treatment neuromuscular diseases. In order to investigate the possible interaction between myostatin and anabolic steroids, as a therapeutic strategy, we studied myostatin expression in the quadriceps femoris of normal mice treated with Decadurabolin® (D), flutamide (F), an antagonist of the androgen receptor, and Decadurabolin administration, post flutamide treatment (FD), as compared to controls, treated with saline (S). We also studied the relative expression of the genes, myogenin, MyoD and Myf5, involved in the pathway of muscle regeneration. We observed significant increase in the body mass in the (D) and (FD) groups, and a decrease in the group (F), when compared to groups (S). Real-time PCR quantitative analysis for myostatin expression showed no statistically significant differences between the studied groups. On the other hand, the groups (D) and (FD) showed a significant decrease in the expression of myogenin, MyoD and Myf5, while animals of the group (F) showed a significant increase in the expression of these genes. We conclude that administration of anabolic steroid, or its inhibition did not alter the expression of the myostatin gene, despite the increase or decrease in the body mass observed in group (D), (FD) and (F). However, the blockade of androgen receptor by flutamide, clearly stimulate the regeneration cascade, by increasing the expression of genes related to proliferation (MyoD and Myf5) and cell differentiation (myogenin). Additional studies will elucidate the possible role of other pathways in this stimulus for regeneration. Financial support: FAPESP-CEPID, CNPq-INCT, FINEP, ABDIM.