Nemaline Myopathy in Brazilian Patients: Molecular and Clinical Characterization.

Nemaline myopathy (NM), a structural congenital myopathy, presents a significant clinical and genetic heterogeneity. Here, we compiled molecular and clinical data of 30 Brazilian patients from 25 unrelated families. Next-generation sequencing was able to genetically classify all patients: sixteen families (64%) with mutation in NEB, five (20%) in ACTA1, two (8%) in KLHL40, and one in TPM2 (4%) and TPM3 (4%). In the NEB-related families, 25 different variants, 11 of them novel, were identified; splice site (10/25) and frame shift (9/25) mutations were the most common. Mutation c.24579 G>C was recurrent in three unrelated patients from the same region, suggesting a common ancestor. Clinically, the "typical" form was the more frequent and caused by mutations in the different NM genes. Phenotypic heterogeneity was observed among patients with mutations in the same gene. Respiratory involvement was very common and often out of proportion with limb weakness. Muscle MRI patterns showed variability within the forms and genes, which was related to the severity of the weakness. Considering the high frequency of NEB mutations and the complexity of this gene, NGS tools should be combined with CNV identification, especially in patients with a likely non-identified second mutation.

Newborn Screening for 5q Spinal Muscular Atrophy: Comparisons between Real-Time PCR Methodologies and Cost Estimations for Future Implementation Programs.

Since the approval of modifying therapies for Spinal Muscular Atrophy (SMA), several protocols aiming to screen SMN1 homozygous deletion in a neonatal context have been published. However, no work has compared different methodologies along with detailed implementation costs for centers where the neonatal screening of SMA has not yet been implemented. Therefore, our work compared different qualitative real-time PCR approaches for SMA screening and the estimated costs of test implementation. Using Brazilian blood samples, the presence and absence (P/A) and melt curve protocols were analyzed. MLPA was used as a confirmatory test. The costs were calculated for the simplex and multiplex tests plus equipment. The test workflow was based on the present experience and literature report. The accuracy of the P/A protocol was 1 (95% CI 0.8677-1) using dried blood spots (DBS). The melt curve protocol also achieved 100% concordance. The consumable costs ranged from USD 1.68 to 4.42 and from USD 2.04 to 12.76 per reaction, for the simplex and multiplex tests, respectively. The equipment acquisition costs ranged from USD 44,817.07 to 467,253.10, with several factors influencing this value presented. Our work presents a framework for decision-making, with a project demonstration of the different assays that will be useful in dealing with the issues of cost and availability of reagents. Moreover, we present a literature review and discussion of important concerns regarding treatment policies. We take the first step towards a future SMA NBS pilot program where it is not yet a reality.

Association of Three Different Mutations in the CLCN1 Gene Modulating the Phenotype in a Consanguineous Family with Myotonia Congenita.

Myotonia congenita is a genetic disease caused by mutations in the CLCN1 gene, which encodes for the major chloride skeletal channel ClC-1, involved in the normal repolarization of muscle action potentials and consequent relaxation of the muscle after contraction. Two allelic forms are recognized, depending on the phenotype and the inheritance pattern: the autosomal dominant Thomsen disease with milder symptoms and the autosomal recessive Becker disorder with a severe phenotype. Before the recent advances of molecular testing, the diagnosis and genetic counseling of families was a challenge due to the large number of mutations in the CLCN1 gene, found both in homozygous or in heterozygous state. Here, we studied a consanguineous family in which three members presented a variable phenotype of myotonia, associated to a combination of three different mutations in the CLCN1 gene. A pathogenic splicing site mutation which causes the skipping of exon 17 was present in homozygosis in one very severely affected son. This mutation was present in compound heterozygosis in the consanguineous parents, but interestingly it was associated to a different second variant in the other allele: c.1453 A > G in the mother and c.1842 G > C in the father. Both displayed variable, but less severe phenotypes than their homozygous son. These results highlight the importance of analyzing the combination of different variants in the same gene in particular in families with patients displaying different phenotypes. This approach may improve the diagnosis, prognosis, and genetic counseling of the involved families.

Manifesting carriers of X-linked myotubular myopathy: Genetic modifiers modulating the phenotype.

OBJECTIVE: To analyze the modulation of the phenotype in manifesting carriers of recessive X-linked myotubular myopathy (XLMTM), searching for possible genetic modifiers. METHODS: Twelve Brazilian families with XLMTM were molecularly and clinically evaluated. In 2 families, 4 of 6 and 2 of 5 manifesting female carriers were identified. These females were studied for X chromosome inactivation. In addition, whole-exome sequencing was performed, looking for possible modifier variants. We also determined the penetrance rate among carriers of the mutations responsible for the condition. RESULTS: Mutations in the MTM1 gene were identified in all index patients from the 12 families, being 4 of them novel. In the heterozygotes, X chromosome inactivation was random in 3 of 4 informative manifesting carriers. The disease penetrance rate was estimated to be 30%, compatible with incomplete penetrance. Exome comparative analyses identified variants within a segment of 4.2 Mb on chromosome 19, containing the killer cell immunoglobulin-like receptor cluster of genes that were present in all nonmanifesting carriers and absent in all manifesting carriers. We hypothesized that these killer cell immunoglobulin-like receptor variants may modulate the phenotype, acting as a protective factor in the nonmanifesting carriers. CONCLUSIONS: Affected XLMTM female carriers have been described with a surprisingly high frequency for a recessive X-linked disease, raising the question about the pattern of inheritance or the role of modifier factors acting on the disease phenotype. We demonstrated the possible existence of genetic mechanisms and variants accountable for the clinical manifestation in these women, which can become future targets for therapies.

Altered in vitro muscle differentiation in X-linked myopathy with excessive autophagy.

X-linked myopathy with excessive autophagy (XMEA) is a genetic disease associated with weakness of the proximal muscles. It is caused by mutations in the VMA21 gene, coding for a chaperone that functions in the vacuolar ATPase (v-ATPase) assembly. Mutations associated with lower content of assembled v-ATPases lead to an increase in lysosomal pH, culminating in partial blockage of macroautophagy, with accumulation of vacuoles of undigested content. Here, we studied a 5-year-old boy affected by XMEA, caused by a small indel in the VMA21 gene. Detection of sarcoplasmic Lc3 (also known as MAP1LC3B)-positive vacuoles in his muscle biopsy confirmed an autophagy defect. To understand how autophagy is regulated in XMEA myogenesis, we used patient-derived muscle cells to evaluate autophagy during in vitro muscle differentiation. An increase in lysosomal pH was observed in the patient's cells, compatible with predicted functional defect of his mutation. Additionally, there was an increase in autophagic flux in XMEA myotubes. Interestingly, we observed that differentiation of XMEA myoblasts was altered, with increased myotube formation observed through a higher fusion index, which was not dependent on lysosomal acidification. Moreover, no variation in the expression of myogenic factors nor the presence of regenerating fibers in the patient's muscle were observed. Myoblast fusion is a tightly regulated process; therefore, the uncontrolled fusion of XMEA myoblasts might generate cells that are not as functional as normal muscle cells. Our data provide new evidence on the reason for predominant muscle involvement in the context of the XMEA phenotype.This article has an associated First Person interview with the first author of the paper.

Dominant or recessive mutations in the RYR1 gene causing central core myopathy in Brazilian patients.

Central Core Disease (CCD) is an inherited neuromuscular disorder characterized by the presence of cores in muscle biopsy. CCD is caused by mutations in the RYR1 gene. This gene encodes the ryanodine receptor 1, which is an intracellular calcium release channel from the sarcoplasmic reticulum to the cytosol in response to depolarization of the plasma membrane. Mutations in this gene are also associated with susceptibility to Malignant Hyperthermia (MHS). In this study, we evaluated 20 families with clinical and histological characteristics of CCD to identify primary mutations in patients, for diagnosis and genetic counseling of the families. We identified variants in the RYR1 gene in 19/20 families. The molecular pathogenicity was confirmed in 16 of them. Most of these variants (22/23) are missense and unique in the families. Two variants were recurrent in two different families. We identified six families with biallelic mutations, five compound heterozygotes with no consanguinity, and one homozygous, with consanguineous parents, resulting in 30% of cases with possible autosomal recessive inheritance. We identified seven novel variants, four of them classified as pathogenic. In one family, we identified two mutations in exon 102, segregating in cis, suggesting an additive effect of two mutations in the same allele. This work highlights the importance of using Next-Generation Sequencing technology for the molecular diagnosis of genetic diseases when a very large gene is involved, associated to a broad distribution of the mutations along it. These data also influence the prevention through adequate genetic counseling for the families and cautions against malignant hyperthermia susceptibility.

Development of a comprehensive noninvasive prenatal test.

Our aim was to develop and apply a comprehensive noninvasive prenatal test (NIPT) by using high-coverage targeted next-generation sequencing to estimate fetal fraction, determine fetal sex, and detect trisomy and monogenic disease without parental genotype information. We analyzed 45 pregnancies, 40 mock samples, and eight mother-child pairs to generate 35 simulated datasets. Fetal fraction (FF) was estimated based on analysis of the single nucleotide polymorphism (SNP) allele fraction distribution. A Z-score was calculated for trisomy of chromosome 21 (T21), and fetal sex detection. Monogenic disease detection was performed through variant analysis. Model validation was performed using the simulated datasets. The novel model to estimate FF was robust and accurate (r2= 0.994, p-value < 2.2e-16). For samples with FF > 0.04, T21 detection had 100% sensitivity (95% CI: 63.06 to 100%) and 98.53% specificity (95% CI: 92.08 to 99.96%). Fetal sex was determined with 100% accuracy. We later performed a proof of concept for monogenic disease diagnosis of 5/7 skeletal dysplasia cases. In conclusion, it is feasible to perform a comprehensive NIPT by using only data from high coverage targeted sequencing, which, in addition to detecting trisomies, also make it possible to identify pathogenic variants of the candidate genes for monogenic diseases.

Complexity of the 5' Untranslated Region of EIF4A3, a Critical Factor for Craniofacial and Neural Development.

Repeats in coding and non-coding regions have increasingly been associated with many human genetic disorders, such as Richieri-Costa-Pereira syndrome (RCPS). RCPS, mostly characterized by midline cleft mandible, Robin sequence and limb defects, is an autosomal-recessive acrofacial dysostosis mainly reported in Brazilian patients. This disorder is caused by decreased levels of EIF4A3, mostly due to an increased number of repeats at the EIF4A3 5'UTR. EIF4A3 5'UTR alleles are CG-rich and vary in size and organization of three types of motifs. An exclusive allelic pattern was identified among affected individuals, in which the CGCA-motif is the most prevalent, herein referred as "disease-associated CGCA-20nt motif." The origin of the pathogenic alleles containing the disease-associated motif, as well as the functional effects of the 5'UTR motifs on EIF4A3 expression, to date, are entirely unknown. Here, we characterized 43 different EIF4A3 5'UTR alleles in a cohort of 380 unaffected individuals. We identified eight heterozygous unaffected individuals harboring the disease-associated CGCA-20nt motif and our haplotype analyses indicate that there are more than one haplotype associated with RCPS. The combined analysis of number, motif organization and haplotypic diversity, as well as the observation of two apparently distinct haplotypes associated with the disease-associated CGCA-20nt motif, suggest that the RCPS alleles might have arisen from independent unequal crossing-over events between ancient alleles at least twice. Moreover, we have shown that the number and sequence of motifs in the 5'UTR region is associated with EIF4A3 repression, which is not mediated by CpG methylation. In conclusion, this study has shown that the large number of repeats in EIF4A3 does not represent a dynamic mutation and RCPS can arise in any population harboring alleles with the CGCA-20nt motif. We also provided further evidence that EIF4A3 5'UTR is a regulatory region and the size and sequence type of the repeats at 5'UTR may contribute to clinical variability in RCPS.

Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers.

Exon skipping uses chemically modified antisense oligonucleotides to modulate RNA splicing. Therapeutically, exon skipping can bypass mutations and restore reading frame disruption by generating internally truncated, functional proteins to rescue the loss of native gene expression. Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the SGCG gene, which encodes the dystrophin-associated protein γ-sarcoglycan. The most common SGCG mutations disrupt the transcript reading frame abrogating γ-sarcoglycan protein expression. In order to treat most SGCG gene mutations, it is necessary to skip 4 exons in order to restore the SGCG transcript reading frame, creating an internally truncated protein referred to as Mini-Gamma. Using direct reprogramming of human cells with MyoD, myogenic cells were tested with 2 antisense oligonucleotide chemistries, 2'-O-methyl phosphorothioate oligonucleotides and vivo-phosphorodiamidate morpholino oligomers, to induce exon skipping. Treatment with vivo-phosphorodiamidate morpholino oligomers demonstrated efficient skipping of the targeted exons and corrected the mutant reading frame, resulting in the expression of a functional Mini-Gamma protein. Antisense-induced exon skipping of SGCG occurred in normal cells and those with multiple distinct SGCG mutations, including the most common 521ΔT mutation. These findings demonstrate a multiexon-skipping strategy applicable to the majority of limb-girdle muscular dystrophy 2C patients.

Concordant utrophin upregulation in phenotypically discordant DMD/BMD brothers.

Utrophin expression was investigated in two phenotypically discordant Duchenne muscular dystrophy half-brothers. The youngest was wheelchair-bound at age 9, while his mildly affected older brother was able to walk without difficulties at age 15. DNA analysis revealed an out-of-frame exon 2 duplication in the DMD gene, associated with muscle dystrophin protein deficiency. Utrophin localization and quantity was analyzed and compared in both sibs to verify whether this could explain the milder phenotype of the older brother. Immunofluorescence analysis showed a clear sarcolemmal labeling for utrophin in both of them, which was present in regenerating as well as in mature fibers. On western blot analysis, utrophin amount was increased 3.4 and 3.3 fold respectively, as compared to normal controls, while it was increased 1.7 to 4.0 fold in a group of DMD patients within the typical range of clinical progression. These data are in accordance with our previous observations suggesting no correlation between phenotype severity and utrophin up-regulation or sarcolemmal localization in dystrophinopathies. Finding the protective mechanisms in patients with milder course is of utmost interest to direct therapeutic targets.

Silent polymorphisms in the RYR1 gene do not modify the phenotype of the p.4898 I>T pathogenic mutation in central core disease: a case report.

BACKGROUND: Central core disease is a congenital myopathy, characterized by presence of central core-like areas in muscle fibers. Patients have mild or moderate weakness, hypotonia and motor developmental delay. The disease is caused by mutations in the human ryanodine receptor gene (RYR1), which encodes a calcium-release channel. Since the RYR1 gene is huge, containing 106 exons, mutation screening has been limited to three 'hot spots', with particular attention to the C-terminal region. Recent next-generation sequencing methods are now identifying multiple numbers of variants in patients, in which interpretation and phenotype prevision is difficult. CASE PRESENTATION: In a Brazilian Caucasian family, clinical, histopathological and molecular analysis identified a new case of central core disease in a 48-year female. Sanger sequencing of the C-terminal region of the RYR1 gene identified two different missense mutations: c.14256 A > C polymorphism in exon 98 and c.14693 T > C in exon 102, which have already been described as pathogenic. Trans-position of the 2 mutations was confirmed because patient's daughter, mother and sister carried only the exon 98's mutation, a synonymous variant that was subsequently found in the frequency of 013-0,05 of alleles. Further next generation sequencing study of the whole RYR1 gene in the patient revealed the presence of additional 5 common silent polymorphisms in homozygosis and 8 polymorphisms in heterozygosis. CONCLUSIONS: Considering that patient's relatives showed no pathologic phenotype, and the phenotype presented by the patient is within the range observed in other central core disease patients with the same mutation, it was concluded that the c.14256 A > C polymorphism alone is not responsible for disease, and the associated additional silent polymorphisms are not acting as modifiers of the primary pathogenic mutation in the affected patient. The case described above illustrates the present reality where new methods for wide genome screening are becoming more accessible and able to identify a great variety of mutations and polymorphisms of unknown function in patients and their families.

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.

Reviewing Large LAMA2 Deletions and Duplications in Congenital Muscular Dystrophy Patients.

BACKGROUND: Congenital muscular dystrophy (CMD) type 1A (MDC1A) is caused by recessive mutations in laminin-α2 (LAMA2) gene. Laminin-211, a heterotrimeric glycoprotein that contains the α2 chain, is crucial for muscle stability establishing a bond between the sarcolemma and the extracellular matrix. More than 215 mutations are listed in the locus specific database (LSDB) for LAMA2 gene (May 2014). OBJECTIVE: A limited number of large deletions/duplications have been reported in LAMA2. Our main objective was the identification of additional large rearrangements in LAMA2 found in CMD patients and a systematic review of cases in the literature and LSDB. METHODS: In four of the fifty-two patients studied over the last 10 years, only one heterozygous mutation was identified, after sequencing and screening for a frequent LAMA2 deletion. Initial screening of large mutations was performed by multiplex ligation-dependent probe application (MLPA). Further characterization implied several techniques: long-range PCR, cDNA and Southern-blot analysis. RESULTS: Three novel large deletions in LAMA2 and the first pathogenic large duplication were successfully identified, allowing a definitive molecular diagnosis, carrier screening and prenatal diagnosis. A total of fifteen deletions and two duplications previously reported were also reviewed. Two possible mutational "hotspots" for deletions may exist, the first encompassing exons 3 and 4 and second in the 3' region (exons 56 to 65) of LAMA2. CONCLUSIONS: Our findings show that this type of mutation is fairly frequent (18.4% of mutated alleles) and is underestimated in the literature. It is important to include the screening of large deletions/duplications as part of the genetic diagnosis strategy.

Assessing pathogenicity for novel mutation/sequence variants: the value of healthy older individuals.

Improvement in DNA technology is increasingly revealing unexpected/unknown mutations in healthy persons and generating anxiety due to their still unknown health consequences. We report a 44-year-old healthy father of a 10-year-old daughter with bilateral coloboma and hearing loss, but without muscle weakness, in whom a whole-genome CGH revealed a deletion of exons 38-44 in the dystrophin gene. This mutation was inherited from her asymptomatic father, who was further clinically and molecularly evaluated for prognosis and genetic counseling (GC). This deletion was never identified by us in 982 Duchenne/Becker patients. To assess whether the present case represents a rare case of non-penetrance, and aiming to obtain more information for prognosis and GC, we suggested that healthy older relatives submit their DNA for analysis, to which several complied. Mutation analysis revealed that his mother, brother, and 56-year-old maternal uncle also carry the 38-44 deletion, suggesting it an unlikely cause of muscle weakness. Genome sequencing will disclose mutations and variants whose health impact are still unknown, raising important problems in interpreting results, defining prognosis, and discussing GC. We suggest that, in addition to family history, keeping the DNA of older relatives could be very informative, in particular for those interested in having their genome sequenced.

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.

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.