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

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

Reviewing Large LAMA2 Deletions and Duplications in Congenital MuscularDystrophy 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.The authors would like to thank all referring clini-cians.UMIB is funded by National Funds through FCT-Foundation for Science and Technology, under thePest-OE/SAU/U10215/2014.MV is founded by FAPESP-CEPID, and CNPq-INCT

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

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

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety ‘Mode of Action’ framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/ mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety ‘Mode of Action’ framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

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.Univ São Paulo, Dept Genet, Human Genome Res Ctr, IB, BR-05508900 São Paulo, BrazilSarah Network Rehabil Hosp, Dept Pathol, Dept Genet, Belo Horizonte, MG, BrazilSarah Network Rehabil Hosp, Neurophysiol Clin, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Pathol, FMUSP, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Anesthesiol, São Paulo, BrazilUniv Fed Minas Gerais, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Pathol, FMUSP, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Anesthesiol, São Paulo, BrazilWeb of Scienc

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

Abstract 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

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

Abstract 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