Next-Generation Sequencing Revealed L-Carnitine Rescues Congenital Myopathy in Transgenic Zebrafish with Tropomyosin 3 Novel Mutation
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ABSTRACT:
PROVIDER: PRJNA627773 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11601820_1.fastq.gz | Fastqsanger.gz | |||
| SRR11601820_2.fastq.gz | Fastqsanger.gz | |||
| SRR11601821_1.fastq.gz | Fastqsanger.gz | |||
| SRR11601821_2.fastq.gz | Fastqsanger.gz | |||
| SRR11601822_1.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: Congenital myopathies (CM) are a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A novel and de novo mutation in tropomyosin 3 [TPM3(E151G)] was identified from a 7-year-old boy with clinically diagnosed CM. TPM3(E151A) mutation was previously reported as causing CM. In this study, we created transgenic zebrafish to investigate the pathogenicity and find potential treatment. The goals of this study are to describe the RNA contents in muscle specimens derived from TPM3(WT), TPM3(E151A), TPM3(E152G) at embryos and adult stages and to compare the differences TPM3(E151A), TPM3(E151G) mutants and TPM3(WT). The biological function of L-carnitine treatment TPM3(E151G) versus without treatment are also predicted and evaluated. Methods: Muscle mRNA profiles of 6 months adult zebrafish from TPM3(WT), TPM3(E151A) and TPM3(E151G) were extracted and performed deep sequencing, in duplicate, using Illumina Novaseq6000. We also obtained the muscle mRNA profiles of 2 day's zebrafish embryos from TPM3(WT), TPM3(E151A), TPM3(E151G) and TPM3(E151G) treated with L-carnitine. RNA profiles of adult and embryos were generated by deep sequencing, using Illumina NovaSeq 6000. Results: After acquiring of clean reads, we mapped about 40 million sequence reads per sample to the zebrafish genome (GRCz11). Saturation rate analysis was then performed evaluating the gene coverage rate along with read depth increase. The transcripts mapping to difference functional region of the genome was then analyzed. Circos software was used to map the consistency of samples with zebrafish genome on a chromosomal level. The quantification of transcript intensity was analyzed using Fragments Per Kilobase of exon modelper Million mapped fragments (FPKM). Conclusions: Our study represents the first detailed analysis of zebrafish muscle transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles between TPM3(WT), TPM3(E151A) and TPM3(E151G) and L-carnitine treatment. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
2020-04-24 | GSE149261 | GEO
Project description:Tropomyosin 3 variant causing adult onset distal myopathy
| PRJEB57384 | ENA
Project description:BackgroundCongenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown.MethodsHistopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated.ResultsWhile TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, L-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by L-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by L-carnitine treatment.ConclusionsThese results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.
| S-EPMC7802209 | biostudies-literature
Project description:The aim of this study was to investigate the molecular mechanisms implicated in this mouse model of nemaline myopathy, and to further compare the molecular disease response in different skeletal muscles. For this purpose, snap frozen skeletla muscle specimens from wild type and transgenic for alpha tropomyosin slow mice were studied. Five different muscle types were used (diaphragm, plantaris, extensor digitorum longus, tibialis anterior, gastrocnemus). Mice were sacrificed between 7 and 10 months. RNA pools from 3-5 animals were created and each pool was hybridized to a U74Av2 Affymetrix GeneChip. Datasets from 36 GeneChips were included in this study. Experiment Overall Design: 36 skeletal mouse muscle RNA pools were used, from 5 different skeletal muscles, in two different conditions (wild type and transgenic)
2007-12-01 | E-GEOD-3384 | biostudies-arrayexpress
Project description:The aim of this study was to investigate the molecular mechanisms implicated in this mouse model of nemaline myopathy, and to further compare the molecular disease response in different skeletal muscles. For this purpose, snap frozen skeletla muscle specimens from wild type and transgenic for alpha tropomyosin slow mice were studied. Five different muscle types were used (diaphragm, plantaris, extensor digitorum longus, tibialis anterior, gastrocnemus). Mice were sacrificed between 7 and 10 months. RNA pools from 3-5 animals were created and each pool was hybridized to a U74Av2 Affymetrix GeneChip. Datasets from 36 GeneChips were included in this study. Keywords: disease mouse model analysis
2006-07-21 | GSE3384 | GEO
Project description:rs07-01_carnitine - carnitine effect - Effect of carnitine on the transcriptome of A. thaliana - Our objective is the description of the existence of an intracellular transport depend on the carnitine at Arabidopsis thaliana. For that, we planned the study of the response of the transcriptome in the presence of exogenic L-carnitine. Seedlings WT are cultivated on medium MS, 6 independent times. At 144h, the seedlings are collected and pools are formed with 3 samples. We proceed in the same way for the seedlings which are cultivated on medium MS containing L-carnitine 5mM. Keywords: treated vs untreated comparison
2008-03-05 | GSE10676 | GEO
Project description:A novel variant in the tropomyosin 3 gene presenting as an adult-onset distal myopathy - a case report
| PRJEB56864 | ENA
Project description:Molecular characterization of collagen-VI related myopathies currently relies on standard sequencing, which yields a detection rate approximating 75-79% in Ullrich congenital muscular dystrophy (UCMD) and 60-65% in Bethlem myopathy (BM) patients as PCR-based techniques tend to miss gross genomic rearrangements as well as copy number variations (CNVs) in both the coding sequence and intronic regions. We have designed a custom oligonucleotide CGH array in order to investigate the presence of CNVs in the coding and non-coding regions of COL6A1, A2, A3, A5 and A6 genes and a group of genes functionally related to collagen VI. A cohort of 12 patients with UCMD/BM negative at sequencing analysis and 2 subjects carrying a single COL6 mutation whose clinical phenotype was not explicable by inheritance were selected and the occurrence of allelic and genetic heterogeneity explored. A deletion within intron 1A of the COL6A2 gene, occurring in compound heterozygosity with a small deletion in exon 28, previously detected by routine sequencing, was identified in a BM patient. RNA studies showed monoallelic transcription of the COL6A2 gene, thus elucidating the functional effect of the intronic deletion. No pathogenic mutations were identified in the remaining analyzed patients, either within COL6A genes, or in genes functionally related to collagen VI. Our custom CGH array may represent a useful complementary diagnostic tool, especially in recessive forms of the disease, when only one mutant allele is detected by standard sequencing. The intronic deletion we identified represents the first example of a pure intronic mutation in COL6A genes. Comparative Genomic Hybridization experiment. Experimental Replicates: twice
2010-01-27 | E-GEOD-20025 | biostudies-arrayexpress
Project description:Microarray analysis of the effect of L-carnitine supplementation on global expression of Sachharomyces cerevisiae cultures in logarithmic growing conditions and after exposure to H2O2 induced oxidative stress; L-Carnitine plays a well documented role in eukaryotic energy homeostasis by acting as a shuttling molecule for activated acyl residues across intracellular membranes. This activity is supported by carnitine acyl-transferases and transporters, and is referred to as the carnitine shuttle. However, several pleiotropic and often beneficial effects of carnitine in humans have been reported that appear to be unrelated to the shuttling activity, but little conclusive evidence regarding the molecular networks that would be affected by carnitine exist. We have recently demonstrated a protective role of carnitine in oxidative stress in yeast that is independent of the carnitine shuttle. A DNA microarray-based global gene expression analysis identified Cyc3p, a cytochrome c heme lyase, as being important for carnitine's protective impact in oxidative stress conditions. These findings establish a direct genetic link to a carnitine-related phenotype that is independent of the shuttle system. The data suggest that the yeast Saccharomyces cerevisiae should provide a useful model for further elucidation of carnitine's physiological roles. Experiment Overall Design: Yeast cultures was grown to mid-log phase with and without carnitine supplementation to a final concentration of 100 mg/L. A second set was grown to mid log phase, with and without carnitine supplementation and exposed to 0.4 mM H2O2 for 30 min. The experiments were performed using biological duplicate.
2009-07-04 | E-GEOD-16346 | biostudies-arrayexpress
Project description:Carnitine is a water soluble quaternary amine which is essential for normal function of all tissues. Besides improving performance characteristics of livestock animals carnitine has attracted scientific interest due to several health-related effects, like protection against neurodegeneration, mitochondrial decay, and oxidative stress, improvement of glucose tolerance and insulin sensitivity as well as reduction of hepatic steatosis. To gain insight into mechanisms through which carnitine exerts its beneficial metabolic effects, we fed piglets either a control or a carnitine supplemented diet, and analyzed the transcriptome in the liver, which plays a central role in whole body metabolism. Total RNA was isolated from pig liver samples for hybridization on Affymetrix GeneChip porcine genome arrays. Two RNA pools, each for the control group and the carnitine group, were used. Each RNA pool comprised RNA from 4 pigs.
2010-07-15 | E-GEOD-22931 | biostudies-arrayexpress