Browsing by Author "Yuo, Chung-Yee"

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Comprehensive target capture/next-generation sequencing as a second-tier diagnostic approach for congenital muscular dystrophy in Taiwan
(2017-02-09)
Liang, Wen-Chen
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Tian, Xia
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Yuo, Chung-Yee
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Chen, Wan-Zi
;
Kan, Tsu-Min
Purpose Congenital muscular dystrophy (CMD) is a heterogeneous disease entity. The detailed clinical manifestation and causative gene for each subgroup of CMD are quite variable. This study aims to analyze the phenotypes and genotypes of Taiwanese patients with CMD as the epidemiology of CMD varies among populations and has been scantly described in Asia. Methods A total of 48 patients suspected to have CMD were screened and categorized by histochemistry and immunohistochemistry studies. Different genetic analyses, including next-generation sequencing (NGS), were selected, based on the clinical and pathological findings. Results We identified 17 patients with sarcolemma-specific collagen VI deficiency (SSCD), 6 patients with merosin deficiency, two with reduced alpha-dystroglycan staining, and two with striking lymphocyte infiltration in addition to dystrophic change on muscle pathology. Fourteen in 15 patients with SSCD, were shown to have COL6A1, COL6A2 or COL6A3 mutations by NGS analysis; all showed marked distal hyperlaxity and normal intelligence but the overall severity was less than in previously reported patients from other populations. All six patients with merosin deficiency had mutations in LAMA2. They showed relatively uniform phenotype that were compatible with previous studies, except for higher proportion of mental retardation with epilepsy. With reduced alpha-dystroglycan staining, one patient was found to carry mutations in POMT1 while another patient carried mutations in TRAPPC11. LMNA mutations were found in the two patients with inflammatory change on muscle pathology. They were clinically characterized by neck flexion limitation and early joint contracture, but no cardiac problem had developed yet. Conclusion Muscle pathology remains helpful in guiding further molecular analyses by direct sequencing of certain genes or by target capture/NGS as a second-tier diagnostic tool, and is crucial for establishing the genotype-phenotype correlation. We also determined the frequencies of the different types of CMD in our cohort which is important for the development of a specific care system for each disease.
High Expression Level of Tra2-beta 1 Is Responsible for Increased SMN2 Exon 7 Inclusion in the Testis of SMA Mice
(2015-03-17)
Chen, Yu-Chia
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Chang, Jan-Gowth
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Jong, Yuh-Jyh
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Liu, Ting-Yuan
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Yuo, Chung-Yee
Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn(-/-) SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-beta 1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-beta 1 among the many tissues examined. Furthermore, overexpression of Tra2-beta 1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-beta 1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-beta 1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-beta 1 may be a modifying factor of SMA disease and a potential target for SMA treatment.
Securinine enhances SMN2 exon 7 inclusion in spinal muscular atrophy cells
(2017-04)
Chen, Yu-Chia
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Chang, Jan-Gowth
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Liu, Ting-Yuan
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Jong, Yuh-Jyh
;
Cheng, Wei-Lin
Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. SMA is caused by deletions or mutations in the survival motor neuron gene (SMN1) on chromosome 5q13. A second copy of the SMN gene (SMN2) also exists on chromosome 5, and both genes can produce functional protein. However, due to alternative splicing of the exon 7, the majority of SMN protein produced by SMN2 is truncated and unable to compensate for the loss of SMN1. Increasing full-length SMN protein production by promoting the exon 7 inclusion in SMN2 mRNA or increasing SMN2 gene transcription could be a therapeutic approach for SMA. In this study, we screened for the compounds that enhance SMN2 exon 7 inclusion by using SMN2 minigene-luciferase reporter system. We found that securinine can increase luciferase activity, indicating that securinine promoted SMN2 exon 7 inclusion. In addition, securinine increased full-length SMN2 mRNA and SMN protein expression in SMA patient-derived lymphoid cell lines. To investigate the mechanism of securinine effect on SMN2 splicing, we compared the protein levels of relevant splicing factors between securinine-treated and untreated cells. We found that securinine downregulated hnRNP A1 and Sam68 and upregulated Tra2-b1 expression. However, securinine, unlike HDAC inhibitors, did not enhance tra2-b1 gene transcription, indicating a post-transcriptional mechanism for Tra2-b1 upregulation. Furthermore, we treated SMA-like mice with securinine by i.p. injection and found that securinine treatment increased SMN2 exon 7 inclusion and SMN protein expression in the brain and spinal cord. According to our results, securinine might have the potential to become a therapeutic drug for SMA disease. (C) 2017 Elsevier Masson SAS. All rights reserved.
Transcribed pseudogene psi PPM1K generates endogenous siRNA to suppress oncogenic cell growth in hepatocellular carcinoma
(2013-04-01)
Chan, Wen-Ling
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Yuo, Chung-Yee
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Yang, Wen-Kuang
;
Hung, Shih-Ya
;
Chang, Ya-Sian
Pseudogenes, especially those that are transcribed, may not be mere genomic fossils, but their biological significance remains unclear. Postulating that in the human genome, as in animal models, pseudogenes may function as gene regulators through generation of endo-siRNAs (esiRNAs), antisense RNAs or RNA decoys, we performed bioinformatic and subsequent experimental tests to explore esiRNA-mediated mechanisms of pseudogene involvement in oncogenesis. A genome-wide survey revealed a partial retrotranscript pseudogene psi PPM1K containing inverted repeats capable of folding into hairpin structures that can be processed into two esiRNAs; these esiRNAs potentially target many cellular genes, including NEK8. In 41 paired surgical specimens, we found significantly reduced expression of two predicted psi PPM1K-specific esiRNAs, and the cognate gene PPM1K, in hepatocellular carcinoma compared with matched non-tumour tissues, whereas the expression of target gene NEK8 was increased in tumours. Additionally, NEK8 and PPM1K were downregulated in stably transfected psi PPM1K-overexpressing cells, but not in cells transfected with an esiRNA1-deletion mutant of psi PPM1K. Furthermore, expression of NEK8 in psi PPM1K-transfected cells demonstrated that NEK8 can counteract the growth inhibitory effects of psi PPM1K. These findings indicate that a transcribed pseudogene can exert tumour-suppressor activity independent of its parental gene by generation of esiRNAs that regulate human cell growth.