Use of RNA‑sequencing to detect abnormal transcription of the collagen α‑2 (VI) chain gene that can lead to Bethlem myopathy

Zhong,Jingzi; Xie,Yanshu; Dang,Yiwu; Zhang,Jiapeng; Song,Yingru; Lan,Dan

doi:10.3892/ijmm.2021.4861

Use of RNA‑sequencing to detect abnormal transcription of the collagen α‑2 (VI) chain gene that can lead to Bethlem myopathy

Authors:
- Jingzi Zhong
- Yanshu Xie
- Yiwu Dang
- Jiapeng Zhang
- Yingru Song
- Dan Lan
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Affiliations: Department of Pediatrics, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China, Department of Pathology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China, Department of Radiology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China
Published online on: January 28, 2021 https://doi.org/10.3892/ijmm.2021.4861
Article Number: 28
Copyright: © Zhong et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Bethlem myopathy (BM) is an autosomal dominant or autosomal recessive disorder and is usually associated with mutations in the collagen VI genes. In the present study, the pathogenicity of a novel splice‑site mutation was explored using RNA‑sequencing in a family with suspected BM, and a myopathy panel was performed in the proband. The genetic status of all family members was confirmed using Sanger sequencing. Clinical data and magnetic resonance imaging (MRI) features were also documented. In silico analysis was performed to predict the effects of the splice mutation. RNA‑sequencing and reverse transcription (RT)‑PCR were used to assess aberrant splicing. Immunocytochemistry was conducted to measure collagen VI protein levels within the gastrocnemius and in cultured skin fibroblasts. The results revealed that three patients in the family shared a similar classic BM presentation. MRI revealed distinct patterns of fatty infiltration in the lower extremities. A novel splicing mutation c.736‑1G>C in the collagen α‑2 (VI) chain (COL6A2) gene was found in all three patients. In silico analysis predicted that the mutation would destroy the normal splice acceptor site. RNA‑sequencing detected two abnormal splicing variants adjacent to the mutation site, and RT‑PCR confirmed the RNA‑sequencing findings. Furthermore, a defect in the collagen protein within cultured fibroblasts was detected using immunocytochemistry. The mutation c.736‑1G>C in the COL6A2 gene caused aberrant splicing and led to premature termination of protein translation. In conclusion, these findings may improve our knowledge of mutations of the COL6A2 gene associated with BM and demonstrated that RNA‑sequencing can be a powerful tool for finding the underlying mechanism of a disease‑causing mutations at a splice site.

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1	Cescon M, Gattazzo F, Chen PW and Bonaldo P: Collagen VI at a glance. J Cell Sci. 128:3525–3531. 2015. View Article : Google Scholar : PubMed/NCBI
2	Bushby KMD, Collins J and Hicks D: Collagen type VI myopathies. Adv Exp Med Biol. 802:185–199. 2014. View Article : Google Scholar : PubMed/NCBI
3	Kim SY, Kim WJ, Kim H, Choi SA, Lee JS, Cho A, Jang SS, Lim BC, Kim KJ, Kim JI, et al: Collagen VI-related myopathy: Expanding the clinical and genetic spectrum. Muscle Nerve. 58:381–388. 2018. View Article : Google Scholar : PubMed/NCBI
4	Foley AR, Hu Y, Zou Y, Columbus A, Shoffner J, Dunn DM, Weiss RB and Bönnemann CG: Autosomal recessive inheritance of classic Bethlem myopathy. Neuromuscular Disord. 19:813–817. 2009. View Article : Google Scholar
5	Gualandi F, Urciuolo A, Martoni E, Sabatelli P, Squarzoni S, Bovolenta M, Messina S, Mercuri E, Franchella A, Ferlini A, et al: Autosomal recessive Bethlem myopathy. Neurology. 73:1883–1891. 2009. View Article : Google Scholar : PubMed/NCBI
6	Norwood FLM, Harling C, Chinnery PF, Eagle M, Bushby K and Straub V: Prevalence of genetic muscle disease in Northern England: In-depth analysis of a muscle clinic population. Brain. 132:3175–3186. 2009. View Article : Google Scholar : PubMed/NCBI
7	Lee JH, Shin HY, Park HJ, Kim SH, Kim SM and Choi YC: Clinical, pathologic, and genetic features of collagen VI-related myopathy in Korea. J Clin Neurol. 13:331–339. 2017. View Article : Google Scholar : PubMed/NCBI
8	Briñas L, Richard P, Quijano-Roy S, Gartioux C, Ledeuil C, Lacène E, Makri S, Ferreiro A, Maugenre S, Topaloglu H, et al: Early onset collagen VI myopathies: Genetic and clinical correlations. Ann Neurol. 68:511–520. 2010. View Article : Google Scholar : PubMed/NCBI
9	Burset M, Seledtsov IA and Solovyev VV: Analysis of canonical and non-canonical splice sites in mammalian genomes. Nucleic Acids Res. 28:4364–4375. 2000. View Article : Google Scholar : PubMed/NCBI
10	Ohno K, Takeda J and Masuda A: Rules and tools to predict the splicing effects of exonic and intronic mutations. Wiley Interdiscip Rev RNA. 9:2018. View Article : Google Scholar
11	Demir K, Kattan WE, Zou M, Durmaz E, BinEssa H, Nalbantoğlu Ö, Al-Rijjal RA, Meyer B, Özkan B and Shi Y: Novel CYP27B1 gene mutations in patients with vitamin D-dependent rickets type 1A. PLoS One. 10:e01313762015. View Article : Google Scholar : PubMed/NCBI
12	Ozsolak F and Milos PM: RNA sequencing: Advances, challenges and opportunities. Nat Rev Genet. 12:87–98. 2011. View Article : Google Scholar :
13	Chen R, Im H and Snyder M: Whole-exome enrichment with the agilent sureselect human all exon platform. Cold Spring Harb Protoc. 2015:626–633. 2015.PubMed/NCBI
14	Li H and Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 25:1754–1760. 2009. View Article : Google Scholar : PubMed/NCBI
15	Martoni E, Urciuolo A, Sabatelli P, Fabris M, Bovolenta M, Neri M, Grumati P, D'Amico A, Pane M, Mercuri E, et al: Identification and characterization of novel collagen VI non-canonical splicing mutations causing ullrich congenital muscular dystrophy. Hum Mutat. 30:E662–E672. 2009. View Article : Google Scholar : PubMed/NCBI
16	Wang C, Yue F and Kuang S: Muscle histology characterization using H&E staining and muscle fiber type classification using immunofluorescence staining. Bio Protoc. 7:e22792017. View Article : Google Scholar :
17	Fu J, Zheng YM, Jin SQ, Yi JF, Liu XJ, Lyn H, Wang ZX, Zhang W, Xiao JX and Yuan Y: 'Target' and 'Sandwich' signs in thigh muscles have high diagnostic values for collagen VI-related myopathies. Chin Med J (Engl). 129:1811–1816. 2016. View Article : Google Scholar
18	Mercuri E, Lampe A, Allsop J, Knight R, Pane M, Kinali M, Bonnemann C, Flanigan K, Lapini I, Bushby K, et al: Muscle MRI in Ullrich congenital muscular dystrophy and Bethlem myopathy. Neuromuscul Disord. 15:303–310. 2005. View Article : Google Scholar : PubMed/NCBI
19	Vanegas OC, Zhang RZ, Sabatelli P, Lattanzi G, Bencivenga P, Giusti B, Columbaro M, Chu ML, Merlini L and Pepe G: Novel COL6A1 splicing mutation in a family affected by mild Bethlem myopathy. Muscle Nerve. 25:513–519. 2002. View Article : Google Scholar : PubMed/NCBI
20	Lucarini L, Giusti B, Zhang RZ, Pan TC, Jimenez-Mallebrera C, Mercuri E, Muntoni F, Pepe G and Chu ML: A homozygous COL6A2 intron mutation causes in-frame triple-helical deletion and nonsense-mediated mRNA decay in a patient with Ullrich congenital muscular dystrophy. Hum Genet. 117:460–466. 2005. View Article : Google Scholar : PubMed/NCBI
21	Zhang RZ, Sabatelli P, Pan TC, Squarzoni S, Mattioli E, Bertini E, Pepe G and Chu ML: Effects on collagen VI mRNA stability and microfibrillar assembly of three COL6A2 mutations in two families with Ullrich congenital muscular dystrophy. J Biol Chem. 277:43557–43564. 2002. View Article : Google Scholar : PubMed/NCBI
22	Jimenez-Mallebrera C, Maioli MA, Kim J, Brown SC, Feng L, Lampe AK, Bushby K, Hicks D, Flanigan KM, Bonnemann C, et al: A comparative analysis of collagen VI production in muscle, skin and fibroblasts from 14 Ullrich congenital muscular dystrophy patients with dominant and recessive COL6A mutations. Neuromuscul Disord. 16:571–582. 2006. View Article : Google Scholar : PubMed/NCBI
23	Norwood F, de Visser M, Eymard B, Lochmüller H and Bushby K; EFNS Guideline Task Force: EFNS guideline on diagnosis and management of limb girdle muscular dystrophies. Eur J Neurol. 14:1305–1312. 2007. View Article : Google Scholar : PubMed/NCBI
24	Lampe AK and Bushby KMD: Collagen VI related muscle disorders. J Med Genet. 42:673–685. 2005. View Article : Google Scholar : PubMed/NCBI
25	Zhang YZ, Zhao DH, Yang HP, Liu AJ, Chang XZ, Hong DJ, Bonnemann C, Yuan Y, Wu XR and Xiong H: Novel collagen VI mutations identified in Chinese patients with Ullrich congenital muscular dystrophy. World J Pediatr. 10:126–132. 2014. View Article : Google Scholar : PubMed/NCBI
26	Yang HP, Zhang YZ, Ding J, Jiao H, Lü JL and Xiong H: Clinical and mutation analyses of a Chinese family with Bethlem myopathy. Zhonghua Yi Xue Za Zhi. 92:2820–2824. 2012.In Chinese.
27	Vaz-Drago R, Custódio N and Carmo-Fonseca M: Deep intronic mutations and human disease. Hum Genet. 136:1093–1111. 2017. View Article : Google Scholar : PubMed/NCBI
28	Tang R, Prosser DO and Love DR: Evaluation of bioinformatic programmes for the analysis of variants within splice site consensus regions. Adv Bioinformatics. 2016:56140582016. View Article : Google Scholar : PubMed/NCBI
29	BinEssa HA, Zou M, Al-Enezi AF, Alomrani B, Al-Faham MSA, Al-Rijjal RA, Meyer BF and Shi Y: Functional analysis of 22 splice-site mutations in the PHEX, the causative gene in X-linked dominant hypophosphatemic rickets. Bone. 125:186–193. 2019. View Article : Google Scholar : PubMed/NCBI
30	Steffensen AY, Dandanell M, Jønson L, Ejlertsen B, Gerdes AM, Nielsen FC and Hansen TV: Functional characterization of BRCA1 gene variants by mini-gene splicing assay. Eur J Hum Genet. 22:1362–1368. 2014. View Article : Google Scholar : PubMed/NCBI
31	Wang Y, Sun Y, Liu M, Zhang X and Jiang T: Functional characterization of argininosuccinate lyase gene variants by mini-gene splicing assay. Front Genet. 10:4362019. View Article : Google Scholar : PubMed/NCBI
32	Zaum AK, Stüve B, Gehrig A, Kölbel H, Schara U, Kress W and Rost S: Deep intronic variants introduce DMD pseudoexon in patient with muscular dystrophy. Neuromuscul Disord. 27:631–634. 2017. View Article : Google Scholar : PubMed/NCBI
33	Lou DI, Hussmann JA, Mcbee RM, Acevedo A, Andino R, Press WH and Sawyer SL: High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing. Proc Natl Acad Sci USA. 110:19872–19877. 2013. View Article : Google Scholar : PubMed/NCBI
34	Lee H, Huang AY, Wang LK, Yoon AJ, Renteria G, Eskin A, Signer RH, Dorrani N, Nieves-Rodriguez S, Wan J, et al: Diagnostic utility of transcriptome sequencing for rare Mendelian diseases. Genet Med. 22:490–499. 2020. View Article : Google Scholar :
35	Alieva M, van Rheenen J and Broekman MLD: Potential impact of invasive surgical procedures on primary tumor growth and metastasis. Clin Exp Metastas. 35:319–331. 2018. View Article : Google Scholar
36	Barker SE, Grosse SM, Siapati EK, Kritz A, Kinnon C, Thrasher AJ and Hart SL: Immunotherapy for neuroblastoma using syngeneic fibroblasts transfected with IL-2 and IL-12. Brit J Cancer. 97:210–217. 2007. View Article : Google Scholar : PubMed/NCBI