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Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family

Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20–40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-indu...

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Autores principales: Sedaghat-Hamedani, Farbod, Rebs, Sabine, Kayvanpour, Elham, Zhu, Chenchen, Amr, Ali, Müller, Marion, Haas, Jan, Wu, Jingyan, Steinmetz, Lars M., Ehlermann, Philipp, Streckfuss-Bömeke, Katrin, Frey, Norbert, Meder, Benjamin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9602549/
https://www.ncbi.nlm.nih.gov/pubmed/36293084
http://dx.doi.org/10.3390/ijms232012230
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author Sedaghat-Hamedani, Farbod
Rebs, Sabine
Kayvanpour, Elham
Zhu, Chenchen
Amr, Ali
Müller, Marion
Haas, Jan
Wu, Jingyan
Steinmetz, Lars M.
Ehlermann, Philipp
Streckfuss-Bömeke, Katrin
Frey, Norbert
Meder, Benjamin
author_facet Sedaghat-Hamedani, Farbod
Rebs, Sabine
Kayvanpour, Elham
Zhu, Chenchen
Amr, Ali
Müller, Marion
Haas, Jan
Wu, Jingyan
Steinmetz, Lars M.
Ehlermann, Philipp
Streckfuss-Bömeke, Katrin
Frey, Norbert
Meder, Benjamin
author_sort Sedaghat-Hamedani, Farbod
collection PubMed
description Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20–40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.
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spelling pubmed-96025492022-10-27 Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family Sedaghat-Hamedani, Farbod Rebs, Sabine Kayvanpour, Elham Zhu, Chenchen Amr, Ali Müller, Marion Haas, Jan Wu, Jingyan Steinmetz, Lars M. Ehlermann, Philipp Streckfuss-Bömeke, Katrin Frey, Norbert Meder, Benjamin Int J Mol Sci Article Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20–40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies. MDPI 2022-10-13 /pmc/articles/PMC9602549/ /pubmed/36293084 http://dx.doi.org/10.3390/ijms232012230 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Sedaghat-Hamedani, Farbod
Rebs, Sabine
Kayvanpour, Elham
Zhu, Chenchen
Amr, Ali
Müller, Marion
Haas, Jan
Wu, Jingyan
Steinmetz, Lars M.
Ehlermann, Philipp
Streckfuss-Bömeke, Katrin
Frey, Norbert
Meder, Benjamin
Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family
title Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family
title_full Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family
title_fullStr Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family
title_full_unstemmed Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family
title_short Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family
title_sort genotype complements the phenotype: identification of the pathogenicity of an lmna splice variant by nanopore long-read sequencing in a large dcm family
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9602549/
https://www.ncbi.nlm.nih.gov/pubmed/36293084
http://dx.doi.org/10.3390/ijms232012230
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