Cracking rare disorders: a new minimally invasive RNA-seq protocol
Abstract RNA sequencing (RNA-seq) has become key to complementing exome and genome sequencing for variant interpretation. We present a minimally invasive RNA-seq protocol using short-term cultured peripheral blood mononuclear cells (PBMCs) with and without cycloheximide treatment, enabling detection...
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| Format: | Article |
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Nature Portfolio
2025-05-01
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| Series: | npj Genomic Medicine |
| Online Access: | https://doi.org/10.1038/s41525-025-00502-7 |
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| author | Laurenz De Cock Erika D’haenens Lies Vantomme Lynn Backers Aude Beyens Kathleen BM Claes Griet De Clercq Robin de Putter Candy Kumps Nika Schuermans Jo Sourbron Hannes Syryn Simon Tavernier Eva Vanbelleghem Olivier Vanakker Bart Vandekerckhove Tim Van Damme Bert Callewaert Annelies Dheedene Sarah Vergult Björn Menten |
| author_facet | Laurenz De Cock Erika D’haenens Lies Vantomme Lynn Backers Aude Beyens Kathleen BM Claes Griet De Clercq Robin de Putter Candy Kumps Nika Schuermans Jo Sourbron Hannes Syryn Simon Tavernier Eva Vanbelleghem Olivier Vanakker Bart Vandekerckhove Tim Van Damme Bert Callewaert Annelies Dheedene Sarah Vergult Björn Menten |
| author_sort | Laurenz De Cock |
| collection | DOAJ |
| description | Abstract RNA sequencing (RNA-seq) has become key to complementing exome and genome sequencing for variant interpretation. We present a minimally invasive RNA-seq protocol using short-term cultured peripheral blood mononuclear cells (PBMCs) with and without cycloheximide treatment, enabling detection of transcripts subject to nonsense-mediated decay. While broadly applicable, this protocol is particularly suited for neurodevelopmental disorders, as up to 80% of the genes in our intellectual disability and epilepsy gene panel are expressed in PBMCs. Applied to 46 affected individuals and 15 parents, RNA-seq revealed splicing defects in six of nine individuals with splice variants, allowing reclassification of seven variants. Targeted cDNA analysis confirmed aberrant splicing in four individuals but missed intron retention in two. Global analyses (FRASER, OUTRIDER, and monoallelic expression) supported findings but did not yield new diagnoses. We propose a flowchart integrating RNA-seq into diagnostic workflows. Overall, our protocol is easily implementable, captures complex splicing events, and enhances variant classification. |
| format | Article |
| id | doaj-art-00045d7b5b794628ab729ea6edb342be |
| institution | DOAJ |
| issn | 2056-7944 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | npj Genomic Medicine |
| spelling | doaj-art-00045d7b5b794628ab729ea6edb342be2025-08-20T03:16:55ZengNature Portfolionpj Genomic Medicine2056-79442025-05-0110111410.1038/s41525-025-00502-7Cracking rare disorders: a new minimally invasive RNA-seq protocolLaurenz De Cock0Erika D’haenens1Lies Vantomme2Lynn Backers3Aude Beyens4Kathleen BM Claes5Griet De Clercq6Robin de Putter7Candy Kumps8Nika Schuermans9Jo Sourbron10Hannes Syryn11Simon Tavernier12Eva Vanbelleghem13Olivier Vanakker14Bart Vandekerckhove15Tim Van Damme16Bert Callewaert17Annelies Dheedene18Sarah Vergult19Björn Menten20Department of Biomolecular Medicine, Ghent UniversityCenter for Medical Genetics Ghent, Ghent University HospitalDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityCenter for Medical Genetics Ghent, Ghent University HospitalCenter for Medical Genetics Ghent, Ghent University HospitalCenter for Medical Genetics Ghent, Ghent University HospitalCenter for Medical Genetics Ghent, Ghent University HospitalDepartment of Biomolecular Medicine, Ghent UniversityCenter for Medical Genetics Ghent, Ghent University HospitalDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityCancer Research Institute Ghent (CRIG)Center for Medical Genetics Ghent, Ghent University HospitalDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityDepartment of Biomolecular Medicine, Ghent UniversityAbstract RNA sequencing (RNA-seq) has become key to complementing exome and genome sequencing for variant interpretation. We present a minimally invasive RNA-seq protocol using short-term cultured peripheral blood mononuclear cells (PBMCs) with and without cycloheximide treatment, enabling detection of transcripts subject to nonsense-mediated decay. While broadly applicable, this protocol is particularly suited for neurodevelopmental disorders, as up to 80% of the genes in our intellectual disability and epilepsy gene panel are expressed in PBMCs. Applied to 46 affected individuals and 15 parents, RNA-seq revealed splicing defects in six of nine individuals with splice variants, allowing reclassification of seven variants. Targeted cDNA analysis confirmed aberrant splicing in four individuals but missed intron retention in two. Global analyses (FRASER, OUTRIDER, and monoallelic expression) supported findings but did not yield new diagnoses. We propose a flowchart integrating RNA-seq into diagnostic workflows. Overall, our protocol is easily implementable, captures complex splicing events, and enhances variant classification.https://doi.org/10.1038/s41525-025-00502-7 |
| spellingShingle | Laurenz De Cock Erika D’haenens Lies Vantomme Lynn Backers Aude Beyens Kathleen BM Claes Griet De Clercq Robin de Putter Candy Kumps Nika Schuermans Jo Sourbron Hannes Syryn Simon Tavernier Eva Vanbelleghem Olivier Vanakker Bart Vandekerckhove Tim Van Damme Bert Callewaert Annelies Dheedene Sarah Vergult Björn Menten Cracking rare disorders: a new minimally invasive RNA-seq protocol npj Genomic Medicine |
| title | Cracking rare disorders: a new minimally invasive RNA-seq protocol |
| title_full | Cracking rare disorders: a new minimally invasive RNA-seq protocol |
| title_fullStr | Cracking rare disorders: a new minimally invasive RNA-seq protocol |
| title_full_unstemmed | Cracking rare disorders: a new minimally invasive RNA-seq protocol |
| title_short | Cracking rare disorders: a new minimally invasive RNA-seq protocol |
| title_sort | cracking rare disorders a new minimally invasive rna seq protocol |
| url | https://doi.org/10.1038/s41525-025-00502-7 |
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