Genetic analysis using long-read sequencing to overcome the difficulties in VWF gene
Background: Genetic defects in von Willebrand factor (VWF) can lead to von Willebrand disease (VWD). Identifying causative or modifier variants of VWF is crucial for the diagnosis, classification, and clinical management of VWF disorders. However, owing to the length (178 kb) and complexity of VWF a...
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Elsevier
2025-05-01
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| Series: | Research and Practice in Thrombosis and Haemostasis |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2475037925002122 |
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| author | Sheng Ye Yuka Eura Masanori Matsumoto Koichi Kokame |
| author_facet | Sheng Ye Yuka Eura Masanori Matsumoto Koichi Kokame |
| author_sort | Sheng Ye |
| collection | DOAJ |
| description | Background: Genetic defects in von Willebrand factor (VWF) can lead to von Willebrand disease (VWD). Identifying causative or modifier variants of VWF is crucial for the diagnosis, classification, and clinical management of VWF disorders. However, owing to the length (178 kb) and complexity of VWF and the presence of the pseudogene VWFP1, Sanger sequencing or short-read next-generation sequencing is often challenging. Objectives: This study aimed to establish a long-read sequencing method using Oxford nanopore technology (ONT) to overcome difficulties associated with VWF gene analysis. Methods: Genetic analyses were established using genomic DNA from a healthy donor and validated using 3 VWF disorder patient samples. Long-range (∼15 kb) polymerase chain reaction was optimized to obtain 21 amplicons covering the entire VWF gene, avoiding unwanted amplification due to repetitive sequences and VWFP1. ONT nanopore sequencing data were analyzed using software programs, including Clair3, Longshot, and Sniffles. The identified candidate variants were verified by several approaches such as Sanger sequencing and haplotyping. Results: The entire VWF gene was successfully read using ONT nanopore sequencing, with >200 variants called in each patient sample. A rare missense variant, p.(Gln2442His) and a rare 2599 bp deletion were identified in patients 2 and 3, respectively. However, the deletion was confirmed as long-range polymerase chain reaction artifacts, which warrant attention when using this method. Conclusion: This study presents an optimal solution using ONT nanopore sequencing to identify variants in VWF, which may improve the diagnosis of VWF disorders. |
| format | Article |
| id | doaj-art-d5de030e8f43402db5ee8bb557a623a6 |
| institution | DOAJ |
| issn | 2475-0379 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Research and Practice in Thrombosis and Haemostasis |
| spelling | doaj-art-d5de030e8f43402db5ee8bb557a623a62025-08-20T03:04:46ZengElsevierResearch and Practice in Thrombosis and Haemostasis2475-03792025-05-019410288810.1016/j.rpth.2025.102888Genetic analysis using long-read sequencing to overcome the difficulties in VWF geneSheng Ye0Yuka Eura1Masanori Matsumoto2Koichi Kokame3Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan; Department of Cardiovascular System Research, Nara Medical University, Kashihara, Nara, JapanDepartment of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Osaka, JapanDepartment of Blood Transfusion Medicine and Hematology, Nara Medical University, Kashihara, Nara, JapanDepartment of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan; Department of Cardiovascular System Research, Nara Medical University, Kashihara, Nara, Japan; Correspondence Koichi Kokame, Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, 6-1 Kishibe–Shimmachi, Suita, Osaka 564-8565, Japan.Background: Genetic defects in von Willebrand factor (VWF) can lead to von Willebrand disease (VWD). Identifying causative or modifier variants of VWF is crucial for the diagnosis, classification, and clinical management of VWF disorders. However, owing to the length (178 kb) and complexity of VWF and the presence of the pseudogene VWFP1, Sanger sequencing or short-read next-generation sequencing is often challenging. Objectives: This study aimed to establish a long-read sequencing method using Oxford nanopore technology (ONT) to overcome difficulties associated with VWF gene analysis. Methods: Genetic analyses were established using genomic DNA from a healthy donor and validated using 3 VWF disorder patient samples. Long-range (∼15 kb) polymerase chain reaction was optimized to obtain 21 amplicons covering the entire VWF gene, avoiding unwanted amplification due to repetitive sequences and VWFP1. ONT nanopore sequencing data were analyzed using software programs, including Clair3, Longshot, and Sniffles. The identified candidate variants were verified by several approaches such as Sanger sequencing and haplotyping. Results: The entire VWF gene was successfully read using ONT nanopore sequencing, with >200 variants called in each patient sample. A rare missense variant, p.(Gln2442His) and a rare 2599 bp deletion were identified in patients 2 and 3, respectively. However, the deletion was confirmed as long-range polymerase chain reaction artifacts, which warrant attention when using this method. Conclusion: This study presents an optimal solution using ONT nanopore sequencing to identify variants in VWF, which may improve the diagnosis of VWF disorders.http://www.sciencedirect.com/science/article/pii/S2475037925002122von Willebrand factorvon Willebrand diseasenanopore sequencingpolymerase chain reaction |
| spellingShingle | Sheng Ye Yuka Eura Masanori Matsumoto Koichi Kokame Genetic analysis using long-read sequencing to overcome the difficulties in VWF gene Research and Practice in Thrombosis and Haemostasis von Willebrand factor von Willebrand disease nanopore sequencing polymerase chain reaction |
| title | Genetic analysis using long-read sequencing to overcome the difficulties in VWF gene |
| title_full | Genetic analysis using long-read sequencing to overcome the difficulties in VWF gene |
| title_fullStr | Genetic analysis using long-read sequencing to overcome the difficulties in VWF gene |
| title_full_unstemmed | Genetic analysis using long-read sequencing to overcome the difficulties in VWF gene |
| title_short | Genetic analysis using long-read sequencing to overcome the difficulties in VWF gene |
| title_sort | genetic analysis using long read sequencing to overcome the difficulties in vwf gene |
| topic | von Willebrand factor von Willebrand disease nanopore sequencing polymerase chain reaction |
| url | http://www.sciencedirect.com/science/article/pii/S2475037925002122 |
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