Mohamed Imran, V R Arvinden, Pabithadevi Balaiah Mehanathan, Raskin Erusan Rajagopal, Suriya Prabha Muthu, Arul Subbiah Arunachalam, Rahul C Bhoyar, Harie Vignesh, Samya Mitra, Ganga Nath Jha, Aayush Gupta, Manoj Kumar, Rohit Bhowmick, Niladri Sekhar Bhunia, Atanu Kumar Dutta, Vinod Scaria, Sridhar Sivasubbu
{"title":"用于家族性高胆固醇血症基因筛查的基于多重 PCR 的下一代扩增片段测序快速可扩展方法。","authors":"Mohamed Imran, V R Arvinden, Pabithadevi Balaiah Mehanathan, Raskin Erusan Rajagopal, Suriya Prabha Muthu, Arul Subbiah Arunachalam, Rahul C Bhoyar, Harie Vignesh, Samya Mitra, Ganga Nath Jha, Aayush Gupta, Manoj Kumar, Rohit Bhowmick, Niladri Sekhar Bhunia, Atanu Kumar Dutta, Vinod Scaria, Sridhar Sivasubbu","doi":"10.1093/jalm/jfae089","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Familial hypercholesterolemia (FH) is a frequently underdiagnosed genetic disorder characterized by elevated low-density lipoprotein (LDL) levels. Genetic testing of LDLR, APOB, and PCSK9 genes can identify variants in up to 80% of clinically diagnosed patients. However, limitations in time, scalability, and cost have hindered effective next-generation sequencing of these genes. Additionally, pharmacogenomic variants are associated with statin-induced adverse effects in FH patients. To address these challenges, we developed a multiplex primer-based amplicon sequencing approach for FH genetic testing.</p><p><strong>Methods: </strong>Multiplex primers were designed for the exons of the LDLR, APOB, and PCSK9 genes, as well as for pharmacogenomic variants rs4149056 (SLCO1B1:c.521T > A), rs2306283 (SLCO1B1:c.388A > G), and rs2231142 (ABCG2:c.421C > A). Analytical validation using samples with known pathogenic variants and clinical validation with 12 FH-suspected probands were conducted. Library preparation was based on a bead-based tagmentation method, and sequencing was conducted on the NovaSeq 6000 platform.</p><p><strong>Results: </strong>Our approach ensured no amplicon dropouts, with over 100× coverage on each amplicon. Known variants in 2 samples were successfully detected. Further, we identified one heterozygous LDLR (p.Glu228Ter) variant and 2 homozygous cases of LDLR (p.Lys294Ter) and LDLR (p.Ser177Leu) variants in patients. Pharmacogenomic analysis revealed that overall 3 patients may require reduced statin doses. Our approach offered reduced library preparation time (approximately 3 h), greater scalability, and lower costs (under $50) for FH genetic testing.</p><p><strong>Conclusions: </strong>Our method effectively sequences LDLR, APOB, and PCSK9 genes including pharmacogenomic variants that will guide appropriate screening and statin dosing, thus increasing both efficiency and affordability.</p>","PeriodicalId":46361,"journal":{"name":"Journal of Applied Laboratory Medicine","volume":null,"pages":null},"PeriodicalIF":1.8000,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"A Rapid and Scalable Multiplex PCR-Based Next-Generation Amplicon Sequencing Method for Familial Hypercholesterolemia Genetic Screening.\",\"authors\":\"Mohamed Imran, V R Arvinden, Pabithadevi Balaiah Mehanathan, Raskin Erusan Rajagopal, Suriya Prabha Muthu, Arul Subbiah Arunachalam, Rahul C Bhoyar, Harie Vignesh, Samya Mitra, Ganga Nath Jha, Aayush Gupta, Manoj Kumar, Rohit Bhowmick, Niladri Sekhar Bhunia, Atanu Kumar Dutta, Vinod Scaria, Sridhar Sivasubbu\",\"doi\":\"10.1093/jalm/jfae089\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Familial hypercholesterolemia (FH) is a frequently underdiagnosed genetic disorder characterized by elevated low-density lipoprotein (LDL) levels. Genetic testing of LDLR, APOB, and PCSK9 genes can identify variants in up to 80% of clinically diagnosed patients. However, limitations in time, scalability, and cost have hindered effective next-generation sequencing of these genes. Additionally, pharmacogenomic variants are associated with statin-induced adverse effects in FH patients. To address these challenges, we developed a multiplex primer-based amplicon sequencing approach for FH genetic testing.</p><p><strong>Methods: </strong>Multiplex primers were designed for the exons of the LDLR, APOB, and PCSK9 genes, as well as for pharmacogenomic variants rs4149056 (SLCO1B1:c.521T > A), rs2306283 (SLCO1B1:c.388A > G), and rs2231142 (ABCG2:c.421C > A). Analytical validation using samples with known pathogenic variants and clinical validation with 12 FH-suspected probands were conducted. Library preparation was based on a bead-based tagmentation method, and sequencing was conducted on the NovaSeq 6000 platform.</p><p><strong>Results: </strong>Our approach ensured no amplicon dropouts, with over 100× coverage on each amplicon. Known variants in 2 samples were successfully detected. Further, we identified one heterozygous LDLR (p.Glu228Ter) variant and 2 homozygous cases of LDLR (p.Lys294Ter) and LDLR (p.Ser177Leu) variants in patients. Pharmacogenomic analysis revealed that overall 3 patients may require reduced statin doses. Our approach offered reduced library preparation time (approximately 3 h), greater scalability, and lower costs (under $50) for FH genetic testing.</p><p><strong>Conclusions: </strong>Our method effectively sequences LDLR, APOB, and PCSK9 genes including pharmacogenomic variants that will guide appropriate screening and statin dosing, thus increasing both efficiency and affordability.</p>\",\"PeriodicalId\":46361,\"journal\":{\"name\":\"Journal of Applied Laboratory Medicine\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.8000,\"publicationDate\":\"2024-11-04\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of Applied Laboratory Medicine\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1093/jalm/jfae089\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"MEDICAL LABORATORY TECHNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Applied Laboratory Medicine","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/jalm/jfae089","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MEDICAL LABORATORY TECHNOLOGY","Score":null,"Total":0}
A Rapid and Scalable Multiplex PCR-Based Next-Generation Amplicon Sequencing Method for Familial Hypercholesterolemia Genetic Screening.
Background: Familial hypercholesterolemia (FH) is a frequently underdiagnosed genetic disorder characterized by elevated low-density lipoprotein (LDL) levels. Genetic testing of LDLR, APOB, and PCSK9 genes can identify variants in up to 80% of clinically diagnosed patients. However, limitations in time, scalability, and cost have hindered effective next-generation sequencing of these genes. Additionally, pharmacogenomic variants are associated with statin-induced adverse effects in FH patients. To address these challenges, we developed a multiplex primer-based amplicon sequencing approach for FH genetic testing.
Methods: Multiplex primers were designed for the exons of the LDLR, APOB, and PCSK9 genes, as well as for pharmacogenomic variants rs4149056 (SLCO1B1:c.521T > A), rs2306283 (SLCO1B1:c.388A > G), and rs2231142 (ABCG2:c.421C > A). Analytical validation using samples with known pathogenic variants and clinical validation with 12 FH-suspected probands were conducted. Library preparation was based on a bead-based tagmentation method, and sequencing was conducted on the NovaSeq 6000 platform.
Results: Our approach ensured no amplicon dropouts, with over 100× coverage on each amplicon. Known variants in 2 samples were successfully detected. Further, we identified one heterozygous LDLR (p.Glu228Ter) variant and 2 homozygous cases of LDLR (p.Lys294Ter) and LDLR (p.Ser177Leu) variants in patients. Pharmacogenomic analysis revealed that overall 3 patients may require reduced statin doses. Our approach offered reduced library preparation time (approximately 3 h), greater scalability, and lower costs (under $50) for FH genetic testing.
Conclusions: Our method effectively sequences LDLR, APOB, and PCSK9 genes including pharmacogenomic variants that will guide appropriate screening and statin dosing, thus increasing both efficiency and affordability.