Vanhoye X, Mouty P, Mouty S, Bargues N, Couprie N, Fayolle E, Géromel V, Taoudi M, Raymond L, Taly J-F
Eurofins Biomnis, Lyon, France.
Pract Lab Med. 2024 Aug 10;41:e00423. doi: 10.1016/j.plabm.2024.e00423. eCollection 2024 Aug.
Long-read sequencing technology, widely used in research, is proving useful in clinical diagnosis, especially for infectious diseases. Despite recent advances, it hasn't been routinely applied to constitutional human diseases. Long-read sequencing detects intronic variants and phases variants, crucial for identifying recessive diseases.
We integrated long-read sequencing into the clinical diagnostic workflow for the MEFV gene, responsible for familial Mediterranean fever (FMF), using a Nanopore-based workflow. This involved long-range PCR amplification, native barcoding kit library preparation, GridION sequencing, and in-house bioinformatics. We compared this new workflow against our validated method using 39 patient samples and 3 samples from an external quality assessment scheme to ensure compliance with ISO15189 standards.
Our evaluation demonstrated excellent performance, meeting ISO15189 requirements for reproducibility, repeatability, sensitivity, and specificity. Since October 2022, 150 patient samples were successfully analyzed with no failures. Among these samples, we identified 13 heterozygous carriers of likely pathogenic (LP) or pathogenic (P) variants, 1 patient with a homozygous LP/P variant in MEFV, and 4 patients with compound heterozygous variants.
This study represents the first integration of long-read sequencing for FMF clinical diagnosis, achieving 100 % sensitivity and specificity. Our findings highlight its potential to identify pathogenic variants without parental segregation analysis, offering faster, cost-effective, and accurate clinical diagnosis. This successful implementation lays the groundwork for future applications in other constitutional human diseases, advancing precision medicine.
长读长测序技术在研究中广泛应用，已证明在临床诊断中有用，特别是对于传染病。尽管最近取得了进展，但它尚未常规应用于人类遗传性疾病。长读长测序可检测内含子变异和相位变异，这对于识别隐性疾病至关重要。
我们使用基于纳米孔的工作流程，将长读长测序整合到负责家族性地中海热（FMF）的MEFV基因的临床诊断工作流程中。这包括长距离PCR扩增、原生条形码试剂盒文库制备、GridION测序和内部生物信息学。我们使用39份患者样本和来自外部质量评估计划的3份样本，将这种新工作流程与我们经过验证的方法进行比较，以确保符合ISO15189标准。
我们的评估显示出优异的性能，符合ISO15189对可重复性、重复性、敏感性和特异性的要求。自2022年10月以来，成功分析了150份患者样本，无失败案例。在这些样本中，我们鉴定出13名可能致病（LP）或致病（P）变异的杂合携带者，1名MEFV基因纯合LP/P变异的患者，以及4名复合杂合变异的患者。
本研究首次将长读长测序整合用于FMF临床诊断，实现了100%的敏感性和特异性。我们的研究结果突出了其在无需亲代分离分析的情况下识别致病变异的潜力，可提供更快、更具成本效益且准确的临床诊断。这一成功实施为未来在其他人类遗传性疾病中的应用奠定了基础，推动了精准医学的发展。
