Long-read Oxford Nanopore sequencing enables rapid, cost-effective, and comprehensive <i>TTR</i> genetic testing for hereditary transthyretin amyloidosis

Abstract Objectives Hereditary transthyretin amyloidosis (ATTRv) is a rare autosomal dominant disorder caused by pathogenic variants in the TTR gene, with marked clinical heterogeneity and frequent underdiagnosis. Genetic confirmation is essential for patient management but is traditionally based on Sanger sequencing or short-read next-generation sequencing (NGS), which are time-consuming or costly. We evaluated the clinical utility of a long-read sequencing strategy using Oxford Nanopore Technologies (ONT) for comprehensive TTR gene analysis. Methods We developed an optimized long-range PCR (6,941 bp) followed by ONT MinION sequencing workflow covering the entire TTR gene. Analytical performance was evaluated in two independent diagnostic laboratories using a total of 89 previously characterized samples (64 from laboratory #1 and 25 from laboratory #2). The method was subsequently implemented for routine clinical testing in 445 additional patients referred for suspected ATTRv amyloidosis. Performance metrics, variant detection rates, turnaround time, and concordance with short-read NGS were assessed. Results All previously identified pathogenic or likely pathogenic TTR variants were correctly detected, and no false-positive calls. As expected, the most frequent variants were p.(Val142Ile) and p.(Val50Met). Results were fully concordant with short-read NGS. Flowcell washing and reuse considerably lowered reagent costs without compromising an accurate TTR genotyping. Implementation of the ONT workflow reduced turnaround time compared with Illumina-based targeted panels. Conclusions Long-read ONT sequencing of the TTR gene provides a robust, rapid, and economical alternative to conventional methods for genetic diagnosis of ATTRv amyloidosis. The approach we developed is well suited for routine clinical laboratories and supports systematic screening of TTR variants in patients with suspected ATTR.