A novel loop-mediated isothermal amplification-based genotyping method and its application for identifying proprotein convertase subtilisin/kexin type 9 variants in familial hypercholesterolemia

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating low-density lipoprotein levels in plasma. While PCSK9 variants are causatively associated with familial hypercholesterolemia (FH), additional genotyping methods for FH targeting PCSK9 variants are required in a clinical setting. Loop-mediated isothermal amplification (LAMP) is a unique amplification method that amplifies a target gene under isothermal conditions (60–65 °C). However, a robust standardized method has not yet been established for LAMP-based genetic screening tests for genetic diseases, including FH. The present study aimed to develop a novel modification of the LAMP method designed to genotype single nucleotide variants (SNVs) and to apply it for the detection of PCSK9 variants. Using short quenching probes (≤ 10 nucleotides) for the loop structures of LAMP amplicons, accurate detection of SNVs was verified separately for each allele, without any additional procedures, within 3 h. The diagnostic performance of this method in detecting PCSK9 variants was validated in FH patients. All PCSK9 variants tested via conventional sequencing in FH patients were successfully detected using this novel LAMP method. We developed a LAMP-based genotyping method to detect PCSK9 variants in FH. Compared to conventional sequencing, our genotyping method is relatively convenient and time-efficient and is suitable for the screening of FH in clinical settings. Future studies on various genes are also warranted. •We developed a new LAMP-based genotyping method using short recognition sites.•This method could detect PCSK9 pathogenic variants commonly seen.•This method has a potential for use as a genetic screening method in FH patients.