Fast and Easy Nanopore Sequencing Workflow for Rapid Genetic Testing of Familial Hypercholesterolemia

Abstract

Familial hypercholesterolemia (FH) is an autosomal dominant lipid metabolism disorder characterized by severely elevated plasma low-density lipoprotein cholesterol levels. The disease is caused by mutations in 3 genes (LDLR, APOB and PCSK9) while over 90% of the mutations are located within the LDLR gene. Thus, genetic analysis of the LDLR gene is the first step in the genetic diagnosis of FH. However, conventional methods like Sanger and NextGen sequencing are still costly and time-consuming. In contrast, Oxford Nanopore technology sequencing is an emerging third-generation sequencing technology featured by easy operability, low cost, small size and the capability of parallel sample sequencing. Here, we present an easy Nanopore-sequencing-based workflow for the rapid genetic testing of FH taking only 3 days and costing less than $50 per sample without the requirement for deep bioinformatic knowledge. Using our workflow, we were able to identify the underlying pathogenic variants of 10 FH patients including one novel, not yet recorded pathogenic variants. Our workflow allows the rapid evaluation of the pathogenic variants by utilizing detailed variant information from Ensembl. Additionally, our workflow is not restricted to sequencing the LDLR gene alone but can be easily adapted to the other FH-causing genes and more importantly, to any desired gene contributing to any hereditary disease. Therefore, our workflow is an attractive opportunity for every diagnostic laboratory to offer fast and easy in-house genetic diagnostics.

Application of Long-Read Nanopore Sequencing to the Search for Mutations in Hypertrophic Cardiomyopathy

“…Our PCR approach is similar to the one utilised by Soufi et al for the LDLR gene, although those authors did not aim to sequence the whole gene [ 11 ]. Instead, they used long-range PCR to amplify batches of exons, thereby precluding determination of whole-gene haplotypes afterwards [ 11 ].…”

“…Our PCR approach is similar to the one utilised by Soufi et al for the LDLR gene, although those authors did not aim to sequence the whole gene [ 11 ]. Instead, they used long-range PCR to amplify batches of exons, thereby precluding determination of whole-gene haplotypes afterwards [ 11 ]. In addition, some deep intronic variants can influence splicing, and, therefore, sequencing the whole gene sequence makes sense, especially for the genes where splice variants are known as disease-causing [ 5 ].…”

“…For example, Leija-Salazar et al applied this method to the whole GBA gene in Gaucher disease, thereby successfully distinguishing it from a pseudogene [ 10 ]. Soufi et al performed Oxford Nanopore Technology (ONT) sequencing of LDLR coding exons in patients with familial hypercholesterolemia [ 11 ]. In another paper, hybridisation capture enrichment for the clinical exome (4800 genes) was used with subsequent MinION sequencing, data processing, and variant calling [ 12 ].…”