Mitochondrial and nuclear disease panel (Mito-aND-Panel): Combined sequencing of mitochondrial and nuclear DNA by a cost-effective and sensitive NGS-based method.

Angela Abicht,Stephanie Kleinle,Rita Horvath,Florentine Scharf,Anna Benet‐Pagès,Elke Holinski‐Feder,Isabel Diebold,Ulrike Schön

doi:10.1002/mgg3.500

Abstract

BackgroundThe diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity of these conditions. Next‐Generation Sequencing (NGS) technology offers a robust high‐throughput platform for nuclear and mitochondrial DNA (mtDNA) analyses.MethodWe developed a custom Agilent SureSelect Mitochondrial and Nuclear Disease Panel (Mito‐aND‐Panel) capture kit that allows parallel enrichment for subsequent NGS‐based sequence analysis of nuclear mitochondrial disease‐related genes and the complete mtDNA genome. Sequencing of enriched mtDNA simultaneously with nuclear genes was compared with the separated sequencing of the mitochondrial genome and whole exome sequencing (WES).ResultsThe Mito‐aND‐Panel permits accurate detection of low‐level mtDNA heteroplasmy due to a very high sequencing depth compared to standard diagnostic procedures using Sanger sequencing/SNaPshot and WES which is crucial to identify maternally inherited mitochondrial disorders.ConclusionWe established a NGS‐based method with combined sequencing of the complete mtDNA and nuclear genes which enables a more sensitive heteroplasmy detection of mtDNA mutations compared to traditional methods. Because the method promotes the analysis of mtDNA variants in large cohorts, it is cost‐effective and simple to setup, we anticipate this is a highly relevant method for sequence‐based genetic diagnosis in clinical diagnostic applications.

Highlights

Mitochondrial disease is highly heterogeneous in cause and features; traditional single‐gene testing strategies have had limited diagnostic success (McCormick, Place, & Falk, 2013)
While a mean coverage depth of 120× is usually enough for detection of nuclear DNA variants, a minimal depth of >1,000× reads may be acceptable for the detection of low‐heteroplasmic variants (Jennings et al, 2017; Matthijs et al, 2016), meaning that much more sequencing reads have to be generated for mitochondrial DNA (mtDNA) compared to nuclear DNA
We established the Mito‐aND‐Panel for parallel sequencing of the entire mtDNA and 1,564 nuclear DNA genes, including 308 nuclear mitochondrial disease‐related genes (MitoCarta v2.0) that achieves variant detection at high resolution for low‐heteroplasmic mtDNA mutations at a mean coverage of >7,000×

Summary

| INTRODUCTION

Mitochondrial disease is highly heterogeneous in cause and features; traditional single‐gene testing strategies have had limited diagnostic success (McCormick, Place, & Falk, 2013). |2 of mitochondrial genomes, as well as the sequences of mitochondrial origin in the nuclear genome (NUMTs, nuclear mitochondrial DNAs), the dual analysis of genomic and mtDNA variants in a single NGS approach is challenging. Recent studies have demonstrated the efficacy of NGS technologies for the genetic diagnosis of inherited mitochondrial diseases (Calvo et al, 2012; Falk et al, 2012, 2015 ; Guo, Li, Li, Shyr, & Samuels, 2013; Legati et al, 2016; Picardi & Pesole, 2012; Vasta, Merritt, Saneto, & Hahn, 2012), these methods do not overcome the limitations and cause ineffectiveness in terms of variant detection accuracy, turnaround time, and costs for separate mtDNA and nuclear DNA analysis. We developed a NGS‐based method with combined sequencing of the complete mtDNA and nuclear genes causing mitochondrial diseases and compared this method with the traditional Sanger sequencing and whole exome sequencing (WES)

| MATERIALS AND METHODS

| RESULTS

Findings

| DISCUSSION