Elsevier

Neurobiology of Aging

Volume 35, Issue 1, January 2014, Pages 261-265
Neurobiology of Aging

Brief communication
Validation of next-generation sequencing technologies in genetic diagnosis of dementia

https://doi.org/10.1016/j.neurobiolaging.2013.07.017Get rights and content

Abstract

Identification of a specific genetic cause of early onset dementia (EOD) is important but can be difficult because of pleiotropy, locus heterogeneity and accessibility of gene tests. Here we assess the use of next generation sequencing (NGS) technologies as a quick, accurate and cost effective method to determine genetic diagnosis in EOD. We developed gene panel based technologies to assess 16 genes known to harbour mutations causal of dementia and combined these with PCR based assessments of the C9orf72 hexanucleotide repeat expansion and the octapeptide repeat region of PRNP. In a blinded study of 95 samples we show very high sensitivity and specificity are achievable using either Ion Torrent or MiSeq sequencing platforms. Modifications to the gene panel permit accurate detection of structural variation in APP. In 2/10 samples which had been selected because they possess a variant of uncertain significance the new technology discovered a causal mutation in genes not previously sequenced. A large proportion (23/85) of samples showed genetic variants of uncertain significance in addition to known mutations. The MRC Dementia Gene Panel and similar technologies are likely to be transformational in EOD diagnosis with a significant impact on the proportion of patients in whom a genetic cause is identified.

Introduction

Genetic diagnosis of the inherited dementias currently relies on sequential Sanger sequencing of genes selected on a clinical basis. This process is costly, time-consuming, and gene tests are variably available, contributing to the limited ascertainment of inherited dementia in the population (Stevens et al., 2011). Here we show that next-generation sequencing (NGS) technology offers a 1-step, accurate, and cost-effective method of screening many causal genes simultaneously, and is likely to be a transformational technology in early-onset dementia diagnosis (Rehm, 2013).

Section snippets

Methods

We used Life Technology's Ion Torrent PGM sequencer with a polymerase chain reaction (PCR) amplicon-based library preparation (AmpliSeq) and Illumina's MiSeq with a PCR amplicon-based (TrueSeq custom amplicon) target enrichment to screen for variants across 16 dementia disease genes (PRNP, PSEN1, PSEN2, APP, GRN, MAPT, TREM2, CHMP2B, CSF1R, FUS, ITM2B, NOTCH3, SERPINI1, TARDBP, TYROBP, and VCP) (Bettens et al., 2013, Rademakers et al., 2012). These sequencing-based technologies were coupled

Results

In the blinded study, the Ion Torrent correctly identified all control samples. Of the 85 case samples, 82 had the expected mutations or polymorphisms identified during primary analysis (sensitivity 96.5%, specificity 100%), including deletion of the entire GRN gene in 1 case. The 3 variants not initially detected were 2 samples with an identical APP p.V717G mutation and an APP duplication. The APP p.V717G mutations were present at the overlap between 2 amplicons and because of a software

Discussion

Using our current testing strategy and allowing for the fact that the APP p.V717G mutations were not identified because of a straightforward and remediable software problem, we demonstrated extremely high sensitivity and specificity. The present recurrent consumable costs are estimated at approximately £60 ($90) per sample using the Ion Torrent and £120 ($180) using the Illumina MiSeq. NGS has many benefits including vastly reduced costs compared with Sanger sequencing of individual genes, a

Disclosure statement

The authors declare no conflicts of interest. All subjects included in this study gave informed consent for genetic research. The work was approved by the London - Queen Square Research Ethics Committee.

Acknowledgements

This work was funded by the Medical Research Council Prion Unit and the NIHR Queen Square Dementia Biomedical Research Unit.

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