The ONDRISeq panel: custom-designed next-generation sequencing of genes related to neurodegeneration

Abstract

The Ontario Neurodegenerative Disease Research Initiative (ONDRI) is a multimodal, multi-year, prospective observational cohort study to characterise five diseases: (1) Alzheimer's disease (AD) or amnestic single or multidomain mild cognitive impairment (aMCI) (AD/MCI); (2) amyotrophic lateral sclerosis (ALS); (3) frontotemporal dementia (FTD); (4) Parkinson's disease (PD); and (5) vascular cognitive impairment (VCI). The ONDRI Genomics subgroup is investigating the genetic basis of neurodegeneration. We have developed a custom next-generation-sequencing-based panel, ONDRISeq that targets 80 genes known to be associated with neurodegeneration. We processed DNA collected from 216 individuals diagnosed with one of the five diseases, on ONDRISeq. All runs were executed on a MiSeq instrument and subjected to rigorous quality control assessments. We also independently validated a subset of the variant calls using NeuroX (a genome-wide array for neurodegenerative disorders), TaqMan allelic discrimination assay, or Sanger sequencing. ONDRISeq consistently generated high-quality genotyping calls and on average, 92% of targeted bases are covered by at least 30 reads. We also observed 100% concordance for the variants identified via ONDRISeq and validated by other genomic technologies. We were successful in detecting known as well as novel rare variants in 72.2% of cases although not all variants are disease-causing. Using ONDRISeq, we also found that the APOE E4 allele had a frequency of 0.167 in these samples. Our optimised workflow highlights next-generation sequencing as a robust tool in elucidating the genetic basis of neurodegenerative diseases by screening multiple candidate genes simultaneously.

Figure 1

Case study: APP variant in AD case. (a) Schematic of the gene and variant discovery process in a neurodegenerative disease case. AD, Alzheimer’s disease, patient 1; *MAF was retrieved using ExAC database. (b) APP protein structure shown from N- to C-terminal, 1: amyloid A4 N-terminal heparin-binding domain; 2: copper-binding of amyloid precursor; 3: Kunitz/Bovine pancreatic trypsin inhibitor domain; 4: E2 domain of amyloid precursor protein; 5: beta-amyloid peptide domain; 6: beta-amyloid precursor protein C-terminus domain. The gold star represents the location of the missense variant. (c) Multiple alignments demonstrate high conservation of wild-type amino-acid residue p.Ala713 (in bold; the variant residue p.Thr173 is not bold) across a set of species-specific APP homologues. The asterisks below indicate fully conserved residues. (d) The ONDRISeq output showing heterozygosity at the position of the genetic variant, 21:27264108G>A. ONDRISeq output produced ×94 coverage. (e) An electropherogram showing the DNA sequence analysis of APP from a patient diagnosed with AD. Our reported cDNA and amino-acid positions are based on NM_000484.3 and NP_000475.1, respectively.