Whole genome sequencing for the diagnosis of neurological repeat expansion disorders in the UK: a retrospective diagnostic accuracy and prospective clinical validation study

Abstract

Background: Repeat expansion disorders affect about 1 in 3000 individuals and are clinically heterogeneous diseases caused by expansions of short tandem DNA repeats. Genetic testing is often locus-specific, resulting in underdiagnosis of people who have atypical clinical presentations, especially in paediatric patients without a previous positive family history. Whole genome sequencing is increasingly used as a first-line test for other rare genetic disorders, and we aimed to assess its performance in the diagnosis of patients with neurological repeat expansion disorders.

Methods: We retrospectively assessed the diagnostic accuracy of whole genome sequencing to detect the most common repeat expansion loci associated with neurological outcomes (AR, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, C9orf72, CACNA1A, DMPK, FMR1, FXN, HTT, and TBP) using samples obtained within the National Health Service in England from patients who were suspected of having neurological disorders; previous PCR test results were used as the reference standard. The clinical accuracy of whole genome sequencing to detect repeat expansions was prospectively examined in previously genetically tested and undiagnosed patients recruited in 2013-17 to the 100 000 Genomes Project in the UK, who were suspected of having a genetic neurological disorder (familial or early-onset forms of ataxia, neuropathy, spastic paraplegia, dementia, motor neuron disease, parkinsonian movement disorders, intellectual disability, or neuromuscular disorders). If a repeat expansion call was made using whole genome sequencing, PCR was used to confirm the result.

Findings: The diagnostic accuracy of whole genome sequencing to detect repeat expansions was evaluated against 793 PCR tests previously performed within the NHS from 404 patients. Whole genome sequencing correctly classified 215 of 221 expanded alleles and 1316 of 1321 non-expanded alleles, showing 97·3% sensitivity (95% CI 94·2-99·0) and 99·6% specificity (99·1-99·9) across the 13 disease-associated loci when compared with PCR test results. In samples from 11 631 patients in the 100 000 Genomes Project, whole genome sequencing identified 81 repeat expansions, which were also tested by PCR: 68 were confirmed as repeat expansions in the full pathogenic range, 11 were non-pathogenic intermediate expansions or premutations, and two were non-expanded repeats (16% false discovery rate).

Interpretation: In our study, whole genome sequencing for the detection of repeat expansions showed high sensitivity and specificity, and it led to identification of neurological repeat expansion disorders in previously undiagnosed patients. These findings support implementation of whole genome sequencing in clinical laboratories for diagnosis of patients who have a neurological presentation consistent with a repeat expansion disorder.

Funding: Medical Research Council, Department of Health and Social Care, National Health Service England, National Institute for Health Research, and Illumina.

Figure 1

Study flow chart (A) Detection of repeat expansions by whole genome sequencing. (B) Validation in patients who had a suspected neurological disorder, undiagnosed with previous genetic testing. NHS=National Health Service.

Figure 2

Performance of repeat expansion detection using whole genome sequencing (A) Swim lane plot showing sizes of repeat expansions predicted by ExpansionHunter across 793 expansion calls. Each genome is represented by two points, one corresponding to each allele for each locus, with the exception of those on the X chromosome (ie, FMR1 and AR) in males, for which only one point is shown. Points indicate the repeat length estimated by ExpansionHunter after visual inspection and the colours indicate the repeat size as assessed by PCR (blue represents non-expanded; red represents expanded). The regions are shaded to indicate non-expanded (blue), premutation (pink), and expanded (red) ranges for each gene, as indicated in the appendix (p 28). Blue points in pink or red shaded regions indicate false positives and red points in blue shaded regions indicate false negatives. The individual calls are provided in the appendix (p 27). (B) Repeat size correlation by locus. Bubble plots show PCR repeat sizes on the x axes and ExpansionHunter repeats sizes on y axes, with the size of each dot showing the number of patients with the same repeat size. The grey points visible for ATXN1, FMR1, FXN≤, and HTT represent ExpansionHunter estimations before visual inspection, whereas the corrected ExpansionHunter sizes after visual inspection are in colour. Red dashed lines represent the premutation cutoff for each locus (appendix p 28). FXN≤ and DMPK≤ show the repeat size correlation when the the size is less than or equal to the read length (ie, 150 bp). FXN> and DMPK> show the repeat size correlation when the size is larger than the read length.

Figure 3

Adult and paediatric patients showing pathogenic expanded repeats Repeat size frequency distribution of genes for which a repeat expansion was detected in paediatric patients (ATN1, ATXN2, ATXN7, and HTT) in 11 631 patients. The number of CAG repeats relative to allele count is shown. The children with large expansions are described in table 3 (ATN1 in patients 46 and 47; ATXN2 in patient 48; ATXN7 in patients 49, 50, and 53; HTT in patients 51 and 52). The dashed red line represents the full mutation threshold, above which the number of repeat expansions is considered to be pathogenic for each locus (appendix p 28). White arrowheads indicate pathogenic expansions detected in adults and red arrowheads indicate pathogenic expansions detected in children.