Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases

Abstract

Background: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome.

Methods: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants.

Results: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving.

Conclusions: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.

Keywords: Bioinformatics pipeline development; Clinical impact; Diagnostic yield; Genome sequencing; Non-coding; Pipeline optimisation; Rare diseases; Splice site variant; Structural variant.

Fig. 1

Overview of OxClinWGS study: workflow, clinical cases and results. A Case selection and referral was mainly done by the Oxford Genomic Medicine Multidisciplinary Team (GM-MDT); a detailed description of this process is provided elsewhere [37]. Selected samples were whole-genome sequenced and analysed by a clinical (yellow) as well as a research pipeline (green). Identified pathogenic candidate variants were validated and reported back to referring clinicians. Unsolved cases were iteratively investigated by a research pipeline incorporating the latest methods for in silico analysis of WGS data. Resulting novel disease candidate variants were regularly discussed by an interdisciplinary expert team and either rejected or forwarded to (functional) validation. B Core statistics of considered pedigrees (n = 122) and individuals (n = 300). Note that some families had more than one affected individual. Criteria for the shown classification of pathogenicity are discussed in the main text. C Considered disease categories, coloured by case status. D Variant types for considered solved cases (including pathogenic, likely pathogenic and cases with evidence of causality, see main text). Small only: all causative variants are SNVs or small INDELs, SSV: at least one causative variant is a splice site variant, SV: at least one causative variant is a large structural variant, Intronic: at least one causative variant is (deep) intronic. E Classification of cases using a previously introduced schema published in [23]: A: variant in novel gene for phenotype with additional (genetic) evidence; B: novel (mechanism) for phenotype; C: known gene for phenotype; D: variant in novel gene for phenotype, further genetic and functional validation studies in progress; SF: secondary finding. Note that two cases have candidate variants in two categories. A detailed description of the categories is provided in Additional file 3: Table S3. Abbreviations: SV, structural variant; SSV, splice site variant

Fig. 2

Overview of the OxClinWGS Study: genetic and clinical results. The OxClinWGS RD cohort included 122 cases, of which 47 were considered solved and a further 12 cases had variants of uncertain significance in lead candidates identified. Two cases had secondary findings. Eight novel disease genes have been identified to date, five of which are confirmed disease genes and three of which have evidence of causality. The asterisk denotes that this group includes novel and putative novel genes. The phenotype for one gene was expanded. Revised clinical diagnoses were provided for six patients, whilst for eight patients, the findings led to changes in their clinical management. Colours denote cases with genes that are considered solved (green), have evidence of causality (light green) or are variants of uncertain significance in lead candidates (brown). Abbreviations: PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne); CNS (central nervous system)

Fig. 3

Validation data for two patients with X-linked structural variants in OxClinWGS study. A–C Patient with ARX deletion: A screenshot showing 125 bp read alignments supporting a de novo deletion of ARX exon 1. Region shown is chrX:25,003,000–25,019,000 (GRCh38). Visualisation is using IGV v2.11.2, with squished and “view as pairs” options. Alignments are coloured by insert size and transcript shown is NM_139058.3. B UCSC genome browser session showing the position of the deletion in relation to the PCR primers and MLPA probes that were used for validation. Also shown is the GC content which rises to > 80% near the distal breakpoint where the coverage drops also in parental genomes. An interactive version is available at https://genome.ucsc.edu/s/AlistairP/ARX_deletion_v6. C The 3 kb deletion was confirmed by the MLPA validation data visualised using coffalyser and shows a drop in signal only for the proband for the exon 1 probe (red arrow). The grey boxes are reference probes and the orange boxes highlight the 95% confidence range of the reference samples used. D–H Complex rearrangement in patient with X-linked neurodevelopmental disorder. D Read count information from short-read sequencing normalised by ngCGH software (https://github.com/seandavi/ngCGH) showing two X chromosome duplications (red arrows). E Split Illumina read-pairs suggest the two duplications are inter-linked. However, two possible configurations can explain the split read pattern. F Circos plot highlights the only SV identified by the Bionano pipeline above the threshold for SV detection. G Genome browser view of the optical maps robustly detects the ~ 600 kb duplication of Xp22.11p21.3 being inserted into Xq27.1, which is present in the carrier mother and both affected male siblings using the Bionano pipeline excluding Complex Multi-Path Regions (CMPR). The red box highlights the duplication inserted into Xq27.1. H The Bionano pipeline without masking CMPR detects ~ 102 kb tandem duplication (red boxes) flanking either side of the 600 kb insertion from Xp22.11-Xp21.3 (blue box), therefore, supporting conformation 1 as suggested in E