Value of Germline WGS in the Analysis of MPTs
Although WGS could arguably offer the most sensitive and comprehensive strategy for detecting germline CPG variants, it is resource intensive in terms of sequencing, data storage, and analytical capacity. In this study, the conservative variant filtering and assessment and the small number of non-coding variants that were used for data interrogation reduced the post-sequencing burden of variants, but small changes to these processes would lead to significant increases with uncertain clinical utility. The approximate cost per sample of WGS as part of the NIHR BioResource Rare Disease project is $1,400, consistent with figures collated by the National Human Genome Research Institute in 2016 and higher than the $1,000 per exome derived from that survey.51 The TCP in our department is currently charged at around $450 per sample. Justification of the extra costs compared with those of other NGS assays, such as panel tests or WES, requires the demonstration that WGS can increase the diagnostic rate over that of other approaches through enhanced detection of coding SNVs and indels, SV identification, or analysis of non-coding regions.
In our analysis, the TCP produced a higher mean depth but a slightly lower percentage of target bases covered at ≥10× than the equivalent regions in WGS data (99.1% versus 100%). WGS identified one TMEM127 SNV (assessed as a VUS) that wasn’t detected by the TCP because the relevant nucleotide was covered by only two reads. Five additional filtered variants from the WGS data weren’t called from panel data, and one of them was assessed as likely pathogenic. This was because the VAF was marginally below the chosen threshold, an issue that also accounted for the calling of two pathogenic variants from TCP data but not from WGS. Non-detection of lower-VAF variants could be resolved through more sensitive bioinformatic filtering of data from either assay. 15 genes on our list of 83 were not targeted by the panel (but no significant variants were detected in them). This illustrates the broader scope of WGS, but our results do not suggest that WGS offers enhanced CPG SNV or indel detection at present.
WGS identified six SVs predicted to affect a gene of interest, and two of these occurred in an individual whose personal or family history included tumors consistent with variants in that gene. The medical record showed no evidence that the individual with the PTEN inversion exhibited other features of constitutional variants in this gene, such as macrocephaly, as well as no record of an examination in a consultation where only BRCA1 and BRCA2 testing was anticipated. Although the numbers of potentially pertinent SVs are small, these aberrations are unlikely to be detected by panel or exome sequencing alone. Copy-number variation can be identified from the analysis of read counts in WES or panel data,52 but most diagnostic laboratories rely on techniques such as multiplex probe ligation assays (MLPAs) to test individual genes. If MLPA analysis is applied to many genes, then the cost could make WGS more economical than WES or panel-based testing, but investigating this would require a detailed cost-benefit analysis. Furthermore, WGS can detect inversions and translocations that are not characterized by MLPA. A note of caution, however, arises from a deletion involving BRCA2 exons 14–16; we were made aware of this deletion by the referring clinician, but it was not detected through our analyses.
Given the limited benefits of WGS over WES and panel analysis demonstrated in this study, a key advantage is the ability to prospectively or retrospectively interrogate regions that are not currently known to be clinically relevant. This includes novel CPGs (many of the P/LP variants in this analysis were detected because the gene or region was not available for testing at the time of consultation). WGS costs should therefore be considered in the context of possible future demand for re-investigation and the consequent resource burden required for this if the region of interest (including non-coding regions) is not sequenced in the first instance. Adequate systems for prioritizing and assessing the multitude of non-coding variants generated by WGS for clinical use do not yet exist.53 Consequently, few clinically non-coding variants are currently known, and we did not identify any of them in this analysis. However, evidence of regulatory elements that influence the expression of any given gene is accumulating,54 and high-throughput functional assays for studying them provide the opportunity to define diagnostically significant variants affecting CPGs.55 If this process were able to elucidate clinically relevant variants, the case for WGS as a first-line investigative tool would become more compelling.
In summary, we have demonstrated that the application of comprehensive CPG testing to a cohort of previously investigated MPT-affected individuals resulted in the detection of multiple pathogenic variants with relevance to the management of those individuals and their relatives. The finding that comprehensive genetic analysis of MPT-affected individuals can frequently result in the identification of pathogenic CPG variants that cannot automatically be attributed as causative for the observed MPT clinical phenotype has important implications both for clinical practice and for future research into the phenotypic consequences of germline CPG variants. Summing together variant detection rates from a previous series of MPT-affected individuals ascertained in a similar manner and the present results suggests that first-line application of WGS (or other strategies for comprehensive CPG variant detection) to a clinical-genetics-referral-based cohort of MPT-affected individuals would detect a deleterious mutation in about a third of individuals, a large proportion of whom would not have a family history of cancer in a first-degree relative.