PMC:6037202 / 37716-56082 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/6037202","sourcedb":"PMC","sourceid":"6037202","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6037202","text":"Discussion\n\nVariant Detection Rates in an MPT Series\nWe previously reported a retrospective series of MPT individuals (defined as having two primary tumors before 60 years of age) referred to a UK clinical genetics service without pre-assessment and observed that 20.7% (44/212) were found to have a molecular diagnosis upon routine targeted molecular genetic testing, including BRCA1 and BRCA2 testing, mismatch-repair gene analysis, or other single-gene testing (APC [MIM: 611731], MUTYH [MIM: 604933], PTEN, TP53 [MIM: 191170], and RB1 [MIM: 614041]).31\nIn the current study, we addressed whether comprehensive genetic analysis in pre-assessed individuals with MPTs might increase the diagnostic yield over routine targeted testing. Thus, we analyzed 460 MPT-affected individuals who had previously undergone routine genetic assessment and/or molecular testing (but without a molecular diagnosis) by using WGS for variants in 83 CPGs and identified a P/LP variant in 67/440 (15.2%) probands (incorporating SNVs, indels, and SVs), including those affected by moderate- and high-risk CPGs.\nBecause the MPT cohort reported here was mostly ascertained from UK genetics centers (and was similar to the previous retrospective cohort that did not have a known genetic cause), we estimate (by assuming that WGS would detect variants identified by routine targeted sequencing approaches) that comprehensive genetic analysis in a genetics-center-referred series of individuals with MPTs (and no prior genetic testing) would detect a P/LP variant in around a third of individuals (20.7% + 12.1% [estimated under the assumption of a diagnostic yield of 15.2% in the 79.3% of individuals without a variant in routine testing] = 32.8%). The estimated proportion of individuals with a P/LP variant and a typical tumor would be ∼27.5% (20.7% [all of those with variants detected by targeted analysis had a typical tumor] + [79.3% × 8.6% = 6.8%]). Therefore, in individuals seen in a genetic clinic, the presence of MPTs (two tumors before 60 years of age or three before 70 years of age) could be taken as an indication for considering genetic testing.\nThe estimates for diagnostic yield are approximate and would be influenced by ascertainment processes but do suggest that a comprehensive testing for CPG variants significantly increases the detection of P/LP variants over the targeted testing that has been routinely employed in most genetics centers.\nMost MPT-affected individuals (38/67 [56.7%] and 38/440 [8.6%] of all pre-assessed probands tested in the current study) with a P/LP variant had been diagnosed with a tumor type characteristically associated with variants in the relevant CPG, findings that have the greatest clinical utility. In, addition, a further 8/440 (1.8%) had a VUS and a previous diagnosis of a characteristic tumor. Such VUSs might eventually be reclassified as LP variants with further investigations (e.g., tumor studies or functional analysis) or additional clinical information (e.g., segregation analysis). However, interpretation of segregation data should be cautious in cancer-predisposition syndromes because of incomplete penetrance and a higher probability of phenocopies. Tumor studies for loss of heterozygosity do not provide absolute confirmation or exclusion of pathogenicity, and together these considerations reinforce the importance of data-sharing initiatives such as ClinVar.20\nA major influence on the number and pattern of variants detected in a study such as this is the tumor phenotypes occurring in the cohort, which in this case reflect both the population incidence and the patterns of referral for genetic assessment and investigation. Compared with MPT-affected individuals in cancer registries, our series is enriched with combinations such as breast-ovary (4.4% versus 1.9%) and breast-colorectal (5.5% versus 2.8%), most likely reflecting common cancers with a significant hereditary component and for which genetic testing has been routinely available for a number of years. Many of these cancers are sex specific, most likely contributing to the uneven sex distribution in this series. Some combination types making up \u003e1% of MPT combinations, e.g., breast-thyroid (3.6% in MPT data), are not observed frequently (\u003c1%) in the population-based cohort used here, which could be accounted for by referral prompted by suspicion of germline PTEN variants.\nBreast cancer accounted for almost a quarter of tumors in our series, and most genes in which deleterious variants were detected are breast CPGs, many of which are not routinely tested in the UK. Pathogenic variants in ATM and CHEK2 are associated with moderate risks,32, 33 and these genes had not been tested by the referring center in any of the individuals with P/LP variants. Six probands had pathogenic variants in PALB2, a gene initially thought to confer moderate risk34 but subsequently reported to have a penetrance somewhere between that of moderate- and high-risk genes such as BRCA1 and BRCA2.35\nGenes can remain uninvestigated by clinicians not only because of uncertainty surrounding risks but also because of recency of discovery. A number of CPGs in which variants were identified, such as MAX and FH, have been relatively recently described as causing pheochromocytoma and paraganglioma. The appearance of these variants in this analysis most likely reflects a lack of availability of testing at the time of consultation and subsequent referral for inclusion in the study. Molecular genetic testing has been available for other genes such as MLH1 and PTEN for a greater period of time, but some individuals appeared not to have fulfilled the clinical testing criteria applied in the referring center. TP53 is a further well-established CPG that is associated with diverse and multiple cancers and has clear clinical testing criteria that are often not fulfilled. Despite this, no pathogenic variants were detected. Germline TP53-variant-related phenotypes (including rare and/or early-onset cancers) are more clearly identifiable clinically and are less likely to appear in cohorts such as ours without specific ascertainment for them. Consistent with this are mutation detection rates of ∼4% in individuals with earlier-onset (≤30 years) breast cancer36 and ∼17% in MPT-affected individuals who were referred for germline TP53 testing and who generally fulfilled criteria for that investigation, had tumors characteristic of Li Fraumeni syndrome, and had an average age at diagnosis (of a first primary tumor) before 30 years.2\nAlthough we report the application of WGS to an adult MPT series, other studies have used agnostic NGS strategies in cohorts with single-site cancer. The detection rate of pathogenic variants in these analyses could be influenced by the assay used, the variant filtering and assessment applied, and the nature of the series in terms of both phenotype and ascertainment. The application of a 76-gene panel to ∼1,000 cancer-affected adults referred for germline genetic testing and ACMG-guideline-based assessment of the resulting variants showed a 17.5% rate,37 whereas tumor-normal sequencing of a similarly sized series with advanced cancer from the same center (regardless of genetic testing referral) reported an equivalent figure of 12.6%.38 The genes containing the most frequent pathogenic variants in both studies were similar to those in the current study (BRCA1, BRCA2, CHEK2, and ATM), but the detection rates were lower than our estimate of around a third of newly referred MPT-affected individuals, most likely reflecting a greater likelihood of a germline pathogenic variant in both genetics referrals and in MPT-affected individuals. Studies of WGS and/or WES applied to unselected pediatric cancer series have also shown pathogenic-variant detection rates close to 10% but a contrasting range of affected genes, suggesting that TP53 and genes associated with embryonal tumors play a far greater role.39, 40, 41\n\nAtypical Tumor-Variant Associations in MPT-Affected Individuals\nIn this study, we applied multi-gene testing in all affected individuals irrespective of the tumor types diagnosed. Strikingly, this resulted in the identification of a large number of probands (29/67 [43.2%]) who harbored a P/LP CPG variant but whose tumor phenotypes were not entirely typical for the relevant CPG. This situation has been frequently reported by other studies of extensive NGS testing of cancer cohorts37, 40, 42 and represents a challenge for clinicians because the relevance of the variant to cancer risk in the consultand (including unaffected family members) is less clear. Specific atypical associations observed in this analysis were heterogeneous, and numbers were small, but some patterns were noted; for example, 5/16 (31.2%) carriers of CHEK2 variants had been previously diagnosed with renal cell carcinoma (RCC) (breast cancer occurred in 8/16 [50%]). An odds ratio of 2.1 for RCC has previously been observed in CHEK2-variant carriers but only in association with the c.470T\u003eC (p.Ile157Thr) founder variant in a Polish population.43 2/6 (33.3%) carriers of PALB2 variants had cutaneous melanoma before the age of 40 years, and 2/10 (20%) individuals with ATM variants had thyroid cancer before that age, but an analysis of 182 melanoma families demonstrated only one pathogenic PALB2 variant,44 and thyroid malignancies have not been reported at increased frequency in carriers of homozygous or heterozygous ATM variants.1, 45\nOne potential interpretation of these atypical tumor phenotypes is that the tumor spectrum associated with some CPGs is wider than currently recognized given that, to date, testing of particular genes has been limited to specific phenotypes. For example, although FH variants were demonstrated to predispose to RCC in 2002, they were shown to predispose to pheochromocytoma and paraganglioma 12 years later.46, 47, 48 We therefore suggest that further “agnostic” research testing of a comprehensive panel of CPGs in MPT-affected individuals could lead to the identification of novel associations between genes and tumor phenotypes. Our observation of a significantly higher rate of loss-of-function variants associated with non-characteristic tumors in our cohort than in the gnomAD dataset suggests that at least some variants identified in individuals with atypical phenotypes are relevant. We would, however, urge caution in automatically linking a pathogenic CPG variant to the observed tumor phenotype without further evidence, such as larger studies of variant carriers or tumor studies that demonstrate a variant’s causative effect.\nAnother possibility is that tumors can occur coincidentally in the presence of a pathogenic constitutional CPG variant. Variants might be considered causative in some contexts or tissues (and would therefore be likely to pass our filtering and assessment) but potentially not in others. For example, an in-frame FH insertion (c.1433−1434insAAA [p.Lys477_Asn478insLys] [Ensembl: ENST00000366560; GenBank: NM_000143.3]) was identified in three individuals, none of whom had been diagnosed with typical hereditary leiomyoma or RCC tumors. This variant causes recessively inherited fumarate hydratase deficiency (MIM: 606812) and has been demonstrated to disrupt enzyme activity.49 However, its significance to cancer predisposition in the heterozygous state is less well defined.\nUnusual MPT-CPG associations can occur when an individual harbors variants in multiple CPGs, either because (at least) one of the variants remains unidentified through diagnostic testing or because an interactive effect exists between them. We have previously reviewed this phenomenon and described it as multiple inherited neoplasia alleles syndrome (MINAS),50 and WGS identified two further examples in our cohort. In the case of PMS2 and BMPR1A variants, the former appears to be penetrant on the basis of tumor studies, whereas the significance of the latter is unclear. Nevertheless, the identification of MINAS cases such as this provides clinicians the opportunity to obtain further evidence. For the individual with FH and MAX variants, it is easier to attribute the diagnosed pheochromocytomas to the truncating MAX variant, but evidence for the role of FH in this tumor type is accumulating, and this variant could have contributed to tumorigenesis.\n\nValue of Germline WGS in the Analysis of MPTs\nAlthough 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.\nIn 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.\nWGS 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.\nGiven 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.\nIn 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 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