The impact of inversions across 33,924 families with rare disease from a national genome sequencing project.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

Improved sensitivity for detection of pathogenic variants in familial NF2-related schwannomatosis.

PURPOSE: To determine the impact of additional genetic screening techniques on the rate of detection of pathogenic variants leading to familial NF2-related schwannomatosis. METHODS: We conducted genetic screening of a cohort of 168 second-generation individuals meeting the clinical criteria for NF2-related schwannomatosis. In addition to the current clinical screening techniques, targeted next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification analysis, we applied additional genetic screening techniques, including karyotype and RNA analysis. For characterisation of a complex structural variant, we also performed long-read sequencing analysis. RESULTS: Additional genetic analysis resulted in increased sensitivity of detection of pathogenic variants from 87% to 95% in our second-generation NF2-related schwannomatosis cohort. A number of pathogenic variants identified through extended analysis had been previously observed after NGS analysis but had been overlooked or classified as variants of uncertain significance. CONCLUSION: Our study indicates there is added value in performing additional genetic analysis for detection of pathogenic variants that are difficult to identify with current clinical genetic screening methods. In particular, RNA analysis is valuable for accurate classification of non-canonical splicing variants. Karyotype analysis and whole genome sequencing analysis are of particular value for identification of large and/or complex structural variants, with additional advantages in the use of long-read sequencing techniques.

Pathogenic variant detection rate varies considerably in male breast cancer families and sporadic cases: minimal additional contribution beyond BRCA2, BRCA1 and CHEK2.

BACKGROUND: Male breast cancer (MBC) affects around 1 in 1000 men and is known to have a higher underlying component of high and moderate risk gene pathogenic variants (PVs) than female breast cancer, particularly in BRCA2. However, most studies only report overall detection rates without assessing detailed family history. METHODS: We reviewed germline testing in 204 families including at least one MBC for BRCA1, BRCA2, CHEK2 c.1100DelC and an extended panel in 93 of these families. Individuals had MBC (n=118), female breast cancer (FBC)(n=80), ovarian cancer (n=3) or prostate cancer-(n=3). Prior probability of having a BRCA1/2 PV was assessed using the Manchester Scoring System (MSS). RESULTS: In the 204 families, BRCA2 was the major contributor, with 51 (25%) having PVs, followed by BRCA1 and CHEK2, with five each (2.45%) but no additional PVs identified, including in families with high genetic likelihood on MSS. Detection rates were 85.7% (12/14) in MSS ≥40 and 65.5% with MSS 30-39 but only 12.8% (6/47) for sporadic breast cancer. PV rates were low and divided equally between BRCA1/2 and CHEK2. CONCLUSION: As expected, BRCA2 PVs predominate in MBC families with rates 10-fold those in CHEK2 and BRCA1. The MSS is an effective tool in assessing the likelihood of BRCA1/2 PVs.

Systematic reanalysis of copy number losses of uncertain clinical significance.

BACKGROUND: Reanalysis of exome/genome data improves diagnostic yield. However, the value of reanalysis of clinical array comparative genomic hybridisation (aCGH) data has never been investigated. Case-by-case reanalysis can be challenging in busy diagnostic laboratories. METHODS AND RESULTS: We harmonised historical postnatal clinical aCGH results from ~16 000 patients tested via our diagnostic laboratory over ~7 years with current clinical guidance. This led to identification of 37 009 copy number losses (CNLs) including 33 857 benign, 2173 of uncertain significance and 979 pathogenic. We found benign CNLs to be significantly less likely to encompass haploinsufficient genes compared with the pathogenic or CNLs of uncertain significance in our database. Based on this observation, we developed a reanalysis pipeline using up-to-date disease association data and haploinsufficiency scores and shortlisted 207 CNLs of uncertain significance encompassing at least one autosomal dominant disease-gene associated with haploinsufficiency or loss-of-function mechanism. Clinical scientist reviews led to reclassification of 15 CNLs of uncertain significance as pathogenic or likely pathogenic. This was ~0.7% of the starting cohort of 2173 CNLs of uncertain significance and 7.2% of 207 shortlisted CNLs. The reclassified CNLs included first cases of CNV-mediated disease for some genes where all previously described cases involved only point variants. Interestingly, some CNLs could not be reclassified because the phenotypes of patients with CNLs seemed distinct from the known clinical features resulting from point variants, thus raising questions about accepted underlying disease mechanisms. CONCLUSIONS: Reanalysis of clinical aCGH data increases diagnostic yield.

Germline testing of BRCA1, BRCA2, PALB2 and CHEK2 c.1100delC in 1514 triple negative familial and isolated breast cancers from a single centre, with extended testing of ATM, RAD51C and RAD51D in over 400.

BACKGROUND: The identification of germline pathogenic gene variants (PGVs) in triple negative breast cancer (TNBC) is important to inform further primary cancer risk reduction and TNBC treatment strategies. We therefore investigated the contribution of breast cancer associated PGVs to familial and isolated invasive TNBC. METHODS: Outcomes of germline BRCA1, BRCA2 and CHEK2_c.1100delC testing were recorded in 1514 women (743-isolated, 771-familial), and for PALB2 in 846 women (541-isolated, 305-familial), with TNBC and smaller numbers for additional genes. Breast cancer free controls were identified from Predicting Risk Of Cancer At Screening and BRIDGES (Breast cancer RIsk after Diagnostic GEne Sequencing) studies. RESULTS: BRCA1_PGVs were detected in 52 isolated (7.0%) and 195 (25.3%) familial cases (isolated-OR=58.9, 95% CI: 16.6 to 247.0), BRCA2_PGVs in 21 (2.8%) isolated and 67 (8.7%) familial cases (isolated-OR=5.0, 95% CI: 2.3 to 11.2), PALB2_PGVs in 9 (1.7%) isolated and 12 (3.9%) familial cases (isolated-OR=8.8, 95% CI: 2.5 to 30.4) and CHEK2_c.1100delC in 0 isolated and 3 (0.45%) familial cases (isolated-OR=0.0, 95% CI: 0.00 to 2.11). BRCA1_PGV detection rate was >10% for all familial TNBC age groups and significantly higher for younger diagnoses (familial: <50 years, n=165/538 (30.7%); ≥50 years, n=30/233 (12.9%); p<0.0001). Women with a G3_TNBC were more likely to have a BRCA1_PGV as compared with a BRCA2 or PALB2_PGV (p<0.0001). 0/743 isolated TNBC had the CHEK2_c.1100delC PGV and 0/305 any ATM_PGV, but 2/240 (0.83%) had a RAD51D_PGV. CONCLUSION: PGVs in BRCA1 are associated with G3_TNBCs. Familial TNBCs and isolated TNBCs <30 years have a >10% likelihood of a PGV in BRCA1. BRCA1_PGVs are associated with younger age of familial TNBC. There was no evidence for any increased risk of TNBC with CHEK2 or ATM PGVs.

Genetic findings in people with schwannomas who do not meet clinical diagnostic criteria for NF2-related schwannomatosis.

BACKGROUND: Most schwannomas are isolated tumours occurring in otherwise healthy people. However, bilateral vestibular schwannomas (BVS) or multiple non-vestibular schwannomas indicate an underlying genetic predisposition. This is most commonly NF2-related schwannomatosis (SWN), but when BVS are absent, this can also indicate SMARCB1-related or LZTR1-related SWN. METHODS: We assessed the variant detection rates for the three major SWN genes (NF2, LZTR1 and SMARCB1) in 154 people, from 150 families, who had at least one non-vestibular schwannoma, but who did not meet clinical criteria for NF2-related SWN at the time of genetic testing. RESULTS: We found that 17 (11%) people from 13 families had a germline SMARCB1 variant and 19 (12%) unrelated individuals had a germline LZTR1 variant. 19 people had an NF2 variant, but 18 of these were mosaic and 17 were only detected when 2 tumours were available for testing. The overall detection rate was 25% using blood alone, but increased to 36% when tumour analysis was included. Another 12 people had a germline variant of uncertain significance (VUS). CONCLUSIONS: There were similar proportions of LZTR1, SMARCB1 or mosaic NF2. However, since an NF2 variant was detected in tumours from 103 people, it is likely that further cases of mosaicism would be detected if more people had additional tumours available for analysis. In addition, if further evidence becomes available to show that the VUSs are pathogenic, this would significantly increase the proportion of people with a genetic diagnosis. Our results indicate the importance of comprehensive genetic testing and improved variant classification.

NF2-related schwannomatosis and other schwannomatosis: an updated genetic and epidemiological study.

OBJECTIVES: New diagnostic criteria for NF2-related schwannomatosis (NF2) were published in 2022. An updated UK prevalence was generated in accordance with these, with an emphasis on the rate of de novo NF2 (a 50% frequency is widely quoted in genetic counselling). The distribution of variant types among de novo and familial NF2 cases was also assessed. METHODS: The UK National NF2 database identifies patients meeting updated NF2 criteria from a highly ascertained population cared for by England's specialised service. Diagnostic prevalence was assessed on 1 February 2023. Molecular analysis of blood and, where possible, tumour specimens for NF2, LZTR1 and SMARCB1 was performed. RESULTS: 1084 living NF2 patients were identified on prevalence day (equivalent to 1 in 61 332). The proportion with NF2 inherited from an affected parent was only 23% in England. If people without a confirmed molecular diagnosis or bilateral vestibular schwannoma are excluded, the frequency of de novo NF2 remains high (72%). Of the identified de novo cases, almost half were mosaic. The most common variant type was nonsense variants, accounting for 173/697 (24.8%) of people with an established variant, but only 18/235 (7.7%) with an inherited NF2 pathogenic variant (p<0.0001). Missense variants had the highest proportion of familial association (56%). The prevalence of LZTR1-related schwannomatosis and SMARCB1-related schwannomatosis was 1 in 527 000 and 1 in 1.1M, respectively, 8.4-18.4 times lower than NF2. CONCLUSIONS: This work confirms a much higher rate of de novo NF2 than previously reported and highlights the benefits of maintaining patient databases for accurate counselling.

The PS4-likelihood ratio calculator: flexible allocation of evidence weighting for case-control data in variant classification.

BACKGROUND: The 2015 American College of Medical Genetics/Association of Molecular Pathology (ACMG/AMP) variant classification framework specifies that case-control observations can be scored as 'strong' evidence (PS4) towards pathogenicity. METHODS: We developed the PS4-likelihood ratio calculator (PS4-LRCalc) for quantitative evidence assignment based on the observed variant frequencies in cases and controls. Binomial likelihoods are computed for two models, each defined by prespecified OR thresholds. Model 1 represents the hypothesis of association between variant and phenotype (eg, OR≥5) and model 2 represents the hypothesis of non-association (eg, OR≤1). RESULTS: PS4-LRCalc enables continuous quantitation of evidence for variant classification expressed as a likelihood ratio (LR), which can be log-converted into log LR (evidence points). Using PS4-LRCalc, observed data can be used to quantify evidence towards either pathogenicity or benignity. Variants can also be evaluated against models of different penetrance. The approach is applicable to balanced data sets generated for more common phenotypes and smaller data sets more typical in very rare disease variant evaluation. CONCLUSION: PS4-LRCalc enables flexible evidence quantitation on a continuous scale for observed case-control data. The converted LR is amenable to incorporation into the now widely used 2018 updated Bayesian ACMG/AMP framework.

Recommendations for laboratory workflow that better support centralised amalgamation of genomic variant data: findings from CanVIG-UK national molecular laboratory survey.

BACKGROUND: National and international amalgamation of genomic data offers opportunity for research and audit, including analyses enabling improved classification of variants of uncertain significance. Review of individual-level data from National Health Service (NHS) testing of cancer susceptibility genes (2002-2023) submitted to the National Disease Registration Service revealed heterogeneity across participating laboratories regarding (1) the structure, quality and completeness of submitted data, and (2) the ease with which that data could be assembled locally for submission. METHODS: In May 2023, we undertook a closed online survey of 51 clinical scientists who provided consensus responses representing all 17 of 17 NHS molecular genetic laboratories in England and Wales which undertake NHS diagnostic analyses of cancer susceptibility genes. The survey included 18 questions relating to 'next-generation sequencing workflow' (11), 'variant classification' (3) and 'phenotypical context' (4). RESULTS: Widely differing processes were reported for transfer of variant data into their local LIMS (Laboratory Information Management System), for the formatting in which the variants are stored in the LIMS and which classes of variants are retained in the local LIMS. Differing local provisions and workflow for variant classifications were also reported, including the resources provided and the mechanisms by which classifications are stored. CONCLUSION: The survey responses illustrate heterogeneous laboratory workflow for preparation of genomic variant data from local LIMS for centralised submission. Workflow is often labour-intensive and inefficient, involving multiple manual steps which introduce opportunities for error. These survey findings and adoption of the concomitant recommendations may support improvement in laboratory dataflows, better facilitating submission of data for central amalgamation.

Extended panel testing in ovarian cancer reveals BRIP1 as the third most important predisposition gene.

PURPOSE: The prevalence of germline pathogenic variants (PVs) in homologous recombination repair (HRR) and Lynch syndrome (LS) genes in ovarian cancer (OC) is uncertain. METHODS: An observational study reporting the detection rate of germline PVs in HRR and LS genes in all OC cases tested in the North West Genomic Laboratory Hub between September 1996 and May 2024. Effect sizes are reported using odds ratios (ORs) and 95% confidence intervals (95% CI) for unselected cases tested between April 2021 and May 2024 versus 50,703 controls from the Breast Cancer Risk after Diagnostic Gene Sequencing study. RESULTS: 2934 women were tested for BRCA1/2 and 433 (14.8%) had a PV. In up to 1572 women tested for PVs in non-BRCA1/2 HRR genes, detection rates were PALB2 = 0.8%, BRIP1 = 1.1%, RAD51C = 0.4% and RAD51D = 0.4%. In 940 unselected cases, BRIP1 (OR = 8.7, 95% CI 4.6-15.8) was the third most common OC predisposition gene followed by RAD51C (OR = 8.3, 95% CI 3.1-23.1), RAD51D (OR = 6.5, 95% CI 2.1-19.7), and PALB2 (OR = 3.9, 95% CI 1.5-10.3). No PVs in LS genes were detected in unselected cases. CONCLUSION: Panel testing in OC resulted in a detection rate of 2% to 3% for germline PVs in non-BRCA1/2 HRR genes, with the largest contributor being BRIP1. Screening for LS in unselected cases of OC is unnecessary.

ATP6V0C variants impair V-ATPase function causing a neurodevelopmental disorder often associated with epilepsy.

The vacuolar H+-ATPase is an enzymatic complex that functions in an ATP-dependent manner to pump protons across membranes and acidify organelles, thereby creating the proton/pH gradient required for membrane trafficking by several different types of transporters. We describe heterozygous point variants in ATP6V0C, encoding the c-subunit in the membrane bound integral domain of the vacuolar H+-ATPase, in 27 patients with neurodevelopmental abnormalities with or without epilepsy. Corpus callosum hypoplasia and cardiac abnormalities were also present in some patients. In silico modelling suggested that the patient variants interfere with the interactions between the ATP6V0C and ATP6V0A subunits during ATP hydrolysis. Consistent with decreased vacuolar H+-ATPase activity, functional analyses conducted in Saccharomyces cerevisiae revealed reduced LysoSensor fluorescence and reduced growth in media containing varying concentrations of CaCl2. Knockdown of ATP6V0C in Drosophila resulted in increased duration of seizure-like behaviour, and the expression of selected patient variants in Caenorhabditis elegans led to reduced growth, motor dysfunction and reduced lifespan. In summary, this study establishes ATP6V0C as an important disease gene, describes the clinical features of the associated neurodevelopmental disorder and provides insight into disease mechanisms.

Differential rates of germline heterozygote and mosaic variants in NF2 may show varying propensity for meiotic or mitotic mutation.

NF2-related schwannomatosis is an autosomal dominant tumour predisposition condition that causes multiple benign tumours of the nervous system, especially schwannomas. This results from germline pathogenic variants in the NF2 gene, which are most commonly de novo NF2 nonsense variants. Over half of these de novo variants occur at just six CpG dinucleotides. In this study, we show that the six NF2 CpG nonsense variants make up 54% (136/252) of de novo nonsense variants, despite constituting <10% of nonsense positions in the germline (total=62), and that this pattern is different from the APC gene, which is also known to have a high rate of mosaicism. In addition, the NF2 c.586C>T; p.(Arg196Ter) has a higher de novo heterozygote to mosaicism ratio than the five other CpG variants (73.1% vs 53.7%, p=0.03) and the neighbouring CpG variant (NF2 c.592C>T; p.(Arg198Ter) 38.5%, p=0.02). This may be due to differences in rates of mutation at meiosis versus mitosis.

Detection of pathogenic variants in breast cancer susceptibility genes in bilateral breast cancer.

PURPOSE: To investigate the frequency of germline pathogenic variants (PVs) in women with bilateral breast cancer. METHODS: We undertook BRCA1/2 and CHEK2 c.1100delC molecular analysis in 764 samples and a multigene panel in 156. Detection rates were assessed by age at first primary, Manchester Score, and breast pathology. Oestrogen receptor (ER) status of the contralateral versus first breast cancer was compared on 1081 patients with breast cancer with BRCA1/BRCA2 PVs. RESULTS: 764 women with bilateral breast cancer have undergone testing of BRCA1/2 and CHEK2; 407 were also tested for PALB2 and 177 for ATM. Detection rates were BRCA1 11.6%, BRCA2 14.0%, CHEK2 2.4%, PALB2 1.0%, ATM 1.1% and, for a subset of mainly very early onset tumours, TP53 4.6% (9 of 195). The highest PV detection rates were for triple negative cancers for BRCA1 (26.4%), grade 3 ER+HER2 for BRCA2 (27.9%) and HER2+ for CHEK2 (8.9%). ER status of the first primary in BRCA1 and BRCA2 PV heterozygotes was strongly predictive of the ER status of the second contralateral tumour since ~90% of second tumours were ER- in BRCA1 heterozygotes, and 50% were ER- in BRCA2 heterozygotes if the first was ER-. CONCLUSION: We have shown a high rate of detection of BRCA1 and BRCA2 PVs in triple negative and grade 3 ER+HER2- first primary diagnoses, respectively. High rates of HER2+ were associated with CHEK2 PVs, and women ≤30 years were associated with TP53 PVs. First primary ER status in BRCA1/2 strongly predicts the second tumour will be the same ER status even if unusual for PVs in that gene.

Differential involvement of germline pathogenic variants in breast cancer genes between DCIS and low-grade invasive cancers.

PURPOSE: To investigate frequency of germline pathogenic variants (PVs) in women with ductal carcinoma in situ (DCIS) and grade 1 invasive breast cancer (G1BC). METHODS: We undertook BRCA1/2 analysis in 311 women with DCIS and 392 with G1BC and extended panel testing (non-BRCA1/2) in 176/311 with DCIS and 156/392 with G1BC. We investigated PV detection by age at diagnosis, Manchester Score (MS), DCIS grade and receptor status. RESULTS: 30/311 (9.6%) with DCIS and 16/392 with G1BC (4.1%) had a BRCA1/2 PV (p=0.003), and 24/176-(13.6%) and 7/156-(4.5%), respectively, a non-BRCA1/2 PV (p=0.004). Increasing MS was associated with increased likelihood of BRCA1/2 PV in both DCIS and G1BC, although the 10% threshold was not predictive for G1GB. 13/32 (40.6%) DCIS and 0/17 with G1BC <40 years had a non-BRCA1/2 PV (p<0.001). 0/16 DCIS G1 had a PV. For G2 and G3 DCIS, PV rates were 10/98 (BRCA1/2) and 9/90 (non-BRCA1/2), and 8/47 (BRCA1/2) and 8/45 (non-BRCA1/2), respectively. 6/9 BRCA1 and 3/26 BRCA2-associated DCIS were oestrogen receptor negative-(p=0.003). G1BC population testing showed no increased PV rate (OR=1.16, 95% CI 0.28 to 4.80). CONCLUSION: DCIS is more likely to be associated with both BRCA1/2 and non-BRCA1/2 PVs than G1BC. Extended panel testing ought to be offered in young-onset DCIS where PV detection rates are highest.

Germline mismatch repair (MMR) gene analyses from English NHS regional molecular genomics laboratories 1996-2020: development of a national resource of patient-level genomics laboratory records.

OBJECTIVE: To describe national patterns of National Health Service (NHS) analysis of mismatch repair (MMR) genes in England using individual-level data submitted to the National Disease Registration Service (NDRS) by the NHS regional molecular genetics laboratories. DESIGN: Laboratories submitted individual-level patient data to NDRS against a prescribed data model, including (1) patient identifiers, (2) test episode data, (3) per-gene results and (4) detected sequence variants. Individualised per-laboratory algorithms were designed and applied in NDRS to extract and map the data to the common data model. Laboratory-level MMR activity audit data from the Clinical Molecular Genetics Society/Association of Clinical Genomic Science were used to assess early years' missing data. RESULTS: Individual-level data from patients undergoing NHS MMR germline genetic testing were submitted from all 13 English laboratories performing MMR analyses, comprising in total 16 722 patients (9649 full-gene, 7073 targeted), with the earliest submission from 2000. The NDRS dataset is estimated to comprise >60% of NHS MMR analyses performed since inception of NHS MMR analysis, with complete national data for full-gene analyses for 2016 onwards. Out of 9649 full-gene tests, 2724 had an abnormal result, approximately 70% of which were (likely) pathogenic. Data linkage to the National Cancer Registry demonstrated colorectal cancer was the most frequent cancer type in which full-gene analysis was performed. CONCLUSION: The NDRS MMR dataset is a unique national pan-laboratory amalgamation of individual-level clinical and genomic patient data with pseudonymised identifiers enabling linkage to other national datasets. This growing resource will enable longitudinal research and can form the basis of a live national genomic disease registry.

High likelihood of actionable pathogenic variant detection in breast cancer genes in women with very early onset breast cancer.

BACKGROUND: While the likelihood of identifying constitutional breast cancer-associated BRCA1, BRCA2 and TP53 pathogenic variants (PVs) increases with earlier diagnosis age, little is known about the correlation with age at diagnosis in other predisposition genes. Here, we assessed the contribution of known breast cancer-associated genes to very early onset disease. METHODS: Sequencing of BRCA1, BRCA2, TP53 and CHEK2 c.1100delC was undertaken in women with breast cancer diagnosed ≤30 years. Those testing negative were screened for PVs in a minimum of eight additional breast cancer-associated genes. Rates of PVs were compared with cases ≤30 years from the Prospective study of Outcomes in Sporadic vs Hereditary breast cancer (POSH) study. RESULTS: Testing 379 women with breast cancer aged ≤30 years identified 75 PVs (19.7%) in BRCA1, 35 (9.2%) in BRCA2, 22 (5.8%) in TP53 and 2 (0.5%) CHEK2 c.1100delC. Extended screening of 184 PV negative women only identified eight additional actionable PVs. BRCA1/2 PVs were more common in women aged 26-30 years than in younger women (p=0.0083) although the younger age group had rates more similar to those in the POSH cohort. Out of 26 women with ductal carcinoma in situ (DCIS) alone, most were high-grade and 11/26 (42.3%) had a PV (TP53=6, BRCA2=2, BRCA1=2, PALB2=1). This PV yield is similar to the 61 (48.8%) BRCA1/2 PVs identified in 125 women with triple-negative breast cancer. The POSH cohort specifically excluded pure DCIS which may explain lower TP53 PV rates in this group (1.7%). CONCLUSION: The rates of BRCA1, BRCA2 and TP53 PVs are high in very early onset breast cancer, with limited benefit from testing of additional breast cancer-associated genes.

Personalised virtual gene panels reduce interpretation workload and maintain diagnostic rates of proband-only clinical exome sequencing for rare disorders.

PURPOSE: The increased adoption of genomic strategies in the clinic makes it imperative for diagnostic laboratories to improve the efficiency of variant interpretation. Clinical exome sequencing (CES) is becoming a valuable diagnostic tool, capable of meeting the diagnostic demand imposed by the vast array of different rare monogenic disorders. We have assessed a clinician-led and phenotype-based approach for virtual gene panel generation for analysis of targeted CES in patients with rare disease in a single institution. METHODS: Retrospective survey of 400 consecutive cases presumed by clinicians to have rare monogenic disorders, referred on singleton basis for targeted CES. We evaluated diagnostic yield and variant workload to characterise the usefulness of a clinician-led approach for generation of virtual gene panels that can incorporate up to three different phenotype-driven gene selection methods. RESULTS: Abnormalities of the nervous system (54.5%), including intellectual disability, head and neck (19%), skeletal system (16%), ear (15%) and eye (15%) were the most common clinical features reported in referrals. Combined phenotype-driven strategies for virtual gene panel generation were used in 57% of cases. On average, 7.3 variants (median=5) per case were retained for clinical interpretation. The overall diagnostic rate of proband-only CES using personalised phenotype-driven virtual gene panels was 24%. CONCLUSIONS: Our results show that personalised virtual gene panels are a cost-effective approach for variant analysis of CES, maintaining diagnostic yield and optimising the use of resources for clinical genomic sequencing in the clinic.

BRCA1/2 in non-mucinous epithelial ovarian cancer: tumour with or without germline testing?

National guidelines recommend testing all cases of non-mucinous epithelial ovarian cancer (NMEOC) for germline (blood) and somatic (tumour) BRCA1/2 pathogenic variants (PVs). We performed paired germline and somatic BRCA1/2 testing in consecutive cases of NMEOC (n = 388) to validate guidelines. Thirty-four somatic BRCA1/2 (sBRCA) PVs (9.7%) were detected in 350 cases with germline BRCA1/2 (gBRCA) wild-type. All sBRCA PVs were detected in non-familial cases. By analysing our regional germline BRCA1/2 database there were 92/1114 (8.3%) gBRCA PVs detected in non-familial cases (only 3% ≥70 years old) and 245/641 (38.2%) in familial cases. Germline non-familial cases were dominated by BRCA2 in older women (8/271 ≥ 70 years old, all BRCA2). The ratio of sBRCA-to-gBRCA was ≤1.0 in women aged <70 years old, compared to 5.2 in women aged ≥70 years old (P = 0.005). The likelihood of missed germline BRCA1/2 PVs (copy-number variants missed on most somatic assays) by testing only tumour DNA was 0.4% in women aged ≥70 years old. We recommend reflex tumour BRCA1/2 testing in all NMEOC cases, and that gBRCA testing is not required for women aged ≥70 years old with no identifiable tumour BRCA1/2 PV and/or family history of breast, ovarian, prostate and/or pancreatic cancer.

Quantifying prediction of pathogenicity for within-codon concordance (PM5) using 7541 functional classifications of BRCA1 and MSH2 missense variants.

PURPOSE: Conditions and thresholds applied for evidence weighting of within-codon concordance (PM5) for pathogenicity vary widely between laboratories and expert groups. Because of the sparseness of available clinical classifications, there is little evidence for variation in practice. METHODS: We used as a truthset 7541 dichotomous functional classifications of BRCA1 and MSH2, spanning 311 codons of BRCA1 and 918 codons of MSH2, generated from large-scale functional assays that have been shown to correlate excellently with clinical classifications. We assessed PM5 at 5 stringencies with incorporation of 8 in silico tools. For each analysis, we quantified a positive likelihood ratio (pLR, true positive rate/false positive rate), the predictive value of PM5-lookup in ClinVar compared with the functional truthset. RESULTS: pLR was 16.3 (10.6-24.9) for variants for which there was exactly 1 additional colocated deleterious variant on ClinVar, and the variant under examination was equally or more damaging when analyzed using BLOSUM62. pLR was 71.5 (37.8-135.3) for variants for which there were 2 or more colocated deleterious ClinVar variants, and the variant under examination was equally or more damaging than at least 1 colocated variant when analyzed using BLOSUM62. CONCLUSION: These analyses support the graded use of PM5, with potential to use it at higher evidence weighting where more stringent criteria are met.

Reclassification of clinically-detected sequence variants: Framework for genetic clinicians and clinical scientists by CanVIG-UK (Cancer Variant Interpretation Group UK).

PURPOSE: Variant classifications may change over time, driven by emergence of fresh or contradictory evidence or evolution in weighing or combination of evidence items. For variant classifications above the actionability threshold, which is classification of likely pathogenic or pathogenic, clinical actions may be irreversible, such as risk-reducing surgery or prenatal interventions. Variant reclassification up or down across the actionability threshold can therefore have significant clinical consequences. Laboratory approaches to variant reinterpretation and reclassification vary widely. METHODS: Cancer Variant Interpretation Group UK is a multidisciplinary network of clinical scientists and genetic clinicians from across the 24 Molecular Diagnostic Laboratories and Clinical Genetics Services of the United Kingdom (NHS) and Republic of Ireland. We undertook surveys, polls, and national meetings of Cancer Variant Interpretation Group UK to evaluate opinions about clinical and laboratory management regarding variant reclassification. RESULTS: We generated a consensus framework on variant reclassification applicable to cancer susceptibility genes and other clinical areas, which provides explicit recommendations for clinical and laboratory management of variant reclassification scenarios on the basis of the nature of the new evidence, the magnitude of evidence shift, and the final classification score. CONCLUSION: In this framework, clinical and laboratory resources are targeted for maximal clinical effect and minimal patient harm, as appropriate to all resource-constrained health care settings.