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.

De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

HNRNPC haploinsufficiency affects alternative splicing of intellectual disability-associated genes and causes a neurodevelopmental disorder.

Heterogeneous nuclear ribonucleoprotein C (HNRNPC) is an essential, ubiquitously abundant protein involved in mRNA processing. Genetic variants in other members of the HNRNP family have been associated with neurodevelopmental disorders. Here, we describe 13 individuals with global developmental delay, intellectual disability, behavioral abnormalities, and subtle facial dysmorphology with heterozygous HNRNPC germline variants. Five of them bear an identical in-frame deletion of nine amino acids in the extreme C terminus. To study the effect of this recurrent variant as well as HNRNPC haploinsufficiency, we used induced pluripotent stem cells (iPSCs) and fibroblasts obtained from affected individuals. While protein localization and oligomerization were unaffected by the recurrent C-terminal deletion variant, total HNRNPC levels were decreased. Previously, reduced HNRNPC levels have been associated with changes in alternative splicing. Therefore, we performed a meta-analysis on published RNA-seq datasets of three different cell lines to identify a ubiquitous HNRNPC-dependent signature of alternative spliced exons. The identified signature was not only confirmed in fibroblasts obtained from an affected individual but also showed a significant enrichment for genes associated with intellectual disability. Hence, we assessed the effect of decreased and increased levels of HNRNPC on neuronal arborization and neuronal migration and found that either condition affects neuronal function. Taken together, our data indicate that HNRNPC haploinsufficiency affects alternative splicing of multiple intellectual disability-associated genes and that the developing brain is sensitive to aberrant levels of HNRNPC. Hence, our data strongly support the inclusion of HNRNPC to the family of HNRNP-related neurodevelopmental disorders.

EyeG2P: an automated variant filtering approach improves efficiency of diagnostic genomic testing for inherited ophthalmic disorders.

BACKGROUND: Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient. METHODS: We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis. RESULTS: EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield. CONCLUSION: Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders.

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.

Multidisciplinary team directed analysis of whole genome sequencing reveals pathogenic non-coding variants in molecularly undiagnosed inherited retinal dystrophies.

The purpose of this paper is to identify likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes, using genome sequencing (GS). Patients with IRD were recruited to the study and underwent comprehensive ophthalmological evaluation and GS. The results of GS were investigated through virtual gene panel analysis, and plausible pathogenic variants and clinical phenotype evaluated by the multidisciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbor a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested by messenger RNA analysis, minigene or luciferase reporter assays. Previously unreported, likely pathogenic, non-coding variants in 7 genes (PRPF31, NDP, IFT140, CRB1, USH2A, BBS10 and GUCY2D), were identified in 11 patients. These were shown to lead to mis-splicing (PRPF31, IFT140, CRB1 and USH2A) or altered transcription levels (BBS10 and GUCY2D). MDT-led, phenotype-driven, non-coding variant re-analysis of GS is effective in identifying the missing causative alleles.

Germline intergenic duplications at Xq26.1 underlie Bazex-Dupré-Christol basal cell carcinoma susceptibility syndrome.

BACKGROUND: Bazex-Dupré-Christol syndrome (BDCS; MIM301845) is a rare X-linked dominant genodermatosis characterized by follicular atrophoderma, congenital hypotrichosis and multiple basal cell carcinomas (BCCs). Previous studies have linked BDCS to an 11·4-Mb interval on chromosome Xq25-q27.1. However, the genetic mechanism of BDCS remains an open question. OBJECTIVES: To investigate the genetic aetiology and molecular mechanisms underlying BDCS. METHODS: We ascertained multiple individuals from eight unrelated families affected with BDCS (F1-F8). Whole-exome (F1 and F2) and genome sequencing (F3) were performed to identify putative disease-causing variants within the linkage region. Array comparative genomic hybridization and quantitative polymerase chain reaction (PCR) were used to explore copy number variations, followed by long-range gap PCR and Sanger sequencing to amplify the duplication junctions and to define the head-tail junctions. Hi-C was performed on dermal fibroblasts from two affected individuals with BDCS and one control. Public datasets and tools were used to identify regulatory elements and transcription factor binding sites within the minimal duplicated region. Immunofluorescence was performed in hair follicles, BCCs and trichoepitheliomas from patients with BDCS and sporadic BCCs. The ACTRT1 variant c.547dup (p.Met183Asnfs*17), previously proposed to cause BDCS, was evaluated with t allele frequency calculator. RESULTS: In eight families with BDCS, we identified overlapping 18-135-kb duplications (six inherited and two de novo) at Xq26.1, flanked by ARHGAP36 and IGSF1. Hi-C showed that the duplications did not affect the topologically associated domain, but may alter the interactions between flanking genes and putative enhancers located in the minimal duplicated region. We detected ARHGAP36 expression near the control hair follicular stem cell compartment, and found increased ARHGAP36 levels in hair follicles in telogen, in BCCs and in trichoepitheliomas from patients with BDCS. ARHGAP36 was also detected in sporadic BCCs from individuals without BDCS. Our modelling showed the predicted maximum tolerated minor allele frequency of ACTRT1 variants in control populations to be orders of magnitude higher than expected for a high-penetrant ultra-rare disorder, suggesting loss of function of ACTRT1 variants to be an unlikely cause for BDCS. CONCLUSIONS: Noncoding Xq26.1 duplications cause BDCS. The BDCS duplications most likely lead to dysregulation of ARHGAP36. ARHGAP36 is a potential therapeutic target for both inherited and sporadic BCCs. What is already known about this topic? Bazex-Dupré-Christol syndrome (BDCS) is a rare X-linked basal cell carcinoma susceptibility syndrome linked to an 11·4-Mb interval on chromosome Xq25-q27.1. Loss-of-function variants in ACTRT1 and its regulatory elements were suggested to cause BDCS. What does this study add? BDCS is caused by small tandem noncoding intergenic duplications at chromosome Xq26.1. The Xq26.1 BDCS duplications likely dysregulate ARHGAP36, the flanking centromeric gene. ACTRT1 loss-of-function variants are unlikely to cause BDCS. What is the translational message? This study provides the basis for accurate genetic testing for BDCS, which will aid precise diagnosis and appropriate surveillance and clinical management. ARHGAP36 may be a novel therapeutic target for all forms of sporadic basal cell carcinomas.

Recommendations for clinical interpretation of variants found in non-coding regions of the genome.

BACKGROUND: The majority of clinical genetic testing focuses almost exclusively on regions of the genome that directly encode proteins. The important role of variants in non-coding regions in penetrant disease is, however, increasingly being demonstrated, and the use of whole genome sequencing in clinical diagnostic settings is rising across a large range of genetic disorders. Despite this, there is no existing guidance on how current guidelines designed primarily for variants in protein-coding regions should be adapted for variants identified in other genomic contexts. METHODS: We convened a panel of nine clinical and research scientists with wide-ranging expertise in clinical variant interpretation, with specific experience in variants within non-coding regions. This panel discussed and refined an initial draft of the guidelines which were then extensively tested and reviewed by external groups. RESULTS: We discuss considerations specifically for variants in non-coding regions of the genome. We outline how to define candidate regulatory elements, highlight examples of mechanisms through which non-coding region variants can lead to penetrant monogenic disease, and outline how existing guidelines can be adapted for the interpretation of these variants. CONCLUSIONS: These recommendations aim to increase the number and range of non-coding region variants that can be clinically interpreted, which, together with a compatible phenotype, can lead to new diagnoses and catalyse the discovery of novel disease mechanisms.

Clinical and genetic findings in TRPM1-related congenital stationary night blindness.

PURPOSE: Congenital stationary night blindness (CSNB) is a heterogeneous group of Mendelian retinal disorders that present in childhood. Biallelic variants altering the protein-coding region of the TRPM1 gene are one of the commonest causes of CSNB. Here, we report the clinical and genetic findings in 10 unrelated individuals with TRPM1-retinopathy. METHODS: Study subjects were recruited through a tertiary clinical ophthalmic genetic service at Manchester, UK. All participants underwent visual electrodiagnostic testing and panel-based genetic analysis. RESULTS: Study subjects had a median age of 8 years (range: 3-20 years). All probands were myopic and had electroretinographic findings in keeping with complete CSNB. Notably, three probands reported no night vision problems. Fourteen different disease-associated TRPM1 variants were detected. One individual was homozygous for the NM_001252024.2 (TRPM1):c.965 + 29G>A variant and a mini-gene assay highlighted that this change results in mis-splicing and premature protein termination. Additionally, two unrelated probands who had CSNB and mild neurodevelopmental abnormalities were found to carry a 15q13.3 microdeletion. This copy number variant encompasses seven genes, including TRPM1, and was encountered in the heterozygous state and in trans with a missense TRPM1 variant in each case. CONCLUSION: Our findings highlight the importance of comprehensive genomic analysis, beyond the exons and protein-coding regions of genes, for individuals with CSNB. When this characteristic retinal phenotype is accompanied by extraocular findings (including learning and/or behavioural difficulties), a 15q13.3 microdeletion should be suspected. Focused analysis (e.g. microarray testing) is recommended to look for large-scale deletions encompassing TRPM1 in patients with CSNB and neurodevelopmental abnormalities.

A basement membrane discovery pipeline uncovers network complexity, regulators, and human disease associations.

Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of 222 human proteins and their animal orthologs localized to BMs. Network analysis and screening in C. elegans and zebrafish uncovered BM regulators, including ADAMTS, ROBO, and TGFß. More than 100 BM network genes associate with human phenotypes, and by screening 63,039 genomes from families with rare disorders, we found loss-of-function variants in LAMA5, MPZL2, and MATN2 and show that they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity of BMs and their impact on in human health.

MRSD: A quantitative approach for assessing suitability of RNA-seq in the investigation of mis-splicing in Mendelian disease.

Variable levels of gene expression between tissues complicates the use of RNA sequencing of patient biosamples to delineate the impact of genomic variants. Here, we describe a gene- and tissue-specific metric to inform the feasibility of RNA sequencing. This overcomes limitations of using expression values alone as a metric to predict RNA-sequencing utility. We have derived a metric, minimum required sequencing depth (MRSD), that estimates the depth of sequencing required from RNA sequencing to achieve user-specified sequencing coverage of a gene, transcript, or group of genes. We applied MRSD across four human biosamples: whole blood, lymphoblastoid cell lines (LCLs), skeletal muscle, and cultured fibroblasts. MRSD has high precision (90.1%-98.2%) and overcomes transcript region-specific sequencing biases. Applying MRSD scoring to established disease gene panels shows that fibroblasts, of these four biosamples, are the optimum source of RNA for 63.1% of gene panels. Using this approach, up to 67.8% of the variants of uncertain significance in ClinVar that are predicted to impact splicing could be assayed by RNA sequencing in at least one of the biosamples. We demonstrate the utility and benefits of MRSD as a metric to inform functional assessment of splicing aberrations, in particular in the context of Mendelian genetic disorders to improve diagnostic yield.

Expanding the genotypic spectrum of TXNL4A variants in Burn-McKeown syndrome.

The developmental disorder Burn-McKeown Syndrome (BMKS) is characterised by choanal atresia and specific craniofacial features. BMKS is caused by biallelic variants in the pre-messenger RNA splicing factor TXNL4A. Most patients have a loss-of-function variant in trans with a 34-base pair (bp) deletion (type 1 Δ34) in the promoter region. Here, we identified two patients with BMKS. One individual has a TXNL4A c.93_94delCC, p.His32Argfs *21 variant combined with a type 1 Δ34 promoter deletion. The other has an intronic TXNL4A splice site variant (c.258-3C>G) and a type 1 Δ34 promoter deletion. We show the c.258-3C>G variant and a previously reported c.258-2A>G variant, cause skipping of the final exon of TXNL4A in a minigene splicing assay. Furthermore, we identify putative transcription factor binding sites within the 56 bp of the TXNL4A promoter affected by the type 1 and type 2 Δ34 and use dual luciferase assays to identify a 22 bp repeated motif essential for TXNL4A expression within this promoter region. We propose that additional variants affecting critical transcription factor binding nucleotides within the 22 bp repeated motif could be relevant to BMKS aetiology. Finally, our data emphasises the need to analyse the non-coding sequence in individuals where a single likely pathogenic coding variant is identified in an autosomal recessive disorder consistent with the clinical presentation.

Molecular diagnoses in the congenital malformations caused by ciliopathies cohort of the 100,000 Genomes Project.

BACKGROUND: Primary ciliopathies represent a group of inherited disorders due to defects in the primary cilium, the 'cell's antenna'. The 100,000 Genomes Project was launched in 2012 by Genomics England (GEL), recruiting National Health Service (NHS) patients with eligible rare diseases and cancer. Sequence data were linked to Human Phenotype Ontology (HPO) terms entered by recruiting clinicians. METHODS: Eighty-three prescreened probands were recruited to the 100,000 Genomes Project suspected to have congenital malformations caused by ciliopathies in the following disease categories: Bardet-Biedl syndrome (n=45), Joubert syndrome (n=14) and 'Rare Multisystem Ciliopathy Disorders' (n=24). We implemented a bespoke variant filtering and analysis strategy to improve molecular diagnostic rates for these participants. RESULTS: We determined a research molecular diagnosis for n=43/83 (51.8%) probands. This is 19.3% higher than previously reported by GEL (n=27/83 (32.5%)). A high proportion of diagnoses are due to variants in non-ciliopathy disease genes (n=19/43, 44.2%) which may reflect difficulties in clinical recognition of ciliopathies. n=11/83 probands (13.3%) had at least one causative variant outside the tiers 1 and 2 variant prioritisation categories (GEL's automated triaging procedure), which would not be reviewed in standard 100,000 Genomes Project diagnostic strategies. These include four structural variants and three predicted to cause non-canonical splicing defects. Two unrelated participants have biallelic likely pathogenic variants in LRRC45, a putative novel ciliopathy disease gene. CONCLUSION: These data illustrate the power of linking large-scale genome sequence to phenotype information. They demonstrate the value of research collaborations in order to maximise interpretation of genomic data.

BBS1 branchpoint variant is associated with non-syndromic retinitis pigmentosa.

BACKGROUND: Inherited retinal diseases (IRDs) can be caused by variants in >270 genes. The Bardet-Biedl syndrome 1 (BBS1) gene is one of these genes and may be associated with syndromic and non-syndromic autosomal recessive retinitis pigmentosa (RP). Here, we identified a branchpoint variant in BBS1 and assessed its pathogenicity by in vitro functional analysis. METHODS: Whole genome sequencing was performed for three unrelated monoallelic BBS1 cases with non-syndromic RP. A fourth case received MGCM 105 gene panel analysis. Functional analysis using a midigene splice assay was performed for the putative pathogenic branchpoint variant in BBS1. After confirmation of its pathogenicity, patients were clinically re-evaluated, including assessment of non-ocular features of Bardet-Biedl syndrome. RESULTS: Clinical assessments of probands showed that all individuals displayed non-syndromic RP with macular involvement. Through detailed variant analysis and prioritisation, two pathogenic variants in BBS1, the most common missense variant, c.1169T>G (p.(Met390Arg)), and a branchpoint variant, c.592-21A>T, were identified. Segregation analysis confirmed that in all families, probands were compound heterozygous for c.1169T>G and c.592-21A>T. Functional analysis of the branchpoint variant revealed a complex splicing defect including exon 8 and exon 7/8 skipping, and partial in-frame deletion of exon 8. CONCLUSION: A putative severe branchpoint variant in BBS1, together with a mild missense variant, underlies non-syndromic RP in four unrelated individuals. To our knowledge, this is the first report of a pathogenic branchpoint variant in IRDs that results in a complex splice defect. In addition, this research highlights the importance of the analysis of non-coding regions in order to provide a conclusive molecular diagnosis.

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.

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.

Extended gene panel testing in lobular breast cancer.

PURPOSE: Lobular breast cancer (LBC) accounts for ~ 15% of breast cancer. Here, we studied the frequency of pathogenic germline variants (PGVs) in an extended panel of genes in women affected with LBC. METHODS: 302 women with LBC and 1567 without breast cancer were tested for BRCA1/2 PGVs. A subset of 134 LBC affected women who tested negative for BRCA1/2 PGVs underwent extended screening, including: ATM, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51D, and TP53. RESULTS: 35 PGVs were identified in the group with LBC, of which 22 were in BRCA1/2. Ten actionable PGVs were identified in additional genes (ATM(4), CDH1(1), CHEK2(1), PALB2(2) and TP53(2)). Overall, PGVs in three genes conferred a significant increased risk for LBC. Odds ratios (ORs) were: BRCA1: OR = 13.17 (95%CI 2.83-66.38; P = 0.0017), BRCA2: OR = 10.33 (95%CI 4.58-23.95; P < 0.0001); and ATM: OR = 8.01 (95%CI 2.52-29.92; P = 0.0053). We did not detect an increased risk of LBC for PALB2, CDH1 or CHEK2. CONCLUSION: The overall PGV detection rate was 11.59%, with similar rates of BRCA1/2 (7.28%) PGVs as for other actionable PGVs (7.46%), indicating a benefit for extended panel genetic testing in LBC. We also report a previously unrecognised association of pathogenic variants in ATM with LBC.

Improving the clinical interpretation of missense variants in X linked genes using structural analysis.

BACKGROUND: Improving the clinical interpretation of missense variants can increase the diagnostic yield of genomic testing and lead to personalised management strategies. Currently, due to the imprecision of bioinformatic tools that aim to predict variant pathogenicity, their role in clinical guidelines remains limited. There is a clear need for more accurate prediction algorithms and this study aims to improve performance by harnessing structural biology insights. The focus of this work is missense variants in a subset of genes associated with X linked disorders. METHODS: We have developed a protein-specific variant interpreter (ProSper) that combines genetic and protein structural data. This algorithm predicts missense variant pathogenicity by applying machine learning approaches to the sequence and structural characteristics of variants. RESULTS: ProSper outperformed seven previously described tools, including meta-predictors, in correctly evaluating whether or not variants are pathogenic; this was the case for 11 of the 21 genes associated with X linked disorders that met the inclusion criteria for this study. We also determined gene-specific pathogenicity thresholds that improved the performance of VEST4, REVEL and ClinPred, the three best-performing tools out of the seven that were evaluated; this was the case in 11, 11 and 12 different genes, respectively. CONCLUSION: ProSper can form the basis of a molecule-specific prediction tool that can be implemented into diagnostic strategies. It can allow the accurate prioritisation of missense variants associated with X linked disorders, aiding precise and timely diagnosis. In addition, we demonstrate that gene-specific pathogenicity thresholds for a range of missense prioritisation tools can lead to an increase in prediction accuracy.

100,000 Genomes Pilot on Rare-Disease Diagnosis in Health Care - Preliminary Report.

BACKGROUND: The U.K. 100,000 Genomes Project is in the process of investigating the role of genome sequencing in patients with undiagnosed rare diseases after usual care and the alignment of this research with health care implementation in the U.K. National Health Service. Other parts of this project focus on patients with cancer and infection. METHODS: We conducted a pilot study involving 4660 participants from 2183 families, among whom 161 disorders covering a broad spectrum of rare diseases were present. We collected data on clinical features with the use of Human Phenotype Ontology terms, undertook genome sequencing, applied automated variant prioritization on the basis of applied virtual gene panels and phenotypes, and identified novel pathogenic variants through research analysis. RESULTS: Diagnostic yields varied among family structures and were highest in family trios (both parents and a proband) and families with larger pedigrees. Diagnostic yields were much higher for disorders likely to have a monogenic cause (35%) than for disorders likely to have a complex cause (11%). Diagnostic yields for intellectual disability, hearing disorders, and vision disorders ranged from 40 to 55%. We made genetic diagnoses in 25% of the probands. A total of 14% of the diagnoses were made by means of the combination of research and automated approaches, which was critical for cases in which we found etiologic noncoding, structural, and mitochondrial genome variants and coding variants poorly covered by exome sequencing. Cohortwide burden testing across 57,000 genomes enabled the discovery of three new disease genes and 19 new associations. Of the genetic diagnoses that we made, 25% had immediate ramifications for clinical decision making for the patients or their relatives. CONCLUSIONS: Our pilot study of genome sequencing in a national health care system showed an increase in diagnostic yield across a range of rare diseases. (Funded by the National Institute for Health Research and others.).

Comparison of in silico strategies to prioritize rare genomic variants impacting RNA splicing for the diagnosis of genomic disorders.

The development of computational methods to assess pathogenicity of pre-messenger RNA splicing variants is critical for diagnosis of human disease. We assessed the capability of eight algorithms, and a consensus approach, to prioritize 249 variants of uncertain significance (VUSs) that underwent splicing functional analyses. The capability of algorithms to differentiate VUSs away from the immediate splice site as being 'pathogenic' or 'benign' is likely to have substantial impact on diagnostic testing. We show that SpliceAI is the best single strategy in this regard, but that combined usage of tools using a weighted approach can increase accuracy further. We incorporated prioritization strategies alongside diagnostic testing for rare disorders. We show that 15% of 2783 referred individuals carry rare variants expected to impact splicing that were not initially identified as 'pathogenic' or 'likely pathogenic'; one in five of these cases could lead to new or refined diagnoses.