Evaluation of the diagnostic accuracy of exome sequencing and its impact on diagnostic thinking for patients with rare disease in a publicly funded health care system: A prospective cohort study.

PURPOSE: To evaluate the diagnostic utility of publicly funded clinical exome sequencing (ES) for patients with suspected rare genetic diseases. METHODS: We prospectively enrolled 297 probands who met eligibility criteria and received ES across 5 sites in Ontario, Canada, and extracted data from medical records and clinician surveys. Using the Fryback and Thornbury Efficacy Framework, we assessed diagnostic accuracy by examining laboratory interpretation of results and assessed diagnostic thinking by examining the clinical interpretation of results and whether clinical-molecular diagnoses would have been achieved via alternative hypothetical molecular tests. RESULTS: Laboratories reported 105 molecular diagnoses and 165 uncertain results in known and novel genes. Of these, clinicians interpreted 102 of 105 (97%) molecular diagnoses and 6 of 165 (4%) uncertain results as clinical-molecular diagnoses. The 108 clinical-molecular diagnoses were in 104 families (35% diagnostic yield). Each eligibility criteria resulted in diagnostic yields of 30% to 40%, and higher yields were achieved when >2 eligibility criteria were met (up to 45%). Hypothetical tests would have identified 61% of clinical-molecular diagnoses. CONCLUSION: We demonstrate robustness in eligibility criteria and high clinical validity of laboratory results from ES testing. The importance of ES was highlighted by the potential 40% of patients that would have gone undiagnosed without this test.

Bridging clinical care and research in Ontario, Canada: Maximizing diagnoses from reanalysis of clinical exome sequencing data.

We examined the utility of clinical and research processes in the reanalysis of publicly-funded clinical exome sequencing data in Ontario, Canada. In partnership with eight sites, we recruited 287 families with suspected rare genetic diseases tested between 2014 and 2020. Data from seven laboratories was reanalyzed with the referring clinicians. Reanalysis of clinically relevant genes identified diagnoses in 4% (13/287); four were missed by clinical testing. Translational research methods, including analysis of novel candidate genes, identified candidates in 21% (61/287). Of these, 24 families have additional evidence through data sharing to support likely diagnoses (8% of cohort). This study indicates few diagnoses are missed by clinical laboratories, the incremental gain from reanalysis of clinically-relevant genes is modest, and the highest yield comes from validation of novel disease-gene associations. Future implementation of translational research methods, including continued reporting of compelling genes of uncertain significance by clinical laboratories, should be considered to maximize diagnoses.

The implementation of an enhanced clinical model to improve the diagnostic yield of exome sequencing for patients with a rare genetic disease: A Canadian experience.

The introduction of clinical exome sequencing (ES) has provided a unique opportunity to decrease the diagnostic odyssey for patients living with a rare genetic disease (RGD). ES has been shown to provide a diagnosis in 29%-57% of patients with a suspected RGD, with as many as 70% remaining undiagnosed. There is a need to advance the clinical model of care by more formally integrating approaches that were previously considered research into an enhanced diagnostic workflow. We developed an Exome Clinic, which set out to evaluate a workflow for improving the diagnostic yield of ES for patients with an undiagnosed RGD. Here, we report the outcomes of 47 families who underwent clinical ES in the first year of the clinic. The diagnostic yield from clinical ES was 40% (19/47). Families who remained undiagnosed after ES had the opportunity for follow-up studies that included phenotyping and candidate variant segregation in relatives, genomic matchmaking, and ES reanalysis. This enhanced diagnostic workflow increased the diagnostic yield to 55% (26/47), predominantly through the resolution of variants and genes of uncertain significance. We advocate that this approach be integrated into mainstream clinical practice and highlight the importance of a coordinated translational approach for patients with RGD.

Homozygous WNT9B variants in two families with bilateral renal agenesis/hypoplasia/dysplasia.

WNT9B plays a key role in the development of the mammalian urogenital system. It is essential for the induction of mesonephric and metanephric tubules, the regulation of renal tubule morphogenesis, and the regulation of renal progenitor cell expansion and differentiation. To our knowledge, WNT9B has not been associated with renal defects in humans; however, WNT9B-/- mice have renal agenesis/hypoplasia and reproductive tract abnormalities. We report four individuals from two unrelated consanguineous families with bilateral renal agenesis/hypoplasia/dysplasia and homozygous variants in WNT9B. The proband from Family 1 has bilateral renal cystic dysplasia and chronic kidney disease. He has two deceased siblings who presented with bilateral renal hypoplasia/agenesis. The three affected family members were homozygous for a missense variant in WNT9B (NM_003396.2: c.949G>A/p.(Gly317Arg)). The proband from Family 2 has renal hypoplasia/dysplasia, chronic kidney disease, and is homozygous for a nonsense variant in WNT9B (NM_003396.2: c.11dupC/p.(Pro5Alafs*52)). Two of her siblings died in the neonatal period, one confirmed to be in the context of oligohydramnios. The proband's unaffected brother is also homozygous for the nonsense variant in WNT9B, suggesting nonpenetrance. We propose a novel association of WNT9B and renal anomalies in humans. Further study is needed to delineate the contribution of WNT9B to genitourinary anomalies in humans.

Leveraging the power of new molecular technologies in the clinical setting requires unprecedented awareness of limitations and drawbacks: experience of one diagnostic laboratory.

BACKGROUND: Advances in molecular technologies and in-silico variant prediction tools offer wide-ranging opportunities in diagnostic settings, yet they also present with significant limitations. OBJECTIVE: Here, we contextualise the limitations of next-generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA) and in-silico prediction tools routinely used by diagnostic laboratories by reviewing specific experiences from our diagnostic laboratory. METHODS: We investigated discordant annotations and/or incorrect variant 'callings' in exons of 56 genes constituting our cardiomyopathy and connective tissue disorder NGS panels. Discordant variants and segmental duplications (SD) were queried using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool and the University of California Santa Cruz genome browser, respectively, to identify regions of high homology. Discrepant variant analyses by in-silico models were re-evaluated using updated file entries. RESULTS: We observed a 5% error rate in MYH7 variant 'calling' using MLPA, which resulted from >90% homology of the MYH7 probe-binding site to MYH6. SDs were detected in TTN, PKP2 and MYLK. SDs in MYLK presented the highest risk (15.7%) of incorrect variant 'calling'. The inaccurate 'callings' and discrepant in-silico predictions were resolved following detailed investigation into the source of error. CONCLUSION: Recognising the limitations described here may help avoid incorrect diagnoses and leverage the power of new molecular technologies in diagnostic settings.

Reinterpretation of sequence variants: one diagnostic laboratory's experience, and the need for standard guidelines.

PurposeThe advent of next-generation sequencing resulted in substantial increases in the number of variants detected, interpreted, and reported by molecular genetics diagnostic laboratories. Recent publications have provided standards for the interpretation of sequence variants, but there are currently no standards regarding reinterpretation of these variants. Recognizing that significant changes in variant classification may occur over time, many genetics diagnostic laboratories have independently developed practices for variant reinterpretation. The purpose of this study is to describe our laboratory approach to variant reinterpretation.MethodsWe surveyed eight genetics diagnostic laboratories in Canada and the United States.ResultsEach laboratory had differing protocols, but most felt that clinically relevant changes to variant classifications should be communicated to ordering providers. Based on results of this survey and our experience, we developed a cost-effective and resource-efficient approach to variant reinterpretation.ConclusionOngoing variant reinterpretation is required to maintain the highest standards for delivering genetics laboratory services. Our approach to variant reinterpretation offers an efficient solution that does not compromise accuracy or timely delivery of genetics laboratory services.

Data sharing as a national quality improvement program: reporting on BRCA1 and BRCA2 variant-interpretation comparisons through the Canadian Open Genetics Repository (COGR).

PurposeThe purpose of this study was to develop a national program for Canadian diagnostic laboratories to compare DNA-variant interpretations and resolve discordant-variant classifications using the BRCA1 and BRCA2 genes as a case study.MethodsBRCA1 and BRCA2 variant data were uploaded and shared through the Canadian Open Genetics Repository (COGR; http://www.opengenetics.ca). A total of 5,554 variant observations were submitted; classification differences were identified and comparison reports were sent to participating laboratories. Each site had the opportunity to reclassify variants. The data were analyzed before and after the comparison report process to track concordant- or discordant-variant classifications by three different models.ResultsVariant-discordance rates varied by classification model: 38.9% of variants were discordant when using a five-tier model, 26.7% with a three-tier model, and 5.0% with a two-tier model. After the comparison report process, the proportion of discordant variants dropped to 30.7% with the five-tier model, to 14.2% with the three-tier model, and to 0.9% using the two-tier model.ConclusionWe present a Canadian interinstitutional quality improvement program for DNA-variant interpretations. Sharing of variant knowledge by clinical diagnostic laboratories will allow clinicians and patients to make more informed decisions and lead to better patient outcomes.

Expanding the clinical spectrum of ocular anomalies in Noonan syndrome: Axenfeld-anomaly in a child with PTPN11 mutation.

Ocular anomalies have been frequently reported in Noonan syndrome. Anterior segment anomalies have been described in 57% of PTPN11 positive patients, with the most common findings being corneal changes and in particular, prominent corneal nerves and cataracts. We report on a neonate with a confirmed PTPN11 mutation and ocular findings consistent with Axenfeld anomaly. The patient initially presented with non-immune hydrops and subsequently developed hypertrophic cardiomyopathy and dysmorphic features typical of Noonan syndrome. While a pathogenic mutation in PTPN11 was confirmed, prior testing for the two common genes associated with Axenfeld-Rieger syndrome, PITX2, and FOXC1 was negative. This finding expands the spectrum of anterior chamber anomalies seen in Noonan syndrome and perhaps suggests a common neural crest related mechanism that plays a critical role in the development of the eye and other organs.

Congenital tremor and myopathy secondary to novel MYBPC1 variant.

Congenital myopathy with tremor (MYOTREM) is a recently described disorder characterized by mild myopathy and a postural and intention tremor present since early infancy. MYOTREM is associated with pathogenic variants in MYBPC1 which encodes slow myosin-binding protein C, a sarcomere protein with regulatory and structural roles. Here, we describe a family with three generations of variably affected members exhibiting a novel variant in MYBPC1 (c.656 T > C, p.Leu219Pro). Among the unique features of affected family members is the persistence of tremor in sleep. We also present the first muscle magnetic resonance images for this disorder, and report muscle atrophy and fatty infiltration.

Protocol for a Prospective, Observational Cost-effectiveness Analysis of Returning Secondary Findings of Genome Sequencing for Unexplained Suspected Genetic Conditions.

PURPOSE: Although costly, genome-wide sequencing (GWS) detects an extensive range of variants, enhancing our ability to diagnose and assess risk for an increasing number of diseases. In addition to detecting variants related to the indication for testing, GWS can detect secondary variants in BRCA1, BRCA2, and other genes for which early intervention may improve health. As the list of secondary findings grows, there is increased demand for surveillance and management by multiple specialists, adding pressure to constrained health care budgets. Secondary finding testing is actively debated because some consider it opportunistic screening for future health risks that may not manifest. Given the economic implications of secondary finding testing and follow-up and its unproven clinical utility, the objective is to assess the incremental cost-effectiveness of secondary finding ascertainment per case detected and per unit of improved clinical utility in families of children with unexplained suspected genetic conditions undergoing clinical GWS. METHODS: Those undergoing trio genome or exome sequencing are eligible for the study. Positive secondary finding index cases will be matched to negative controls (1:2) based on age group, primary result(s) type, and clinical indication. During the 2-year study, 71 cases and 142 matched controls are expected. Health service use will be collected in patients and 1 adult family member every 6 months. The per-child and per-dyad total cost will be determined by multiplying use of each resource by a corresponding unit price and summing all cost items. Costs will be estimated from the public and societal payer perspectives. The mean cost per child and per dyad for secondary finding-positive and secondary finding-negative groups will be compared statistically. If important demographic differences are observed between groups, ordinary least-squares regression, log transformation, or other nonparametric technique will be used to compare adjusted mean costs. The ratio of the difference in mean cost to the secondary finding yield will be used to estimate incremental cost-effectiveness. In secondary analyses, effectiveness will be estimated using the number of clinical management changes due to secondary findings or the Clinician-Reported Genetic Testing Utility Index (C-GUIDE) score, a validated measure of clinical utility. Sensitivity analysis will be undertaken to assess the robustness of the findings to variation in key parameters. IMPLICATIONS: This study generates key evidence to inform clinical practice and funding allocation related to secondary finding testing. The inclusion of family members and a new measure of clinical utility represent important advancements in economic evaluation in genomics.

Comparing genome sequencing technologies to improve rare disease diagnostics: a protocol for the evaluation of a pilot project, Genome-wide Sequencing Ontario.

BACKGROUND: Genome-wide sequencing has emerged as a promising strategy for the timely diagnosis of rare diseases, but it is not yet available as a clinical test performed in Canadian diagnostic laboratories. We describe the protocol for evaluating a 2-year pilot project, Genome-wide Sequencing Ontario, to offer high-quality clinical genome-wide sequencing in Ontario, Canada. METHODS: The Genome-wide Sequencing Ontario protocol was codesigned by the Ontario Ministry of Health, the Hospital for Sick Children in Toronto and the Children's Hospital of Eastern Ontario in Ottawa. Enrolment of a prospective cohort of patients began on Apr. 1, 2021. Eligible cases with blood samples available for the index case and both parents (i.e., trios) are randomized to receive exome sequencing or genome sequencing. We will collect patient-level data and ascertain costs associated with the laboratory workflow for exome sequencing and genome sequencing. We will compare point estimates for the diagnostic utility and timeliness of exome sequencing and genome sequencing, and we will determine an incremental cost-effectiveness ratio (expressed as the incremental cost of genome sequencing versus exome sequencing per additional patient with a causal variant detected). INTERPRETATION: Findings from this work will provide robust evidence for the diagnostic utility, cost-effectiveness and timeliness of exome sequencing and genome sequencing, and will be disseminated via academic publications and policy briefs. Findings will inform provincial and cross-provincial policy related to the long-term organization, delivery and reimbursement of clinical-grade genome diagnostics for rare disease.

Adopting High-Resolution Allele Frequencies Substantially Expedites Variant Interpretation in Genetic Diagnostic Laboratories.

A cohort of 1242 individuals tested in a clinical diagnostic laboratory was used to test whether the filtering allele frequencies (FAFs)-based framework, recently recommended for MHY7-associated cardiomyopathy, is extendable to 45 cardiomyopathy genes. Statistical analysis revealed a threshold of 0.00164% for the extreme outlier allele frequencies (AFs), based on the Genome Aggregation Database (exome fraction) total AFs of 138 unique pathogenic and likely pathogenic variants; 135 of them (97.8%) had AFs of <0.004%, the recommended threshold to apply moderate pathogenicity evidence for MYH7-associated cardiomyopathy. Of the 460 cases reported with only variant(s) of unknown clinical significance (VUCSs), 97 (21%) solely had VUCSs with FAFs >0.03%, frequencies above which were estimated herein as strong evidence against pathogenicity. Interestingly, 74.5% (172/231) of the unique VUCSs with FAFs >0.03% had Genome Aggregation Database maximum allele frequencies across all populations AFs >0.1%, deemed herein as stand-alone evidence against pathogenicity. Accordingly, using an FAF threshold of >0.1%, compared with AF >1%, led us to issue considerably more (25.9% versus 41.3%) negative patient reports. Also, 82.7% (N = 629) of the unique classified benign or likely benign variants with AFs <1% had FAFs >0.1%, reinforcing the use of this filtering strategy. Together, these data demonstrate that implementing FAF thresholds may considerably decrease the amount of variant interpretations and significantly reduce the cost of genetic testing for clinical genetic laboratories, without compromising the accuracy of genetic diagnostic services.

Genetic Diagnostic Testing for Inherited Cardiomyopathies: Considerations for Offering Multi-Gene Tests in a Health Care Setting.

Inherited cardiomyopathies (ICs) are a major cause of heart disease. Given their marked clinical and genetic heterogeneity, the content and clinical utility of IC multi-gene panels has been the topic of continuous debate. Our genetics diagnostic laboratory has been providing clinical diagnostic testing for ICs since 2012. We began by testing nine genes and expanded our panel by fivefold in 2015. Here, we describe the implementation of a cost-effective next-generation sequencing (NGS)-based assay for testing of IC genes, including a protocol that minimizes the amount of Sanger sequencing required to confirm variants identified by NGS, which reduces the cost and time of testing. The NGS assay was developed for the simultaneous analysis of 45 IC genes and was assessed for the impact of panel expansion on variant detection, turnaround time, and cost of testing in a cohort of 993 patients. The assay led to a considerable reduction in test cost and turnaround time. However, only a marginal increase was observed in the diagnostic yield, whereas the rate of inconclusive findings increased considerably. These findings suggest that the ongoing evaluation of gene content and monitoring of clinical utility for multi-gene tests are essential to achieve maximum clinical utility of multi-gene tests in a publicly funded health care setting.