Improved diagnostics by exome sequencing following raw data reevaluation by clinical geneticists involved in the medical care of the individuals tested

PurposeReanalysis of exome sequencing data when results are negative may yield additional diagnoses. We sought to estimate the contribution of clinical geneticists to the interpretation of sequencing data of their patients.MethodsThe cohort included 84 probands attending a tertiary genetics institute (2015–2018) with a nondiagnostic result on clinical exome sequencing performed in one of five external laboratories. The raw data were uploaded to the Emedgene bioinformatics and interpretation platform for reanalysis by a team of two clinical geneticists, the geneticist directly involved in the patient’s care, and a bioinformatician.ResultsIn ten probands (11.9%), a new definitive diagnosis was reached based on genes that were known to be associated with the phenotype at the time the original report was issued. The main reasons for a negative exome result were incorrect interpretation of the clinical context and absence of OMIM entry. Pathogenic variants in genes with previously unknown gene–disease associations were discovered to be causative in three probands. In total, new diagnoses were established in 13/84 individuals (15.5%).ConclusionDirect access to complete clinical data and shortening of time to including gene–phenotype associations in databases can assist the analytics team and reduce the need for additional unnecessary tests.     

Assessing the necessity of confirmatory testing for exome-sequencing results in a clinical molecular diagnostic laboratory

PurposeSanger sequencing is currently considered the gold standard methodology for clinical molecular diagnostic testing. However, next-generation sequencing has already emerged as a much more efficient means to identify genetic variants within gene panels, the exome, or the genome. We sought to assess the accuracy of next-generation sequencing variant identification in our clinical genomics laboratory with the goal of establishing a quality score threshold for confirmatory Sanger-based testing.MethodsConfirmation data for reported results from 144 sequential clinical exome-sequencing cases (94 unique variants) and an additional set of 16 variants from comparable research samples were analyzed.ResultsOf the 110 total single-nucleotide variants analyzed, 103 variants had a quality score ≥Q500, 103 (100%) of which were confirmed by Sanger sequencing. Of the remaining seven variants with quality scores <Q500, six were confirmed by Sanger sequencing (85%).ConclusionFor single-nucleotide variants, we predict that going forward, we will be able to reduce our Sanger confirmation workload by 70–80%. This serves as a proof of principle that as long as sufficient validation and quality control measures are implemented, the volume of Sanger confirmation can be reduced, alleviating a significant amount of the labor and cost burden on clinical laboratories wishing to use next-generation sequencing technology. However, Sanger confirmation of low-quality single-nucleotide variants and all insertions or deletions <10 bp remains necessary at this time in our laboratory.Genet Med16 7, 510–515.     

Interface design recommendations for computerised clinical audit and feedback: Hybrid usability evidence from a research-led system

BackgroundAudit and Feedback (A&F) is a widely used quality improvement technique that measures clinicians’ clinical performance and reports it back to them. Computerised A&F (e-A&F) system interfaces may consist of four key components: (1) Summaries of clinical performance; (2) Patient lists; (3) Patient-level data; (4) Recommended actions. There is a lack of evidence regarding how to best design e-A&F interfaces; establishing such evidence is key to maximising usability, and in turn improving patient safety.AimTo evaluate the usability of a novel theoretically-informed and research-led e-A&F system for primary care (the Performance Improvement plaN GeneratoR: PINGR).Objectives(1) Describe PINGR’s design, rationale and theoretical basis; (2) Identify usability issues with PINGR; (3) Understand how these issues may interfere with the cognitive goals of end-users; (4) Translate the issues into recommendations for the user-centred design of e-A&F systems.MethodsEight experienced health system evaluators performed a usability inspection using an innovative hybrid approach consisting of five stages: (1) Development of representative user tasks, Goals, and Actions; (2) Combining Heuristic Evaluation and Cognitive Walkthrough methods into a single protocol to identify usability issues; (3) Consolidation of issues; (4) Severity rating of consolidated issues; (5) Analysis of issues according to usability heuristics, interface components, and Goal-Action structure.ResultsA final list of 47 issues were categorised into 8 heuristic themes. The most error-prone heuristics were ‘Consistency and standards’ (13 usability issues; 28% of the total) and ‘Match between system and real world’ (n = 10, 21%). The recommended actions component of the PINGR interface had the most usability issues (n = 21, 45%), followed by patient-level data (n = 5, 11%), patient lists (n = 4, 9%), and summaries of clinical performance (n = 4, 9%). The most error-prone Actions across all user Goals were: (1) Patient selection from a list; (2) Data identification from a figure (both population-level and patient-level); (3) Disagreement with a system recommendation.ConclusionsBy contextualising our findings within the wider literature on health information system usability, we provide recommendations for the design of e-A&F system interfaces relating to their four key components, in addition to how they may be integrated within a system.     

The implications of familial incidental findings from exome sequencing: the NIH Undiagnosed Diseases Program experience

PurposeUsing exome sequence data from 159 families participating in the National Institutes of Health Undiagnosed Diseases Program, we evaluated the number and inheritance mode of reportable incidental sequence variants.MethodsFollowing the American College of Medical Genetics and Genomics recommendations for reporting of incidental findings from next-generation sequencing, we extracted variants in 56 genes from the exome sequence data of 543 subjects and determined the reportable incidental findings for each participant. We also defined variant status as inherited or de novo for those with available parental sequence data.ResultsWe identified 14 independent reportable variants in 159 (8.8%) families. For nine families with parental sequence data in our cohort, a parent transmitted the variant to one or more children (nine minor children and four adult children). The remaining five variants occurred in adults for whom parental sequences were unavailable.ConclusionOur results are consistent with the expectation that a small percentage of exomes will result in identification of an incidental finding under the American College of Medical Genetics and Genomics recommendations. Additionally, our analysis of family sequence data highlights that genome and exome sequencing of families has unavoidable implications for immediate family members and therefore requires appropriate counseling for the family.     

“Possibly positive or certainly uncertain?”: participants’ responses to uncertain diagnostic results from exome sequencing

PurposeClinical genome sequencing produces uncertain diagnostic results, raising concerns about how to communicate the method’s inherent complexities in ways that reduce potential misunderstandings and harm. This study investigates clinicians’ communications and patient/participant responses to uncertain diagnostic results arising from a clinical exome sequencing research study, contributing empirical data to the debate surrounding disclosure of uncertain genomic information.MethodsWe investigated the communication and impact of uncertain diagnostic results using ethnographic observations of result disclosures with 21 adults and 11 parents of child patients, followed by two semistructured interviews with these same participants.ResultsParticipants understood their uncertain results in ways that were congruent with clinical geneticists’ communications. They followed recommendations for further consultation, although family testing to resolve uncertainty was not always done. Participants were prepared for learning an uncertain result and grasped the key concept that it should not be used to guide health-care or other decisions. They did not express regret for having learned the uncertain result; most regarded it as potentially valuable in the future.ConclusionThis study suggests that uncertain diagnostic results from genome sequencing can be relayed to patients in ways they can understand and consistent with providers’ interpretations, without causing undue harm.     

Development of DNA Confirmatory and High-Risk Diagnostic Testing for Newborns Using Targeted Next-Generation DNA Sequencing

PurposeGenetic testing is routinely used for second-tier confirmation of newborn sequencing results to rule out false positives and to confirm diagnoses in newborns undergoing inpatient and outpatient care. We developed a targeted next-generation sequencing panel coupled with a variant processing pipeline and demonstrated utility and performance benchmarks across multiple newborn disease presentations in a retrospective clinical study.MethodsThe test utilizes an in silico gene filter that focuses directly on 126 genes related to newborn screening diseases and is applied to the exome or a next-generation sequencing panel called NBDx. NBDx targets the 126 genes and additional newborn-specific disorders. It integrates DNA isolation from minimally invasive biological specimens, targeted next-generation screening, and rapid characterization of genetic variation.ResultsWe report a rapid parallel processing of 8 to 20 cases within 105 hours with high coverage on our NBDx panel. Analytical sensitivity of 99.8% was observed across known mutation hotspots. Concordance calls with or without clinical summaries were 94% and 75%, respectively.ConclusionRapid, automated targeted next-generation sequencing and analysis are practical in newborns for second-tier confirmation and neonatal intensive care unit diagnoses, laying a foundation for future primary DNA-based molecular screening of additional disorders and improving existing molecular testing options for newborns.Genet Med 17 5, 337–347.     

Solving the molecular diagnostic testing conundrum for Mendelian disorders in the era of next-generation sequencing: single-gene,gene panel,or exome/genome sequencing

Next-generation sequencing is changing the paradigm of clinical genetic testing. Today there are numerous molecular tests available, including single-gene tests, gene panels, and exome sequencing or genome sequencing. As a result, ordering physicians face the conundrum of selecting the best diagnostic tool for their patients with genetic conditions. Single-gene testing is often most appropriate for conditions with distinctive clinical features and minimal locus heterogeneity. Next-generation sequencing–based gene panel testing, which can be complemented with array comparative genomic hybridization and other ancillary methods, provides a comprehensive and feasible approach for heterogeneous disorders. Exome sequencing and genome sequencing have the advantage of being unbiased regarding what set of genes is analyzed, enabling parallel interrogation of most of the genes in the human genome. However, current limitations of next-generation sequencing technology and our variant interpretation capabilities caution us against offering exome sequencing or genome sequencing as either stand-alone or first-choice diagnostic approaches. A growing interest in personalized medicine calls for the application of genome sequencing in clinical diagnostics, but major challenges must be addressed before its full potential can be realized. Here, we propose a testing algorithm to help clinicians opt for the most appropriate molecular diagnostic tool for each scenario.Genet Med17 6, 444–451.     

Patient decisions for disclosure of secondary findings among the first 200 individuals undergoing clinical diagnostic exome sequencing

PurposeExome sequencing of a single individual for a clinical indication may result in the identification of incidental deleterious variants unrelated to the indication for testing (secondary findings). Given the recent availability of clinical exome testing, there is a limited knowledge regarding the disclosure preferences and impact of secondary findings in a clinical diagnostic setting. In this article, we provide preliminary data regarding the preferences for secondary findings results disclosure based on the first 200 families referred to Ambry Genetics for diagnostic exome sequencing.MethodsSecondary findings were categorized into four groups in the diagnostic exome sequencing consent: carrier status of recessive disorders, predisposition to later-onset disease, predisposition to increased cancer risk, and early-onset disease. In this study, we performed a retrospective analysis of patient responses regarding the preferences for secondary findings disclosure.ResultsThe majority of patients (187/200; 93.5%) chose to receive secondary results for one or more available categories. Adult probands were more likely than children to opt for blinding of secondary data (16 vs. 4%, respectively). Among responses for blinding, preferences were evenly scattered among categories.ConclusionThese data represent the unprecedented results of a large reference laboratory providing clinical exome sequencing. We report, for the first time, the preferences of patients and families for the receipt of secondary findings based on clinical genome sequencing. Overwhelmingly, families undergoing exome sequencing opt for the disclosure of secondary findings. The data may have implications regarding the development of guidelines for secondary findings reporting among patients with severe and/or life-threatening disease undergoing clinical genomic sequencing.     

The use of fault reporting of medical equipment to identify latent design flaws

Background and purposePoor device design that fails to adequately account for user needs, cognition, and behavior is often responsible for use errors resulting in adverse events. This poor device design is also often latent, and could be responsible for “No Fault Found” (NFF) reporting, in which medical devices sent for repair by clinical users are found to be operating as intended. Unresolved NFF reports may contribute to incident under reporting, clinical user frustration, and biomedical engineering technologist inefficacy. This study uses human factors engineering methods to investigate the relationship between NFF reporting frequency and device usability.Material and methodsAn analysis of medical equipment maintenance data was conducted to identify devices with a high NFF reporting frequency. Subsequently, semi-structured interviews and heuristic evaluations were performed in order to identify potential usability issues. Finally, usability testing was conducted in order to validate that latent usability related design faults result in a higher frequency of NFF reporting.ResultsThe analysis of medical equipment maintenance data identified six devices with a high NFF reporting frequency. Semi-structured interviews, heuristic evaluations and usability testing revealed that usability issues caused a significant portion of the NFF reports. Other factors suspected to contribute to increased NFF reporting include accessory issues, intermittent faults and environmental issues. Usability testing conducted on three of the devices revealed 23 latent usability related design faults.ConclusionsThese findings demonstrate that latent usability related design faults manifest themselves as an increase in NFF reporting and that devices containing usability related design faults can be identified through an analysis of medical equipment maintenance data.     

Clinical utility of exome sequencing in infantile heart failure

PurposePediatric cardiomyopathy is rare, has a broad differential diagnosis, results in high morbidity and mortality, and has suboptimal diagnostic yield using next-generation sequencing panels. Exome sequencing has reported diagnostic yields ranging from 30% to 57% for neonates in intensive care units. We aimed to characterize the clinical utility of exome sequencing in infantile heart failure.MethodsInfants diagnosed with acute heart failure prior to 1 year old over a period of 34 months at a large tertiary children’s hospital were recruited. Demographic and diagnostic information was obtained from medical records. Fifteen eligible patients were enrolled.ResultsDilated cardiomyopathy was the predominant cardiac diagnosis, seen in 60% of patients. A molecular diagnosis was identified in 66.7% of patients (10/15). Of those diagnoses, 70% would not have been detected using multigene next-generation sequencing panels focused on cardiomyopathy or arrhythmia disease genes. Genetic testing changed medical decision-making in 53% of all cases and 80% of positive cases, and was especially beneficial when testing was expedited.ConclusionGiven the broad differential diagnosis and critical status of infants with heart failure, rapid exome sequencing provides timely diagnoses, changes medical management, and should be the first-tier molecular test.     

Clinical whole-exome sequencing: are we there yet?

BackgroundClinical laboratories began offering whole-exome sequencing in 2011 at a cost between $4,500 and $9,000. Reported detection rates for deleterious mutations range from 25 to 50%. Based on the experience of our clinical genetics service, actual success rates may be lower than estimated rates. We report results from our own experience along with a survey of clinical geneticists to ascertain (i) current success rates for causal gene detection in a clinical setting; (ii) if there are insurance authorization issues; and (iii) if turnaround times quoted by the clinical laboratories are accurate; we also gauge provider opinions toward clinical whole-exome sequencing.MethodsWe reviewed our results and the results of a survey that was electronically distributed to 47 clinical genetics centers.ResultsA total of 35 exome reports were available. If all positive results are collated, we observe a success rate of 22.8%. One result incorrectly identified a known benign variant as pathogenic. Some insurers covered all testing, whereas others denied any insurance coverage. Only three (23.1%) of our reports were available within the laboratory’s quoted turnaround times. More than 50% of clinicians queried in our survey had not ordered whole-exome sequencing at the current time, many stating concerns regarding interpretation, insurance coverage, and cost.ConclusionClinical whole-exome sequencing has proven diagnostic utility; however, currently many clinicians have concerns regarding interpretation of results, insurance coverage, and cost.     

The importance of automation in genetic diagnosis: Lessons from analyzing an inherited retinal degeneration cohort with the Mendelian Analysis Toolkit (MATK)

PurposeIn Mendelian disease diagnosis, variant analysis is a repetitive, error-prone, and time consuming process. To address this, we have developed the Mendelian Analysis Toolkit (MATK), a configurable, automated variant ranking program.MethodsMATK aggregates variant information from multiple annotation sources and uses expert-designed rules with parameterized weights to produce a ranked list of potentially causal solutions. MATK performance was measured by a comparison between MATK-aided and human-domain expert analyses of 1060 families with inherited retinal degeneration (IRD), analyzed using an IRD-specific gene panel (589 individuals) and exome sequencing (471 families).ResultsWhen comparing MATK-assisted analysis with expert curation in both the IRD-specific gene panel and exome sequencing (1060 subjects), 97.3% of potential solutions found by experts were also identified by the MATK-assisted analysis (541 solutions identified with MATK of 556 solutions found by conventional analysis). Furthermore, MATK-assisted analysis identified 114 additional potential solutions from the 504 cases unsolved by conventional analysis.ConclusionMATK expedites the process of identification of likely solving variants in Mendelian traits, and reduces variability stemming from human error and researcher bias. MATK facilitates data reanalysis to keep up with the constantly improving annotation sources and next-generation sequencing processing pipelines. The software is open source and available at https://gitlab.com/matthew_maher/mendelanalysis.     

ACMG clinical laboratory standards for next-generation sequencing

Next-generation sequencing technologies have been and continue to be deployed in clinical laboratories, enabling rapid transformations in genomic medicine. These technologies have reduced the cost of large-scale sequencing by several orders of magnitude, and continuous advances are being made. It is now feasible to analyze an individual’s near-complete exome or genome to assist in the diagnosis of a wide array of clinical scenarios. Next-generation sequencing technologies are also facilitating further advances in therapeutic decision making and disease prediction for at-risk patients. However, with rapid advances come additional challenges involving the clinical validation and use of these constantly evolving technologies and platforms in clinical laboratories. To assist clinical laboratories with the validation of next-generation sequencing methods and platforms, the ongoing monitoring of next-generation sequencing testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics has developed the following professional standards and guidelines.Genet Med15 9, 733–747.     

Enhanced utility of family-centered diagnostic exome sequencing with inheritance model–based analysis: results from 500 unselected families with undiagnosed genetic conditions

PurposeDiagnostic exome sequencing was immediately successful in diagnosing patients in whom traditional technologies were uninformative. Herein, we provide the results from the first 500 probands referred to a clinical laboratory for diagnostic exome sequencing.MethodsFamily-based exome sequencing included whole-exome sequencing followed by family inheritance−based model filtering, comprehensive medical review, familial cosegregation analysis, and analysis of novel genes.ResultsA positive or likely positive result in a characterized gene was identified in 30% of patients (152/500). A novel gene finding was identified in 7.5% of patients (31/416). The highest diagnostic rates were observed among patients with ataxia, multiple congenital anomalies, and epilepsy (44, 36, and 35%, respectively). Twenty-three percent of positive findings were within genes characterized within the past 2 years. The diagnostic rate was significantly higher among families undergoing a trio (37%) as compared with a singleton (21%) whole-exome testing strategy.ConclusionOverall, we present results from the largest clinical cohort of diagnostic exome sequencing cases to date. These data demonstrate the utility of family-based exome sequencing and analysis to obtain the highest reported detection rate in an unselected clinical cohort, illustrating the utility of diagnostic exome sequencing as a transformative technology for the molecular diagnosis of genetic disease.Genet Med 17 7, 578–586.     

Lifting the lid on unborn lethal Mendelian phenotypes through exome sequencing

PurposeMendelian phenotypes in humans vary from benign variants to lethal disorders. Embryonic lethal phenotypes that are similar to what has been known for a long time in mice have remained largely unknown because of the difficulty in arriving at a molecular diagnosis. The purpose of this study is to test whether next generation sequencing can reveal the underlying etiology of recurrent fetal loss.MethodsWe hypothesized that exome sequencing combined with autozygome analysis can reveal the underlying mutation in a family in which recurrent fetal loss was likely to be autosomal recessive in origin.ResultsA novel mutation in CHRNA1 was identified. This gene is known to cause multiple pterygium and fetal akinesia syndrome.ConclusionThis is the first report of exome sequencing to identify the cause of recurrent fetal loss and reveal the diagnosis of a lethal human phenotype. Our results should inspire a systematic examination of the extent of “unborn” Mendelian phenotypes in humans using next-generation sequencing.Genet Med 2013:15(4):307–309     

Detection and characterization of usability problems in structured data entry interfaces in dentistry

BackgroundPoor usability is one of the major barriers for optimally using electronic health records (EHRs). Dentists are increasingly adopting EHRs, and are using structured data entry interfaces to enter data such that the data can be easily retrieved and exchanged. Until recently, dentists have lacked a standardized terminology to consistently represent oral health diagnoses.ObjectivesIn this study we evaluated the usability of a widely used EHR interface that allow the entry of diagnostic terms, using multi-faceted methods to identify problems and work with the vendor to correct them using an iterative design method.MethodsFieldwork was undertaken at two clinical sites, and dental providers as subjects participated in user testing (n = 32), interviews (n = 36) and observations (n = 24).ResultsUser testing revealed that only 22–41% of users were able to successfully complete a simple task of entering one diagnosis, while no user was able to complete a more complex task. We identified and characterized 24 high-level usability problems reducing efficiency and causing user errors. Interface-related problems included unexpected approaches for displaying diagnosis, lack of visibility, and inconsistent use of UI widgets. Terminology related issues included missing and mis-categorized concepts. Work domain issues involved both absent and superfluous functions. In collaboration with the vendor, each usability problem was prioritized and a timeline set to resolve the concerns.DiscussionMixed methods evaluations identified a number of critical usability issues relating to the user interface, underlying terminology of the work domain. The usability challenges were found to prevent most users from successfully completing the tasks. Our further work we will determine if changes to the interface, terminology and work domain do result in improved usability.     

Phenotype-driven gene target definition in clinical genome-wide sequencing data interpretation

PurposeGenome-wide sequencing approaches are increasingly being used in place of disease gene panel sequencing approaches. Despite the well-recognized benefits of these approaches, they also carry with them an increased burden of analyzing overwhelmingly large gene targets and an increased possibility of detecting incidental findings.MethodsWe propose a novel approach for design of individualized phenotype gene panels using the set of signs and symptoms observed and selecting relevant genes on the basis of known phenotype–gene associations.ResultsWe used results of diagnostic exome sequencing in 405 cases submitted to our institution to show retrospectively that using the phenotype gene panel increases the sensitivity of masked exome analysis (increase from 25.4 to 29.7% in overall diagnostic yield). We also show that such a strategy enables the possibility of masked analysis of genome-wide sequencing data in patients with poorly defined and multifaceted clinical presentations. Ultimately, we show that this approach enables control over the incidental findings rate (0.25% in phenotype gene panels). Finally, we provide a Web tool for customized phenotype panel creation (available at http://www.kimg.eu/generator).ConclusionIn conclusion, we present a novel approach to a phenotype-driven diagnostic process of genome scale sequencing data that harnesses the sensitivity of these approaches while restricting the analysis to genes relevant to clinical presentation in patient.Genet Med 18 11, 1102–1110.     

Genomic answers for children: Dynamic analyses of >1000 pediatric rare disease genomes

PurposeThis study aimed to provide comprehensive diagnostic and candidate analyses in a pediatric rare disease cohort through the Genomic Answers for Kids program.MethodsExtensive analyses of 960 families with suspected genetic disorders included short-read exome sequencing and short-read genome sequencing (srGS); PacBio HiFi long-read genome sequencing (HiFi-GS); variant calling for single nucleotide variants (SNV), structural variant (SV), and repeat variants; and machine-learning variant prioritization. Structured phenotypes, prioritized variants, and pedigrees were stored in PhenoTips database, with data sharing through controlled access the database of Genotypes and Phenotypes.ResultsDiagnostic rates ranged from 11% in patients with prior negative genetic testing to 34.5% in naive patients. Incorporating SVs from genome sequencing added up to 13% of new diagnoses in previously unsolved cases. HiFi-GS yielded increased discovery rate with >4-fold more rare coding SVs compared with srGS. Variants and genes of unknown significance remain the most common finding (58% of nondiagnostic cases).ConclusionComputational prioritization is efficient for diagnostic SNVs. Thorough identification of non-SNVs remains challenging and is partly mitigated using HiFi-GS sequencing. Importantly, community research is supported by sharing real-time data to accelerate gene validation and by providing HiFi variant (SNV/SV) resources from >1000 human alleles to facilitate implementation of new sequencing platforms for rare disease diagnoses.