Beyond gene-disease validity: capturing structured data on inheritance, allelic requirement, disease-relevant variant classes, and disease mechanism for inherited cardiac conditions.

BACKGROUND: As the availability of genomic testing grows, variant interpretation will increasingly be performed by genomic generalists, rather than domain-specific experts. Demand is rising for laboratories to accurately classify variants in inherited cardiac condition (ICC) genes, including secondary findings. METHODS: We analyse evidence for inheritance patterns, allelic requirement, disease mechanism and disease-relevant variant classes for 65 ClinGen-curated ICC gene-disease pairs. We present this information for the first time in a structured dataset, CardiacG2P, and assess application in genomic variant filtering. RESULTS: For 36/65 gene-disease pairs, loss of function is not an established disease mechanism, and protein truncating variants are not known to be pathogenic. Using the CardiacG2P dataset as an initial variant filter allows for efficient variant prioritisation whilst maintaining a high sensitivity for retaining pathogenic variants compared with two other variant filtering approaches. CONCLUSIONS: Access to evidence-based structured data representing disease mechanism and allelic requirement aids variant filtering and analysis and is a pre-requisite for scalable genomic testing.

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.

Beyond gene-disease validity: capturing structured data on inheritance, allelic-requirement, disease-relevant variant classes, and disease mechanism for inherited cardiac conditions.

Background: As availability of genomic testing grows, variant interpretation will increasingly be performed by genomic generalists, rather than domain-specific experts. Demand is rising for laboratories to accurately classify variants in inherited cardiac condition (ICC) genes, including as secondary findings. Methods: We analyse evidence for inheritance patterns, allelic requirement, disease mechanism and disease-relevant variant classes for 65 ClinGen-curated ICC gene-disease pairs. We present this information for the first time in a structured dataset, CardiacG2P, and assess application in genomic variant filtering. Results: For 36/65 gene-disease pairs, loss-of-function is not an established disease mechanism, and protein truncating variants are not known to be pathogenic. Using CardiacG2P as an initial variant filter allows for efficient variant prioritisation whilst maintaining a high sensitivity for retaining pathogenic variants compared with two other variant filtering approaches. Conclusions: Access to evidence-based structured data representing disease mechanism and allelic requirement aids variant filtering and analysis and is pre-requisite for scalable genomic testing.

Novel Homozygous Variant in COQ7 in Siblings With Hereditary Motor Neuropathy.

Background and Objectives: Coenzyme Q10 (CoQ10) is an important electron carrier and antioxidant. The COQ7 enzyme catalyzes the hydroxylation of 5-demethoxyubiquinone-10 (DMQ10), the second-to-last step in the CoQ10 biosynthesis pathway. We report a consanguineous family presenting with a hereditary motor neuropathy associated with a homozygous c.1A > G p.? variant of COQ7 with abnormal CoQ10 biosynthesis. Methods: Affected family members underwent clinical assessments that included nerve conduction testing, histologic analysis, and MRI. Pathogenicity of the COQ7 variant was assessed in cultured fibroblasts and skeletal muscle using a combination of immunoblots, respirometry, and quinone analysis. Results: Three affected siblings, ranging from 12 to 24 years of age, presented with a severe length-dependent motor neuropathy with marked symmetric distal weakness and atrophy with normal sensation. Muscle biopsy of the quadriceps revealed chronic denervation pattern. An MRI examination identified moderate to severe fat infiltration in distal muscles. Exome sequencing demonstrated the homozygous COQ7 c.1A > G p.? variant that is expected to bypass the first 38 amino acid residues at the n-terminus, initiating instead with methionine at position 39. This is predicted to cause the loss of the cleavable mitochondrial targeting sequence and 2 additional amino acids, thereby preventing the incorporation and subsequent folding of COQ7 into the inner mitochondrial membrane. Pathogenicity of the COQ7 variant was demonstrated by diminished COQ7 and CoQ10 levels in muscle and fibroblast samples of affected siblings but not in the father, unaffected sibling, or unrelated controls. In addition, fibroblasts from affected siblings had substantial accumulation of DMQ10, and maximal mitochondrial respiration was impaired in both fibroblasts and muscle. Discussion: This report describes a new neurologic phenotype of COQ7-related primary CoQ10 deficiency. Novel aspects of the phenotype presented by this family include pure distal motor neuropathy involvement, as well as the lack of upper motor neuron features, cognitive delay, or sensory involvement in comparison with cases of COQ7-related CoQ10 deficiency previously reported in the literature.

Fetal akinesia deformation sequence syndrome associated with recessive TTN variants.

Arthrogryposis multiplex congenita (AMC) [also known as multiple joints contracture or Fetal Akinesia Deformation Sequence (FADS)] is etiologically a heterogeneous condition with an estimated incidence of approximately 1 in 3000 live births and much higher incidence when prenatally diagnosed cases are included. The condition can be acquired or secondary to fetal exposures and can also be caused by a variety of single-gene disorders affecting the brain, spinal cord, peripheral nerves, neuromuscular junction, muscle, and a variety of disorders affecting the connective tissues (Niles et al., Prenatal Diagnosis, 2019; 39:720-731). The introduction of next-generation gene sequencing uncovered many genes and causative variants of AMC but also identified genes that cause both dominant and recessive inherited conditions with the variability of clinical manifestations depending on the genes and variants. Molecular diagnosis in these cases is not only important for prognostication but also for the determination of recurrence risk and for providing reproductive options including preimplantation and prenatal diagnosis. TTN, the largest known gene in the human genome, has been known to be associated with autosomal dominant dilated cardiomyopathy. However, homozygote and compound heterozygote pathogenic variants with recessive inheritance have rarely been reported. We report the effect of recessive variants located within the fetal IC and/or N2BA isoforms in association with severe FADS in three families. All parents were healthy obligate carriers and none of them had cardiac or skeletal muscle abnormalities. This report solidifies FADS as an alternative phenotypic presentation associated with homozygote/compound heterozygous pathogenic variants in the TTN.

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.

Evidence-Based Assessment of Genes in Dilated Cardiomyopathy.

BACKGROUND: Each of the cardiomyopathies, classically categorized as hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy, has a signature genetic theme. Hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy are largely understood as genetic diseases of sarcomere or desmosome proteins, respectively. In contrast, >250 genes spanning >10 gene ontologies have been implicated in DCM, representing a complex and diverse genetic architecture. To clarify this, a systematic curation of evidence to establish the relationship of genes with DCM was conducted. METHODS: An international panel with clinical and scientific expertise in DCM genetics evaluated evidence supporting monogenic relationships of genes with idiopathic DCM. The panel used the Clinical Genome Resource semiquantitative gene-disease clinical validity classification framework with modifications for DCM genetics to classify genes into categories on the basis of the strength of currently available evidence. Representation of DCM genes on clinically available genetic testing panels was evaluated. RESULTS: Fifty-one genes with human genetic evidence were curated. Twelve genes (23%) from 8 gene ontologies were classified as having definitive (BAG3, DES, FLNC, LMNA, MYH7, PLN, RBM20, SCN5A, TNNC1, TNNT2, TTN) or strong (DSP) evidence. Seven genes (14%; ACTC1, ACTN2, JPH2, NEXN, TNNI3, TPM1, VCL) including 2 additional ontologies were classified as moderate evidence; these genes are likely to emerge as strong or definitive with additional evidence. Of these 19 genes, 6 were similarly classified for hypertrophic cardiomyopathy and 3 for arrhythmogenic right ventricular cardiomyopathy. Of the remaining 32 genes (63%), 25 (49%) had limited evidence, 4 (8%) were disputed, 2 (4%) had no disease relationship, and 1 (2%) was supported by animal model data only. Of the 16 evaluated clinical genetic testing panels, most definitive genes were included, but panels also included numerous genes with minimal human evidence. CONCLUSIONS: In the curation of 51 genes, 19 had high evidence (12 definitive/strong, 7 moderate). It is notable that these 19 genes explain only a minority of cases, leaving the remainder of DCM genetic architecture incompletely addressed. Clinical genetic testing panels include most high-evidence genes; however, genes lacking robust evidence are also commonly included. We recommend that high-evidence DCM genes be used for clinical practice and that caution be exercised in the interpretation of variants in variable-evidence DCM genes.

Harmonizing the Collection of Clinical Data on Genetic Testing Requisition Forms to Enhance Variant Interpretation in Hypertrophic Cardiomyopathy (HCM): A Study from the ClinGen Cardiomyopathy Variant Curation Expert Panel.

Diagnostic laboratories gather phenotypic data through requisition forms, but there is no consensus as to which data are essential for variant interpretation. The ClinGen Cardiomyopathy Variant Curation Expert Panel defined a phenotypic data set for hypertrophic cardiomyopathy (HCM) variant interpretation, with the goal of standardizing requisition forms. Phenotypic data elements listed on requisition forms from nine leading cardiomyopathy testing laboratories were compiled to assess divergence in data collection. A pilot of 50 HCM cases was implemented to determine the feasibility of harmonizing data collection. Laboratory directors were surveyed to gauge potential for adoption of a minimal data set. Wide divergence was observed in the phenotypic data fields in requisition forms. The 50-case pilot showed that although demographics and assertion of a clinical diagnosis of HCM had 86% to 98% completion, specific phenotypic features, such as degree of left ventricular hypertrophy, ejection fraction, and suspected syndromic disease, were completed only 24% to 44% of the time. Nine data elements were deemed essential for variant classification by the expert panel. Participating laboratories unanimously expressed a willingness to adopt these data elements in their requisition forms. This study demonstrates the value of comparing and sharing best practices through an expert group, such as the ClinGen Program, to enhance variant interpretation, providing a foundation for leveraging cumulative case-level data in public databases and ultimately improving patient care.

Interplay between probe design and test performance: overlap between genomic regions of interest, capture regions and high quality reference calls influence performance of WES-based assays.

Whole exome sequencing (WES)-based assays undergo rigorous validation before being implemented in diagnostic laboratories. This validation process generates experimental evidence that allows laboratories to predict the performance of the intended assay. The NA12878 Genome in a Bottle (GIAB) HapMap reference sample is commonly used for validation in diagnostic laboratories. We investigated what data points should be taken into consideration when validating WES-based assays using the GIAB reference in a diagnostic setting. We delineate specific factors that require special consideration and identify OMIM genes associated with diseases that may 'bypass' validation. Four replicates of the NA12878 sample were sequenced at the CHEO Genetics Diagnostic Laboratory on a NextSeq 500; the data were analyzed using the bcbio_nexgen v1.1.2 pipeline. The hap.py validation engine, Real Time Genomics vcfeval tool, and high confidence (HC) variant calls in HC regions available for the GIAB sample were used to validate the obtained variant calls. The same validation process was then used to evaluate variant calls obtained for the same sample by two other clinical diagnostic laboratories. We showed that variant calls in NA12878 can be confidently measured only in the regions that intersect between the GIAB HC regions and the target regions of exome capture. Of the 4139 (as of October 2019) OMIM genes associated with a phenotype and having a known molecular basis of disease, 84 were fully outside of the GIAB HC regions and many of the remaining OMIM genes were only partially covered by the HC regions. A significant proportion of variants identified in the NA12878 sample outside of the HC regions have unknown (UNK) status due to the absence of HC reference alleles. Verification of such calls is possible either by an alternative truth set or by orthogonal testing. Similarly, many variants outside of exome capture regions, if not accounted for, will be deemed false negatives due to insufficient probe coverage. Our results demonstrate the importance of the intersection between genomic regions of interest, capture regions, and the high confidence regions. If not considered, false and ambiguous variant calls could have a negative impact on diagnostic accuracy of the intended WES-based diagnostic assay and increase the need for confirmatory testing. To enable laboratories to identify 'problematic' regions and optimize validation efforts, we have made our VCF and BED files available in UCSC Genome Browser: NA12878 WES Benchmark. Relevant genes and genome annotations are evolving, we implemented a general purpose algorithm to cross-reference OMIM genes with the genomic regions of interest that can be applied to capture genes/regions outside HC regions (see repository of data material section).

Implementation of Epilepsy Multigene Panel Testing in Ontario, Canada.

BACKGROUND: Epilepsy is a common neurological condition that shows a marked genetic predisposition. The advent of next-generation sequencing (NGS) has transformed clinical genetic testing by allowing the rapid screen for causative variants in multiple genes. There are currently no NGS-based multigene panel diagnostic tests available for epilepsy as a licensed clinical diagnostic test in Ontario, Canada. Eligible patient samples are sent out of country for testing by commercial laboratories, which incurs significant cost to the public healthcare system. OBJECTIVE: An expert Working Group of medical geneticists, pediatric neurologists/epileptologists, biochemical geneticists, and clinical molecular geneticists from Ontario was formed by the Laboratories and Genetics Branch of the Ontario Ministry of Health and Long-Term Care to develop a programmatic approach to implementing epilepsy panel testing as a provincial service. RESULTS: The Working Group made several recommendations for testing to support the clinical delivery of care in Ontario. First, an extension of community healthcare outcomes-based program should be incorporated to inform and educate ordering providers when requesting and interpreting a genetic panel test. Second, any gene panel testing must be "evidence-based" and takes into account varied clinical indications to reduce the chance of uncertain and secondary results. Finally, an ongoing evaluative process was recommended to ensure continued test improvement for the future. CONCLUSION: This epilepsy panel testing implementation plan will be a model for genetic care directed toward a specific set of conditions in the province and serve as a prototype for genetic testing for other genetically heterogeneous diseases.

Mise en œuvre d'un test diagnostique permettant en Ontario l'analyse d'un panel de plusieurs gènes liés à l'épilepsie.Contexte:L'épilepsie demeure un trouble neurologique fréquent dont la prédisposition génétique apparaît notable. L'émergence du séquençage de nouvelle génération (SNG) a aussi transformé les tests génétiques en permettant un dépistage rapide des variantes causales que l'on retrouve dans de nombreux gènes. À l'heure actuelle, il n'existe pas, pour l'épilepsie, de tests diagnostiques homologués qui permettent en Ontario l'analyse d'un panel de gènes en vertu du SNG. Les échantillons de patients admissibles sont alors envoyés à l'extérieur du Canada afin d'être analysés par des laboratoires commerciaux, ce qui pèse lourd dans les budgets des systèmes publics de santé. Objectif : Un groupe de travail formé d'experts (généticiens médicaux, neurologues pédiatriques et spécialistes en épileptologie, généticiens biochimiques et généticiens moléculaires cliniques) a été formé par le service des laboratoires et de la génétique des ministères de la Santé et des Soins de Longue durée de l'Ontario afin d'élaborer une démarche programmatique visant à mettre en œuvre des tests diagnostiques basés sur un panel de plusieurs gènes. Ces tests seraient ensuite reconnus à titre de service public. Résultats:En matière de dépistage, ce groupe de travail a ainsi émis plusieurs recommandations visant à accompagner la prestation clinique en Ontario. Tout d'abord, un programme s'inspirant du projet « ECHO ¼ (Extension of Community Healthcare Outcomes) devrait être ajouté dans le but de renseigner et de sensibiliser les prestataires de soins de santé qui demandent et qui interprètent ces tests basés sur un panel de plusieurs gènes. Ensuite, tout test de ce type doit reposer sur des preuves et tenir compte d'une panoplie d'indications cliniques afin de réduire les possibilités d'incertitude et de résultats secondaires. Enfin, il a été recommandé de procéder à un processus continu d'évaluation pour s'assurer que ces tests puissent être améliorés dans le futur. Conclusion:Ce plan de mise en œuvre de tests basés sur un panel de plusieurs gènes deviendra un modèle pour les soins destinés à un ensemble spécifique de problèmes de santé en Ontario. Outre l'épilepsie, il pourra servir comme prototype pour le dépistage d'autres maladies hétérogènes sur le plan génétique.

ALU transposition induces familial hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy (LVH) in the absence of predisposing cardiovascular conditions. Pathogenic variants in at least 16 cardiac sarcomeric genes have been implicated in HCM, most of which act in a dominant-negative fashion. However loss-of-function (haploinsufficiency) is the most common disease mechanism for pathogenic variants in MYBPC3, suggesting that MYBPC3 complete deletion may play a role in HCM pathogenesis. Here, we investigate MYBPC3 complete deletion as a disease mechanism in HCM by analyzing two unrelated patients with confirmed diagnosis of HCM that tested negative by Sanger sequencing analysis. METHODS: MYBPC3 complete deletion was investigated by Multiplex ligation-dependent probe amplification (MLPA) and microarray analyses. The mechanism of deletion was investigated by interrogating the SINEBase database. RESULTS: Patient-1 was diagnosed with nonobstructive HCM in his mid-40s while undergoing assessment for palpitations, and patient-2 with obstructive HCM in his late-20s while undergoing systolic heart murmur assessment for an unrelated illness. MLPA testing revealed a heterozygous deletion of all MYBPC3 exons in both patients. Subsequent microarray testing confirmed these deletions which extended beyond the 5' and 3' ends of MYBPC3. Genomic assessment suggested that these deletions resulted from Alu/Alu-homologous recombination. CONCLUSION: Our results demonstrate that haploinsufficiency resulting from MYBPC3 complete deletion, potentially mediated by Alu recombination, is an important disease mechanism in cardiomyopathy and emphasizes the importance of copy number variation analysis in patients clinically suspected of HCM.

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.

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.

Scaling resolution of variant classification differences in ClinVar between 41 clinical laboratories through an outlier approach.

ClinVar provides open access to variant classifications shared from many clinical laboratories. Although most classifications are consistent across laboratories, classification differences exist. To facilitate resolution of classification differences on a large scale, clinical laboratories were encouraged to reassess outlier classifications of variants with medically significant differences (MSDs). Outliers were identified by first comparing ClinVar submissions from 41 clinical laboratories to detect variants with MSDs between the laboratories (650 variants). Next, MSDs were filtered for variants with ≥3 classifications (244 variants), of which 87.6% (213 variants) had a majority consensus in ClinVar, thus allowing for identification of outlier classifications in need of reassessment. Laboratories with outlier classifications were sent a custom report and encouraged to reassess variants. Results were returned for 204 (96%) variants, of which 62.3% (127) were resolved. Of those 127, 64.6% (82) were resolved due to reassessment prompted by this study and 35.4% (45) resolved by a previously completed reassessment. This study demonstrates a scalable approach to classification resolution and capitalizes on the value of data sharing within ClinVar. These activities will help the community move toward more consistent variant classifications, which will improve the care of patients with, or at risk for, genetic disorders.

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.

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.

Next-generation sequencing for diagnosis of rare diseases in the neonatal intensive care unit.

BACKGROUND: Rare diseases often present in the first days and weeks of life and may require complex management in the setting of a neonatal intensive care unit (NICU). Exhaustive consultations and traditional genetic or metabolic investigations are costly and often fail to arrive at a final diagnosis when no recognizable syndrome is suspected. For this pilot project, we assessed the feasibility of next-generation sequencing as a tool to improve the diagnosis of rare diseases in newborns in the NICU. METHODS: We retrospectively identified and prospectively recruited newborns and infants admitted to the NICU of the Children's Hospital of Eastern Ontario and the Ottawa Hospital, General Campus, who had been referred to the medical genetics or metabolics inpatient consult service and had features suggesting an underlying genetic or metabolic condition. DNA from the newborns and parents was enriched for a panel of clinically relevant genes and sequenced on a MiSeq sequencing platform (Illumina Inc.). The data were interpreted with a standard informatics pipeline and reported to care providers, who assessed the importance of genotype-phenotype correlations. RESULTS: Of 20 newborns studied, 8 received a diagnosis on the basis of next-generation sequencing (diagnostic rate 40%). The diagnoses were renal tubular dysgenesis, SCN1A-related encephalopathy syndrome, myotubular myopathy, FTO deficiency syndrome, cranioectodermal dysplasia, congenital myasthenic syndrome, autosomal dominant intellectual disability syndrome type 7 and Denys-Drash syndrome. INTERPRETATION: This pilot study highlighted the potential of next-generation sequencing to deliver molecular diagnoses rapidly with a high success rate. With broader use, this approach has the potential to alter health care delivery in the NICU.