Recommendations for Next-Generation Sequencing Germline Variant Confirmation: A Joint Report of the Association for Molecular Pathology and National Society of Genetic Counselors.

Clinical laboratory implementation of next-generation sequencing (NGS)-based constitutional genetic testing has been rapid and widespread. In the absence of widely adopted comprehensive guidance, there remains substantial variability among laboratories in the practice of NGS. One issue of sustained discussion in the field is whether and to what extent orthogonal confirmation of genetic variants identified by NGS is necessary or helpful. The Association for Molecular Pathology Clinical Practice Committee convened the NGS Germline Variant Confirmation Working Group to assess current evidence regarding orthogonal confirmation and to establish recommendations for standardizing orthogonal confirmation practices to support quality patient care. On the basis of the results of a survey of the literature, a survey of laboratory practices, and subject expert matter consensus, eight recommendations are presented, providing a common framework for clinical laboratory professionals to develop or refine individualized laboratory policies and procedures regarding orthogonal confirmation of germline variants detected by NGS.

Diagnostic testing laboratories are valuable partners for disease gene discovery: 5-year experience with GeneMatcher.

Although the rates of disease gene discovery have steadily increased with the expanding use of genome and exome sequencing by clinical and research laboratories, only ~16% of genes in the genome have confirmed disease associations. Here we describe our clinical laboratory's experience utilizing GeneMatcher, an online portal designed to promote disease gene discovery and data sharing. Since 2016, we submitted 246 candidates from 243 unique genes to GeneMatcher, of which 111 (45%) are now clinically characterized. Submissions meeting our candidate gene-reporting criteria based on a scoring system using patient and molecular-weighted evidence were significantly more likely to be characterized as of October 2021 versus genes that did not meet our clinical-reporting criteria (p = 0.025). We reported relevant findings related to these newly characterized gene-disease associations in 477 probands. In 218 (46%) instances, we issued reclassifications after an initial negative or candidate gene (uncertain) report. We coauthored 104 publications delineating gene-disease relationships, including descriptions of new associations (60%), additional supportive evidence (13%), subsequent descriptive cohorts (23%), and phenotypic expansions (4%). Clinical laboratories are pivotal for disease gene discovery efforts and can screen phenotypes based on genotype matches, contact clinicians of relevant cases, and issue proactive reclassification reports.

Misattributed parentage identified through diagnostic exome sequencing: Frequency of detection and reporting practices.

Access to genetic testing, namely, diagnostic exome sequencing (DES), has significantly improved, subsequently increasing the likelihood of discovering incidental findings, such as misattributed relationships and specifically misattributed parentage (MP). Until the recently published ACMG statement, there had been no consensus for laboratories and clinicians to follow when addressing such findings. Family-based genomic testing is valuable for accurate variant interpretation but has the potential to uncover misattributed familial relationships. Here, we present the first published data on the frequency of MP identified through DES at a clinical laboratory. We also investigated clinicians' decisions on how to proceed with analysis, reporting, and disclosure. A database of 6,752 families who underwent parent-proband ('trio') DES was retrospectively reviewed for molecular identification of MP and clinicians' MP disclosure decisions. Among 6,752 trios, 39 cases of MP were detected (0.58%). Non-paternity was detected in all cases, and in one instance, non-maternity was also identified. All clinicians decided to proceed by omitting the MP individual from the analysis. Clinicians chose to proceed with duo analysis (87.2%), modify information on the report (74.4%), and communicate MP results to the mother (71.8%), suggesting a trend toward not disclosing to the putative father or proband. The data show that trio DES involves a chance of detecting MP and that clinician disclosure practices do not appear to routinely include direct disclosure to the putative father. MP identified in our parent-proband trios sent in for DES is lower than the reported frequency of MP in the general population due in part to ascertainment bias as families with known or suspected MP are presumably less likely to pursue trio testing. These data may inform laboratory policies and clinician practices for addressing incidental findings such as MP.

Facing the challenge of genetic counselors' need for rapid continuing education about genomic technologies.

The last couple of decades have seen the rapid advancement of genomic technologies (GT) and their equally rapid adoption into clinical testing. Regardless of specialty, all genetic counselors are unified by the fundamental goal to aid in diagnosing patient's genetic disease underscoring the importance for genetic counselors to maintain an in-depth understanding of GT. The National Society of Genetic Counselors' (NSGC) GT Special Interest Group conducted an online survey of NSGC members to assess current genomic technologies knowledge gaps. A total of 171 individuals from a variety of primary work settings completed the survey sufficiently to be included in the analysis. The majority of respondents received their degree in genetic counseling in more recent years (2000-2015). On average across all technologies, >70% of respondents deemed knowledge of GTs as important for successful job performance, 55% responded that additional job training in GTs is needed to successfully perform job functions, and only 28% responded that graduate training in GTs was good. Overall, the data show that participating genetic counselors perceive that their knowledge of GTs is inadequate while it is a key component of their jobs. These results have implications both for training programs and for continuing education efforts. These data can be used as a starting point for additional research into GT educational needs of genetic counselors.

Clinical diagnostic exome sequencing in dystonia: Genetic testing challenges for complex conditions.

Patients with dystonia are particularly appropriate for diagnostic exome sequencing (DES), due to the complex, diverse features and genetic heterogeneity. Personal and family history data were collected from test requisition forms and medical records from 189 patients with reported dystonia and available family members received for clinical DES. Of them, 20.2% patients had a positive genetic finding associated with dystonia. Detection rates for cases with isolated and combined dystonia were 22.4% and 25.0%, respectively. 71.4% of the cohort had co-occurring non-movement-related findings and a detection rate of 24.4%. Patients with childhood-onset dystonia trended toward higher detection rates (31.8%) compared to infancy (23.6%), adolescence (12.5%), and early-adulthood onset (16%). Uncharacterized gene findings were found in 6.7% (8/119) of cases that underwent analysis for genes without an established disease relationship. Patients with intellectual disability/developmental delay, seizures/epilepsy and/or multifocal dystonia were more likely to have positive findings (P = .0093, .0397, .0006). Four (2.1%) patients had findings in two genes, and seven (3.7%) had reclassification after the original report due to new literature, new clinical information or reanalysis request. Pediatric patients were more likely to have positive findings (P = .0180). Our observations show utility of family-based DES in patients with dystonia and illustrate the complexity of testing.

When moments matter: Finding answers with rapid exome sequencing.

BACKGROUND: When time is of the essence in critical care cases, a fast molecular diagnosis is often necessary to help health care providers quickly determine best next steps for treatments, prognosis, and counseling of their patients. In this paper, we present the diagnostic rates and improved quality of life for patients undergoing clinical rapid exome sequencing. METHODS: The clinical histories and results of 41 patients undergoing rapid exome sequencing were retrospectively reviewed. RESULTS: Clinical rapid exome sequencing identified a definitive diagnosis in 13/41 (31.7%) and other relevant findings in 17 of the patients (41.5%). The average time to verbal report was 7 days; to written report was 11 days. CONCLUSIONS: Our observations demonstrate the utility and effectiveness of rapid family-based diagnostic exome sequencing in improving patients care.

A retrospective review of multiple findings in diagnostic exome sequencing: half are distinct and half are overlapping diagnoses.

PURPOSE: We evaluated clinical and genetic features enriched in patients with multiple Mendelian conditions to determine which patients are more likely to have multiple potentially relevant genetic findings (MPRF). METHODS: Results of the first 7698 patients who underwent exome sequencing at Ambry Genetics were reviewed. Clinical and genetic features were examined and degree of phenotypic overlap between the genetic diagnoses was evaluated. RESULTS: Among patients referred for exome sequencing, 2% had MPRF. MPRF were more common in patients from consanguineous families and patients with greater clinical complexity. The difference in average number of organ systems affected is small: 4.3 (multiple findings) vs. 3.9 (single finding) and may not be distinguished in clinic. CONCLUSION: Patients with multiple genetic diagnoses had a slightly higher number of organ systems affected than patients with single genetic diagnoses, largely because the comorbid conditions affected overlapping organ systems. Exome testing may be beneficial for all cases with multiple organ systems affected. The identification of multiple relevant genetic findings in 2% of exome patients highlights the utility of a comprehensive molecular workup and updated interpretation of existing genomic data; a single definitive molecular diagnosis from analysis of a limited number of genes may not be the end of a diagnostic odyssey.

Clinical validity assessment of genes for inclusion in multi-gene panel testing: A systematic approach.

BACKGROUND: Advances in sequencing technology have led to expanded use of multi-gene panel tests (MGPTs) for clinical diagnostics. Well-designed MGPTs must balance increased detection of clinically significant findings while mitigating the increase in variants of uncertain significance (VUS). To maximize clinical utililty, design of such panels should include comprehensive gene vetting using a standardized clinical validity (CV) scoring system. METHODS: To assess the impact of CV-based gene vetting on MGPT results, data from MGPTs for cardiovascular indications were retrospectively analyzed. Using our CV scoring system, genes were categorized as having definitive, strong, moderate, or limited evidence. The rates of reported pathogenic or likely pathogenic variants and VUS were then determined for each CV category. RESULTS: Of 106 total genes, 42% had definitive, 17% had strong, 29% had moderate, and 12% had limited CV. The detection rate of variants classified as pathogenic or likely pathogenic was higher for genes with greater CV, while the VUS rate showed an inverse relationship with CV score. No pathogenic or likely pathogenic findings were observed in genes with a limited CV. CONCLUSION: These results demonstrate the importance of a standardized, evidence-based vetting process to establish CV for genes on MGPTs. Using our proposed system may help to increase the detection rate while mitigating higher VUS rates.

Misattributed parentage as an unanticipated finding during exome/genome sequencing: current clinical laboratory practices and an opportunity for standardization.

PURPOSE: Clinical laboratories performing exome or genome sequencing (ES/GS) are familiar with the challenges associated with proper consenting for and reporting of medically actionable secondary findings based on recommendations from the American College of Medical Genetics and Genomics (ACMG). Misattributed parentage is another type of unanticipated finding a laboratory may encounter during family-based ES/GS; however, there are currently no professional recommendations related to the proper consenting for and reporting of misattributed parentage encountered during ES/GS. METHODS: We surveyed 10 clinical laboratories offering family-based ES/GS regarding their consent language, discovery, and reporting of misattributed parentage. RESULTS: Many laboratories have already developed their own practices/policies for these issues, which do not necessarily agree with those from other labs. CONCLUSION: There are several other possibilities besides true misattributed parentage that could result in similar laboratory findings, and laboratories often feel they lack sufficient information to make formal conclusions on a report regarding the true genetic relatedness of the submitted samples. However, understanding the genetic relatedness (or lack thereof) of the samples submitted for family-based ES/GS has medical relevance. Therefore, professional recommendations for the appropriate handling of suspected misattributed parentage encountered during ES/GS are needed to help standardize current clinical laboratory practices.

Detection of structural variation using target captured next-generation sequencing data for genetic diagnostic testing.

PURPOSE: Structural variation (SV) is associated with inherited diseases. Next-generation sequencing (NGS) is an efficient method for SV detection because of its high-throughput, low cost, and base-pair resolution. However, due to lack of standard NGS protocols and a limited number of clinical samples with pathogenic SVs, comprehensive standards for SV detection, interpretation, and reporting are to be established. METHODS: We performed SV assessment on 60,000 clinical samples tested with hereditary cancer NGS panels spanning 48 genes. To evaluate NGS results, NGS and orthogonal methods were used separately in a blinded fashion for SV detection in all samples. RESULTS: A total of 1,037 SVs in coding sequence (CDS) or untranslated regions (UTRs) and 30,847 SVs in introns were detected and validated. Across all variant types, NGS shows 100% sensitivity and 99.9% specificity. Overall, 64% of CDS/UTR SVs were classified as pathogenic/likely pathogenic, and five deletions/duplications were reclassified as pathogenic using breakpoint information from NGS. CONCLUSION: The SVs presented here can be used as a valuable resource for clinical research and diagnostics. The data illustrate NGS as a powerful tool for SV detection. Application of NGS and confirmation technologies in genetic testing ensures delivering accurate and reliable results for diagnosis and patient care.

Clinical whole-exome sequencing results impact medical management.

BACKGROUND: Clinical diagnostic whole-exome sequencing (WES) is a powerful tool for patients with undiagnosed genetic disorders. To demonstrate the clinical utility, we surveyed healthcare providers (HCP) about changes in medical management and treatment, diagnostic testing, reproductive planning, and use of educational services subsequent to WES testing. METHODS: For a period of 18 months, an 18-question survey was sent to HCPs attached to the WES reports. We analyzed the molecular diagnosis, patient clinical features, and the medical management changes reported in the returned surveys. RESULTS: A total of 62 (2.2% of 2,876) surveys were returned, consisting of 37.1% patients with a positive or likely positive pathogenic alteration, 51.6% negative results, 9.7% uncertain findings, and 1 patient (1.6%) with a novel candidate finding. Overall, 100% of the HCPs of patients with positive or likely positive WES results (n = 23) and HCPs of patients with uncertain WES results (n = 6) responded positively to one of the 18 queries. Of note, 37.5% of the HCPs of patients with negative WES results (n = 32) responded positively to at least one query. CONCLUSION: Overall, these data clearly demonstrate the clinical utility of WES by demonstrating the impact on medical management irrespective of the exome result.

Clinical germline diagnostic exome sequencing for hereditary cancer: Findings within novel candidate genes are prevalent.

Clinical diagnostic exome sequencing (DES) has been effective in diagnosing individuals with suspected genetic conditions; nevertheless little has been described regarding its clinical utility in individuals with a personal and family history of cancer. This study aimed to assess diagnostic yield and clinical characteristics of pediatric and adult patients undergoing germline DES for hereditary cancer. We retrospectively reviewed 2171 patients referred for DES; cases with a personal and/or family history of cancer were further studied. Of 39 cancer patients, relevant alterations were found in eight individuals (21%), including one (3%) positive pathogenic alteration within a characterized gene, two (5%) uncertain findings in characterized genes, and five (13%) alterations in novel candidate genes. Two of the 5 pediatric patients, undergoing testing, (40%) had findings in novel candidate genes, with the remainder being negative. We include brief case studies to illustrate the variety of challenging issues related to these patients. Our observations demonstrate utility of family-based exome sequencing in patients for suspected hereditary cancer, including familial co-segregation analysis, and comprehensive medical review. DES may be particularly useful when traditional approaches do not result in a diagnosis or in families with unique phenotypes. This work also highlights the importance and complexity of analysis of uncharacterized genes in exome sequencing for hereditary cancer.

Exome sequencing in neonates: diagnostic rates, characteristics, and time to diagnosis.

PURPOSE: Neonatal patients are particularly appropriate for utilization of diagnostic exome sequencing (DES), as many Mendelian diseases are known to present in this period of life but often with complex, heterogeneous features. We attempted to determine the diagnostic rates and features of neonatal patients undergoing DES. METHODS: The clinical histories and results of 66 neonatal patients undergoing DES were retrospectively reviewed. RESULTS: Clinical DES identified potentially relevant findings in 25 patients (37.9%). The majority of patients had structural anomalies such as birth defects, dysmorphic features, cardiac, craniofacial, and skeletal defects. The average time for clinical rapid testing was 8 days. CONCLUSION: Our observations demonstrate the utility of family-based exome sequencing in neonatal patients, including familial cosegregation analysis and comprehensive medical review.

Correction: Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases.

In the published version of this paper, some of the columns in the last three rows of Table 3 were mistakenly transposed. The corrected table appears below. In col. 6 of the row for DNMT3A, "S3" was published in the original article. However, in the revised table for the corrigendum, it has been corrected to "S1". In col. 6 of the row for SON, "S3" was published in the original article. However, in the revised table for the corrigendum, it has been corrected to "S2".

Outcomes of Diagnostic Exome Sequencing in Patients With Diagnosed or Suspected Autism Spectrum Disorders.

BACKGROUND: Exome sequencing has recently been proved to be a successful diagnostic method for complex neurodevelopmental disorders. However, the diagnostic yield of exome sequencing for autism spectrum disorders has not been extensively evaluated in large cohorts to date. MATERIALS AND METHODS: We performed diagnostic exome sequencing in a cohort of 163 individuals with autism spectrum disorder (66.3%) or autistic features (33.7%). RESULTS: The diagnostic yield observed in patients in our cohort was 25.8% (42 of 163) for positive or likely positive findings in characterized disease genes, while a candidate genetic etiology was reported for an additional 3.3% (4 of 120) of patients. Among the positive findings in the patients with autism spectrum disorder or autistic features, 61.9% were the result of de novo mutations. Patients presenting with psychiatric conditions or ataxia or paraplegia in addition to autism spectrum disorder or autistic features were significantly more likely to receive positive results compared with patients without these clinical features (95.6% vs 27.1%, P < 0.0001; 83.3% vs 21.2%, P < 0.0001, respectively). The majority of the positive findings were in recently identified autism spectrum disorder genes, supporting the importance of diagnostic exome sequencing for patients with autism spectrum disorder or autistic features as the causative genes might evade traditional sequential or panel testing. CONCLUSIONS: These results suggest that diagnostic exome sequencing would be an efficient primary diagnostic method for patients with autism spectrum disorders or autistic features. Moreover, our data may aid clinicians to better determine which subset of patients with autism spectrum disorder with additional clinical features would benefit the most from diagnostic exome sequencing.

Classification of Genes: Standardized Clinical Validity Assessment of Gene-Disease Associations Aids Diagnostic Exome Analysis and Reclassifications.

Ascertaining a diagnosis through exome sequencing can provide potential benefits to patients, insurance companies, and the healthcare system. Yet, as diagnostic sequencing is increasingly employed, vast amounts of human genetic data are produced that need careful curation. We discuss methods for accurately assessing the clinical validity of gene-disease relationships to interpret new research findings in a clinical context and increase the diagnostic rate. The specifics of a gene-disease scoring system adapted for use in a clinical laboratory are described. In turn, clinical validity scoring of gene-disease relationships can inform exome reporting for the identification of new or the upgrade of previous, clinically relevant gene findings. Our retrospective analysis of all reclassification reports from the first 4 years of diagnostic exome sequencing showed that 78% were due to new gene-disease discoveries published in the literature. Among all exome positive/likely positive findings in characterized genes, 32% were in genetic etiologies that were discovered after 2010. Our data underscore the importance and benefits of active and up-to-date curation of a gene-disease database combined with critical clinical validity scoring and proactive reanalysis in the clinical genomics era.

Candidate-gene criteria for clinical reporting: diagnostic exome sequencing identifies altered candidate genes among 8% of patients with undiagnosed diseases.

PURPOSE: Diagnostic exome sequencing (DES) is now a commonly ordered test for individuals with undiagnosed genetic disorders. In addition to providing a diagnosis for characterized diseases, exome sequencing has the capacity to uncover novel candidate genes for disease. METHODS: Family-based DES included analysis of both characterized and novel genetic etiologies. To evaluate candidate genes for disease in the clinical setting, we developed a systematic, rule-based classification schema. RESULTS: Testing identified a candidate gene among 7.7% (72/934) of patients referred for DES; 37 (4.0%) and 35 (3.7%) of the genes received evidence scores of "candidate" and "suspected candidate," respectively. A total of 71 independent candidate genes were reported among the 72 patients, and 38% (27/71) were subsequently corroborated in the peer-reviewed literature. This rate of corroboration increased to 51.9% (27/52) among patients whose gene was reported at least 12 months previously. CONCLUSIONS: Herein, we provide transparent, comprehensive, and standardized scoring criteria for the clinical reporting of candidate genes. These results demonstrate that DES is an integral tool for genetic diagnosis, especially for elucidating the molecular basis for both characterized and novel candidate genetic etiologies. Gene discoveries also advance the understanding of normal human biology and more common diseases.Genet Med 19 2, 224-235.

A survey of current practices for genomic sequencing test interpretation and reporting processes in US laboratories.

PURPOSE: While the diagnostic success of genomic sequencing expands, the complexity of this testing should not be overlooked. Numerous laboratory processes are required to support the identification, interpretation, and reporting of clinically significant variants. This study aimed to examine the workflow and reporting procedures among US laboratories to highlight shared practices and identify areas in need of standardization. METHODS: Surveys and follow-up interviews were conducted with laboratories offering exome and/or genome sequencing to support a research program or for routine clinical services. The 73-item survey elicited multiple choice and free-text responses that were later clarified with phone interviews. RESULTS: Twenty-one laboratories participated. Practices highly concordant across all groups included consent documentation, multiperson case review, and enabling patient opt-out of incidental or secondary findings analysis. Noted divergence included use of phenotypic data to inform case analysis and interpretation and reporting of case-specific quality metrics and methods. Few laboratory policies detailed procedures for data reanalysis, data sharing, or patient access to data. CONCLUSION: This study provides an overview of practices and policies of experienced exome and genome sequencing laboratories. The results enable broader consideration of which practices are becoming standard approaches, where divergence remains, and areas of development in best practice guidelines that may be helpful.Genet Med advance online publication 03 Novemeber 2016.

Diagnostic exome sequencing provides a molecular diagnosis for a significant proportion of patients with epilepsy.

PURPOSE: To assess the yield of diagnostic exome sequencing (DES) and to characterize the molecular findings in characterized and novel disease genes in patients with epilepsy. METHODS: In an unselected sample of 1,131 patients referred for DES, overall results were compared between patients with and without epilepsy. DES results were examined based on age of onset and epilepsy diagnosis. RESULTS: Positive/likely positive results were identified in 112/293 (38.2%) epilepsy patients compared with 210/732 (28.7%) patients without epilepsy (P = 0.004). The diagnostic yield in characterized disease genes among patients with epilepsy was 33.4% (105/314). KCNQ2, MECP2, FOXG1, IQSEC2, KMT2A, and STXBP1 were most commonly affected by de novo alterations. Patients with epileptic encephalopathies had the highest rate of positive findings (43.4%). A likely positive novel genetic etiology was proposed in 14/200 (7%) patients with epilepsy; this frequency was highest in patients with epileptic encephalopathies (17%). Three genes (COQ4, DNM1, and PURA) were initially reported as likely positive novel disease genes and were subsequently corroborated in independent peer-reviewed publications. CONCLUSION: DES with analysis and interpretation of both characterized and novel genetic etiologies is a useful diagnostic tool in epilepsy, particularly in severe early-onset epilepsy. The reporting on novel genetic etiologies may further increase the diagnostic yield.Genet Med 18 9, 898-905.