The American College of Medical Genetics and Genomics (ACMG) previously developed guidance for the interpretation of sequence variants.1 In the past decade, sequencing technology has evolved rapidly with the advent of high-throughput next generation sequencing. By adopting and leveraging next generation sequencing, clinical laboratories are now performing an ever increasing catalogue of genetic testing spanning genotyping, single genes, gene panels, exomes, genomes, transcriptomes and epigenetic assays for genetic disorders. By virtue of increased complexity, this paradigm shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context, the ACMG convened a workgroup in 2013 comprised of representatives from the ACMG, the Association for Molecular Pathology (AMP) and the College of American Pathologists (CAP) to revisit and revise the standards and guidelines for the interpretation of sequence variants. The group consisted of clinical laboratory directors and clinicians. This report represents expert opinion of the workgroup with input from ACMG, AMP and CAP stakeholders. These recommendations primarily apply to the breadth of genetic tests used in clinical laboratories including genotyping, single genes, panels, exomes and genomes. This report recommends the use of specific standard terminology: ‘pathogenic’, ‘likely pathogenic’, ‘uncertain significance’, ‘likely benign’, and ‘benign’ to describe variants identified in Mendelian disorders. Moreover, this recommendation describes a process for classification of variants into these five categories based on criteria using typical types of variant evidence (e.g. population data, computational data, functional data, segregation data, etc.). Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends that clinical molecular genetic testing should be performed in a CLIA-approved laboratory with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or equivalent.

In clinical exome and genome sequencing, there is potential for the recognition and reporting of incidental or secondary findings unrelated to the indication for ordering the sequencing but of medical value for patient care. The American College of Medical Genetics and Genomics (ACMG) recently published a policy statement on clinical sequencing, which emphasized the importance of disclosing the possibility of such results in pretest patient discussions, clinical testing, and reporting of results. The ACMG appointed a Working Group on Incidental Findings in Clinical Exome and Genome Sequencing to make recommendations about responsible management of incidental findings when patients undergo exome or genome sequencing. This Working Group conducted a year-long consensus process, including review by outside experts, and produced recommendations that have been approved by the ACMG Board. Specific and detailed recommendations, and the background and rationale for these recommendations, are described herein. We recommend that laboratories performing clinical sequencing seek and report mutations of the specified classes or types in the genes listed here. This evaluation and reporting should be performed for all clinical germline (constitutional) exome and genome sequencing, including the ‘normal’ of tumor-normal subtractive analyses in all subjects, irrespective of age, but excluding fetal samples. We recognize that there are insufficient data on clinical utility to fully support these recommendations and we encourage the creation of an ongoing process for updating these recommendations at least annually as further data are collected.

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Importance Clinical exome sequencing (CES) is rapidly becoming a common molecular diagnostic test for individuals with rare genetic disorders. Objective To report on initial clinical indications for CES referrals and molecular diagnostic rates for different indications and for different test types. Design, Setting, and Participants Clinical exome sequencing was performed on 814 consecutive patients with undiagnosed, suspected genetic conditions at the University of California, Los Angeles, Clinical Genomics Center between January 2012 and August 2014. Clinical exome sequencing was conducted as trio-CES (both parents and their affected child sequenced simultaneously) to effectively detect de novo and compound heterozygous variants or as proband-CES (only the affected individual sequenced) when parental samples were not available. Main outcomes and Measures Clinical indications for CES requests, molecular diagnostic rates of CES overall and for phenotypic subgroups, and differences in molecular diagnostic rates between trio-CES and proband-CES. Results Of the 814 cases, the overall molecular diagnosis rate was 26% (213 of 814; 95% CI, 23%-29%). The molecular diagnosis rate for trio-CES was 31% (127 of 410 cases; 95% CI, 27%-36%) and 22% (74 of 338 cases; 95% CI, 18%-27%) for proband-CES. In cases of developmental delay in children (<5 years, n = 138), the molecular diagnosis rate was 41% (45 of 109; 95% CI, 32%-51%) for trio-CES cases and 9% (2of 23, 95% CI, 1%-28%) for proband-CES cases. The significantly higher diagnostic yield (P value = .002; odds ratio, 7.4 [95% CI, 1.6-33.1]) of trio-CES was due to the identification of de novo and compound heterozygous variants. Conclusions and Relevance In this sample of patients with undiagnosed, suspected genetic conditions, trio-CES was associated with higher molecular diagnostic yield than proband-CES or traditional molecular diagnostic methods. Additional studies designed to validate these findings and to explore the effect of this approach on clinical and economic outcomes are warranted.

ACMG previously developed recommendations for standards for interpretation of sequence variations. We now present the updated revised recommendations. Here, we describe six interpretative categories of sequence variations: (1) sequence variation is previously reported and is a recognized cause of the disorder; (2) sequence variation is previously unreported and is of the type which is expected to cause the disorder; (3) sequence variation is previously unreported and is of the type which may or may not be causative of the disorder; (4) sequence variation is previously unreported and is probably not causative of disease; (5) sequence variation is previously reported and is a recognized neutral variant; and (6) sequence variation is previously not known or expected to be causative of disease, but is found to be associated with a clinical presentation. We emphasize the importance of appropriate reporting of sequence variations using standardized terminology and established databases, and of clearly reporting the limitations of sequence-based testing. We discuss follow-up studies that may be used to ascertain the clinical significance of sequence variations, including the use of additional tools (such as predictive software programs) that may be useful in variant classification. As more information becomes available allowing the interpretation of a new sequence variant, it is recommended that the laboratory amend previous reports and provide updated results to the physician. The ACMG strongly recommends that the clinical and technical validation of sequence variation detection be performed in a CLIA-approved laboratory and interpreted by a board-certified clinical molecular geneticist or equivalent. Genet Med 2008:10(4):294-300.These recommendations for the standardization of interpretation and reporting of sequence variations identified in the course of providing clinical laboratory services are intended (1) to provide a framework for laboratories for the interpretation and reporting of such test results, and (2) to aid referring clinicians by educating them as to possible testing outcomes so that they may inform their patients and families appropriately. These revised recommendations are based on the foundation laid by the previous ACMG practice guideline recommendations in 2000. 1 I. INTERPRETATIVE CATEGORIES AND DEFINITIONS OF SEQUENCE VARIATIONSIncreasingly, clinical molecular laboratories are detecting novel sequence variations in the course of testing patient spec-

Purpose:To determine whether maternal plasma cell–free DNA sequencing can effectively identify trisomy 18 and 13.Methods:Sixty-two pregnancies with trisomy 18 and 12 with trisomy 13 were selected from a cohort of 4,664 pregnancies along with matched euploid controls (including 212 additional Down syndrome and matched controls already reported), and their samples tested using a laboratory-developed, next-generation sequencing test. Interpretation of the results for chromosome 18 and 13 included adjustment for CG content bias.Results:Among the 99.1% of samples interpreted (1,971/1,988), observed trisomy 18 and 13 detection rates were 100% (59/59) and 91.7% (11/12) at false-positive rates of 0.28% and 0.97%, respectively. Among the 17 samples without an interpretation, three were trisomy 18. If z-score cutoffs for trisomy 18 and 13 were raised slightly, the overall false-positive rates for the three aneuploidies could be as low as 0.1% (2/1,688) at an overall detection rate of 98.9% (280/283) for common aneuploidies. An independent academic laboratory confirmed performance in a subset.Conclusion:Among high-risk pregnancies, sequencing circulating cell–free DNA detects nearly all cases of Down syndrome, trisomy 18, and trisomy 13, at a low false-positive rate. This can potentially reduce invasive diagnostic procedures and related fetal losses by 95%. Evidence supports clinical testing for these aneuploidies.

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