Wayne W. Grody

Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology

Disclaimer: These ACMG Standards and Guidelines were developed primarily as an educational resource for clinical laboratory geneticists to help them provide quality clinical laboratory services. Adherence to these standards and guidelines is voluntary and does not necessarily assure a successful medical outcome. These Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient’s record the rationale for the use of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They also are advised to take notice of the date any particular guideline was adopted and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.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 shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context the ACMG convened a workgroup in 2013 comprising representatives from the ACMG, the Association for Molecular Pathology (AMP), and the College of American Pathologists 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 College of American Pathologists 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 genes that cause Mendelian disorders. Moreover, this recommendation describes a process for classifying variants into these five categories based on criteria using typical types of variant evidence (e.g., population data, computational data, functional data, segregation data). 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 Clinical Laboratory Improvement Amendments–approved laboratory, with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or the equivalent.Genet Med 17 5, 405–423.

ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing

In clinical exome and genome sequencing, there is a 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 that emphasized the importance of alerting the patient to 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 an open forum at the 2012 Annual Meeting and 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. The ACMG recommends 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 penetrance and 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.Genet Med 2013:15(7):565–574

DNA sequencing of maternal plasma to detect Down syndrome: An international clinical validation study

Purpose: Prenatal screening for Down syndrome has improved, but the number of resulting invasive diagnostic procedures remains problematic. Measurement of circulating cell-free DNA in maternal plasma might offer improvement.Methods: A blinded, nested case-control study was designed within a cohort of 4664 pregnancies at high risk for Down syndrome. Fetal karyotyping was compared with an internally validated, laboratory-developed test based on next-generation sequencing in 212 Down syndrome and 1484 matched euploid pregnancies. None had been previously tested. Primary testing occurred at a CLIA-certified commercial laboratory, with cross validation by a CLIA-certified university laboratory.Results: Down syndrome detection rate was 98.6% (209/212), the false-positive rate was 0.20% (3/1471), and the testing failed in 13 pregnancies (0.8%); all were euploid. Before unblinding, the primary testing laboratory also reported multiple alternative interpretations. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving performance.Conclusion: When applied to high-risk pregnancies, measuring maternal plasma DNA detects nearly all cases of Down syndrome at a very low false-positive rate. This method can substantially reduce the need for invasive diagnostic procedures and attendant procedure-related fetal losses. Although implementation issues need to be addressed, the evidence supports introducing this testing on a clinical basis.

ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007

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.

DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study.

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.Genet Med 2012:14(3):296–305

Laboratory standards and guidelines for population- based cystic fibrosis carrier screening

In 1997, the National Institutes of Health convened a Consensus Development Conference on Cystic Fibrosis (CF).1 The Consensus Conference recommended that genetic screening for CF mutations should be offered to identify carriers among adults with a positive family history of CF, partners of individuals with CF, couples currently planning a pregnancy, and couples seeking prenatal care. A second NIH-sponsored conference that focused on the implementation of the Consensus Conference recommendations was held in 1998.2 Shortly thereafter, the American College of Medical Genetics (ACMG) and the American College of Obstetricians and Gynecologists (ACOG), in conjunction with the National Human Genome Research Institute, formed a Steering Committee to coordinate the implementation of population-based CF carrier screening and to develop “Clinical and Laboratory Provider Guidelines” for (1) provider education; (2) laboratory testing, interpretation, and genetic counseling; and (3) patient education and informed consent. The ACMG charged the Accreditation of Genetic Services Committee, chaired by Dr. Robert Desnick, to establish a Subcommittee on Cystic Fibrosis Carrier Screening (henceforth the “Committee”) to develop recommendations and guidelines for optimal laboratory testing, interpretation, and counseling. The Subcommittee, cochaired by Drs. Wayne Grody and Garry Cutting, met twice yearly since October 1998. The issues considered by the Committee included (1) the target population to be screened (universal vs. limited to certain high-risk ethnic groups); (2) the screening model to be used (couple-based vs. sequential); (3) criteria for and selection of the standard mutation testing panel; (4) potential value and use of an extended testing panel with additional mutations; (5) whether to test for mutations and variants associated with mild or nonclassical phenotypes (such as congenital bilateral absence of the vas deferens); (6) test interpretation, reporting, and genetic counseling; and (7) laboratory quality assurance. The recommendations detailed here have been incorporated into a joint ACMG/ACOG/NIH Steering Committee document entitled “Preconceptual and Prenatal Carrier Screening for Cystic Fibrosis” which will be widely distributed. This document also will include guidelines for providers, patient education, and informed consent. Patient education materials will include two pamphlets, entitled “Cystic Fibrosis Carrier Testing. . . The Decision is Yours” and “Cystic Fibrosis Testing: What Happens if Both My Partner and I are Carriers?” It is important to note that these guidelines were prepared for population CF carrier screening and that different testing and counseling strategies would be employed for the identification of the mutation(s) in patients diagnosed with CF or in relatives of CF patients. Such diagnostic and prenatal mutation analyses should be referred to a genetics center for appropriate testing and counseling.

Clinical Exome Sequencing for Genetic Identification of Rare Mendelian Disorders

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% (2 of 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.

Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel

Keeping up with the next generation: massively parallel sequencing in clinical diagnostics.

The speed, accuracy, efficiency, and cost-effectiveness of DNA sequencing have been improving continuously since the initial derivation of the technique in the mid-1970s. With the advent of massively parallel sequencing technologies, DNA sequencing costs have been dramatically reduced. No longer is it unthinkable to sequence hundreds or even thousands of genes in a single individual with a suspected genetic disease or complex disease predisposition. Along with the benefits offered by these technologies come a number of challenges that must be addressed before wide-scale sequencing becomes accepted medical practice. Molecular diagnosticians will need to become comfortable with, and gain confidence in, these new platforms, which are based on radically different technologies compared to the standard DNA sequencers in routine use today. Experience will determine whether these instruments are best applied to sequencing versus resequencing. Perhaps most importantly, along with increasing read lengths inevitably comes increased ascertainment of novel sequence variants of uncertain clinical significance, the postanalytical aspects of which could bog down the entire field. But despite these obstacles, and as a direct result of the promises these sequencing advances present, it will likely not be long before next-generation sequencing begins to make an impact in molecular medicine. In this review, technical issues are discussed, in addition to the practical considerations that will need to be addressed as advances push toward personal genome sequencing.

Infection of the heart by the human immunodeficiency virus.

Heart muscle disease in the acquired immune deficiency syndrome (AIDS), characterized by electrocardiographic changes or congestive cardiomyopathy, is a documented clinical problem, but its pathogenesis is obscure. In AIDS the heart is known to be involved by a variety of opportunistic infections as well as Kaposis sarcoma, but no causative relation with the development of cardiomyopathy has been established. This study reports evidence for direct infection of the heart in AIDS, not by an opportunistic pathogen but by the AIDS, not by an opportunistic pathogen but by the AIDS virus itself, the human immunodeficiency virus (HIV). For this study the technique of in situ deoxyribonucleic acid hybridization was applied to cardiac tissues obtained at autopsy from AIDS patients. Using sulfur-35-labeled ribonucleic acid probes encompassing the entire HIV genome, HIV nucleic acid sequences were detected in cardiac tissue sections from 6 of 22 patients examined who died of AIDS. The hybridization targets appeared to be cardiac myocytes, although their precise morphology was often obscured by the intensity of the signal. The myocardial cells showing a positive hybridization signal were sparse, often comprising only 1 or a few cells per section, and their number and location did not correlate obviously with any histopathologic or clinical evidence of heart muscle disease in these patients. It is conceivable that the presence of HIV nucleic acid sequences may represent a preclinical marker of impending AIDS-associated heart muscle disease. This sequela would not be recognized in many patients, including those in this series, who died rapidly of Pneumocystis carinii pneumonia, Kaposis sarcoma and other well-documented manifestations of AIDS.(ABSTRACT TRUNCATED AT 250 WORDS)