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.

Atopic disease, including atopic dermatitis (eczema), allergy and asthma, has increased in frequency in recent decades and now affects approximately 20% of the population in the developed world. Twin and family studies have shown that predisposition to atopic disease is highly heritable. Although most genetic studies have focused on immunological mechanisms, a primary epithelial barrier defect has been anticipated. Filaggrin is a key protein that facilitates terminal differentiation of the epidermis and formation of the skin barrier. Here we show that two independent loss-of-function genetic variants (R510X and 2282del4) in the gene encoding filaggrin (FLG) are very strong predisposing factors for atopic dermatitis. These variants are carried by approximately 9% of people of European origin. These variants also show highly significant association with asthma occurring in the context of atopic dermatitis. This work establishes a key role for impaired skin barrier function in the development of atopic disease.

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Ichthyosis vulgaris (OMIM 146700) is the most common inherited disorder of keratinization and one of the most frequent single-gene disorders in humans. The most widely cited incidence figure is 1 in 250 based on a survey of 6,051 healthy English schoolchildren. We have identified homozygous or compound heterozygous mutations R501X and 2282del4 in the gene encoding filaggrin (FLG) as the cause of moderate or severe ichthyosis vulgaris in 15 kindreds. In addition, these mutations are semidominant; heterozygotes show a very mild phenotype with incomplete penetrance. The mutations show a combined allele frequency of approximately 4% in populations of European ancestry, explaining the high incidence of ichthyosis vulgaris. Profilaggrin is the major protein of keratohyalin granules in the epidermis. During terminal differentiation, it is cleaved into multiple filaggrin peptides that aggregate keratin filaments. The resultant matrix is cross-linked to form a major component of the cornified cell envelope. We find that loss or reduction of this major structural protein leads to varying degrees of impaired keratinization.

Purpose:We report the diagnostic yield of whole-exome sequencing (WES) in 3,040 consecutive cases at a single clinical laboratory.Methods: WES was performed for many different clinical indications and included the proband plus two or more family members in 76% of cases. Results:The overall diagnostic yield of WES was 28.8%. The diagnostic yield was 23.6% in proband-only cases and 31.0% when three family members were analyzed. The highest yield was for patients who had disorders involving hearing (55%, N = 11), vision (47%, N = 60), the skeletal muscle system (40%, N = 43), the skeletal system (39%, N = 54), multiple congenital anomalies (36%, N = 729), skin (32%, N = 31), the central nervous system (31%, N = 1,082), and the cardiovascular system (28%, N = 54). Of 2,091 cases in which secondary findings were analyzed for 56

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

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-

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