Precision Medicine in Internal Medicine

Key Summary Points Genetic testing can be helpful in 2 clinical situations: diagnostic testing in persons who are symptomatic, and predisposition testing in those who are asymptomatic but at risk on the basis of family history and personal factors. Diagnostic testing is currently the most common type of genetic testing in internal medicine practice and includes targeted Sanger sequencing for suspected monogenic disorders and focused panel sequencing of genes for hereditary cancer and cardiac diseases. Exome sequencing (ES) targets all protein-coding segments (exons) of the genome and can reliably establish a molecular diagnosis for known genetic disorders. Unlike targeted testing, ES can identify a previously unsuspected molecular diagnosis. Genome sequencing (GS) involves sequencing of the entire human genome, providing information on noncoding regions and copy number variants and enabling derivation of polygenic risk scores for complex traits. It is not routinely used in clinical practice. In addition to diagnostic information for monogenic disorders, both ES and GS provide information on actionable secondary findings. Interpretation of genetic variation detected by sequencing is challenging but continues to improve with data sharing and accelerated gene discovery for monogenic diseases. Barriers to widespread implementation of diagnostic ES include uncertainty related to variant interpretation, insufficient data on persons of diverse ancestries, unwillingness of some insurers to cover testing, a small workforce of genetic professionals, limited genetic literacy among patients and physicians, concerns about privacy and genetic discrimination, and a lack of standards for reinterpretation of genomic data over time. In general, clinicians might consider genetic testing in 2 situations: to establish a diagnosis in symptomatic persons (diagnostic testing), or to assess predisposition for disease in asymptomatic persons who have increased risk due to family history or personal characteristics (predisposition or predictive testing). In some circumstances, population-wide genetic testing may be used for newborn screening or universal carrier screening for reproductive purposes. Diagnostic genetic testing in symptomatic persons can clarify the diagnosis and prognosis, suggest the most appropriate management strategies, and indicate other associated features for which medical surveillance or intervention may be helpful. Identifying an underlying molecular genetic cause may also help in family planning and counseling of blood relatives. To determine disease risk for unaffected relatives in a family with a medical condition (such as colon cancer), it is best to start genetic testing in an affected family member to determine whether there is an identifiable hereditary factor. If the affected person has a familial mutation, targeted mutation analysis in unaffected family members allows for the most cost-effective and informative risk stratification. When a familial mutation is identified, a normal genetic test result in asymptomatic family members is informative and reduces their disease risk to the level in the general population. However, when the affected family member is unavailable for or unwilling to undergo testing, normal results in asymptomatic family members are uninformative. Clinicians and patients must recognize that for common diseases with substantial risk in the general population, such as breast cancer, no onenot even those with an informative genetic test resultis risk-free. What Genetic Sequencing Strategies Are Available in Clinical Practice? The most common genetic testing strategies that are available in clinical practice are targeted gene sequencing, gene panel sequencing, and clinical exome sequencing (ES). Targeted gene sequencing using the Sanger method is useful for diagnostic testing when the clinician suspects a mutation in a specific gene. It is not easily scalable, so it is limited to sequencing of a small number of genes. Panel sequencing and ES use next-generation sequencing, which can sequence many genes simultaneously and provides reliable, rapid, and cost-effective detection of genetic variants. Panel sequencing interrogates a preselected set of genes known to be involved in a particular condition, such as cancer or cardiomyopathy, for which mutations in any one of several genes can cause similar phenotypes. It enables coverage of all relevant regions of the genes and is usually optimized to also capture a range of variants that are not easily detectable by Sanger sequencing, such as insertions, deletions, and other rearrangements. One major disadvantage of this strategy is that the panels require frequent updating with the discovery of new relevant genes. Exome sequencing involves sequencing of the coding regions (exons) of all genes, and genome sequencing (GS) involves sequencing of both coding and noncoding regions. The exome represents about 1% of the genome. Currently, ES is available for clinical diagnostics for some indications and GS is used predominantly in the research setting. Because ES involves sequence analysis of all genes in the genome, it can identify mutations in genes that are not suspected on the basis of clinical presentation or are not yet known to cause disease. When initial analysis is unable to establish a diagnosis, ES data can be reinterrogated as new genes for a given condition are discovered and new exome analysis methods are developed. Genome sequencing provides information on nonprotein coding variation; gives more complete coverage of the coding regions; and enables more accurate detection of structural variants, such as translocations, deletions, and duplications. However, despite its comprehensiveness, GS is infrequently used in clinical settings because of its higher cost and greater computational requirements compared with ES (13). Figure 1 summarizes the approximate numbers of genetic variants found in a typical human exome. On average, ES detects approximately 10000 protein-altering variants, including 150 to 180 protein-truncating variants, 20 to 30 known disease-causing (mainly recessive) variants, and 1 to 4 de novo variants that are not present in parents. The key challenge relates to interpretation of genetic variants. As of 1 February 2019, the Online Mendelian Inheritance in Man database indicated that out of 20000 protein-coding genes, only 3652 (approximately 18%) had been found to cause known single-gene disorders. The process of establishing associations of the other genes with diseases remains laborious and continues to evolve (6). Moreover, because some genes have been studied more extensively than others, the level of evidence is highly variable for different Mendelian disorders. Figure 1. Expected findings from genome and exome sequencing for an individual patient. The numbers shown are approximate and depend on specific sequencing platforms and populations being studied. Protein-altering variants are variants that alter amino acid sequence in any way. Truncating variants are a subset of protein-altering variants that lead to a premature stop codon or truncation of a protein. Knocked-out genes are genes carrying homozygous loss-of-function variants. ClinVar disease variants are known pathogenic variants that cause human diseases according to ClinVar (Table 2). De novo variants are new mutational events that, by definition, are not inherited from parents. (Adapted from references 4 and 5.) In practice, routine application of bioinformatic methods narrows the search for a diagnostic variant in the clinical analysis of ES data (Figure 2). Expert review is usually required to interpret variant pathogenicity and to assess the concordance of molecular findings with clinical features. The final determination of the molecular diagnosis may require genetic testing of other family members to assess whether the clinical phenotype travels with the genetic variant within the family (the procedure known as segregation analysis). Figure 2. Steps in the diagnostic sequence analysis of an individual exome. gnomAD= Genome Aggregation Database; HGMD= Human Gene Mutation Database; OMIM= Online Mendelian Inheritance in Man. How Should Clinicians Interpret Genetic Test Results? Guidelines from the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology classify genetic variants for Mendelian disorders into 1 of 5 categories: pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, or benign (Table 1) (7). Pathogenic variants are considered to be disease-causing and should be acted on as such. Likely pathogenic variants have a 90% estimated probability of being pathogenic, and clinical geneticists typically act on them as if they are pathogenic. Variants of uncertain significance are common and should not trigger clinical action unless they are reclassified as likely pathogenic or pathogenic. However, they are more likely to eventually be reclassified as likely benign or benign. Variants that are likely benign or benign are often not included on genetic test reports because of their lack of clinical significance. Table 1. Variant Classifications ClinVar (Table 2) is a free open resource that provides classifications of all clinically relevant variants. Each entry includes a 4-star scale about the level of confidence in the classification. Clinicians and patients can check ClinVar to see the current classification of a variant if they are considering action in light of that variant. Over time, ClinVar will reclassify variants using data from the general population (especially allele frequency in unaffected persons), information within families that demonstrates that the variants are de novo or segregate with disease, and additional research. To assist with correct reclassification, some laboratories offer free genetic testing to family members for segregation analysis when the index