Fig. 2: Rubric for categorizing screen-positive results.

A genomic screening test must first have an established set of criteria that define a ‘positive’ or ‘negative’ screen (1), typically variant-level thresholds (for example, pathogenic/likely pathogenic versus VUS) and zygosity requirements for any given monogenic condition. Variant confirmation in a new sample using an orthogonal method (2) can identify rare cases of sample swap (estimated at 1:1,000 for clinical laboratory workflows) or artifactual variant calls (probability depending on the type of variant). Both types of laboratory errors could be considered ‘technical’ false positives that will happen at a somewhat predictable frequency. Family studies to determine de novo occurrence or to phase putative biallelic variants (3) may reveal that two variants reported as a positive screen are actually in cis and therefore not a disease-causing genotype, considered an ‘allelic’ false positive or, alternatively, may reveal inheritance of a heterozygous variant from an unaffected parent, potentially causing the clinical laboratory to downgrade the variant, considered a ‘classification’ false positive. A molecular diagnosis is thereby established (4) with varying degrees of confidence (Table 3). With general population screening, many people will initially be asymptomatic. Subsequent clinical correlation results in accumulation of laboratory findings, clinical features and family history (5). People with a definite molecular diagnosis are considered a true positive even if they never develop signs or symptoms of the condition (nonpenetrant). If clinical findings consistent with the putative molecular diagnosis emerge, then it can be confirmed as a true positive with penetrance established (though expressivity may vary). Sometimes, the phenotypic consequence of the variant(s) identified may end up being different than the intent of the screening program, such as hypomorphic variants with milder phenotypes, or variants having a different molecular mechanism that cause a phenotype not intended as part of the screen. These could be considered ‘phenotypic’ false positives, even though such findings may ultimately have clinical relevance. People with probable, likely or possible molecular diagnoses who remain asymptomatic over time make up a category in which the screening result could represent a false positive or a true positive (with incomplete penetrance and/or variable expressivity). Longitudinal follow-up and management of these people will be needed, thus creating additional burden with unclear benefit. Rarely, a person with a negative screen may develop symptoms of a monogenic disease over time, indicating a potential false negative (6). As with ‘technical’ false positives, a sample swap or low confidence variant call could result in a false negative. Similarly, a single heterozygous variant in a gene associated with a recessive condition could result in an ‘allelic’ false negative if the other variant was not detected, or a ‘classification’ false negative if the other variant was a VUS and therefore not reported. The category of VUS in general presents a challenge for screening in both recessive and dominant conditions, potentially resulting in ‘classification’ false positives and/or false negatives, depending on whether this category of variant is returned in a screening program. In some cases, disease manifestations may be due to locus heterogeneity (where the gene responsible for symptoms was not included in the screen) or because symptoms are caused by a nonmonogenic etiology; these would be ‘phenotypic’ false negatives. Each of these potential scenarios is unlikely in genomic screening due to the rarity of the screened conditions, but clinicians must remain alert to the possibility of a false negative as they are for any condition included in standard public health NBS. If a person remains asymptomatic over time, a negative becomes increasingly likely to be a true negative. FN, false negative; FP, false positive; TN, true negative; TP, true positive.