The landscape of reported VUS in multi-gene panel and genomic testing: Time for a change.

PURPOSE: Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact. METHODS: Rates of inconclusive results due to VUS were collected from over 1.5 million sequencing test results from 19 clinical laboratories in North America from 2020 to 2021. RESULTS: We found a lower rate of inconclusive test results due to VUSs from ES/GS (22.5%) compared with MGPs (32.6%; P < .0001). For MGPs, the rate of inconclusive results correlated with panel size. The use of trios reduced inconclusive rates (18.9% vs 27.6%; P < .0001), whereas the use of GS compared with ES had no impact (22.2% vs 22.6%; P = ns). CONCLUSION: The high rate of VUS observed in diagnostic MGP testing warrants examining current variant reporting practices. We propose several approaches to reduce reported VUS rates, while directing clinician resources toward important VUS follow-up.

Parent-of-Origin Effects in 15q11.2 BP1-BP2 Microdeletion (Burnside-Butler) Syndrome.

To identify whether parent-of-origin effects (POE) of the 15q11.2 BP1-BP2 microdeletion are associated with differences in clinical features in individuals inheriting the deletion, we collected 71 individuals reported with phenotypic data and known inheritance from a clinical cohort, a research cohort, the DECIPHER database, and the primary literature. Chi-squared and Mann-Whitney U tests were used to test for differences in specific and grouped clinical symptoms based on parental inheritance and proband gender. Analyses controlled for sibling sets and individuals with additional variants of uncertain significance (VOUS). Among all probands, maternal deletions were associated with macrocephaly (p = 0.016) and autism spectrum disorder (ASD; p = 0.02), while paternal deletions were associated with congenital heart disease (CHD; p = 0.004). Excluding sibling sets, maternal deletions were associated with epilepsy as well as macrocephaly (p < 0.05), while paternal deletions were associated with CHD and abnormal muscular phenotypes (p < 0.05). Excluding sibling sets and probands with an additional VOUS, maternal deletions were associated with epilepsy (p = 0.019) and paternal deletions associated with muscular phenotypes (p = 0.008). Significant gender-based differences were also observed. Our results supported POEs of this deletion and included macrocephaly, epilepsy and ASD in maternal deletions with CHD and abnormal muscular phenotypes seen in paternal deletions.

Differential Kv1.3, KCa3.1, and Kir2.1 expression in "classically" and "alternatively" activated microglia.

Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) promote differentiation into classically activated M1-like microglia, which produce high levels of pro-inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL-4 in contrast induces a phenotype associated with anti-inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K+ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL-4) microglia and studying their K+ channel expression by whole-cell patch-clamp, quantitative PCR and immunohistochemistry. We identified three major types of K+ channels based on their biophysical and pharmacological fingerprints: a use-dependent, outwardly rectifying current sensitive to the KV 1.3 blockers PAP-1 and ShK-186, an inwardly rectifying Ba2+ -sensitive Kir 2.1 current, and a Ca2+ -activated, TRAM-34-sensitive KCa 3.1 current. Both KV 1.3 and KCa 3.1 blockers inhibited pro-inflammatory cytokine production and iNOS and COX2 expression demonstrating that KV 1.3 and KCa 3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN-γ microglia exhibited high KV 1.3 current densities (∼50 pA/pF at 40 mV) and virtually no KCa 3.1 and Kir currents, while microglia differentiated with IL-4 exhibited large Kir 2.1 currents (∼ 10 pA/pF at -120 mV). KCa 3.1 currents were generally low but moderately increased following stimulation with IFN-γ or ATP (∼10 pS/pF). This differential K+ channel expression pattern suggests that KV 1.3 and KCa 3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106-121.

An integrated framework for examining groundwater vulnerability in the Mekong River Delta region.

The Mekong River provides water, food security, and many other valuable benefits to the more than 60 million Southeast Asian residents living within its basin. However, the Mekong River Basin is increasingly stressed by changes in climate, land cover, and infrastructure. These changes can affect water quantity and quality and exacerbate related hazards such as land subsidence and saltwater intrusion, resulting in multiple compounding risks for neighboring communities. In this study, we demonstrate the connection between climate change, groundwater availability, and social vulnerability by linking the results of a numerical groundwater model to land cover and socioeconomic data at the Cambodia-Vietnam border in the Mekong River Delta region. We simulated changes in groundwater availability across 20 years and identified areas of potential water stress based on domestic and agriculture-related freshwater demands. We then assessed adaptive capacity to understand how communities may be able to respond to this stress to better understand the growing risk of groundwater scarcity driven by climate change and overextraction. This study offers a novel approach for assessing risk of groundwater scarcity by linking the effects of climate change to the socioeconomic context in which they occur. Increasing our understanding of how changes in groundwater availability may affect local populations can help water managers better plan for the future, leading to more resilient communities.

NeuroSCORE is a genome-wide omics-based model that identifies candidate disease genes of the central nervous system.

To identify candidate disease genes of central nervous system (CNS) phenotypes, we created the Neurogenetic Systematic Correlation of Omics-Related Evidence (NeuroSCORE). We identified five genome-wide metrics highly associated with CNS phenotypes to score 19,601 protein-coding genes. Genes scored one point per metric (range: 0-5), identifying 8298 scored genes (scores ≥ 1) and 1601 "high scoring" genes (scores ≥ 3). Using logistic regression, we determined the odds ratio that genes with a NeuroSCORE from 1 to 5 would be associated with known CNS-related phenotypes compared to genes that scored zero. We tested NeuroSCORE using microarray copy number variants (CNVs) in case-control cohorts and aggregate mouse model data. High scoring genes are associated with CNS phenotypes (OR = 5.5, p < 2E-16), enriched in case CNVs, and mouse ortholog genes that cause behavioral and nervous system abnormalities. We identified 1058 high scoring genes with no disease association in OMIM. Transforming the logistic regression results indicates high scoring genes have an 84-92% chance of being associated with a CNS phenotype. Top scoring genes include GRIA1, MAP4K4, SF1, TNPO2, and ZSWIM8. Finally, we interrogated CNVs in the Clinical Genome Resource, finding the majority of clinically significant CNVs contain high scoring genes. These findings can direct future research and improve molecular diagnostics.

Testing a best practices risk result format to communicate genetic risks.

OBJECTIVE: To investigate the effect of a genetic report format using risk communication "best-practices" on risk perceptions, in part to reduce risk overestimates. METHODS: Adults (N = 470) from the Coriell Personalized Medicine Collaborative (CPMC) were randomized to a 2 × 2 experimental design to receive a hypothetical "personalized" genetic risk result for leukemia (relative risk = 1.5 or 2.5) through either the standard CPMC report (N = 232) or an enriched report informed by best practices (N = 238). A one-time, online survey assessed numeracy and risk perceptions including "feelings of risk" and a numerical estimate. RESULTS: Regardless of numeracy, participants who received the enriched report had fewer overestimates of their lifetime risk estimate (LRE; odds ratio = 0.19, p < .001) and lower feelings of risk on two of three measures (p < .001). Participants with higher numeracy scores had fewer overestimates of LRE (OR = 0.66, p < .001) and lower feelings of risk on two out of three measures (p ≤ .01); the interaction between numeracy and report format was non-significant. CONCLUSION: The enriched report produced more accurate LRE and lower risk perceptions regardless of numeracy level, suggesting the enriched format was helpful to individuals irrespective of numeracy ability. PRACTICE IMPLICATIONS: Best practice elements in risk reports may help individuals form more accurate risk perceptions.

Critical exon indexing improves clinical interpretation of copy number variants in neurodevelopmental disorders.

OBJECTIVE: To evaluate a new tool to aid interpretation of copy number variants (CNVs) in individuals with neurodevelopmental disabilities. METHODS: Critical exon indexing (CEI) was used to identify genes with critical exons (CEGs) from clinically reported CNVs, which may contribute to neurodevelopmental disorders (NDDs). The 742 pathogenic CNVs and 1,363 variants of unknown significance (VUS) identified by chromosomal microarray analysis in 5,487 individuals with NDDs were subjected to CEI to identify CEGs. CEGs identified in a subsequent random series of VUS were evaluated for relevance to CNV interpretation. RESULTS: CEI identified a total of 2,492 unique CEGs in pathogenic CNVs and 953 in VUS compared with 259 CEGs in 6,965 CNVs from 873 controls. These differences are highly significant (p < 0.00001) whether compared as frequency, average, or normalized by CNV size. Twenty-one percent of VUS CEGs were not represented in Online Mendelian Inheritance in Man, highlighting limitations of existing resources for identifying potentially impactful genes within CNVs. CEGs were highly correlated with other indices and known pathways of relevance. Separately, 136 random VUS reports were reevaluated, and 76% of CEGs had not been commented on. In multiple cases, further investigation yielded additional relevant literature aiding interpretation. As one specific example, we discuss GTF2I as a CEG, which likely alters interpretation of several reported duplication VUS in the Williams-Beuren region. CONCLUSIONS: Application of CEI to CNVs in individuals with NDDs can identify genes of potential clinical relevance, aid laboratories in effectively searching the clinical literature, and support the clinical reporting of poorly annotated VUS.