ClinGen guidance for use of the PP1/BS4 co-segregation and PP4 phenotype specificity criteria for sequence variant pathogenicity classification.

The 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology variant classification publication established a standard employed internationally to guide laboratories in variant assessment. Those recommendations included both pathogenic (PP1) and benign (BS4) criteria for evaluating the inheritance patterns of variants, but details of how to apply those criteria at appropriate evidence levels were sparse. Several publications have since attempted to provide additional guidance, but anecdotally, this issue is still challenging. Additionally, it is not clear that those prior efforts fully distinguished disease-gene identification considerations from variant pathogenicity considerations nor did they address autosomal-recessive and X-linked inheritance. Here, we have taken a mixed inductive and deductive approach to this problem using real diseases as examples. We have developed a practical heuristic for genetic co-segregation evidence and have also determined that the specific phenotype criterion (PP4) is inseparably coupled to the co-segregation criterion. We have also determined that negative evidence at one locus constitutes positive evidence for other loci for disorders with locus heterogeneity. Finally, we provide a points-based system for evaluating phenotype and co-segregation as evidence types to support or refute a locus and show how that can be integrated into the Bayesian framework now used for variant classification and consistent with the 2015 guidelines.

Advanced variant classification framework reduces the false positive rate of predicted loss-of-function variants in population sequencing data.

Predicted loss of function (pLoF) variants are often highly deleterious and play an important role in disease biology, but many pLoF variants may not result in loss of function (LoF). Here we present a framework that advances interpretation of pLoF variants in research and clinical settings by considering three categories of LoF evasion: (1) predicted rescue by secondary sequence properties, (2) uncertain biological relevance, and (3) potential technical artifacts. We also provide recommendations on adjustments to ACMG/AMP guidelines' PVS1 criterion. Applying this framework to all high-confidence pLoF variants in 22 genes associated with autosomal-recessive disease from the Genome Aggregation Database (gnomAD v.2.1.1) revealed predicted LoF evasion or potential artifacts in 27.3% (304/1,113) of variants. The major reasons were location in the last exon, in a homopolymer repeat, in a low proportion expressed across transcripts (pext) scored region, or the presence of cryptic in-frame splice rescues. Variants predicted to evade LoF or to be potential artifacts were enriched for ClinVar benign variants. PVS1 was downgraded in 99.4% (162/163) of pLoF variants predicted as likely not LoF/not LoF, with 17.2% (28/163) downgraded as a result of our framework, adding to previous guidelines. Variant pathogenicity was affected (mostly from likely pathogenic to VUS) in 20 (71.4%) of these 28 variants. This framework guides assessment of pLoF variants beyond standard annotation pipelines and substantially reduces false positive rates, which is key to ensure accurate LoF variant prediction in both a research and clinical setting.

Using the ACMG/AMP framework to capture evidence related to predicted and observed impact on splicing: Recommendations from the ClinGen SVI Splicing Subgroup.

The American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) framework for classifying variants uses six evidence categories related to the splicing potential of variants: PVS1, PS3, PP3, BS3, BP4, and BP7. However, the lack of guidance on how to apply such codes has contributed to variation in the specifications developed by different Clinical Genome Resource (ClinGen) Variant Curation Expert Panels. The ClinGen Sequence Variant Interpretation Splicing Subgroup was established to refine recommendations for applying ACMG/AMP codes relating to splicing data and computational predictions. We utilized empirically derived splicing evidence to (1) determine the evidence weighting of splicing-related data and appropriate criteria code selection for general use, (2) outline a process for integrating splicing-related considerations when developing a gene-specific PVS1 decision tree, and (3) exemplify methodology to calibrate splice prediction tools. We propose repurposing the PVS1_Strength code to capture splicing assay data that provide experimental evidence for variants resulting in RNA transcript(s) with loss of function. Conversely, BP7 may be used to capture RNA results demonstrating no splicing impact for intronic and synonymous variants. We propose that the PS3/BS3 codes are applied only for well-established assays that measure functional impact not directly captured by RNA-splicing assays. We recommend the application of PS1 based on similarity of predicted RNA-splicing effects for a variant under assessment in comparison with a known pathogenic variant. The recommendations and approaches for consideration and evaluation of RNA-assay evidence described aim to help standardize variant pathogenicity classification processes when interpreting splicing-based evidence.

A calibrated functional patch-clamp assay to enhance clinical variant interpretation in KCNH2-related long QT syndrome.

Modern sequencing technologies have revolutionized our detection of gene variants. However, in most genes, including KCNH2, the majority of missense variants are currently classified as variants of uncertain significance (VUSs). The aim of this study was to investigate the utility of an automated patch-clamp assay for aiding clinical variant classification in KCNH2. The assay was designed according to recommendations proposed by the Clinical Genome Sequence Variant Interpretation Working Group. Thirty-one variants (17 pathogenic/likely pathogenic, 14 benign/likely benign) were classified internally as variant controls. They were heterozygously expressed in Flp-In HEK293 cells for assessing the effects of variants on current density and channel gating in order to determine the sensitivity and specificity of the assay. All 17 pathogenic variant controls had reduced current density, and 13 of 14 benign variant controls had normal current density, which enabled determination of normal and abnormal ranges for applying evidence of moderate or supporting strength for VUS reclassification. Inclusion of functional assay evidence enabled us to reclassify 6 out of 44 KCNH2 VUSs as likely pathogenic. The high-throughput patch-clamp assay can provide moderate-strength evidence for clinical interpretation of clinical KCNH2 variants and demonstrates the value of developing automated patch-clamp assays for functional characterization of ion channel gene variants.

Calibration of computational tools for missense variant pathogenicity classification and ClinGen recommendations for PP3/BP4 criteria.

Recommendations from the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) for interpreting sequence variants specify the use of computational predictors as "supporting" level of evidence for pathogenicity or benignity using criteria PP3 and BP4, respectively. However, score intervals defined by tool developers, and ACMG/AMP recommendations that require the consensus of multiple predictors, lack quantitative support. Previously, we described a probabilistic framework that quantified the strengths of evidence (supporting, moderate, strong, very strong) within ACMG/AMP recommendations. We have extended this framework to computational predictors and introduce a new standard that converts a tool's scores to PP3 and BP4 evidence strengths. Our approach is based on estimating the local positive predictive value and can calibrate any computational tool or other continuous-scale evidence on any variant type. We estimate thresholds (score intervals) corresponding to each strength of evidence for pathogenicity and benignity for thirteen missense variant interpretation tools, using carefully assembled independent data sets. Most tools achieved supporting evidence level for both pathogenic and benign classification using newly established thresholds. Multiple tools reached score thresholds justifying moderate and several reached strong evidence levels. One tool reached very strong evidence level for benign classification on some variants. Based on these findings, we provide recommendations for evidence-based revisions of the PP3 and BP4 ACMG/AMP criteria using individual tools and future assessment of computational methods for clinical interpretation.

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.

Homing tasks performed using variations of crawling gait patterns reveal a role for attention in podokinetic path integration.

Self-motion can be perceived via podokinetic information, that is, based upon the movements of the legs during legged locomotion. This information can be integrated in order to perceive a path of travel through the environment (i.e., via podokinetic path integration). Two types of podokinetic information have been distinguished by analyzing the patterns of bias that result from manipulating the gait patterns used in direct-route homing tasks. Each type of podokinetic information has been associated specific groupings of gaits that support equivalent perceptual measurements of self-motion. Specifically, gaits are grouped if they can be varied across the outbound and inbound phases of a homing task (e.g., walking outbound and jogging inbound) and the accuracy of homing task performances does not differ from matched-gait control conditions. Recently, it was theorized that different types of podokinetic information are related to the differences in the kinematic form of limb motions in these groupings of gaits. Here we test an alternative hypothesis, namely that attention plays a role in selecting the type of podokinetic information. In three experiments, we manipulated the crawling gait patterns used in direct-route homing tasks. Consistent with our hypotheses, we observe that self-motion is equivalently measured via crawling movement patterns that (1) have distinct kinematic forms, but that similarly direct participants' attention onto controlling the swing phase trajectories of their arms, and (2) have distinct inter-limb coordination patterns (i.e., pace vs. trot), but do not require attention to be specifically focused upon swing phase arm trajectories.

Acoustic information about upper limb movement in voicing.

We show that the human voice has complex acoustic qualities that are directly coupled to peripheral musculoskeletal tensioning of the body, such as subtle wrist movements. In this study, human vocalizers produced a steady-state vocalization while rhythmically moving the wrist or the arm at different tempos. Although listeners could only hear and not see the vocalizer, they were able to completely synchronize their own rhythmic wrist or arm movement with the movement of the vocalizer which they perceived in the voice acoustics. This study corroborates recent evidence suggesting that the human voice is constrained by bodily tensioning affecting the respiratory-vocal system. The current results show that the human voice contains a bodily imprint that is directly informative for the interpersonal perception of another's dynamic physical states.

Harmonizing variant classification for return of results in the All of Us Research Program.

The All of Us Research Program (AoURP) is a historic effort to accelerate research and improve healthcare by generating and collating data from one million people in the United States. Participants will have the option to receive results from their genome analysis, including actionable findings in 59 gene-disorder pairs for which disorder-associated variants are recommended for return by the American College of Medical Genetics and Genomics. To ensure consistent reporting across the AoURP, in a prelaunch study the four participating clinical laboratories shared all variant classifications in the 59 genes of interest from their internal databases. Of the 11,813 unique variants classified by at least two of the four laboratories, classifications were concordant with regard to reportability for 99.1% (11,711), with only 0.9% (102) having reportability differences. Through variant reassessment, data sharing, and discussion of rationale, participating laboratories resolved all 102 reportable differences. These approaches will be maintained during routine AoU reporting to ensure continuous classification harmonization and consistent reporting within AoURP.

Prognostic Neurobiomarkers in Neonatal Encephalopathy.

Therapeutic hypothermia (TH) is now a standard treatment for infants with moderate-to-severe neonatal encephalopathy (NE), and improves brain damage on neuroimaging and neurodevelopmental outcomes. Critically, for effective neuroprotection, hypothermia should be started within 6 h from birth. There is compelling evidence to suggest that a proportion of infants with mild NE have material risk of developing brain damage and poor outcomes. This cohort is increasingly being offered TH, despite lack of trial evidence for its benefit. In current practice, infants need to be diagnosed within 6 h of birth for therapeutic treatment, compared to retrospective NE grading in the pre-hypothermia era. This presents challenges as NE is a dynamic brain disorder that can worsen or resolve over time. Neurological symptoms of NE can be difficult to discern in the first few hours after birth, and confounded by analgesics and anesthetic treatment. Using current enrolment criteria, a significant number of infants with NE that would benefit from hypothermia are not treated, and vice versa, some infants receive hypothermia when its benefit will be limited. Better biomarkers are needed to further improve management and treatment of these neonates. In the present review, we examine the latest research, and highlight a central limitation of most current biomarkers: that their predictive value is consistently greatest after most neuroprotective therapies are no longer effective.

Homing tasks and distance matching tasks reveal different types of perceptual variables associated with perceiving self-motion during over-ground locomotion.

Self-motion perception refers to the ability to perceive how the body is moving through the environment. Perception of self-motion has been shown to depend upon the locomotor action patterns used to move the body through the environment. Two separate lines of enquiry have led to the establishment of two distinct theories regarding this effect. One theory has proposed that distances travelled during locomotion are perceived via higher order perceptual variables detected by the haptic perceptual system. This theory proposes that two higher order haptic perceptual variables exist, and that the implication of one of these variables depends upon the type of gait pattern that is used. A second theory proposes that self-motion is perceived via a higher order perceptual variable termed multimodally specified energy expenditure (MSEE). This theory proposes that the effect of locomotor actions patterns upon self-motion perception is related to changes in the metabolic cost of locomotion per unit of perceptually specified traversed distance. Here, we test the hypothesis that the development of these distinct theories is the result of different choices in methodology. The theory of gait type has been developed based largely on the results of homing tasks, whereas the effect of MSEE has been developed based on the results of distance matching tasks. Here we test the hypothesis that the seemly innocuous change in experimental design from using a homing task to using a distance matching task changes the type of perceptual variables implicated in self-motion perception. To test this hypothesis, we closely replicated a recent study of the effect of gait type in all details bar one-we investigated a distance matching task rather than a homing task. As hypothesized, this change yielded results consistent with the predictions of MSEE, and distinct from gait type. We further show that, unlike the effect of gait type, the effect of MSEE is unaffected by the availability of vision. In sum, our findings support the existence of two distinct types of higher order perceptual variables in self-motion perception. We discuss the roles of these two types of perceptual variables in supporting effective human wayfinding.

Plasma Mucin-1 (CA15-3) Levels in Autosomal Dominant Tubulointerstitial Kidney Disease due to MUC1 Mutations.

INTRODUCTION: Patients with ADTKD-MUC1 have one allele producing normal mucin-1 (MUC1) and one allele producing mutant MUC1, which remains intracellular. We hypothesized that ADTKD-MUC1 patients, who have only 1 secretory-competent wild-type MUC1 allele, should exhibit decreased plasma mucin-1 (MUC1) levels. To test this hypothesis, we repurposed the serum CA15-3 assay used to measure MUC1 in breast cancer to measure plasma MUC1 levels in ADTKD-MUC1. METHODS: This cross-sectional study analyzed CA15-3 levels in a reference population of 6,850 individuals, in 85 individuals with ADTKD-MUC1, and in a control population including 135 individuals with ADTKD-UMOD and 114 healthy individuals. RESULTS: Plasma CA15-3 levels (mean ± standard deviation) were 8.6 ± 4.3 U/mL in individuals with ADTKD-MUC1 and 14.6 ± 5.6 U/mL in controls (p < 0.001). While there was a significant difference in mean CA15-3 levels, there was substantial overlap between the 2 groups. Plasma CA15-3 levels were <5 U/mL in 22% of ADTKD-MUC1 patients, in 0/249 controls, and in 1% of the reference population. Plasma CA15-3 levels were >20 U/mL in 1/85 ADTKD-MUC1 patients, in 18% of control individuals, and in 25% of the reference population. Segregation of plasma CA15-3 levels by the rs4072037 genotype did not significantly improve differentiation between affected and unaffected individuals. CA15-3 levels were minimally affected by gender and estimated glomerular filtration rate. DISCUSSION/CONCLUSIONS: Plasma CA15-3 levels in ADTKD-MUC1 patients are approximately 40% lower than levels in healthy individuals, though there is significant overlap between groups. Further investigations need to be performed to see if plasma CA15-3 levels would be useful in diagnosis, prognosis, or assessing response to new therapies in this disorder.

Assessing the relative contribution of vision to odometry via manipulations of gait in an over-ground homing task.

The visual, vestibular, and haptic perceptual systems are each able to detect self-motion. Such information can be integrated during locomotion to perceive traversed distances. The process of distance integration is referred to as odometry. Visual odometry relies on information in optic flow patterns. For haptic odometry, such information is associated with leg movement patterns. Recently, it has been shown that haptic odometry is differently calibrated for different types of gaits. Here, we use this fact to examine the relative contributions of the perceptual systems to odometry. We studied a simple homing task in which participants travelled set distances away from an initial starting location (outbound phase), before turning and attempting to walk back to that location (inbound phase). We manipulated whether outbound gait was a walk or a gallop-walk. We also manipulated the outbound availability of optic flow. Inbound reports were performed via walking with eyes closed. Consistent with previous studies of haptic odometry, inbound reports were shorter when the outbound gait was a gallop-walk. We showed that the availability of optic flow decreased this effect. In contrast, the availability of optic flow did not have an observable effect when the outbound gait was walking. We interpreted this to suggest that visual odometry and haptic odometry via walking are similarly calibrated. By measuring the decrease in shortening in the gallop-walk condition, and scaling it relative to the walk condition, we estimated a relative contribution of optic flow to odometry of 41%. Our results present a proof of concept for a new, potentially more generalizable, method for examining the contributions of different perceptual systems to odometry, and by extension, path integration. We discuss implications for understanding human wayfinding.

BRCA Challenge: BRCA Exchange as a global resource for variants in BRCA1 and BRCA2.

The BRCA Challenge is a long-term data-sharing project initiated within the Global Alliance for Genomics and Health (GA4GH) to aggregate BRCA1 and BRCA2 data to support highly collaborative research activities. Its goal is to generate an informed and current understanding of the impact of genetic variation on cancer risk across the iconic cancer predisposition genes, BRCA1 and BRCA2. Initially, reported variants in BRCA1 and BRCA2 available from public databases were integrated into a single, newly created site, www.brcaexchange.org. The purpose of the BRCA Exchange is to provide the community with a reliable and easily accessible record of variants interpreted for a high-penetrance phenotype. More than 20,000 variants have been aggregated, three times the number found in the next-largest public database at the project's outset, of which approximately 7,250 have expert classifications. The data set is based on shared information from existing clinical databases-Breast Cancer Information Core (BIC), ClinVar, and the Leiden Open Variation Database (LOVD)-as well as population databases, all linked to a single point of access. The BRCA Challenge has brought together the existing international Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium expert panel, along with expert clinicians, diagnosticians, researchers, and database providers, all with a common goal of advancing our understanding of BRCA1 and BRCA2 variation. Ongoing work includes direct contact with national centers with access to BRCA1 and BRCA2 diagnostic data to encourage data sharing, development of methods suitable for extraction of genetic variation at the level of individual laboratory reports, and engagement with participant communities to enable a more comprehensive understanding of the clinical significance of genetic variation in BRCA1 and BRCA2.

Fitting a naturally scaled point system to the ACMG/AMP variant classification guidelines.

Recently, we demonstrated that the qualitative American College of Medical Genetics and Genomics/Association for Medical Pathology (ACMG/AMP) guidelines for evaluation of Mendelian disease gene variants are fundamentally compatible with a quantitative Bayesian formulation. Here, we show that the underlying ACMG/AMP "strength of evidence categories" can be abstracted into a point system. These points are proportional to Log(odds), are additive, and produce a system that recapitulates the Bayesian formulation of the ACMG/AMP guidelines. The strengths of this system are its simplicity and that the connection between point values and odds of pathogenicity allows empirical calibration of the strength of evidence for individual data types. Weaknesses include that a narrow range of prior probabilities is locked in and that the Bayesian nature of the system is inapparent. We conclude that a points-based system has the practical attribute of user-friendliness and can be useful so long as the underlying Bayesian principles are acknowledged.

Prune belly syndrome in surviving males can be caused by Hemizygous missense mutations in the X-linked Filamin A gene.

BACKGROUND: Prune belly syndrome (PBS) is a rare, multi-system congenital myopathy primarily affecting males that is poorly described genetically. Phenotypically, its morbidity spans from mild to lethal, however, all isolated PBS cases manifest three cardinal pathological features: 1) wrinkled flaccid ventral abdominal wall with skeletal muscle deficiency, 2) urinary tract dilation with poorly contractile smooth muscle, and 3) intra-abdominal undescended testes. Despite evidence for a genetic basis, previously reported PBS autosomal candidate genes only account for one consanguineous family and single cases. METHODS: We performed whole exome sequencing (WES) of two maternal adult half-brothers with syndromic PBS (PBS + Otopalatodigital spectrum disorder [OPDSD]) and two unrelated sporadic individuals with isolated PBS and further functionally validated the identified mutations. RESULTS: We identified three unreported hemizygous missense point mutations in the X-chromosome gene Filamin A (FLNA) (c.4952 C > T (p.A1448V), c.6727C > T (p.C2160R), c.5966 G > A (p.G2236E)) in two related cases and two unrelated sporadic individuals. Two of the three PBS mutations map to the highly regulatory, stretch-sensing Ig19-21 region of FLNA and enhance binding to intracellular tails of the transmembrane receptor ß-integrin 1 (ITGß1). CONCLUSIONS: FLNA is a regulatory actin-crosslinking protein that functions in smooth muscle cells as a mechanosensing molecular scaffold, transmitting force signals from the actin-myosin motor units and cytoskeleton via binding partners to the extracellular matrix. This is the first evidence for an X-linked cause of PBS in multiple unrelated individuals and expands the phenotypic spectrum associated with FLNA in males surviving even into adulthood.

For humans navigating without vision, navigation depends upon the layout of mechanically contacted ground surfaces.

Navigation can be haptically guided. In specific, tissue deformations arising from both limb motions during locomotion (i.e., gait patterns) and mechanical interactions between the limbs and the environment can convey information, detected by the haptic perceptual system, about how the body is moving relative to the environment. Here, we test hypotheses concerning the properties of mechanically contacted environments relevant to navigation of this kind. We studied blindfolded participants implicitly learning to perceive their location within environments that were physically encountered via walking on, stepping on, and probing ground surfaces with a cane. Environments were straight-line paths with elevated sections where the path either narrowed or remained the same width. We formed hypotheses concerning how these two environments would affect spatial updating and reorientation processes. In the constant pathwidth environment, homing task accuracy was higher and a manipulation of the elevated surface, to be either unchanged or (unbeknown to participants) shortened, biased the performance. This was consistent with our hypothesis of a metric recalibration scaled to elevated surface extent. In the narrowing pathwidth environment, elevated surface shortening did not bias performance. This supported our hypothesis of positional recalibration resulting from contact with the leading edge of the elevated surface. We discuss why certain environmental properties, such as path-narrowing, have significance for how one becomes implicitly oriented the surrounding environment.

Energy flows in gesture-speech physics: The respiratory-vocal system and its coupling with hand gestures.

Expressive moments in communicative hand gestures often align with emphatic stress in speech. It has recently been found that acoustic markers of emphatic stress arise naturally during steady-state phonation when upper-limb movements impart physical impulses on the body, most likely affecting acoustics via respiratory activity. In this confirmatory study, participants (N = 29) repeatedly uttered consonant-vowel (/pa/) mono-syllables while moving in particular phase relations with speech, or not moving the upper limbs. This study shows that respiration-related activity is affected by (especially high-impulse) gesturing when vocalizations occur near peaks in physical impulse. This study further shows that gesture-induced moments of bodily impulses increase the amplitude envelope of speech, while not similarly affecting the Fundamental Frequency (F0). Finally, tight relations between respiration-related activity and vocalization were observed, even in the absence of movement, but even more so when upper-limb movement is present. The current findings expand a developing line of research showing that speech is modulated by functional biomechanical linkages between hand gestures and the respiratory system. This identification of gesture-speech biomechanics promises to provide an alternative phylogenetic, ontogenetic, and mechanistic explanatory route of why communicative upper limb movements co-occur with speech in humans.

Updated recommendation for the benign stand-alone ACMG/AMP criterion.

The Clinical Genome Resource (ClinGen) Sequence Variant Interpretation Working Group set out to refine the American College of Medical Genetics and Genomics and the Association of Molecular Pathologists (ACMG/AMP) variant pathogenicity recommendations for stand-alone rule BA1 (a variant with minor allele frequency [MAF] > 0.05 is benign), by clarifying how it should be used and specifying a set of variants that should be exempted from this rule. We cross-referenced ClinVar and Exome Aggregation Consortium data to identify variants for which there was a plausible argument for pathogenicity and the variant exists in one or more population data sets at MAF > 0.05. We identified nine such variants that were present in these data sets that may not be benign. The ACMG/AMP criteria were applied to these variants that resulted in four pathogenic and five variants of uncertain significance. We have refined benign rule BA1 by clarifying terms used to describe its use, which databases we recommend using, and assumptions made about this rule. We also recognized an initial list of nine variants for which there was some evidence of pathogenicity even though the MAF was high for these variants. We specify processes whereby individuals can petition ClinGen for amendments to our variant-specific assertions and the criteria experts should use when setting a numerically lower threshold for BA1 for specific genes.