Genetics in Medicine

Various professional organizations recommend screening prospective parents for autosomal recessive (AR) and X-linked (XL) conditions, which is reflected in commercial screening panels. There is merit to developing a distinct reproductive gene-list and analytic framework inclusive of genes based on available perinatal intervention, defined as possible prenatal intervention (including investigational) for the fetus or necessary early initiation of approved postnatal treatments. We evaluated a reproductive genetic screening framework that incorporates perinatal actionability across AR, XL, and selected autosomal dominant (AD) genes. Using a curated list of genetic conditions with perinatal intervention, we evaluated five subset gene lists to determine the individual-level number-needed-to-screen (NNS) to identify one individual with at least one qualifying heterozygous variant, defined as a heterozygous pathogenic or likely pathogenic (P/LP) variant in a gene on the specified list. To conduct NNS analyses, we sourced carrier frequency and allele frequency data for each gene and their respective ClinVar-curated high-confidence (>=2 star) P/LP variants, from two population databases -- gnomAD v4.1 and All of Us (AoU) v8. The analyses produced an individual-level NNS of 3.20 (CI: 3.193, 3.212) using gnomAD and 3.62 (CI: 3.606, 3.640) using AoU. These estimates do not represent couple-level reproductive risk, affected-pregnancy yield, clinical diagnostic yield, or validation of a clinical screening test. These findings support further evaluation of a perinatal-actionability framework, with clinical value dependent on which genes drive yield, and whether the relevant gene, variant, mechanism, and phenotype combinations are actionable in a reproductive or perinatal context for both the pregnant woman and her future offspring.

Background: The ClinGen ENIGMA BRCA1/BRCA2 Variant Curation Expert Panel (VCEP) has adapted the ACMG/AMP framework into gene-specific specifications. However, applying these specifications manually remains labour-intensive and prone to inconsistency, requiring integration of population, computational, functional, and clinical evidence through gene-specific decision trees and a points-based classification system. Methods: We developed HECTOR, a free web-based tool that implements the complete ENIGMA VCEP v1.2 specifications for BRCA1 and BRCA2. HECTOR automatically populates all evidence codes derivable from public data, routes curator-dependent evidence to a manual input layer and returns a transparent five-tier classification with code-level evidence. We validated HECTOR against two independent reference datasets: the 143-variant ENIGMA Evidence Repository, used as a clinical-grade reference standard, and 134 manually curated in-house variants of uncertain significance. HECTOR was then applied to the complete ClinVar BRCA1/BRCA2 catalogue (n = 34,077). Results: At the criterion level, HECTOR exactly reproduced 326 of 413 VCEP-assigned criteria (78.9%). The discordance arising predominantly from curator-dependent evidence rather than implementation errors whereas computationally accessible criteria showed perfect concordance. Across ClinVar, HECTOR classified 33,913 variants (99.5%). Agreement with definitive ClinVar classifications was 96.7% for pathogenic variants overall. Among variants for which HECTOR generated a definitive classification, directional concordance reached 99.7% for pathogenic and 99.9% for benign variants. HECTOR also resolved a substantial proportion of variants classified as uncertain (67.3%) or conflicting (88.7%), predominantly toward benign classifications. Conclusions: HECTOR provides a faithful, transparent implementation of the ENIGMA VCEP v1.2 specifications for BRCA1 and BRCA2, enabling rapid, standardized, and reproducible application of gene-specific variant classification guidelines while reducing the burden of manual curation.

Background: Wolfram syndrome is a rare neurodegenerative disorder, most commonly caused by pathogenic variants in WFS1, while cases due to CISD2 are exceedingly rare. The estimated prevalence is 1 in 160,000 to 770,000 individuals worldwide. In these clinical guidelines, disorders caused by WFS1 are referred to as WFS1-Wolfram syndrome, and those caused by CISD2 as CISD2-Wolfram syndrome. Historically, it has been characterized by early-onset, antibody-negative, insulin-dependent diabetes mellitus, progressive optic atrophy, sensorineural hearing loss, arginine vasopressin deficiency, and brainstem and cerebellar atrophy. More recently, partial and late onset forms have been identified. There are currently no licensed disease-modifying treatments, and international clinical guidelines have not previously been established. Methods: An international steering committee systematically reviewed 273 peer-reviewed publications and generated draft consensus statements across six clinical domains. These statements were evaluated by international specialists in endocrinology, clinical genetics, neurology, ophthalmology and neuro-ophthalmology, psychiatry, and urology, drawn from North America, Europe, Latin America, Oceania, and Asia, using a modified three-round Delphi process. Additional feedback was incorporated from nurses specializing in multidisciplinary Wolfram syndrome care, from leaders of international patient organizations, and from specialists in the genetic diagnosis of monogenic diabetes. Structured feedback from patients and families was gathered through multiple international patient advocacy organizations. Consensus was defined as [≥]80% agreement. Results: All 35 final consensus statements reached the pre-specified consensus threshold of [≥]80% agreement, spanning diagnosis and genetic testing, multidisciplinary care organization, neuro-ophthalmology, neurology, endocrinology, urology, gastroenterology, and psychiatry. Conclusions: These guidelines are the first international clinical consensus for Wolfram syndrome and provide actionable recommendations for clinicians worldwide. Implementation should be accompanied by a prospective audit to expand the evidence base and support future iterations.

Importance: Genomic newborn screening (gNBS) is a potential public health intervention, but its positive predictive value (PPV) remains uncertain. Estimating the prevalence and penetrance of pathogenic and likely pathogenic (P/LP) variants in genes prioritized for screening may clarify the long-term PPV and clinical utility of gNBS. Objective: To compare ICD-based ascertainment, electronic medical record (EMR) review, and clinical assessment of genetic disorders in adults with P/LP variants in 54 genes prioritized for gNBS. Design: Two-cohort observational study with EMR review and clinical assessment in the hospital-based cohort. Setting: The U.K. Biobank (UKB) and Mass General Brigham Biobank (MGBB). Participants: 451,877 adults from the UKB and 53,371 from the MGBB, all with exome sequencing data. Exposures: P/LP variants in 54 genes prioritized through expert consensus for gNBS, in genotypes consistent with each gene's inheritance pattern. Main outcomes and measures: The primary outcome was the absolute difference in the proportion of MGBB participants identified as affected by ICD versus EMR ascertainment. Secondary outcomes included findings from clinical assessments of undiagnosed MGBB participants, corrected UKB penetrance estimates, and extrapolation to U.S.. annual birth cohorts and living adults. Results: P/LP variants were identified in 665 UKB participants (0.15%) and 82 MGBB participants (0.15%), approximately 1 in 650. In MGBB, EMR review revealed that 58/82 individuals (70.7%) were undiagnosed, although 25 of 58 (43.1%) had documented symptoms. Disease-associated ICD codes were found in 39.0% (32/82) of participants, whereas EMR review identified symptoms in 59.8% (49/82, McNemar P<.001). Applied to UKB, this correction yielded a penetrance of 28.4% (95% CI, 18.6% to 38.2%), implying that 73 to 203 participants beyond the 51 identified by ICD codes may have clinical features of disease. Extrapolated to U.S. birth cohorts, 4,900 to 5,700 newborns per year may harbor P/LP variants in these genes and survive into adulthood. Approximately 355,000 to 410,000 U.S. adults may have P/LP variants in these genes. Conclusions and relevance: Penetrance of P/LP variants in genes prioritized for gNBS is substantially higher than ICD estimates suggest. Many adults with P/LP variants are symptomatic but undiagnosed, supporting inclusion of these genes in gNBS.

Background: Clear guidance is lacking regarding how 'truthset' variants should be used for clinical validation of functional assays, namely determining the allocatable evidence points (EPs) towards clinical classification. It is argued that assays should be validated using truthsets of missense variants, as this is the variant type for which classification is most impacted by functional data. EPs will be influenced by both the number of available 'truthset' variants and their concordance with assay readouts. Methods: We first reviewed 112 sets of ClinGen gene-specific classification specifications (CSPECs) to assess methodologies they applied for truthset assembly and clinical validation of assays. We then proposed differing rules regarding variant type and stringency of classification by which truthsets might be assembled using ClinVar-extracted classifications. We then examined augmentation of ClinVar-classified truthsets with 'proxy-clinical' benign-classified missense variants systematically assembled applying ACMG/AMP rules (of differing stringencies). In total, these constituted 70 basic approaches to ClinVar-based truthset assembly, which we applied to VHL, BRCA1, BRCA2 and RAD51C. We additionally analysed the impact on the size of the truthsets of changing the specified phenotypes against which ClinVar classification had been submitted. We then applied these truthsets to quantify concordance and allocatable EPs for five large-scale multiplexed functional assays for VHL, BRCA1, BRCA2, and RAD51C. Results The EPs from clinical validation of each assay varied widely according to which truthset was used across 2,120 permutations of gene-truthset-assay combinations. For example, sequentially applying 700 different ClinVar-based truthsets to 2,268 VHL assay variant readouts (70 basic ClinVar-based approaches, augmented by examining 5 different phenotypes for each basic approach, and separate validation against two defined deleterious zones), the evidence strength allocatable for pathogenicity ranged from nil to strong evidence (0.0 to 5.6 EPs); for benignity it ranged from supporting to strong evidence (-1.5 to -6.4 EPs). Clinical validation using truthsets comprising just ClinVar-classified missense variants typically resulted in lower EPs than truthsets comprising protein truncating (PTV) and synonymous variants; this was more due to paucity of ClinVar-classified missense truthset variants than poorer concordance. Augmentation with larger 'proxy-clinical' benign-classified missense truthsets typically improved evidence allocatable for pathogenicity, with improved power negating modest reduction in concordance. Conclusions EPs can be improved by augmentation with systematically-generated 'proxy-clinical' benign-classified missense variants and/or reduction of truthset stringency. Explicit prescriptive clinical guidance is urgently required to improve consistency in clinical validation of functional assays and consequent evidence application for clinical variant classification.

Background Large-scale functional assays, including multiplex assays of variant effect, have substantial potential to resolve variants of uncertain significance (VUS), particularly for rare missense variants where clinical and population evidence are limited. The ClinGen assay-level clinical validation framework described by Brnich et al provided baseline guidance for the use of functional data for variant classification. However, clear consensus regarding construction of variant 'truthsets' by which to clinically validate functional data remains lacking. Methods CanVIG-UK developed consensus recommendations for truthset construction through an iterative national consultation process involving the CanVIG Steering Advisory Group (CStAG), wider CanVIG-UK membership, and engagement with international functional genomics experts. Consultation was based on previous analyses of 2,120 truthset constructions examining the impact of truthset composition on evidence point allocation within the ClinGen assay-level clinical validation framework. Results Across several consultations, CanVIG-UK established nine guiding principles and seven best-practice recommendations for assay-level clinical validation, using the assumed context of an assay for a cancer susceptibility gene where loss-of-function is the mechanism of pathogenicity. The principal recommendation stipulates, where assays are intended for use in interpretation of largely missense variants, the truthset used to validate should comprise only missense variants. Rather than mixtures of different variant types which may serve to over-estimate assay performance. Additional recommendations support option for relaxation of truthset stringency to improve power, augmentation of benign missense truthsets with systematically derived 'proxy-clinical' benign variants, independent clinical validation separate from assayist-defined validation, and careful evaluation of missense score distributions against that of protein-truncating and synonymous variants. Guidance is also provided for scenarios with limited pathogenic truthset availability and for assays reporting multiple deleterious zones or readouts. Conclusions The CanVIG-UK principles and recommendations for truthset construction upon the ClinGen assay-level clinical validation framework, while aiming to form a baseline for future discussion regarding other functional and disease contexts and helping to address the gap between publication of new data and routine clinical implementation.

Background: Genetic testing is a Class I recommendation for patients with hypertrophic cardiomyopathy (HCM). As knowledge and frameworks continue to evolve, genetic variant classifications may change with new evidence over time. Classifications rely on evidence sought from publicly available case data, improved classification rules, and gene-disease validity. We evaluated the frequency and reasons for variant reclassification from a large multi-center international HCM registry (Sarcomeric Human Cardiomyopathy Registry; SHaRe). Methods: Participants were clinically evaluated at specialised HCM centres. Genetic variants were sought from the genetic test report, with classifications based on either the initial report, an updated report or some underwent further SHaRe adjudication. All variants were computationally reannotated and reevaluated. Variants underwent expedited curation if no new evidence was present. The remainder underwent full manual curation using accepted criteria and classified as pathogenic/likely pathogenic (P/LP), variant of uncertain significance (VUS) and benign/likely benign (B/LB). Results: Of 12,187 HCM patients, 8,054 (66%) had genetic testing between 1989-2020, and 4,923 (61%) had a variant identified in one of 29 HCM genes (1606 unique variants). Expedited curation was performed for 704 (44%) variants and 902 (56%) underwent manual curation. There were 1275 (79%) variants that retained their classification: 146 B/LB, 660 VUS, and 468 P/LP. While 276 (17%) variants (n=672 patients) were reclassified (n=276), including 73 upgrades: 61 from VUS to P/LP (199 patients), and 12 from B/LB to VUS. There were 203 downgrades: 108 from P/LP to VUS (n=196 patients), and 95 from P/LP or VUS to B/LB. VUS were additionally subclassified: 90 VUS-High, 129 VUS-Mid, 115 VUS-Low. Sub-classification of VUS resulted in less uncertainty, with 369 (40.6%) variants reclassified as VUS-Low or B/LB, indicating a very strong probability of not being HCM associated. Conclusions: Clinically meaningful reclassification occurred in 10% of variants identified in HCM probands. Most VUS were unlikely to be causal, and sub-classification has potential to reduce their burden on clinicians and families. Periodic reevaluation is essential for accurate clinical interpretation.

Objectives To determine whether boys with VUS detected through newborn screening (NBS) for adrenoleukodystrophy (ALD) develop adrenal insufficiency (aiALD) and cerebral ALD (cALD) at rates comparable to those with pathogenic variants, and to evaluate the relationship between C26:0-lysophosphatidylcholine (C26:0-LPC) levels and clinical outcomes. Methods We conducted a retrospective multicenter cohort study (2013 - 2025) across six US centers, including 201 males identified through NBS in 19 states. Variants were classified as pathogenic (n=65), likely pathogenic (n=45), or VUS (n=88). Primary outcomes were development of aiALD and cALD; secondary outcomes included C26:0-LPC levels. Statistical analyses included Kaplan-Meier, mixed-effects regression, and Cox models. Results 201 males with ABCD1 variants identified through NBS for ALD. Median age at last follow-up was 4.2 years (IQR 2.5 - 7.9). Overall, 26% developed aiALD (54% pathogenic, 16% likely pathogenic, 11% VUS), and 8% developed cALD (11%, 9%, and 4.5%, respectively). Pathogenic/likely pathogenic variants were associated with higher odds of aiALD than VUS (OR 5.8; 95% CI 2.16 - 15.58; p=0.001). At 150 months, 39% of individuals with pathogenic/likely pathogenic variants remained free of aiALD versus 85% with VUS. C26:0-LPC levels were higher in pathogenic variants and correlated with genotype (p=0.0006). Higher levels were associated with increased aiALD risk and earlier onset (HR 1.38 per 0.1 umol/L; 95% CI 1.20 - 1.59; p<0.0001). Conclusions Boys with VUS had lower rates of aiALD and lower C26:0-LPC levels than those with pathogenic variants, although some developed disease. C26:0-LPC correlates with genotype and risk, supporting its role in variant classification and risk-stratified surveillance.

IMPORTANCE: Most United States children with neurodevelopmental disorders have not received genetic testing aligned with current guidelines. Integration of genetic counselors into non-genetics departments is a potential strategy to improve uptake, but prevalence and details of integrated care models are unknown. OBJECTIVE: To characterize availability, utilization, and perceived need for genetic counselors across non-genetics departments caring for patients with neurodevelopmental disorders DESIGN: Cross-sectional observational department-level survey SETTING: Child neurology, adult neurology, developmental pediatrics, child psychiatry, and adult psychiatry departments at Intellectual and Developmental Disabilities Research Centers PARTICIPANTS: The survey was distributed to 67 departments across 15 institutions. The departmental response rate was 52% (35/67), with at least one response from 87% (13/15) of institutions. EXPOSURE: Presence/absence of dedicated genetic counselor(s), where "dedicated" was defined as hired by the department MAIN OUTCOME(S) AND MEASURE(S): This was a descriptive study only, with no comparative statistical analyses due to the exploratory nature. RESULTS: One third of departments (34%; 12/35) reported having dedicated clinical genetic counselors. Prevalence was highest in child neurology (67%; 8/12), followed by adult neurology (40%; 2/5) and developmental pediatrics (22%; 2/9), with none in child psychiatry (0/7) or adult psychiatry (0/2). In almost all departments with genetic counselors (92%; 11/12), they directly billed for their services, which universally included pre-test counseling/consent and post-test counseling. In departments without genetic counselors, only 39% (9/23) reported providers ordered their own genetic testing. Among all departments, over half (57%) were interested in adding/increasing genetic counseling support, while 26% were unsure and 17% uninterested. Insufficient funding was the most cited barrier; only one department reported insufficient need. CONCLUSIONS AND RELEVANCE: Though currently implemented in only one third of departments, our findings suggest those with dedicated genetic counselors directly pursue genetic testing (without referring to genetics) more than those without genetic counselors. Interest in increasing or adding genetic counseling support was high, and though funding was a reported barrier, feasible funding models were described. In the context of limited medical geneticists and expanding precision therapies, alternate delivery models for neurodevelopmental genetic testing including genetic counselor integration in non-genetics departments may help to scale and sustain uptake.

Purpose: Elective genomic sequencing (EGS) returns monogenic disease findings in multiple genes, including potentially novel variants, and may also provide participants with carrier status, pharmacogenomic and other health-related information. The PeopleSeq Study assessed participants' motivations for and concerns about EGS and the associated clinical and psychosocial outcomes across diverse EGS providers. Methods: We administered a shared questionnaire to participants who chose to undergo EGS via 18 academic, clinical, or commercial EGS platforms. Results: We enrolled 1575 participants, of whom 1147 (72.8%) completed a questionnaire after receiving their EGS results. A majority (60.3%) of the participants who completed a post-result questionnaire self-reported receiving results they assessed as important, including negative findings, and 75.9% reported a form of health-related utility. Among a subset (19.4%) who shared their EGS reports, 16.6% (37 of n=223) received a monogenic finding and self-reported results deemed "important" were consistent with EGS reports. Most participants (74.1%) discussed their results with their family, but fewer discussed their results with a healthcare provider other than the site team (41.7%) or had one or more medical visits as a direct result of their EGS testing (23.1%). Participants expressed diverse motivations for EGS, with 91.4% expressing interest in their personal disease risk and 54% who expressed quasi-indication-based motivations related to family medical history. Individuals motivated by family history reported important results at a significantly higher rate. Conclusions: Early adopters of EGS are motivated by general interest in their health as well as quasi-indication-based considerations such as family history. A majority of participants learned results they considered medically important, but a much smaller segment engaged healthcare providers with their results.

Introduction Tandem repeats (TRs), including short tandem repeats (1-6 bp motifs) and variable number tandem repeats (7+ bp motifs), have been linked to more than 50 Mendelian diseases. However, current frameworks for evaluating gene-disease relationships do not adequately address TR-specific complexities. As a result, proposed TR locus-disease relationships are often incorrectly classified, under-evaluated, or excluded entirely, limiting discovery and leading to underdiagnosis of TR disorders. Methods We developed criTRia, a scoring framework designed to accurately evaluate TR locus-disease relationships at the locus level rather than the gene level. Building on ClinGen best practices, criTRia introduces TR-specific evidence categories and reweighted scoring. We applied criTRia to curate 65 loci from STRchive, a database of disease-associated TRs. Results We compared criTRia curations with gene-level curations from nine Gene Curation Coalition (GenCC) groups. Of 65 newly scored loci, 7 had not been previously evaluated by GenCC and 17 showed significant disagreement across groups. These differences have direct implications for whether a disease is recommended for inclusion in a diagnostic gene panel. The criTRia framework also enabled curation of previously unassessed associations, bringing the total to 77 curated TR locus-disease associations and identifying four contradictory associations. Discussion By incorporating TR-specific evidence, criTRia provides a reproducible methodology for assessing TR locus-disease relationships, improving classification consistency and establishing a foundation for better integrating tandem repeats into clinical genetic medicine and providing more accurate diagnoses.

Background: Many at-risk patients lack access to genetic services due to a genetic counselor (GC) workforce shortage. Little is known about how digital alternatives impact patients with and without cancer who meet criteria for genetic testing. Methods: eREACH2 is a randomized 4-arm non-inferiority trial where pre-test (visit 1) and/or return of results (visit 2) GC counseling was replaced with a patient-centered digital intervention. Arms include: A (GC/GC), B (GC/digital), C (digital/GC) and D (digital/digital). Primary outcomes were non-inferiority in uptake of genetic services and change in genetic knowledge and general anxiety from baseline to post-disclosure of results (T0-T2). Secondary cognitive and affective outcomes were assessed using non-inferiority ANOVAs and equivalency chi-squared tests in intention-to-treat and per-protocol analyses. Findings: 773 participants were recruited nationwide; 46.6% from rural areas. Mean age was 51 years (range 20-87), 13% male, 12% non-white, 29% had less than a college education, and 33% had a personal history of cancer. 584 (76%) patients completed testing (14% had a positive result, 16% had a VUS). In the primary ITT analyses, we met the non-inferiority for uptake of genetic services and anxiety, but results were inconclusive for knowledge. Secondary outcomes were heterogeneous across arms. Arm C demonstrated consistently favorable effects, while Arms B and D showed less favorable outcomes in select domains (e.g. satisfaction and MICRA). Patients who received positive or VUS results via digital disclosure had significantly higher MICRA scores - indicating greater negative response to testing. Interpretation: In this large, randomized trial of patients with and without cancer, the eREACH intervention was effective for pre-test counseling, but inconclusive for digital disclosure of results. Exploratory analyses suggest that digital delivery could be a reasonable alternative for individuals receiving negative results, while those receiving positive or VUS results may derive some short-term psychosocial benefit from GC disclosure.

Purpose As genomics permeates all healthcare specialties, genetic counseling in conjunction with genetic testing is broadly recommended. Improving access to genetics professionals is crucial for Americans insured by Medicaid. The purpose of this study was to conduct a comprehensive review of Medicaid policies for genetic counseling performed by Certified Genetic Counselors (CGC(C)). Methods Fee-for-service Medicaid policies across 50 states and Washington DC were reviewed and coded. Four states with exemplary policies were identified, and CGC managers in two of these were surveyed regarding the real-world effects of these policies. Results As of 2024, 20 states (39%) had a published policy for genetic counseling with most (N=16, 80%) expressly covering genetic counseling in connection with any covered genetic test. Of the states without a policy, 12 (24%) mention genetic counseling in the context of scenario-specific policies, and 19 (37%) have no published policy. Twenty states explicitly cover CGC services, while 2 exclude CGCs as service providers. CGC managers in Indiana and Michigan confirmed the policies identified as exemplary successfully led to reimbursement of CGC services. Conclusion There is significant variability in Medicaid coverage for genetic counseling. Comprehensive policies are needed to support patient access to genetics professionals, including CGCs.

Accurate interpretation of rare germline variants remains a major challenge in developmental disorders (DD). Somatic mutation data represent a largely untapped source of evidence for germline variant classi-fication. Identical or nearby mutations that drive positive selection when present in somatic tissues can cause developmental disorders when present in the germline. We integrated somatic mutation data from the Catalogue Of Somatic Mutations In Cancer (COSMIC), and healthy tissues (sperm and buccal epithelium) with germline variant datasets from ClinVar and large studies of de novo mutations in DD patients. Across 970 dominant DD genes, 195 have evidence of somatic selection, with a majority demonstrating concordant mechanisms between germline and somatic contexts. We benchmark the ability of somatic data to discriminate pathogenic from benign germline missense variation across dominant DD genes, identifying 145 genes in which somatic data are informative. The strongest utility is in altered-function genes where germline and somatic mechanisms are concordant, for example the RASopathy genes. In these genes, codon-level aggregation of somatic missense counts yields predictive performance comparable to computational predictors or MAVE assays (AUC-ROC 0.895 for somatic data, versus 0.893 for REVEL). Combining somatic features with computational scores improves discrimination further. Using likelihood ratios, we map COSMIC missense codon count thresholds onto American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP)-style evidence strengths, showing that somatic data can reach strong levels of evidence in germline variant interpretation in DD and enable reclassification of variants of uncertain significance. Together, these results establish somatic mutation data as a scalable and clinically actionable evidence source for germline variant interpretation in select DD genes. Graphical abstract(Generated using FigureLabs) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/732808v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@150bec9org.highwire.dtl.DTLVardef@1dacf5org.highwire.dtl.DTLVardef@46121dorg.highwire.dtl.DTLVardef@4f5c38_HPS_FORMAT_FIGEXP M_FIG C_FIG

Genetic testing for cardiomyopathy and arrhythmia (CM/ARRH) provides diagnostic information and informs screening for at-risk relatives. Clinical guidelines recommend genetic testing for these conditions; however, data on how genetic test results influence clinical management recommendations are limited. Here, we determined the frequency of cardiologist-recommended management changes for patients following CM/ARRH genetic testing. This was a retrospective cross-sectional study of patients referred for multigene panel testing between April 2016 and April 2024. Genetically-experienced cardiologists at multicenter academic clinical practices were recruited for participation to complete surveys indicating clinical decision making on patients who had genetic testing. Cases for review were randomly selected to have both positive and non-positive results. Among 249 patients (138 positive, 111 non-positive), 136 (54.6%) received clinical management recommendations for their own or their at-risk relatives? care. Of these, 75 (55.1%) received recommendations for the patient?s own care, most frequently additional diagnostic tests/procedures (n=33). Compared to non-positive results, patients with positive results were more likely to receive recommendations for their own management (66/138, 47.8% vs 9/111, 8.1%; P<0.00001). Patients with positive results in arrhythmogenic cardiomyopathy genes had 263% higher odds of recommended management changes compared to those with TTN (OR=3.63, CI:1.40-9.84, P=0.009). The results from CM/ARRH genetic testing on affected patients influenced cardiologists? medical decision making and management recommendations. Additional research is needed to evaluate how genetic testing for CM/ARRH impact health outcomes.

Long-read whole genome sequencing (lrWGS) shows promise as an all-in-one test to detect clinically relevant variants and variants difficult to detect by current short-read whole genome sequencing (srWGS) pipelines. Comparisons between lrWGS and srWGS (or exome sequencing) pipelines will become commonplace as lrWGS is more widely adopted for clinical testing, particularly for individuals not diagnosed by srWGS. However, the sensitivity of lrWGS for detecting variants previously identified and prioritized by clinical srWGS has yet to be assessed. As part of the SeqFirst-neo study, a subset of critically ill newborns and their parents who underwent clinical srWGS also underwent lrWGS on the Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) platforms. In total, 134 families were sequenced across multiple technologies including 128 families with clinical srWGS who were sequenced on both lrWGS platforms. We compared the variants reported by clinical testing with the variants identified by lrWGS. Among the 128 families sequenced on all three platforms, 89 SNV/indels and 14 SV/CNVs clinically reported by the srWGS testing pipeline were evaluated. All variants assessed in probands were ultimately detected by both lrWGS platforms, although three events were not detected prior to application of an updated variant caller, highlighting the rapid evolution of lrWGS variant calling. Additionally, breakpoint coordinates and event sizes often differed substantially between calls from srWGS and events called in lrWGS data. Our work demonstrates that while most clinically reported variants from srWGS can be detected by lrWGS pipelines, challenges remain when attempting direct comparisons, particularly for SV/CNVs.

Objective Beckwith-Wiedemann spectrum (BWSp) is an overgrowth and cancer predisposition disorder caused by genetic and epigenetic alterations of chromosome 11p15. The 2018 international consensus produced a clinical scoring system to capture the phenotypic variability of BWSp and guide genetic testing and clinical management, including tumor screening, in patients without molecular confirmation. In this study, we evaluated BWSp predictors to identify the most informative features. Methods Supervised machine learning analyzed 25 phenotypic features in 555 patients with BWSp and 150 controls. Logistic regression, combined with a purposeful stepwise selection algorithm, identified a subset of features that can accurately classify subjects. Model performance was evaluated in a testing set and validated externally. Results The final model included six predictors: macroglossia, lateralized overgrowth, midface flattening, hepatomegaly, omphalocele, and developmental delay. Developmental delay was the only negative predictor; macroglossia (OR 46.10) and lateralized overgrowth (OR 27.87) were the strongest predictors. The proposed model and 2018 system did not differ in classification performance for testing (P = .39) or external (P = .15) sets. Conclusion A simplified diagnostic model, driven by macroglossia and lateralized overgrowth, differentiates between patients with BWSp and controls with performance comparable to the 2018 system. And may help physicians prioritize BWSp evaluation.

Purpose: Pathogenic or likely pathogenic (P/LP) variants are increasingly identified in genes more commonly associated with adult-onset cancer predisposition, but their prevalence and relevance to children who present with cancer remain unclear. Methods: We retrospectively analyzed 1,280 consecutive pediatric patients with cancer who underwent clinical germline sequencing, using a virtual panel, from 2021 to 2024. Genes with P/LP variants were categorized as aoCPG or pediatric-onset cancer predisposition genes (poCPG) according to cancer risk before age 18 years and pediatric surveillance recommendations. Variant relevance was adjudicated using tumor diagnosis/histopathology, immunohistochemistry, and tumor molecular features and classified as primary, secondary, or indeterminate. Results: Among 1,280 patients, 197 (15.4%) harbored 211 P/LP variants across 54 genes. Sixty-six variants (31.3%) occurred in aoCPG, 87 (41.2%) in poCPG, and 58 (27.5%) were heterozygous variants in autosomal recessive genes. Among adult-onset variants, 7 (10.6%) were primary, 54 (81.8%) secondary, and 5 (7.6%) indeterminate. Among pediatric-onset variants, 77 (88.5%) were primary and 10 (11.5%) secondary. Six patients (3 adult-onset variants; 3 pediatric-onset variants) received targeted therapy informed by germline/somatic sequencing results. Conclusion: In pediatric oncology, most variants in aoCPG are secondary rather than tumor-related findings. Tumor-informed interpretation, beyond variant classification, may improve reporting, counseling, and therapeutic decision-making

Germline mutations in PTPN11 cause Noonan syndrome (NS) and NS with multiple lentigines (NSML), yet how specific variants drive divergent clinical outcomes through distinct signaling and developmental mechanisms remains unclear. We find that germline and somatic mutations converge on N-SH2 and PTP domains but diverge at residue-level hotspots, reflecting distinct selective pressures. Clinical stratification of 18 pediatric patients reveals four distinct phenotypic classes including (i) the NSML-associated c.1403C>T (T468M) variant, characterized by lentigines, moderate growth impairment, and distinctive facial features; (ii) variants including the VUS c.1282G>A (V428M) and c.1432A>G (I478V), which were associated with cognitive deficits and variable growth impairment; (iii) c.1471C>A (P491T) and c.1472C>T (P491L), predominantly affecting cardiac and growth phenotypes with limited neurocognitive features; and (iv) a severe, multisystem class comprising c.172A>G (N58D), c.178G>A (G60S), c.844A>G (I282V), c.922A>G (N308D), and c.923A>G (N308S), spanning cardiac, growth, cognitive, and craniofacial abnormalities. Biochemical profiling in HEK293T cells revealed that PTPN11 variants stratify beyond simple gain/loss-of-function dichotomies into strong ERK-dependent hyperactivation, moderate ERK activation with variable protein stability and the paradoxical c.1282G>A variant, which did not increase ERK phosphorylation. In vivo, this variant drove excessive neural crest cell migration in chick embryos, suggesting that its effects on NCC migration may involve ERK-independent mechanisms or context-dependent signaling not captured by steady-state assays. ERK activation did not strictly correlate with clinical severity, yet these functional differences were associated with distinct growth, cardiac, pigmentation, and neurodevelopmental outcomes. Our data suggest lineage-specific sensitivity to SHP2 dosage, with dorsal root ganglia neurons appearing more vulnerable to reduced SHP2 stability than melanocyte precursors. Although direct correlations between specific signaling defects and individual clinical features remain complex, our findings provide a refined framework for PTPN11 variant classification, and reveal unexpected SHP2 functions in neural crest development.

Purpose: Autosomal Dominant Polycystic Kidney Disease is the most common monogenic kidney disease and largely due to variants in PKD1. We aimed to assess pathogenicity evidence for PKD1 missense variants in disease databases and evaluate in silico pathogenicity prediction tool performance. Methods: PKD1 missense variants reported as pathogenic, likely pathogenic or likely benign were extracted from ClinVar and PKDB. Variants were re-classified using ACMG/AMP criteria to identify "truth sets" of pathogenic and benign variants. In silico scores were obtained from five tools (SIFT, PolyPhen-2, CADD, REVEL, AlphaMissense) and evaluated using established thresholds. A Receiver Operating Characteristic curve analysis was performed using the PKD1 variant truth sets. Results: 346/389 (89%) reported disease-causing missense variants in PKD1 were downgraded to Variants of Unknown Significance (VUS) using current classification criteria. Based on current thresholds, REVEL achieved the highest sensitivity of 62%, with specificity of 79%. AlphaMissense was the only tool not to misclassify any truth set variants, but many of the variant scores were between the pathogenic and benign thresholds. Conclusion: A large majority of PKD1 missense variants are classified as VUS with current pathogenicity criteria. Commonly used in silico tools, applied with established genome-wide thresholds, do not reliably distinguish pathogenic and benign missense variants in PKD1.