Genetics in Medicine Open

Introduction: Genetic testing is increasingly central to the diagnosis and management of cardiovascular genetic conditions. However, use and follow-through vary across patient populations. Examining clinician perspectives on sociocultural and structural factors influencing testing is important for understanding these differences and informing public health genomics research and implementation efforts. Methods: We conducted semi-structured interviews with 15 cardiologists from health systems across the United States who have integrated cardiogenetics in their practice. Interviews explored experiences diagnosing cardiovascular genetic conditions among patients from underrepresented backgrounds, as well as approaches to incorporating social and contextual information into care. Data were coded thematically and analyzed using a framework analysis guided by the Health Equity Implementation Framework and Social Determinants of Health domains. Results: Clinicians described multi-level factors shaping genetic testing practices, including patient-provider interactions, clinical workflows, health system infrastructure, and broader policy contexts. Key themes included challenges communicating complex genetic information across language and literacy differences; patient trust shaped by prior healthcare experiences; fragmented insurance coverage separating genetic testing from genetic counseling; and challenges interpreting variants of uncertain significance, particularly for populations underrepresented in genomic reference databases. Clinicians also described adaptive strategies, such as interdisciplinary collaboration, telehealth, and patient assistance programs, that supported testing in some settings but were often inconsistent or resource-dependent. Conclusion: Among cardiologists using genetic testing, system-level and sociocultural factors shape the feasibility and downstream use of cardiovascular genetic testing. Findings highlight considerations for public health-informed genomic infrastructure that accounts for social context, supports communication, and reduces reliance on individual clinician workarounds, with implications for clinical decision support and related public health genomics initiatives.

Advances in high-throughput sequencing and genetic research have expanded the role of genetics in medicine and society. Population-based screening programs, including neonatal and preconception testing, are increasingly implemented globally, alongside the rise of direct-to-consumer (DTC) genetic testing. The "Genetics and the General Public" Ethics Working Group of the French Federation of Human Genetics (FFGH) assessed knowledge and awareness of genetics within the French population through a nationally representative survey (n=3,013) conducted by the polling firm Ipsos bva. Results indicated that 69% of respondents report an interest in genetics, although their level of knowledge remains limited. Most respondents expressed positive attitudes toward genetics, perceiving it as a major source of hope in healthcare. While a majority indicated willingness to undergo genetic testing for medical purposes, they also reported legitimate concerns regarding the potential results. Despite legal restrictions, 12% reported having ordered a DTC genetic test (5% for genealogical; 5% for medical and 2% for both purposes), and 45% of non-users expressed strong interest in this type of test. Notably, there is a substantial lack of awareness regarding the limitations of these tests and the French legal framework governing their use. These findings highlight critical gaps in public knowledge, emphasizing the need for improved genetic education, including incorporating genetics into school curricula and launching targeted awareness campaigns. These initiatives should help clarify the distinctions between clinically validated genetic tests and DTC genetic testing services, addressing both their benefits and their ethical, legal, and scientific limitations, in order to promote informed decision-making.

Objectives: To assess genetic counselors perspectives on ambient AI adoption and its impact on counselor burnout. Materials and Methods: We utilized a mixed methods approach, surveying burnout using the validated Stanford Professional Fulfilment Index (PFI) before and after ambient AI adoption and exploring adoption perspectives through semi-structured interviews. Results: 64% of participants (16/25) completed the pre-survey, with eleven completing post-surveys (69% response rate for completion of all three surveys). 14/25 participants completed interviews. Ambient AI use was associated with reduction in burnout after 90 days; respondents who reported using ambient AI (vs. non-use) had burnout scores 1.05 points lower, on average (p=0.008). Benefits of adoption included effective use with interpreters, memory aid, summarization of non-templated note sections (e.g. family/social history), and improved patient engagement. Challenges included template customization, variable accuracy, oversimplified medical language, and rapport disruption during consent. Ethical and regulatory considerations included data privacy, bias, awareness of training resources, and concerns about job displacement. Discussion: Ambient AI documentation can reduce documentation burden and burnout among genetic counselors. By evaluating both outcomes and real world implementation considerations, our study provides evidence to guide scalable integration of AI enabled documentation tools in clinical genomic medicine. Conclusion: Ambient AI can help support the sustainability of the clinical genetics workforce as genomic medicine initiatives are scaled across health systems. Addressing genetics-specific documentation needs while prioritizing patient trust, transparency, and provider oversight is essential for responsible ambient AI implementation.

Genetic testing for inherited cardiovascular conditions is recommended by multiple national guidelines to inform medical management. However, access to genetic counseling and testing is often limited particularly in the inpatient setting. Cardiologists cite lack of access to genetic counselors as a reason for not pursuing genetic testing. Genetic test education videos have been successfully implemented in the outpatient setting to increase patient volumes and decrease wait times, but they have not been studied in the inpatient setting.

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.

Background: Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion in the DMPK gene and represents the most common adult-onset myopathy. Current molecular diagnostics rely on labor-intensive assays that limit accessibility and scalability. Haplotype-based approaches offer a promising alternative for detecting pathogenic expansions indirectly. Methods: We performed genome-wide genotyping in 226 genetically confirmed DM1 patients from the Saguenay-Lac-Saint-Jean founder population and reconstructed haplotypes surrounding the DMPK pathogenic repeat expansion. Based on these haplotypes, we performed a phylogenetic analysis that was further integrated with genealogical reconstruction from the BALSAC database to investigate the origin and transmission of DM1 haplotypes. To evaluate epidemiological utility, we implemented gene dropping simulations within the SLSJ extended genealogies (>80,000 starting individuals) to estimate DM1 incidence at birth. Results: A DM1-associated haplotype was identified in all patients (226/226), consistent with a single major ancestral origin in the SLSJ population. This complete concordance supports the robustness of haplotype-based approaches to infer carrier status without direct repeat sizing. Integrating phylogenetic analysis and genealogical data identified a single couple as the most likely entry point of DM1 in Quebec. Simulation-based estimates of incidence at birth exceeded observed prevalence, suggesting underdiagnosis in the region. Marked geographic heterogeneity in the SLSJ is also observed. Conclusions: Our results demonstrate that haplotype-based approaches can provide a reliable, cost-effective alternative to conventional pathogenic DM1 repeat carriers identification and familial screening strategies.

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.

Genetic screening for rare pathogenic variants facilitates early detection and prevention of disease manifestations in medically actionable disorders, but sequencing costs limit widespread use. We introduce DoBSeq, a low-cost, high-throughput screening framework for detecting rare, single-nucleotide variants and indels. The framework includes: extraction of DNA from dried blood spots used in neonatal screening, automation of two-dimensional DNA pooling and library preparation, high-depth targeted sequencing using a 582-gene custom panel, and a probabilistic model to assign rare pathogenic variants to individuals. Benchmarked against whole-genome sequencing across 582 genes in a batch of 576 individuals, the framework detected 95% of all variants and recovered all clinically relevant pathogenic single-nucleotide variants in American College of Medical Genetics and Genomics (ACMG) actionable genes. Applied to 2304 anonymised blood donors, it yielded variant frequencies consistent with existing population estimates. At a sample cost of 29 USD, including 11 USD running costs, this framework provides a cost-efficient approach to population-level genetic screening.

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.

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.

Rare diseases collectively affect 1 in 10 individuals, yet current genetic testing fails to identify a causal variant for most cases. At present, cytogenetic methods and/or sequencing approaches such as exome (ES) or short-read genome sequencing (srGS) represent the state-of-the-art for comprehensive clinical discovery of sequence and structural variants (SVs), including copy number variants, balanced SVs, complex SVs, and tandem repeats (TRs). Recently, long-read genome sequencing (lrGS), coupled with multiomics data, has presented great promise to resolve variation in genomic regions recalcitrant to characterization by srGS such as highly repetitive simple repeat sequences and segmental duplications. However, there are few guidelines to enable clinical interpretation of genetic variation in these highly repetitive genomic regions, and the enthusiasm of the field in adopting lrGS has made it difficult to assess the true added diagnostic yield of this technology due to widely variable and inconsistently applied analytic pipelines and variable degrees of pre-screening by ES or srGS. Here, we investigated the contribution of SVs to rare diseases using srGS as a front-line strategy when paired with highly sensitive SV discovery and evaluate the added diagnostic yield of incorporating lrGS for a subset of cases. Our srGS analysis encompassed 1,462 families (3,450 individuals) recruited through the Broad Institute Center for Mendelian Genetics and the Genomics Research to Elucidate the Genetics of Rare Diseases (GREGoR) programs. Diagnostic SVs were identified in 5.4% of cases (79/1,462), of which 80% were uniquely detectable by srGS compared to standard cytogenetic techniques. For 96 families (including 10 families with a heterozygous variant observed in a known recessive gene of clinical relevance), we performed lrGS with methylation profiling, as well as long-read transcriptomic analyses in a subset of 20 trios. Analyses with lrGS yielded over 25,000 SVs per genome, 63% of which were not captured by srGS, along with an additional ~200 rare SNV/indels per genome not previously captured and 12 differentially methylated regions per genome. Among these, we identified only one diagnostic variant not interpreted by srGS, an apparently mosaic de novo SNV in CASK that was absent in the srGS callset due to allelic imbalance. No new diagnoses were supported by long-read transcriptomics or episignatures. In this well characterized rare disease cohort, the added diagnostic yield was thus 1.04% (1/96 families). Following a systematic literature review of prior lrGS studies, we find that most reported diagnoses were detectable by srGS and that our added diagnostic yield is consistent with those prior studies. These studies emphasize the significant impact of comprehensive SV discovery in rare disease cases and further demonstrate the power for increased discovery of novel genomic variation and episignatures from lrGS. Nonetheless, they also serve to temper expectations of dramatic diagnostic advances in rare disease patients until there is more extensive annotation of the functional and clinical impact of all coding and noncoding variation uniquely accessible to lrGS with extensive reference databases spanning highly repetitive genomic sequencing that could be enabled by this transformative technology.

Early glaucoma detection and treatment are critical to prevent irreversible blindness. Glaucoma polygenic risk scores (PRS) offer an effective approach for stratifying disease risk and are increasingly available in clinical practice. However, the psychosocial impact of receiving glaucoma PRS results is currently unknown. As such, this study investigated short-term psychosocial outcomes of disclosing glaucoma PRS to individuals over 50 years from the general population. Individuals from the bottom 10%, middle 45 to 55%, and top 10% of PRS scores were invited to receive their results and complete surveys before and 2 weeks after receiving results to assess anxiety, test-related distress, decisional regret, recall and understanding. Of invited participants, 51.7% (136/263) enrolled with 133 completing both surveys. Two weeks after disclosure, PRS recall was high (78.2%), although PRS knowledge remained limited. Privacy concerns were moderate, not differing across PRS groups (X^2 = 4.17, p = .124). Small reductions in glaucoma-related anxiety (z = -2.93, p = .003), generalised anxiety (z = -3.75, p < .001) and stress (z = -2.49, p = .013) were observed following disclosure. While scores remained within normal ranges, higher glaucoma-related anxiety (t = -2.36, p = .020), higher negative emotions (X^2 = 20.80, p < .001), and lower positive experience (F = 5.70, p = .004) were seen for high-risk participants compared to lower-risk participants. Decisional regret was low and did not differ across PRS groups (X^2 = 0.28, p = .869). These findings support the psychosocial safety of glaucoma PRS testing while highlighting the need for improved education and longer-term follow-up to support clinical implementation.

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.

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.

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.

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 and Objectives: Leukodystrophies are rare genetic diseases affecting the central nervous system white matter, leading to progressive disabilities and death. Although early diagnosis is critical for therapies, the penetrance and phenotypic spectrum of many leukodystrophies remain poorly defined. Here, we integrate sequencing population screening with longitudinal electronic health record (EHR) data. Our goals were to assess the prevalence of undiagnosed leukodystrophy, characterize phenotypic variability among genotype-positive individuals, and estimate penetrance across multiple leukodystrophies. Methods: We analyzed 19 genes associated with 13 leukodystrophies in pediatric and adult individuals recruited via the HerediGene Population Study, a 5-year study conducted primarily of healthy individuals in the U.S. intermountain west. Sequencing was performed on 210,983 individuals, consisting of genome sequencing for 34,033 and SNP panel imputation for 176,950. Variant results were cross-referenced to comprehensive and longitudinal (20+ years) clinical data in the Intermountain Health Enterprise Data Warehouse and to the Utah Leukodystrophy Program. Results: Pathogenic variants were identified in 4 genes (CSF1R, PLP1, POLR3A, SNORD118) in 9 individuals, none of whom had a clinical leukodystrophy diagnosis or characteristic MRI findings. These findings suggest that missed clinical diagnoses of most leukodystrophies are uncommon in a centralized healthcare system, but also demonstrate that for some leukodystrophies there may be variable or reduced penetrance, or broader phenotypic spectra than recognized. We used published incidence estimates and the observed leukodystrophy-associated genotypes to infer penetrance ranges that varied from wide for ultra-rare leukodystrophies, to tightly bounded for more prevalent conditions. Discussion: In this predominantly healthy population, we did not find any patients with leukodystrophy who had been genetically undiagnosed but then identified by sequencing. However, we identified 9 individuals with genotypes previously reported to result in leukodystrophy, but none of whom had clinical symptoms or MRI features associated with the specific leukodystrophy. Our results support a revised model in which leukodystrophies exist along a continuum of penetrance and expressivity, with implications for newborn screening, variant interpretation, and risk stratification.

Type 2 diabetes (T2D) is a highly heritable, polygenic disease with over 600 loci identified through genome-wide association studies (GWAS). However, despite possessing unique genetic variation shaped by demographic history and admixture, Latin American populations remain markedly underrepresented in global genomic research. To address this gap, we conducted genome- and exome-wide analyses of 19,431 T2D cases and 105,611 controls from the Mexico City Prospective Study (MCPS). We identified 86 independent GWAS associations, including 21 novel signals, 15 of which replicated in external cohorts. Risk alleles at novel loci were enriched in individuals with Indigenous American ancestry. Exome analyses revealed rare and ultra-rare missense variants with substantial risk effects at HNF1A and GCK, as well as a protein-damaging variant in SLC30A8 that reduced T2D risk by 45% in carriers. Integrative analyses indicate that T2D genetic architecture in Mexico is predominantly driven by common regulatory variation acting in the endocrine pancreas. Polygenic risk scores strongly stratified T2D risk and transferred to Indigenous Mexican populations. These findings demonstrate the power of large-scale genetic discovery in diverse populations to refine disease architecture and identify loci with potential therapeutic relevance.

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.

Aims Metformin remains the primary treatment for type 2 diabetes, yet over 40% of patients fail to maintain glycaemic control. We aimed to identify patients unlikely to respond to metformin prior to treatment initiation and to evaluate whether on-treatment management can improve glycaemic outcomes in suboptimal responders, informing early treatment decisions. Materials and Methods We analyzed 59,881 longitudinal HbA1c measurements from 7,105 patients with type 2 diabetes receiving metformin monotherapy using real-world electronic health records from Kaiser Permanente Northern California with up to six years of follow-up. We integrated demographic, clinical, genetic, and pharmacological factors to characterize metformin responder phenotypes and quantify the impact of adherence and weight control on time to glycaemic failure. Results Three distinct trajectory-based phenotypes were identified: good (63.6%), poor (8.9%), and non-responders (27.5%). Poor responders initially achieved glycaemic targets but lost control within 2.5 years, while non-responders showed minimal HbA1c reduction and failed within 1 year. Five baseline factors-HbA1c, age at diagnosis, body mass index, sex, and estimated glomerular filtration rate-classified phenotypes with good discrimination (area under the receiver operating characteristic curve = 0.84). Incorporating on-treatment HbA1c further enhanced identification of non-responders. Among suboptimal responders, weight control and improved adherence delayed glycaemic failure by approximately 7 months; however, eventual glycaemic failure remained likely. Conclusions We characterized three clinically relevant metformin responder phenotypes and showed that suboptimal responders can be identified early using baseline features. Poor and non-responders are unlikely to achieve durable glycaemic control with metformin alone and may require alternative treatment strategies.