Genetics in Medicine

Purpose: Genetic variant reclassification is increasingly common in clinical genomics, yet limited data describe how patients experience re-contact and variant reclassification in routine clinical care. Methods: We conducted semi-structured qualitative interviews with 20 adult patients who received a variant reclassification following routine clinical genetic testing. Interviews explored emotional responses, communication experiences, and perceived value of genetic testing. Data were analyzed using Template Analysis, a form of thematic analysis. Results: Three overarching themes were identified. Participants identified a need for improved communication of reclassified results, particularly with respect to timing, modality, and contextualization (Theme 1). Experiences with reclassification also shaped perceptions of the value of genetic testing, with most participants viewing testing as worthwhile despite its evolving nature (Theme 2). Finally, many participants interpreted reclassification as evidence of personalized and ongoing care, reinforcing trust in genetic testing and biomedical research (Theme 3). Participants generally preferred to be informed of reclassified results regardless of reclassification type, although the direction of reclassification influenced emotional responses and preferred modes of communication. Downgrades from variants of uncertain significance to benign or likely benign were widely viewed as meaningful by participants. Conclusion: Variant reclassification was experienced as a signal of personalized, ongoing care. Timely, contextualized, patient-centered re-contact practices may reduce uncertainty, strengthen trust, and help patients not feel forgotten.

Purpose Multiplexed assays of variant effect (MAVEs) are transforming clinical variant interpretation. However, many genes are associated with more than one disease, making it unclear whether functional data generated in one disease context may be directly applicable to another. For example, germline BAP1 missense variants are associated with both BAP1 tumor predisposition syndrome (BAP1-TPDS) and Kury-Isidor syndrome (KURIS), a rare neurodevelopmental disorder. Here, we demonstrate how phenotype-specific calibration of BAP1 MAVE data enables disease-specific variant classification. Methods Saturation genome editing (SGE) data for BAP1 were recalibrated using either BAP1-TPDS- or KURIS-associated missense variants as pathogenic controls. Functional evidence strength was quantified using the Odds of Pathogenicity (OddsPath) framework and mapped to ACMG/AMP PS3/BS3 criteria. Recalibrated functional evidence was integrated with standard clinical criteria for variant classification. A workshop was developed to teach phenotype-specific MAVE recalibration to clinicians and variant curators and evaluated for educational impact. Results Phenotype-specific recalibration using BAP1-TPDS and KURIS controls yielded OddsPath values consistent with PS3_Strong evidence in both contexts. Application of KURIS-specific recalibration enabled the diagnosis of KURIS in an individual with a previously uncertain BAP1 missense variant. The educational workshop enabled quantitatively improved understanding in applying functional evidence. Conclusion Phenotype-specific recalibration enables appropriately calibrated reuse of MAVE datasets across distinct disease contexts, increasing the clinical utility of MAVE datasets and the interpretability of variants in pleiotropic genes. This framework expands the diagnostic utility of existing functional datasets without requiring new experimental assays.

Background: RNA sequencing (RNA-seq) is increasingly recognized as a complementary tool to DNA-based sequencing for improving the diagnostic yield in Mendelian disorders. However, how the diagnostic performance of RNA-seq varies across molecularly and phenotypically distinct patient subgroups remains poorly defined. This study aimed to evaluate and compare the diagnostic utility of RNA-seq across three stratified groups of patients with non-diagnostic exome sequencing. Methods: We performed RNA-seq on whole blood samples from 90 patients with suspected Mendelian disease in whom clinical exome or whole-exome sequencing had failed to establish a molecular diagnosis. Patients were prospectively stratified into three groups of 30: (i) patients with a candidate variant of uncertain significance (VUS) with predicted splicing impact (Group 1), (ii) patients with a specific clinical pre-diagnosis but no identified pathogenic variant (Group 2), and (iii) patients without a specific pre-diagnosis or candidate variant (Group 3). Aberrant splicing, gene expression outliers, and allele-specific expression were analyzed using multiple bioinformatic tools and compared against a GTEx-derived control cohort. Results: RNA-seq contributed to a molecular diagnosis in 29 of 88 evaluable patients (32.9%). Diagnostic yield differed substantially across groups: 82.8% (24/29) in Group 1, 6.9% (2/29) in Group 2, and 10% (3/30) in Group 3. In Group 1, RNA-seq enabled reclassification of candidate VUS through direct demonstration of aberrant splicing events. In Group 2, RNA-seq identified a somatic mosaic ACTB variant missed by exome sequencing and reclassified a previously deprioritized APPL1 VUS. In Group 3, a deep intronic pseudoexon-activating variant in IGBP1 was identified in two siblings with severe microcephaly, providing evidence for a candidate X-linked microcephaly gene, and a pathogenic RNU4-2 variant was detected in a patient with ReNU syndrome, a non-protein-coding gene not captured by standard exome sequencing. Conclusions: RNA-seq has the highest diagnostic utility when applied to evaluate candidate splice variants identified by prior DNA testing but also provides independent diagnostic value in patients without candidate variants. The systematic comparison across stratified patient groups supports the integration of RNA-seq into clinical genomic workflows and highlights the need for standardized analytic frameworks.

Yield of reported results from genetic testing provides a proximal measure of clinical usefulness. While ACMG/AMP guidelines provide representations of uncertainty for individual genetic variant classification, additional factors are considered when determining whether results explain a patient's presentation. To standardize cross-consortium analysis, a working group of the Clinical Sequencing Evidence-Generating Research (CSER2) consortium iteratively identified factors used when contextualizing variant-level results to case-level interpretation (i.e., interpretation of an individual's genetic data with respect to the indication for testing). Sites independently categorized results; complex cases were discussed collaboratively, leading to revision of classification categories. Our metric incorporates factors beyond classification of reported variants. Analogous to variant-level results, "Definitive Positive" and "Probable Positive" represent certainty that results may be clinically explanatory. The category "Inconclusive" applies when results may or may not fully explain the patient presentation, with subdivision into multiple (non-exclusive) subcategories. Cases falling outside all of the other categories are considered "Negative". The overall diagnostic yield by this metric and use of categories for inconclusive results varied by CSER project, in part paralleling study design differences. This case-level categorization provides a meaningful assessment of diagnostic yield, and for inconclusive cases identifies potentially resolvable factors for case resolution.

Background: Pretest genetic counseling (GC) is recommended in conjunction with genetic testing (GT) for cardiovascular (CV) indications, yet access to CVGC is limited leading to delayed GT. Posttest GC could increase GC and GT access but requires efficient pretest education that supports both informed GT decision-making and robust GT uptake. Methods: We developed four indication-tailored online CV genetics education videos and deployed them in a 3-arm randomized trial comparing pretest vs. posttest outpatient CVGC (RESEQUENCE-GC, NCT05422573). Participants were 1:1:1 randomized to pretest video education plus an optional (efficiency arm) or required (flipped arm) phone call with a genetic counselor and planned posttest CVGC or to standard pretest CVGC (SOC arm). Questionnaires administered at baseline and post-education included the CV Multidimensional Model of Informed Choice [MMIC] to quantify GT knowledge and informed GT choice. Results: 389/767 (50.7%) adults aged 18-80 (mean 51.2{+/-}14.9 years) scheduling a first CVGC appointment consented to RESEQUENCE-GC and completed the baseline questionnaire. Efficiency arm participants (video education + optional phone call) were most likely to complete pretest education (134, 97.4% efficiency; 107, 85.6% flipped; 111, 87.4% SOC, p=0.0012) and elect GT (131, 95.6% efficiency; 105, 84.0% flipped; 107, 84.2% SOC, p=0.0036). Few (4, 2.9%) efficiency arm participants requested an optional pretest phone call. Most flipped arm participants (90, 84.1%) had no post-video questions, consistent with the 97 second [IQR: 65s-145s] median call duration. CV genetics knowledge was high post-education (median 8 [IQR 7,8]/8 MMIC items correct). Only video-based pretest education was associated with a significant increase in knowledge (p<0.0001). Nearly all participants made an informed GT choice with no difference between intervention (95.6%) and SOC (90.4%) arms (p=0.074). Conclusions: Tailored, online video pretest education can enhance CV GT uptake, support informed GT decision-making, and be integrated into efficient pretest workflows, suggesting utility in scalable posttest CVGC.

Purpose. Periodic reanalysis of genome sequencing data can yield additional diagnoses as knowledge evolves, yet manual reanalysis is labour-intensive. We compared automated and manual reanalysis approaches in rare disease genomics. Methods. We reanalyzed 377 rare disease cases: 158 with pathogenic or likely pathogenic (P/LP) findings, 49 with variants of uncertain significance (VUS) findings, and 170 had no findings. Manual reanalysis used standard diagnostic workflow for all cases without prior P/LP diagnoses (219 cases). An automated pipeline using Talos was benchmarked on the 158 P/LP cases before application to the 219-case reanalysis cohort. The mean reanalysis interval was 660 days. Results. Manual reanalysis identified three additional P/LP cases and two newly classified as VUS, increasing P/LP cases from 158 (41.9%) to 161 (42.7%). Talos recovered all three P/LP findings but only identified one of the two new VUS findings. Benchmarking showed 80.0% singleton concordance and 75.2% (82.8% proband-only) trio concordance, with approximately three variants per case. Conclusion. Reanalysis at 1.8 years yields modest but clinically meaning- ful gain. Automated reanalysis closely approximates manual performance while reducing hands-on effort, supporting scalable reanalysis in routine genomic care. Keywords: rare disease genomics, genome sequencing, automated reanalysis, variant prioritization, Talos, diagnostic yield

Background Orofacial clefts (OFCs) and other palate abnormalities (PAs) are among the most common birth defects worldwide and are characterized by the abnormal formation of the lip and/or palate. Genetic studies have traditionally classified OFC cases as either syndromic, involving OFCs alongside other congenital anomalies, or nonsyndromic, which represent the majority of cases and occur in isolation. Emerging genomic evidence indicates that genes traditionally associated with syndromic forms of OFC can also harbor variants contributing to isolated cases, challenging the notion of a strict dichotomy between these categories and supporting their integration for gene discovery. Methods In this study, we applied multiple analytic approaches to characterize the genetic architecture of OFC and PAs by integrating genomic data from 2,497 trios with an OFC (n=2080) and PA (n=417) affected proband. We compared these findings across OFC subtypes and syndromic status with those from 5,515 control trios to identify enriched biological pathways and mechanisms and to prioritize candidate genes using variant burden testing. Results We observed a significant enrichment of de novo protein-truncating and damaging missense variants in cases compared to controls (OR = 2.17, p = 1.21x10-32), with particularly strong signals in biologically relevant gene sets involving OFC-associated, constrained, Mendelian disorder, and mouse candidate genes. Variant burden testing identified 39 OFC risk genes at FDR [&le;] 0.05, which we then integrated with 593 established OFC genes to interrogate the functional underpinnings of OFC via network analysis. This analysis revealed 309 high-order interactor genes not previously associated with OFC. Notably, this OFC network clustered into ten distinct biological pathways, with nucleosome-associated genes showing significant enrichment among cases in our cohort (OR = 14.8, p = 8.1x10-4). In a final integrative step, we combined evidence across all analyses to nominate 231 candidate genes, 32 of which contained at least two deleterious de novo variants in our cohort. Conclusions These findings underscore the value of integrating diverse OFC and PA subtypes, syndromic status, and variant classes to refine the genetic architecture of these disorders, highlighting both phenotypic expansion of known disease genes and the emergence of novel gene-phenotype associations.

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

Congenital heart defects (CHD) are a common congenital anomaly and a leading cause of neonatal mortality. Even in ostensibly isolated cases, genetic testing can reveal monogenic causes of isolated CHD or identify syndromic conditions before additional features become clinically apparent. A timely and accurate genetic diagnosis can inform medical management and surveillance, reduce the need for unnecessary investigations, and offer families valuable information about prognosis, recurrence risk, and anticipatory guidance. In September of 2023, Primary Childrens Hospital introduced a universal genetic testing protocol that implemented whole genome sequencing for all neonates admitted to the cardiac intensive care unit (CICU) undergoing cardiac surgery before 30 days of life, with the goal of increasing the number of patients who receive a timely genetic diagnosis and improving clinical care. This is a retrospective chart review of patients who underwent whole genome sequencing (WGS) under the new universal genetic testing protocol at Primary Childrens Hospital from its initiation in September 2023 to February 2026. Over the study period, 217 neonates with CHD participated in the universal WGS protocol. Of these patients, 23 (10.6%) received a genetic diagnosis that was causative of their CHD, of which 11 patients (48%) had no major extracardiac features at the time testing was ordered. Twenty patients were diagnosed with a syndromic condition, and three patients were diagnosed with a non-syndromic condition. All of these patients received additional referrals to specialists following their new diagnosis, and six families used results to inform decisions regarding continuation of care. An additional 19 patients (8.8%) received WGS results that were clinically relevant but non-diagnostic for their CHD, including partial diagnoses, secondary findings, and carrier status. In total, 19.4% of patients (n=42) had clinically relevant variants identified on their WGS.

AI-driven chatbots have been utilized in healthcare to automate administrative tasks, improve patient education, and expand access to medical information; however, their role in genetic counseling remains underexplored. To investigate the adoption, perceptions, and potential utility of AI-based chatbots in genetic counseling practice, 217 genetic counselors and genetic counseling students from across North America were surveyed regarding chatbot usage, confidence in their application, and perceived benefits and limitations. While most participants (166/217; 76.5%) reported using general AI chatbots outside of clinical settings, far fewer (18/204; 8.8%) reported using or recommending clinical genetics chatbots in clinical practice. For those that used clinical genetics chatbots, the primary purpose was for communication with at-risk family members (11/18; 61.1%) and patient education (10/18; 55.6%). Confidence in chatbot technology varied, with highest confidence in gathering family history information (81/199; 40.7%) and lowest confidence in their ability to disclose variants of uncertain significance or positive genetic testing results (5/199; 2.5%). The greatest perceived benefits included reducing repetitive tasks (165/195, 84.6%) and allowing for time for other tasks (141/195; 72.3%), while major concerns revolved around patient comprehension (167/195; 85.6%) and having accurate, up-to-date information (145/195; 74.4%). Despite some concern about AI replacing human counselors, most participants reported they felt there was potential for chatbots to enhance workflow efficiency (128/195; 65.6%) if properly integrated and regulated. Limited AI training was identified as a barrier to adoption (16/195; 8.2% received training), highlighting a need for structured education on AI applications in genetic counseling. These findings suggest that AI chatbots hold promise as supplementary tools, but significant challenges must be addressed before widespread implementation in genetic counseling practice.

Background: For many evidence criteria within v3.0 of the ACMG/AMP guidelines, methodologies have been developed to empower their use outside the stipulated evidence strengths. However, no such methodology has been established for case-control data (PS4). With the release of large-scale unselected case-control datasets and expansion of nationally-collected laboratory datasets enriched for pathogenic variant carriers, there is potential to combine datasets across ascertainment contexts in a more quantitative manner using novel likelihood ratio tools. Methods: Using our published PS4-LR-Calculator, we calculated a combined log likelihood ratio (PS4-LLR) across five datasets (three unselected, and two enriched), and estimated enrichment of pathogenic variants in clinically-ascertained laboratory data using truncating variant prevalence. Results: Data were combined for 10,817 missense variants from 325,345 female breast cancer patients and 671,006 controls of Western European ancestry for five breast cancer susceptibility genes (BRCA1, BRCA2, PALB2, ATM, CHEK2). A combined LLR was produced for 4,690 missense variants; 927 variants received evidence towards pathogenicity (LLR[&ge;]1), and 3,242 received evidence towards benignity (LLR[&le;]-1). Conclusion: This flexible, variant-level methodology combines nationally-collected 'enriched' datasets with unselected case-control cohorts, expanding the available information for case-control analysis, boosting power, enabling exploration of atypical penetrance and empowering variant classification.

Importance BRCA genetic testing is critical for cancer risk assessment, treatment and personalization, yet substantial underutilization persists. Socioeconomic and clinical factors may strongly influence testing uptake; therefore, identifying the potential drivers to BRCA testing and treatment is essential for addressing gaps in access, increasing retention into care, and improving cancer outcomes. Objective To quantify the putatively causal effects of SDoH on BRCA genetic testing among individuals with breast, ovarian, pancreatic, and prostate cancers and to develop a predictive model to identify patients at risk for underuse of testing. Design, Setting, and Participants This observational case-control study used data from a large multistate clinical research data network covering southern US (2012-2023). The network contained records of more than 26 million individuals and was linked with ZIP code-level SDoH variables derived from national socioeconomic datasets. Adults diagnosed with breast, ovarian, pancreatic, or prostate cancer were eligible for cases (received BRCA testing) or controls (did not receive BRCA testing, matched by cancer diagnosis). Exposure SDoH categories, including economic conditions, education, healthcare access, neighborhood conditions, and social connectedness. Main Outcomes and Measures The primary outcome was receipt of BRCA genetic testing after cancer diagnosis. Results Among 3,279 people diagnosed with cancer, 748 received BRCA testing and 2,531 served as controls. Study population mean [SD] age was 66.8 [15.7] years; 1,758 were women [53.6%], 2,238 [69.6%] were White and 616 [18.8%] were Black or African American. Breast (1,420 [42.8%]) and prostate (1,342 [40.9%]) cancers were the most common diagnoses, followed by pancreatic (242 [7.4%]), ovarian (238 [7.2%]), and multiple cancers (55 [1.7%]). Upon adjusting for potential confounding, higher educational attainment (odds ratio [OR], 1.19), public-sector employment (OR, 1.42), neighborhood safety (OR, 1.28), and social participation (OR, 1.72) showed an increased likelihood of undergoing BRCA testing, whereas economic instability, including housing cost burden and reliance on public insurance, had an effect of reduced testing. A random forest classifier demonstrated good discriminative performance (AUROC, 0.776) to predict cancer patients who were likely to take BRCA testing, where nativity, language, and residential stability ranked among the most influential social determinants according to SHapley Additive exPlanations (SHAP) analysis. Conclusions and Relevance In this observational case-control study, SDoHs were strongly associated with receipt of BRCA genetic testing among people with cancer. These findings suggest that disparities in genetic testing may reflect structural and social barriers rather than differences in clinical eligibility alone. Efforts to improve equitable access to genetic testing may benefit from integrating social-context information into clinical workflows and targeting outreach or navigation strategies toward socially disadvantaged populations.

Purpose Patients carrying Germline Pathogenic Variants (GPVs) in multiple cancer susceptibility genes (CSGs) can be described within the context of Multi-locus Inherited Neoplasia Allele Syndrome (MINAS). The role of each GPV is typically interpreted based on clinical phenotypes. Here, we used tumor sequencing, particularly mutational signatures, to investigate the contribution of GPVs in MUTYH and PALB2 to colorectal polyposis and breast cancer in a single patient at a molecular level. Methods We analyzed tumor sequencing data, including mutational signatures and genomic scars, of a breast tumor and a colorectal polyp from a patient with biallelic GPVs in MUTYH and a heterozygous GPV in PALB2. Results The colorectal polyp showed a dominant contribution of MUTYH-associated Base Excision Repair deficiency (BERd) mutational signatures, with no evidence of Homologous Recombination Repair Deficiency (HRD). In contrast, the breast tumor showed both MUTYH-driven BERd and HRD-associated signatures, including SBS3, ID6 and an elevated HRD score, despite the absence of a detectable second hit in PALB2. These findings suggest a differential contribution from the CSGs, with MUTYH contributing to both lesions and PALB2 contributing specifically to the breast tumor. The observed pattern does not align with the additive or synergistic models described in MINAS. Conclusions Our study provides evidence that mutational signatures can elucidate the contribution of multiple CSGs to tumorigenesis within a single patient. These findings extend current interpretations of MINAS beyond additive or synergistic phenotypes, which may help to better understand tumor etiology, with potential clinical implications, including eligibility for targeted therapies.

Background: Biallelic variants in GFM2, encoding mitochondrial elongation factor G2 (mtEFG2), a GTPase involved in the termination stage of mitochondrial translation, cause autosomal recessive combined oxidative phosphorylation deficiency. Noncoding structural variants may be missed by exome sequencing but can disrupt splicing and provide opportunities for variant-specific therapeutic rescue. We investigated the molecular mechanism underlying suspected Leigh syndrome in an infant with mitochondrial disease and evaluated whether splice-switching oligonucleotide (SSO) treatment could correct the pathogenic splicing defect. Methods: The proband underwent exome sequencing followed by short-read and long-read whole genome sequencing. RNA sequencing, reverse-transcription PCR, quantitative PCR, and cycloheximide treatment were used to characterize the effect of the identified intronic duplication on GFM2 splicing and transcript stability. Patient-derived fibroblasts were treated with SSOs targeting the aberrant splice junction. Rescue was assessed by RNA studies, western blotting, and spectrophotometric measurement of cytochrome c oxidase (COX). Results: Whole genome sequencing identified a paternally-inherited GFM2 missense variant, NM_032380.5:c.2195C>T p.(Pro732Leu), in trans to a maternally-inherited 221-nucleotide intronic duplication, NM_032380.5:c.2029-741_2029-521dup. RNA studies revealed a 87-nucleotide pseudoexon, generated by activation of a cryptic acceptor splice site within the duplicated sequence. The resulting transcript harbored a premature termination codon (PTC) and underwent nonsense-mediated decay, as confirmed by cycloheximide rescue. Together with reduced mtEFG2 protein levels on western blot, the findings supported a loss-of-function mechanism. Enzymatic analysis of affected fibroblasts showed reduced activity of the mtDNA-dependent complex IV subunit COX, with preservation of the nuclear-encoded complex II enzyme succinate dehydrogenase and the control enzyme citrate synthase, consistent with impaired mitochondrial translation. A SSO targeting the aberrant intron-pseudoexon junction nearly abolished pseudoexon inclusion, restored correctly spliced GFM2 transcript from the duplication-containing allele, increased mtEFG2 protein levels, and significantly improved COX activity. Conclusions: This study identifies a pathogenic intronic GFM2 duplication that causes mitochondrial disease through pseudoexon activation and nonsense-mediated decay. The findings demonstrate the value of integrated genome and transcriptome analysis for exome-negative mitochondrial disease and provide in-vitro proof of concept that SSOs can restore transcript processing, protein expression, and mitochondrial respiratory-chain function in patient-derived cells.

Background: Neurological, neuropsychiatric, and neurodevelopmental disorders cluster in ALS families, sharing a common genetic architecture with ALS. Pathogenic variants in genes associated with other neurological, neurodevelopmental, or neuropsychiatric disorders may also co-occur in ALS and modify phenotype. We have sought to determine the prevalence and clinical pattern of likely-pathogenic/pathogenic (LP/P) non-ALS neurological, neurodevelopmental, and neuropsychiatric variants, alone and in combination with ALS-gene variants, in two large ALS cohorts. Methods: Whole-genome sequencing (WGS) of 469 Irish and 774 Answer ALS people with ALS (pwALS) was analysed for ClinVar LP/P variants associated with other neurological (n = 15541), neurodevelopmental (n = 9761), and neuropsychiatric (n = 321) phenotypes. Inheritance patterns for associated genes (autosomal recessive/autosomal dominant) along with the associated phenotype were validated using OMIM. Standardised clinical data included family history, site and age of onset, El Escorial category, survival, motor decline, and cognitive and behavioural assessments. Known ALS-gene variants and C9orf72 repeat expansion status were included for each cohort. Results: Non-ALS neurological variants were identified in 47/469 (10.0%) Irish and 69/774 (8.9%) Answer ALS participants, most frequently in hereditary spastic paraplegia-associated genes (3.2% Irish; 2.8% Answer ALS). Irish neurological variant carriers showed higher frequency of respiratory onset (10.6% vs 1.2%, Fisher's exact p = 0.002, {Phi} = 0.20) and fewer premorbid behavioural symptoms (0.92 +/- 0.56 vs 3.08 +/- 0.97, Cohen's d = -0.40). Neurodevelopmental variants occurred in 12/469 (2.6%) Irish and 20/774 (2.6%) Answer ALS participants. In the Irish cohort, neurodevelopmental variant carriers had significantly shorter survival in Cox proportional hazards model (log-rank p = 0.005), corresponding to a more than two-fold increased hazard of death (HR = 2.25, 95% CI 1.26-4.00), and had significantly increased familial burden of neuropsychiatric disorders among first- and second-degree relatives (negative binomial IRR for carriers = 2.41, 95% CI: 1.12-5.18, p = 0.025). Across combined cohorts, 18 individuals (Irish n = 8; Answer ALS n = 10) carried [&ge;]2 LP/P variants spanning ALS and non-ALS genes. Conclusion: Rare LP/P variants in genes associated with other neurological and neurodevelopmental disorders occur in up to 12% of pwALS across two independent cohorts. Carriers show distinct phenotypes, shorter survival, and characteristic family history patterns. These findings suggest that extended pleiotropic and oligogenic architectures may contribute to ALS heterogeneity.

BackgroundThe diagnostic yield for 46,XY disorders of sex development (DSD) remains limited. Whole-genome sequencing (WGS) improves detection of both coding and non-coding variants that may be missed by routine testing. Cytochrome b5, encoded by CYB5A, is an essential co-factor for CYP17A1-mediated 17,20-lyase activity. We report on WGS on a Vietnamese family with 46,XY DSD with two siblings presenting with female external genitalia. MethodsClinical assessment and hormone profiling were conducted. WGS was conducted on peripheral blood DNA, in two affected siblings followed by variant annotation and ACMG-based classification. A minigene RNA splicing assay in HEK293 cells was used to evaluate the functional impact of the CYB5A intronic variant. ResultsThe patients hormone profile showed low testosterone and estradiol. WGS identified compound-heterozygous CYB5A variants: a paternally inherited missense variant (p.Val34Glu, likely pathogenic) and a maternally inherited deep intronic deletion (c.129+862_129+863del) for which SpliceAI predicted aberrant splicing. Minigene assays confirmed that the intronic deletion creates cryptic splice sites, resulting in pseudoexon inclusion and a premature stop codon, consistent with nonsense-mediated decay. The intronic variant meets ACMG criteria for pathogenicity. ConclusionThis family expands the spectrum of CYB5A-related DSD and demonstrates that compound-heterozygous variants, including deep intronic defects, can lead to a disruption in 17,20-lyase activity. These findings highlight the importance of WGS and functional assays for identifying clinically relevant non-coding variants in DSD.

ObjectiveAccurate classification of ion channel variants of uncertain significance (VUS) remains a persistent challenge in clinical genomics, limiting diagnostic resolution in neurological disorders. MethodsWe developed a calibrated electrophysiological framework to generate functional evidence for clinical interpretation of CACNA1G variants encoding the low-voltage-activated calcium channel Cav3.1. Functional metrics derived from automated patchclamp recordings were calibrated against benign/likely benign (B/LB) and pathogenic/likely pathogenic (P/LP) reference variants to enable conservative application of ACMG/AMP functional criteria within clinical variant interpretation workflows. ResultsCalibration using 25 B/LB and 16 P/LP CACNA1G variants showed that more than 80% of P/LP variants exhibited reduced current density (CD). Deactivation kinetics ({tau}Deact) provided complementary discriminatory information by identifying gating abnormalities in variants with preserved CD. Application of this dual-metric framework to five VUS identified in Australian patients revealed two variants (Cav3.1-R186Q and R1394Q) with abnormal functional profiles consistent with voltage-sensor perturbation, supporting reassessment as likely pathogenic under ACMG/AMP guidelines. The remaining VUS displayed functional properties overlapping the benign reference distribution. ConclusionThese findings establish a calibrated functional framework for generating electrophysiological evidence that supports clinical interpretation of CACNA1G missense variants under ACMG/AMP guidelines. When applied as external functional evidence, this approach improves resolution of CACNA1G-associated VUS while maintaining conservative standards for variant classification.

Abstract Purpose: Published clinical outcome data on preconception carrier screening (PCS) in Central Asia are limited. We report the first clinical implementation study from Uzbekistan of a whole-exome sequencing (WES)-based multi-platform PCS program combining exome sequencing with targeted SMA, FMR1, and DMD assays. Methods: We retrospectively analyzed anonymized data from 65 individuals (19 couples, 27 singletons) screened at IMC Genomics, Tashkent, between January 2024 and May 2026. WES covering the protein-coding regions of approximately 20,000 genes was followed by exome-wide bioinformatics filtering and clinical geneticist interpretation. Partly overlapping cohorts underwent SMA carrier screening (n=179), FMR1 CGG-repeat analysis in females (n=155), and DMD deletion/duplication testing in preconception females (n=29). Variants were classified by ACMG/AMP criteria against gnomAD v4.1. Results: Sixty-one of 65 WES-screened individuals (93.8%; 95% CI 85.2 - 97.6%) carried at least one reportable variant (152 instances across 126 genes). Four of 19 couples (21.1%; 95% CI 8.5 - 43.3%) were concordant for pathogenic or likely pathogenic variants in the same autosomal recessive gene; two were referred for preimplantation genetic testing for monogenic disease. SMA screening identified four carriers, including two 2+0 silent carriers; FMR1 analysis identified one intermediate allele; DMD MLPA identified no exonic rearrangements. Conclusion: This first reported WES-based multi-platform PCS program in Uzbekistan was feasible and clinically informative, identifying actionable couple-level reproductive risks and supporting structured implementation of reproductive genetic screening in Central Asia.

Background: Polygenic risk scores (PRS) for breast cancer are increasingly used for risk stratification to inform screening and prevention. However, for PRSs to be equitable and clinically useful, they need to perform well across diverse populations. While PRS performance is known to be ancestry-dependent, it is not well understood how environmental context, such as that of socioeconomic status (SES), affects PRS transferability. Here, we assess whether SES, measured via self-reported household income, modifies breast cancer PRS performance and, if so, whether socioeconomic context contributes predictive information beyond genetic risk alone. Methods: We used the US-based All of Us biobank to evaluate how SES impacts breast cancer PRS performance. First, we quantified changes in breast cancer PRS performance by modeling a commonly-cited polygenic score for breast cancer previously described by Mavaddat et al. with SES. We then reestimated the genetic effect sizes of the 3,820 variants from Mavaddat et al. in All of Us with and without income as a covariate. Because social determinants of health affect breast cancer detection and outcomes, we stratified analyses by socially defined populations on the basis of self-identified race and ethnicity. We further stratified individuals whose self-identified race is White (''White'') into three SES groups (high, middle, low) based on self-reported income and re-estimated genetic effect sizes to create SES-specific PRSs. We then applied these PRSs to White participants, the largest group in the study, and to Black or African American (''Black'') and Hispanic or Latino (''Hispanic'') participants, groups underrepresented in breast cancer research. Model discrimination between cases and controls was measured by area under the curve (AUC). Results: We analyzed 163,715 women from the All of Us biobank, which included 8,833 breast cancer cases (6,619 White, 1,178 Black, and 1,036 Hispanic), with relative income available for a subset of these cases (5,525 White, 848 Black, and 566 Hispanic). The ancestry-dependent performance of the breast cancer PRS described in Mavaddat et al. was replicated in All of Us. In Black individuals, this PRS (AUC and 95% CI: 0.576 [0.571, 0.582]) produced a similar increase in AUC as relative income (AUC: 0.573 [0.568, 0.577]) when added to an age-only model. Incorporating income with PRS, age, and genetic PCs 1-3 improved AUC by 0.007 in White Americans and 0.018 in Black Americans (both p < 10-11), while attenuating the contribution of PRS in the full model. PRS performance also varied among SES categories. Notably, PRSs with variant effect sizes that were recalibrated in low-SES White participants performed best in low-SES White participants (AUC: 0.605 [0.583, 0.628]) and Black Americans (AUC: 0.588 [0.586, 0.591]), both better than performance in high-SES White Americans (AUC: 0.579 [0.577, 0.580]) and middle-SES White Americans (AUC: 0.578 [0.569, 0.586]). Conclusion: Socioeconomic context, measured by income, significantly impacts the transferability of a PRS for breast cancer within and among groups defined by self-identified race and ethnicity. Accounting for SES improves PRS performance, most notably in Black Americans and low-SES White individuals.

Introduction Spinal muscular atrophy (SMA) is a monogenic neuromuscular disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Onasemnogene abeparvovec is a U.S. FDA-approved single-dose gene therapy for SMA. Both its intravenous formulation (Zolgensma, approximately USD 2.13 million per patient) and intrathecal formulation (Itvisma, around USD 2.59 million per patient) are prohibitively expensive, substantially limiting accessibility in low- and middle-income countries (LMICs). We conducted a clinical study of vesemnogene lantuparvovec, an alternative to onasemnogene abeparvovec developed for use in LMIC settings. Methods Sixteen patients with SMA, including 8 with type 1 SMA and 8 with type 2 SMA, received a single intrathecal administration of vesemnogene lantuparvovec. Eleven patients were treated with a low dose (1.5 * 10^14 vg) and five with a high dose (3.0 * 10^14 vg). The primary endpoints were safety and efficacy, assessed by changes from baseline in developmental gross motor milestones according to the World Health Organization criteria. Overall survival was primarily evaluated in type 1 SMA patients. This trial was registered with ClinicalTrials.gov NCT06288230. Results As of the March 2026 cutoff date, 15 of 16 treated patients had completed at least 12 months of follow-up after treatment, while the remaining one type 1 SMA patient died of disease progression at month 6 post-treatment. At 12 months post-treatment, among the surviving 7 patient with type 1 SMA, the median age was 21.6 months (range, 16.1 to 32.3 months). Among the 16 treated patients, the median age at diagnosis was 4.4 months (range, 0.0 to 18.0 months), and the median age at dosing was 10.7 months (range, 2.8 to 22.5 months). All patients experienced at least one AE. Thirty-one AESIs were reported in 13 patients, including hepatotoxicity, thrombocypenia-related events and cardiac events. No patient required prolonged prednisolone prophylaxis. SAEs, including pneumonia, lower respiratory tract infection, upper respiratory tract infection, and haemorrhagic diarrhoea, occurred in 5 of 8 (63%) patients with type 1 SMA and 2 of 8 (25%) patients with type 2 SMA. Two patients with type 1 SMA required invasive ventilation, and one of whom subsequently died. At 12 months post-treatment, 11 of 16 treated patients (69%) gained at least one new WHO motor milestone versus baseline, including 3 type 1 and 8 type 2 SMA patients; one type 2 patient gained six WHO motor milestones and achieved independent walking. Conclusions In patients younger than 24 months of age with type 1 or type 2 SMA, a single intrathecal dose of vesemnogene lantuparvovec was safe and generally well tolerated and was associated with improvements in developmental gross motor milestones compared with outcomes observed among referred but untreated patients. Additional studies are required to further evaluate the long-term safety and efficacy of this gene therapy.