Introduction: Charcot-Marie-Tooth disease (CMT) is one of the most common inherited Neuromuscular Disorders (NMDs), classified under peripheral neuropathies and characterised by progressive motor and sensory dysfunction. Although Wholeexome Sequencing (WES), gene panels, and conventional methods have improved detection rates, they often miss deep intronic, regulatory, and Structural Variants (SVs). Wholegenome Sequencing (WGS), with its comprehensive coverage of coding and non coding regions, enables the identification of variants that are often overlooked by other approaches. Aim: To assess the diagnostic utility of WGS in CMT cases that remain unresolved by WES, analysing both coding and non coding variants. Materials and Methods: The present cross-sectional diagnostic study was conducted between July 2023 and January 2025 at the Neuberg Center for Genomic Medicine (NCGM), Ahmedabad, Gujarat, India. WGS was performed on 31 clinically suspected CMT patients, including two who had previously tested negative by WES. Both coding and non coding variants including missense, nonsense, frameshift, in-frame, intronic, and 5’ Untranslated Region (UTR) mutations were analysed. Variants were classified according to American College of Medical Genetics and Genomics (ACMG) guidelines, incorporating Combined Annotation Dependent Depletion (CADD) scores and Minor Allele Frequency (MAF) thresholds. They were interpreted based on pathogenicity, inheritance patterns, and genotypephenotype correlations. Selected non coding variants in the Gap Junction Beta-1 (GJB1; c.-16-511G>C) and Lamin A/C (LMNA; c.-142C>A) genes were validated by Sanger sequencing. Results: Sequencing data from 31 participants were processed using a standardised bioinformatics pipeline. Variants were classified according to ACMG guidelines, and their frequencies were calculated. WES and WGS results were compared to determine the additional diagnostic yield. WGS identified clinically significant non coding variants in GJB1 (intronic) and LMNA (5’ UTR) in two cases, yielding a 6.5% increase over WES. Overall, 31 variants were detected: 11 (35.5%) classified as pathogenic, 2 (6.5%) as likely pathogenic, and 18 (58.0%) as Variants of Uncertain Significance (VUS), reflecting the genetic heterogeneity of CMT. Conclusion: The WGS enhances diagnostic accuracy in CMT by detecting clinically relevant non coding variants often missed by WES. This is the first report from India confirming a GJB1 intronic variant and a Lamin A/C (LMNA) 5´ UTR variant using WGS in CMT patients. These findings support the integration of WGS into routine diagnostic workflows and highlight the value of comprehensive variant analysis for early and precise genetic diagnosis.

Sample tracking in whole exome sequencing.

Background/Objectives: Malignant hyperthermia (MH) is a life-threatening pharmacogenetic disorder of skeletal muscle calcium regulation and commonly associated with pathogenic variants in the RYR1 gene. Interpretation of rare RYR1 variants remains challenging, particularly when classified as variants of uncertain significance (VUS). This study describes the clinical, functional, and genetic evaluation of a patient with suspected MH susceptibility carrying a rare RYR1 mutation. Methods: We report a retrospective case evaluation of a 32-year-old female referred for MH assessment following a prior peri-operative hypermetabolic event. Clinical records were reviewed, and MH susceptibility was assessed using the caffeine–halothane contracture test (CHCT). Genetic testing was performed using a targeted MH susceptibility gene panel, including RYR1, CACNA1S, and STAC3. Variant classification was conducted following American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines. Results: The patient demonstrated a positive CHCT, consistent with MH susceptibility. Genetic analysis identified a rare heterozygous RYR1 missense variant. No pathogenic or likely pathogenic variants were detected in CACNA1S or STAC3. Based on ACMG/AMP criteria, the RYR1 p.Gln474His variant is currently classified as a VUS. However, its localization within the N-terminal regulatory region of RyR1 and concordance with abnormal CHCT findings provide supportive functional context. Conclusions: This case underscores the importance of integrating clinical history, functional contracture testing, and genetic data in the evaluation of MH susceptibility. While functional findings may support biological plausibility, definitive pathogenic classification of rare RYR1 variants requires additional segregation data or mechanistic studies.

Background The IL2RB gene encodes the beta subunit of the interleukin-2 (IL2) receptor, which transmits signals from the cytokines IL-2 and IL-15. IL2RB shows constitutive or induced expression on various types of immune cells, including CD4+ T regulatory cells, CD4+ and CD8+ T cells, B cells, and natural killer cells. IL2RB cytoplasmic domain is essential for JAK1/3 signaling. Biallelic loss-of-function variants typically cause autosomal recessive immunodeficiency. Here, we describe a patient with a novel heterozygous splice site variant and severe immune dysregulation. Methods Clinical evaluation and whole exome sequencing were performed. Variant classification followed the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines. In silico predictions were used to assess the molecular consequences. Results A 4-year-old girl born to non-consanguineous parents presented with neonatal diabetes requiring insulin-pump therapy (anti-glutamic acid decarboxylase antibodies positive), autoimmune hypothyroidism (anti-thyroglobulin and anti-thyroperoxidase antibodies positive), autoimmune hemolytic anemia (direct Coombs positive), chronic diarrhea consistent with inflammatory bowel disease, and bullous pemphigoid (anti-bullous pemphigoid antibodies positive). This last manifestation was refractory to high-dose intravenous immunoglobulin, corticosteroids, dapsone, and rituximab, with only partial improvement; clinical stabilization was achieved after dupilumab plus palliative care. Whole exome sequencing identified a heterozygous IL2RB splice site variant (c.903+1G>T), absent from gnomAD and classified as likely pathogenic (PVS1, PM2). The variant disrupts the canonical donor site of intron 9, and in silico splicing predictors indicate activation of a cryptic donor site 26 nt upstream in exon 9, generating a frameshift and premature stop codon located within the last exon, thereby predicted to escape nonsense-mediated decay. The predicted truncated protein lacks most of the intracellular domain, likely compromising JAK1/3-mediated signaling. Conclusion This case raises the possibility of a dominant-negative or haploinsufficient mechanism of IL2RB-related disease. Functional studies are needed to confirm the impact on receptor signaling and clarify the inheritance pattern.

The objective of this study was to provide the first genomic diagnosis of chronic granulomatous disease (CGD) in a referral hospital in Southern Brazil, a rare and underdiagnosed disease with limited data in the country. Six male patients (P1-P6) were clinically and genetically analyzed, through medical reports review and massively parallel sequencing by a panel for the CYBB, CYBA, NCF1, NCF2, and NCF4 genes and whole genome sequencing. The gene-scan technique was used to detect the Tyr26HisfsTer variant (ΔGT) in NCF1 and to distinguish it of its pseudogenes (ΨNCF1), which naturally have ΔGT. Variants were classified according to the American College of Medical Genetics and Genomics guidelines. Structural modelling was performed for missense variants using PyMOL and I-TASSER to verify their potential impact on the NADPH oxidase complex, which is defective in CGD. Among the clinical manifestations, the most commonly affected organs were the lungs, skin, and lymph nodes, with all patients presenting with recurrent infections and pneumonia. Adverse reactions to BCG vaccination were observed in two patients. Four patients carried variants in CYBB: (P1) p.Cys257Ser, a novel variant, and (P2) p.Cys257Arg, both classified as likely pathogenic and predicted to significantly affect the structure of NADPH oxidase, with Gibbs free energy values of 6.66 and 6.23 kcal/mol, respectively (reference value: >1.6 kcal/mol); p.Arg157Ter (P3), and p.Trp483Ter (P4), both classified as pathogenic and predicted to undergo nonsense-mediated mRNA decay. Gene-scan analysis revealed the ΔGT in two siblings: P5 (homozygous) and P6 (heterozygous). It was hypothesized that P6 may have an NCF1-related pseudogene lacking the ΔGT originated by unequal recombination with NCF1, resulting in the absence of functional alleles in P6. This study underscores the importance of genetic characterization for accurate diagnosis, reducing diagnosis odysseys and supporting the indication of bone marrow transplantation to prevent fatal outcomes, and reinforcing the contraindication of Bacillus Calmette–Guérin vaccination in patients with CGD.

Background NF-κB1 is a key transcription factor involved in immune regulation, and pathogenic variants in this gene can lead to a spectrum of primary immunodeficiencies, including common variable immunodeficiency (CVID). We report a family with a novel heterozygous NFKB1 variant segregating across three generations with variable expressivity of immunodeficiency and chronic fungal infections. Case Presentation The index case is a 6.5-year-old girl who presented with severe hypogammaglobulinemia affecting all three immunoglobulin isotypes, impaired polysaccharide vaccine response, recurrent respiratory infections, chronic diarrhea, and candida onychomycosis. B cells were present but showed a disrupted maturation profile with reduced memory B cells and abnormal pre- and post-switched subsets. His 43-year-old father had recurrent infections, moderate hypogammaglobulinemia (IgG and IgM), facial fungal infections with hyaline branching hyphae on biopsy, chronic diarrhea, and severe intestinal polyposis. The 68-year-old grandmother also had a history of recurrent respiratory infections and hypogammaglobulinemia. Whole exome sequencing identified a novel heterozygous NFKB1 variant (c.202G>A, p.Gly68Ser) in the index case, which was confirmed in both the father and grandmother. Discussion The variant lies within the functional Rel homology DNA-binding domain (PM1_Moderate), a region enriched in known pathogenic variants. It is absent from population databases (PM2_Supporting), and in silico prediction using REVEL yields a high pathogenicity score (0.797; PP3_Moderate). The phenotype across three generations is consistent with reported clinical manifestations of NFKB1 haploinsufficiency, including CVID, impaired class-switch recombination, and chronic infections (PP4_Supporting). The variant cosegregates with the disease in the family (PP1_Supporting), supporting a pathogenic role. Based on the American College of Medical Genetics and Genomics/ClinGen guidelines, the variant is classified as a likely pathogenic variant. Conclusion We report a novel familial NFKB1 variant associated with a CVID-like phenotype and chronic fungal infections. Functional validation is needed to confirm pathogenicity and to guide targeted therapeutic approaches.

CARD11 is a component of the CBM complex, which plays a critical role in the antigen-dependent activation of B and T lymphocytes via the NF-κB. Mutations in CARD11 have been associated with three distinct inborn errors of immunity. One of these forms is caused by dominant-negative loss-of-function mutations, characterized by atopy, hyper-IgE, and hypogammaglobulinemia. However, clinical presentations could be heterogeneous, as reported in the literature. We describe the case of a female patient, born to non-consanguineous parents, who presented with early-onset severe atopic dermatitis occasionally associated with secondary bacterial skin infections beginning at two months of age. Laboratory evaluation revealed hyper-IgE syndrome and hypogammaglobulinemia, with reduced levels of IgM and IgG. Immunoglobulin replacement was initiated at 10 months of age. By this time, she had also been diagnosed with food allergy. Within one year, the patient underwent two procedures for the removal of disseminated molluscum contagiosum lesions, with one curettage involving more than 300 lesions. Despite no evidence of lymphopenia in blood count, immunophenotyping revealed mildly reduced levels of natural killer cells. Four months later, a primary immunodeficiency orientation tube (PIDOT) analysis identified lymphopenia in both naive and post-germinal center B cell, with or without rearrangement. Whole exome sequencing revealed a heterozygous loss-of-function variant in CARD11:c.C119A/p.A40D. This variant was classified as likely pathogenic following functional assay, in accordance with the American College of Medical Genetics and Genomics guidelines. Family segregation analysis showed that the patient’s mother also carries the same variant. She presented with a milder phenotype, including psoriasis, skin allergic manifestations, and asthma particularly during childhood. This family case highlights the phenotypic variability associated with loss-of-function dominant-negative CARD11 mutations. While the proband exhibited a severe atopic and infectious phenotype requiring early intervention, her mother displayed only mild symptoms.

There is clear evidence that deleterious germline variants in CHEK2 increase risk for breast and prostate cancers; there is limited or conflicting evidence for other cancers. To quantify the prevalence of as well as cancer risk and survival associated with CHEK2 germline pathogenic and likely pathogenic variants using genomic ascertainment. This case-control study used 2 electronic health record-linked and exome-sequenced biobanks: UK Biobank (n = 469 765) and Geisinger MyCode (adults only; n = 167 050). Variants were classified according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology criteria. Cases were defined as individuals with heterozygous CHEK2, harboring pathogenic or likely pathogenic variants; controls as individuals with a benign or likely benign CHEK2 variation or wildtype CHEK2. Cancer registry (MyCode since approximately 1943; UK Biobank since approximately 1970) and demographic data were retrieved; to adjust for relatedness, association analysis was performed with SAIGE-GENE+ with Bonferroni correction. Prevalence of as well as cancer risk and survival in adults with CHEK2 germline variants. Of 469 765 individuals in the UK Biobank, there were 3232 case participants (mean [SD] age, 70.8 [8.0] years; 3139 [97.1%] White; 1744 [54.0%] women); of 167 050 individuals with MyCode, there were 3153 case participants (mean [SD] age, 60.5 [17.8] years; 3123 [98.8%] White; 1935 [61.5%] women). In case participants in both MyCode and UKBB, there was a significant excess risk of all cancers (odds ratio [OR], 1.33 [95% CI, 1.18-1.49]; OR, 1.41 [95% CI, 1.26-1.59], respectively), breast (OR, 1.54 [95% CI, 1.18-2.00]; OR, 1.84 [95% CI, 1.49-2.27], respectively), prostate (OR, 1.62 [95% CI, 1.27-2.07]; OR, 1.78 [95% CI, 1.48-2.16], respectively), kidney (OR, 1.58 [95% CI, 1.03-2.41]; OR, 1.84 [95% CI, 1.22-2.77], respectively), and bladder (OR, 1.50 [95% CI, 1.01-2.23]; OR, 1.64 [95% CI, 1.17-2.31], respectively) cancers as well as lymphoid leukemia (OR, 2.08 [95% CI, 1.17-3.69]; OR, 2.21 [95% CI, 1.19-4.08], respectively). Compared with control participants, time to cancer in case participants was significantly shorter in both cohorts; no significant difference was observed between the age-dependent penetrance of truncating and missense variants for cancer in either cohort. Overall survival was significantly decreased in case participants in UK Biobank; however, the primary consequence was seen after 75 years. There was no statistical difference in survival in MyCode. There were no differences in survival between case participants with cancer and control participants with cancer. In this case-control study of genomic ascertainment of individuals with heterozygous CHEK2 pathogenic or likely pathogenic variants in 2 population-scale cohorts, there was a significant excess risk of breast, prostate, kidney, bladder, and lymphoid leukemia cancer. The conferred excess mortality and cancer risk was low (ORs <2). This has clinical implications for individuals ascertained this way (vs with a family history of cancer).

ABSTRACTFamilial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive renal tubular disorder, caused by mutations in the Claudin‐16 or Claudin‐19 genes. It is characterized by renal wasting of calcium and magnesium, bilateral nephrocalcinosis, and progression to kidney failure eventually. We had a pediatric patient with hypomagnesemia, hypercalciuria, and nephrocalcinosis with the presence of a novel missense variant of c.175C>T (p.Arg59Cys) in the CLDN16 gene on whole exome sequencing (WES). This variant falls in the category of variants of uncertain significance as per the American College of Medical Genetics and Genomics guideline. MutationTaster, Sorting Intolerant From Tolerant, and Combined Annotation‐Dependent Depletion support its deleterious effect, putting it into the pathogenic category. The variant found in our case is compatible with the patient phenotype. WES is a powerful tool to confirm clinical, biochemical, and radiological diagnoses in cases of rare genetic disorders, like FHHNC.

Heterozygous variant in HOMER2 is associated with autosomal dominant nonsyndromic hearing loss (ADNSHL), designated as the locus of DFNA68. Only five pathogenic variants in HOMER2 have been identified to date. The aim of this study is to investigate the molecular etiology of ADNSHL in a four-generation Chinese family. Whole exome sequencing analysis was conducted to detect the disease-causing variant. Co-segregation analysis was performed using Sanger sequencing. The family exhibited autosomal dominant, progressive, post-lingual, nonsyndromic sensorineural hearing loss, similar to that observed in previously reported DFNA68 families. Unlike the initially high-frequency hearing loss observed in the previously reported families, the young proband in our study (IV:4, 18 years old) exhibited typical low-frequency hearing loss. A novel frameshift variant, c.1023_1029del (p.Asp342ArgfsTer54), in HOMER2 was identified, which co-segregated with the hearing loss phenotype in the family. The variant deletes 7 nucleotides, leading to an extended incorrect protein C terminus. Based on the American College of Medical Genetics and Genomics guidelines, the variant c.1023_1029del (p.Asp342ArgfsTer54) is classified as likely pathogenic. These findings may help expand the spectrum of pathogenic variants of the HOMER2 gene and provide a molecular interpretation for these patients with ADNSHL.

HCSeeker: A Classification Tool for Human Genetic Variant Hot and Cold Spots Designed for PM1 and Benign Criteria in the ACMG-AMP Guideline.

Common and rare genetic variants explain distinct diagnostic variance in pediatric attention deficit hyperactivity disorder.

ABSTRACTIntroductionMeckel‐Gruber syndrome (MKS, OMIM 24,900), also known as Meckel syndrome, is a rare and severe autosomal recessive disorder. The syndrome is typically characterized by a triad of occipital encephalocele, bilateral renal cystic dysplasia, and postaxial polydactyly. MKS shows significant clinical heterogeneity, which poses challenges for accurate prenatal diagnosis. Prenatal ultrasound is an important tool for detecting potential cases, but the complexity of MKS often requires additional advanced techniques such as prenatal whole‐exome sequencing (WES) to provide more accurate molecular genetic evidence.MethodsIn this study, we used whole‐exome sequencing (WES) to analyze the genetic causes of suspected MKS in a Chinese fetus. Sanger sequencing was used to confirm the origin of the variants. The classification of variants was carried out in accordance with the guidelines of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP).ResultsA 26‐year‐old pregnant woman was referred to our antenatal centre for genetic diagnosis at 13 + 5 weeks of gestation due to fetal occipital encephalocele and renal cysts detected by ultrasound. Two novel heterozygous variants, c.1047delA (p.Val351fs*1) and c.1336C>T (p.Arg446*), were identified in TCTN2. Sanger sequencing revealed that the c.1047delA (p.Val351fs*1) variant was inherited from the mother and the c.1336C>T (p.Arg446*) variant was inherited from the father. According to the ACMG/AMP guidelines, these two variants were evaluated as pathogenic.ConclusionsThis study further expands the genetic mutation spectrum of TCTN2 and is conducive to further clarifying the relationship between the genotype and phenotype of MKS8. Severe variants in the TCTN2 gene appear to be more likely to lead to MKS8. Clinically, the triad is an important basis for the diagnosis of MKS8, while other variable phenotypes of MKS8 can provide additional information for prenatal diagnosis. The combination of prenatal ultrasound and WES can provide a more comprehensive and accurate diagnosis of MKS8, which will greatly aid support for early intervention and treatment.

How do the genetic complexities of neurofibromatosis type 1 (NF1) impact reproductive counseling, preimplantation genetic testing (PGT) design, and PGT treatment? We established association between both incidence and tissue mosaicism with multiple exon deletions and specific single-nucleotide variants (SNVs) in neurofibromin 1 (NF1), a clinical actionable finding that we structured as a flowchart outlining challenges in and an approach for reproductive counseling, PGT design, and PGT treatment for NF1. NF1 has a prevalence of 1 in 2500-3000 and is one of the most frequently requested autosomal dominant indications for PGT. NF1 is a large gene with a high mutation rate, resulting in a 50% de novo occurrence, many different reported variants scattered across the gene and relatively frequent mosaicism. We conducted a retrospective, observational cohort study on PGT molecular design for NF1 in three large PGT centers (n = 281 couples), starting from the first assay for NF1 developed in 2004 until 2022. A PGT assay was developed for 281 couples with 218 different variants in NF1. Newly described variants (n = 76) were scored using the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) classification system and submitted prior to publication in the Leiden Open Variation Database (LOVD). The employed techniques were PCR-based PGT using short tandem repeat markers (n = 230), SNP-array-based PGT (n = 39), and next-generation sequencing (NGS)-based PGT (n = 12). Minisequencing (SNAPshot) or double amplification refractory mutation system (D-ARMS) was used to incorporate SNVs. Small deletions and insertions were incorporated using fragment length analysis. All PGT assays were designed and validated according to local protocols and ESHRE guidelines. Mosaicism was present in 8% of the sporadic cases (n = 13/168), of which about half were unknown prior to PGT (n = 6/13). Mosaicism was significantly higher in patients with multiple exon deletions (n = 4/6) as compared to patients with SNVs (n = 9/162) (P < 0.001, Fisher's exact test). Additionally, two recurrent SNVs were significantly associated with mosaicism (P <0.0167, Fisher's exact test). Importantly, three unrelated families with different NF1 variants in close relatives were identified. Due to its retrospective design, not all details on the genetic test results and clinical phenotype could be retrieved for some cases (n = 6). The extent to which our findings are applicable to centers worldwide depends on their local procedures and legislation. Our findings substantially impact reproductive counseling for couples with NF1, enabling informed reproductive decision-making. For couples affected with NF1 proceeding with PGT, our findings alert colleagues worldwide on NF1-specific pitfalls in PGT molecular design and treatment. There was no funding involved in the research for this publication. M.Z.E. is an inventor on two patent applications: ZL910050-PCT/EP2011/060211-WO/2011/157846 'Methods for haplotyping single cells' and ZL913096-PCT/EP2014/068315-WO/2015/028576 'Haplotyping and copy-number typing using polymorphic variant allelic frequencies'. The other authors have no competing interests to disclose. N/A.

Clinical laboratories are increasingly implementing pharmacogenomic (PGx) testing. Although PGx is similar to genetic testing for other indications, there are unique aspects that laboratories should consider. To aid clinical laboratories that are implementing clinical PGx testing by describing characteristics of PGx test design and validation, as well as approaches to reporting. Resources that are useful for clinical laboratories performing PGx testing will be highlighted. The College of American Pathologists formed a workgroup composed of laboratorians with expertise in clinical PGx testing. The workgroup included representatives from the Association for Molecular Pathology and the American College of Medical Genetics and Genomics. The workgroup reviewed pertinent literature, as well as experience from proficiency testing and from members' laboratories. The workgroup recommends that laboratories implementing PGx consider the following concepts: testing platform, test design (ie, selection of pharmacogenes and variants/alleles), use of reference materials during test development and as controls during clinical runs, star allele and standard nomenclature systems, translations from genotype to predicted phenotype, and considerations for result reporting including making medication recommendations. The workgroup provides considerations when using report vendors, emphasizing the clinical laboratory's role and responsibility when implementing such reporting tools from vendors. Clinical laboratories should be familiar with the fundamentals of PGx, ensure that PGx testing meets the applicable regulatory requirements for all aspects of the clinical laboratory testing process, and follow recommendations for standardization of nomenclature and reporting.

Referral to genetics specialists plays a pivotal role in the diagnostic journey of pediatric patients with conditions such as autism spectrum disorder (ASD), developmental delay, intellectual disability, and hearing loss. Although there are referral guidelines in place from the American College of Medical Genetics and Genomics (ACMG), not all eligible children receive referrals. Among individuals with ASD, developmental delay, or intellectual disability, only about 20-50% of eligible patients are referred for genetic testing. Additionally, further barriers hinder those who are referred from accessing care. Our study delves into these often-overlooked barriers impeding pediatric patients' access to genetics services. We invited the parents of young children with one of the aforementioned conditions, identified through early intervention and related programs, including family-professional organizations, in South Carolina and Florida, to complete an online survey about referral to genetic services and barriers to attending a genetics consultation. Insurance coverage for genetic testing was a barrier both for referral to genetics and attendance at a genetics consultation. The child's race was a predictor of attendance at a genetics consultation after referral. Our study highlights the critical need to address barriers, such as insurance coverage and racial disparities, that prevent pediatric patients from accessing genetics services. We investigated barriers to genetic services for pediatric patients who were not referred or who were referred but never attended, study populations that are under-represented in the literature. This study sheds light on key factors influencing referral to and attendance at genetics consultations for pediatric patients, including race and health insurance coverage for genetic testing. Our findings emphasize the need for genetics education and improved accessibility to genetics services.

Brugada Syndrome (BrS) is an inherited arrhythmia disorder that causes an elevated risk of sudden cardiac death. Approximately 20% of patients with BrS have rare variants in SCN5A, which encodes the cardiac sodium channel NaV1.5. Genetic workup of BrS is often complicated by SCN5A variants of uncertain significance (VUS) and/or incomplete penetrance. This study deployed an SCN5A-BrS functional assay at cohort scale to facilitate the implementation of genetic and precision medicine. All 252 missense and in-frame insertion/deletion SCN5A variants from a previously published large cohort of BrS cases (n = 3335 patients) were analysed using a calibrated high-throughput automated patch-clamp (APC) assay. Variant functional Z-scores were assigned evidence levels ranging from BS3_moderate (normal function) to PS3_strong (loss-of-function), as defined by American College of Medical Genetics and Genomics criteria. Functional evidence was combined with population frequency, hotspot, case counts, protein-length changes, and in silico predictions. Odds ratios of BrS case-control enrichment and penetrance for BrS were calculated from variant frequencies in the BrS cohort and in gnomAD. Most variants (146/252) were functionally abnormal (Z ≤ -2), with 100 having severe loss-of-function (Z ≤ -4). Functional evidence enabled the reclassification of 110 of 225 VUS; 104 to likely pathogenic and 6 to likely benign. SCN5A variants with loss-of-function were mainly localized to the transmembrane domains, especially the regions comprising the central pore. SCN5A variant penetrance was proportional to the severity of loss-of-function; variants with Z ≤ -6 had penetrance of 24.5% (15.9%-37.7% CI) and an odds ratio of 501 for BrS. This cohort-scale APC dataset stratifies SCN5A variants found in BrS patients into normal function 'bystander' variants that have a low risk of BrS and loss-of-function variants that have a high risk for BrS. Functional data can be integrated with other criteria to reclassify a substantial fraction of VUS. The dataset helps clarify the SCN5A-BrS relationship and will improve the diagnosis and clinical management of BrS probands and their families.

GBA1 variants cause Gaucher's disease (GD) in biallelic forms and increase Parkinson's disease (PD) risk in heterozygous carriers. Carriers of mild or severe variants (causing GD type 1 or types 2-3) can enroll in clinical trials, whereas those with GBA1 variants classified as unknown are typically excluded. We assessed the contribution of unknown variants to PD risk and their relevance for trial stratification. We meta-analyzed 34 case-control studies (24,060 PD cases, 14,465 controls). Odds ratios (ORs) were estimated using random-effects models and stratified by the American College of Medical Genetics and Genomics (ACMG) criteria. Unknown variants also classified as variants of uncertain significance (VUSs) per ACMG criteria were associated with PD (OR = 1.59, 95% confidence interval [CI]: 1.25-2.02; I2 = 0%). VUSs + likely pathogenic + pathogenic also showed an association (OR = 1.63, 95% CI: 1.28-2.06; I2 = 0%). Unknown GBA1 variants may be considered provisionally in clinical trials if also classified as VUS, likely pathogenic, or pathogenic per ACMG criteria. © 2025 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Background:Expanded newborn genetic screening can identify a wide range of inherited conditions early in life. However, the prevalence and distribution of pathogenic and likely pathogenic variants in large-scale cohorts remain underexplored in the Chinese population.Methods:We screened 33,894 newborns using targeted sequencing of 465 genes associated with monogenic disorders. Variants were classified with locally optimized American College of Medical Genetics and Genomics guidelines, focusing on pathogenic and likely pathogenic variants. We analyzed cumulative carrier rates, disease-specific prevalence, and regional differences.Results:Here we show that among 33,894 newborns, 16,687 (49.2%) carry at least one pathogenic/likely pathogenic variant. In total, 22,457 such alleles are detected across 427 (91.8%) genes. Detection rates are higher in the south (52.1%) than in the north (48.5%). On average, each newborn carries 0.7 variants. The most frequent genes (allele frequency >1%) include GJB2 (5.56%), PAH (1.41%), and SLC26A4 (1.30%). The cumulative predicted incidence of 57 inborn errors of metabolism included in newborn tandem mass spectrometry screening is 1/2,177, with PAH, MMACHC, SLC22A5, MMUT and SLC25A13 as the main contributors. Regional analysis reveales significant geographic variation, with three inborn errors of metabolism (e.g., methylmalonic aciduria, phenylketonuria) more common in the north, five disorders (e.g. G6PD deficiency, thalassemia) more common in the south.Conclusions:This large-scale study highlights the utility of targeted genetic screening for identifying carrier status and early-onset disease risks in newborns. The findings provide a critical foundation for integrating genetic screening into routine newborn care and for optimizing public health strategies.

Background: Channelopathies are among the major causes of sudden cardiac death (SCD). Mutations in ion channel genes have been associated with life-threatening arrhythmias. This study aimed to characterize genetic spectrum of Kazakhstani patients with channelopathies using targeted next-generation sequencing (NGS), which may facilitate personalized therapeutic strategies. Material and methods: Genomic DNA was extracted from whole blood samples of patients with clinically diagnosed channelopathies. Targeted NGS was performed on the NovaSeq 6000 platform (Illumina) using the TruSight Cardio panel, which covers 174 genes associated with inherited cardiovascular disorders. Bioinformatic analysis was performed using standard pipelines, and variants were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines. International databases (ClinVar, MutationTaster, gnomAD, Polyphen-2) were additionally used for variant interpretation. Results: Genetic screening revealed 23 rare variants. We found five disease-causing pathogenic (P) and likely pathogenic (LP) variants in our study. Namely, disease-causing mutations are detected in SCN5A, KCNQ2, CACNB2, KCNQ1. In addition, 11 variants of unknown significance (VUS) with suggestive evidence of pathogenicity were identified in patients. Moreover, several variants were located in genes previously implicated in arrhythmia susceptibility, warranting further investigation. The results of genetic screening were reported to clinicians to support further clinical decision-making and patient management. Conclusion: Our study demonstrated genetic profiles of patients with channelopathies by using the TruSight Cardio panel. Genetic screening revealed clinically significant variants within ion channel genes. Acknowledgement: The study was funded by grants from the Committee of the Ministry of Science and Higher Education, Republic of Kazakhstan (AP23490249, BR24993023) and Nazarbayev University CRP - 211123CRP1608. Keywords: channelopathies, mutations, targeted sequencing