The Journal of Molecular Diagnostics

PurposeCarrier screening for hereditary conditions is challenged by genes with complex genomic architecture, where short-read sequencing can fail to detect clinically relevant variants. This study evaluated a unified, amplification-based nanopore sequencing workflow across multiple laboratories for comprehensive analysis of such loci. MethodsA modular long-read sequencing assay was evaluated across five laboratories using targeted PCR enrichment, Oxford Nanopore sequencing, and automated variant analysis. The workflow interrogated genes associated with spinal muscular atrophy, thalassemia, cystic fibrosis, fragile X syndrome, congenital adrenal hyperplasia, Gaucher disease, and hemophilia A. Performance was assessed against orthogonal methods for single nucleotide variants (SNVs), indels, copy-number variants, repeat expansions, and structural rearrangements. ResultsAcross 882 unique samples (1,266 tests), overall agreement with comparator methods exceeded 96% for variant-level detection and 97% for genotype status classification. Long-read sequencing enabled phasing of paralogous loci, integrated sizing and interruption analysis for FMR1 repeats, and simultaneous detection of SNVs and structural variants in globin loci and CYP21A2-TNXB region, reducing reliance on multiple workflows. ConclusionThis multisite evaluation suggests that targeted long-read sequencing can consolidate complex variant detection into a single workflow, improving analytical completeness and operational efficiency for carrier screening.

ContextPancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy often diagnosed at advanced stages due to the lack of early clinical symptoms. DNA methylation alterations arise early in PDAC tumorigenesis and may serve as promising biomarkers for blood-based cancer detection. ObjectiveTo evaluate the performance of EPISEEK, a laboratory-developed blood-based multi-cancer early detection (MCED) assay, for detecting PDAC across disease stages. DesignA retrospective cohort study included 97 patients with stage I-IV PDAC and 201 asymptomatic healthy controls. Sensitivity, specificity, area under the curve (AUC), and stage-specific performance were assessed. EPISEEK-MCED performance was also compared with CA 19-9 alone and in combination with CA 19-9. ResultsEPISEEK-MCED classified 65 of 97 PDAC cases as positive, corresponding to an observed sensitivity of 70.1% (95% CI, 60.3% - 78.3%) at 99.5% specificity. The assay demonstrated strong discrimination between PDAC cases and healthy controls, with an AUC of 0.916 (95% CI, 0.88 - 0.952). Sensitivity increased with advancing stage while remaining substantial in early-stage disease, measuring 53.6% for stage I and 65.1% for stage II PDAC, 100% for stage III and 94.7% for stage IV. Across stages, EPISEEK-MCED outperformed CA 19-9 alone, particularly in early-stage disease. Combined analysis of EPISEEK-MCED and CA 19-9 further improved detection performance, achieving sensitivity of 57.1% and 81.4% for stage I and II, respectively. ConclusionsEPISEEK-MCED demonstrated high specificity and sensitivity for PDAC detection across disease stages, including early-stage disease. Combining EPISEEK-MCED with CA19-9 further improved performance, supporting its clinical utility for PDAC detection.

BackgroundFacioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic dysregulation at the chromosome 4q35 D4Z4 repeat array under specific permissive genetic conditions. Due to the complexity, expense, and general inaccessibility of FSHD genetic testing, many individuals displaying characteristic muscle weakness are never genetically confirmed and at-risk relatives cannot get screened. We previously developed a targeted bisulfite sequencing (BSS) protocol using the Sanger method to determine DNA methylation levels at specific D4Z4 loci relevant to distinguishing forms of FSHD from non-FSHD that can be used with DNA isolated from saliva, thereby reducing cost and increasing accessibility compared to traditional D4Z4 deletion testing that uses DNA isolated from blood. MethodsHere, we adapt the D4Z4 BSS protocol to next-generation sequencing (NGS) to increase sequencing depth and further reduce cost, validate both sequencing technologies against several cohorts of genetically defined samples, and introduce the D4Z4caster software for computing DNA methylation signatures with diagnostic utility from raw sequencing data. ResultsBoth Sanger and NGS BSS methods using D4Z4caster were validated as providing high sensitivity and specificity, with geometric mean of sensitivity and specificity (G-mean) >95% and area-under-the ROC curve (AUC) of 0.99. The NGS method allows for higher throughput and increased read depth, while the Sanger method allows faster processing of individual samples. Importantly, the NGS method could identify FSHD1 cases that are likely mosaic and would otherwise be missed. ConclusionsD4Z4caster methylation signatures can accurately detect contracted FSHD1-permissive chromosome 4q35 alleles, hypomethylation of D4Z4 arrays indicative of FSHD2, and SNPs that are important for diagnostic use. This workflow is amenable to transitioning to clinical settings for an accurate, low-cost FSHD molecular diagnostic test that could be accessible worldwide. What is already known on this topicCurrently accepted genetic diagnostics for FSHD1 are complex and expensive and can mischaracterize certain complex genetic cases. These diagnostics all require high molecular weight genomic DNA typically freshly isolated from blood, highly specialized equipment, and additional testing for FSHD2, making FSHD diagnostics the most expensive among neuromuscular diseases and inaccessible to much of the world. However, the epigenetic status of the 4q35 and 10q26 D4Z4 repeat arrays, as determined by DNA methylation status using our bisulfite sequencing-based protocol, distinguishes genetically FSHD1, FSHD2, and non-FSHD samples. Additionally, since our protocol is PCR-based, it can utilize DNA isolated from multiple sources, including saliva and buccal swabs. What this study addsThis study validates the relevant DNA methylation signatures against several large cohorts of genetically-confirmed FSHD and non-FSHD samples and optimizes the DNA methylation data analysis for the greater accuracy required for diagnostic utility, including the exclusion of nonpathogenic chromosome 10q or 4A166 contractions. In addition, we introduce the D4Z4caster analysis software, which runs in a portable and scalable Docker container, and provides increased quantitative accuracy important for: 1) confirming likely clinical cases of FSHD that do not meet the currently accepted genetic definition of FSHD1 or FSHD2, 2) identifying FSHD1 somatic mosaicism, and 3) potential prognostic applications. How this study might affect research, practice or policyFSHD1 is genetically defined by a D4Z4 array at the 4q35 locus that is contracted to 1-10 repeat units. However, disease penetrance is influenced by repeat number, epigenetic modifications, and genetic background, causing a misalignment of current genetic diagnosis with clinical diagnosis. This study will improve the accuracy of epigenetic analysis for determining cases of genetic FSHD, help broaden the definition of genetic FSHD to more accurately correspond to clinical FSHD, and allow identification of those at risk for developing clinical FSHD in affected families and in large population studies now being performed and proposed. In addition, it will better inform how an individuals epigenetic status is interpreted for potential prognostic value. Overall, this methodology is: 1) significantly less expensive than current clinically-approved FSHD diagnostic technologies, 2) more accessible due to compatibility with DNA isolated from multiple sources including saliva, and 3) compatible with the current sequencing equipment and workflow for DNA isolation used in commercial clinical laboratories. Together, these advantages will help move the technology toward becoming an approved molecular diagnostic test for FSHD in the USA, Europe, and countries currently lacking clear access to testing.

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.

BackgroundInduced pluripotent stem cells (iPSCs) are an important model for studying human diseases in vitro. However, previous studies have shown that iPSC reprogramming and extended cell culture can introduce genomic structural variants (SVs). Technologies like karyotyping, CNV microarrays, and whole-genome sequencing have limitations in resolution, sensitivity, or the ability to detect large and complex structural variants compared to optical genome mapping (OGM). OGM is a genome-wide structural variant detection method that analyzes fluorescently labeled ultra-high-molecular-weight DNA molecules to identify copy-number and balanced rearrangements. At sufficient coverage, OGM can detect SVs at approximately [≥]2 kbp and identify mosaic events supported by molecule-level evidence, offering higher resolution than conventional karyotyping or SNP-array-based QC. Here, we compared iPSC clones derived from peripheral blood mononuclear cells (PBMCs) and fibroblasts (FBCs) to determine whether starting somatic cell source is associated with differences in structural variant burden and SV-type profiles after nuclear reprogramming into iPSCs. ResultsWe analyzed 73 low-passage iPSC clones generated from 25 parental lines using OGM. Compared with PBMC-iPSCs, FBC-iPSCs showed higher SV burden with the enrichment of duplications [≥]100 kbp, more frequent overlap with protein-coding genes, fragile sites, and recurrent chromosomal hotspot regions. In contrast, PBMC-iPSCs showed fewer SVs overall, and a higher proportion of clones without detectable clone-specific SVs. ConclusionsOGM provides a high-resolution approach for post-reprogramming genomic quality control by detecting clone-specific structural variants at approximately [≥]2 kbp, including events below the resolution of conventional cytogenetic and SNP-array-based assays. In these early passage iPSCs, SVs overlapped protein-coding genes, fragile sites, and recurrent culture-associated chromosomal regions, underscoring the need for clone-level genomic assessment before downstream applications. FBC-derived iPSCs showed a higher SV burden, including more frequent and larger duplications, whereas PBMC-derived iPSCs more often lacked detectable clone-specific SVs. These findings suggest that PBMC-iPSCs and FBC-iPSCs can differ in post-reprogramming SV profiles and support the use of OGM as a QC strategy during iPSC generation and selection.

Molecular syndromic panels such as the BioFire FilmArray Gastrointestinal Panel (BF-GIP) have been widely adopted for gastrointestinal illness diagnosis due to their fast turnaround times and broad pathogen coverage. Recently, the BF-GIP demonstrated increased rates of norovirus false-positive detections, prompting a Class II recall of more than two million tests in February 2024. We examined the prevalence of BF-GIP norovirus false positives across four hospitals from December 2024 to June 2025. Among 185 BF-GIP norovirus-positive results confirmed with the BD MAX Enteric Viral Panel, the false discovery rate ranged from 31 to 74% across sites, with the highest rate seen at a specialized cancer care hospital. Deep sequencing of BF-GIP pouches (n=42) confirmed the Noro-1 assay as the primary source of off-target amplification, identifying 78 off-target species, predominantly commensal stool bacteria, compared to only two species for the Noro-2 assay. Off-target species amplified by the Noro-1 assay were recovered from both false-positive and true-negative pouches, suggesting no single species accounted for the false-positive results. Partial primer complementarity at off-target loci and amplicon Tm values within the acceptable range support mispriming of gut microbiota as the underlying cause. False-positive pouches exhibited significantly higher Cp values than true positives for both assays (Noro-1: 26.6 vs. 11.1, p=0.013; Noro-2: 30.0 vs. 13.1, p<0.001), consistent with low-level off-target amplification. These findings highlight the high false discovery rate of the Noro-1 assay, identify bacterial species involved in mispriming, and demonstrate the need to redesign this assay to ensure reliable testing and improved patient care.

Numbers of research articles studying circRNAs have increased rapidly since 2017. Previous analyses of human circRNA articles in two high impact factor cancer research journals identified papers with wrongly identified nucleotide sequence reagents and circRNAs whose identities could not be independently verified. In the present study, verification of human nucleotide sequence reagent and cell line identities in retracted circRNA articles published from 2017-2021 in high impact factor journals found wrongly identified nucleotide sequences and/or cell lines in all 13 retracted papers. Similar analyses of human circRNA papers published in high impact factor journals in 2022 found wrongly identified, non-verifiable and/or questionable reagents in 71% (84/118) papers, where 51% (60/118) papers described at least one wrongly identified reagent. When individual error types and features of concern were considered, 2022 circRNA papers described wrongly identified nucleotide sequence reagents (52/118, 44%), questionable circRNA probes that did not meet accepted targeting requirements (34/118, 29%), non-verifiable nucleotide sequences (25/118, 21%), wrongly identified cell lines (22/118, 19%), and/or non-verifiable cell line identifiers (6/118, 5%). In summary, wrongly identified, non-verifiable and/or questionable reagents were unexpectedly frequent in human circRNA papers in high impact journals, highlighting the need for critical engagement with the circRNA literature.

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

Background Thalassemia is one of the most common monogenic disorders worldwide, current screening strategies combining hematological testing with molecular assays still carry a risk of missed diagnoses and undesirable efficiency, particularly for complex structural variants and rare mutations. Methods In this prospective double-blind, multicenter cohort study of 3,842 participants (3,362 pregnant women and 480 male partners), we conducted a head-to-head comparison to systematically evaluate the incremental clinical value and detection performance of single-molecule nanopore sequencing in thalassemia (SMITH) against conventional hematological testing and next-generation sequencing (NGS). Findings The overall concordance rate between NGS and SMITH was 98.6% (3789/3842). The discrepant cases (n=53) were directly attributed to the superior detection capabilities of SMITH, which successfully identified complex structural rearrangements-including 45 -globin gene triplications and four HK alleles-that were missed by NGS. Furthermore, SMITH accurately detected four rare variants (c.134_135insT/, c.-22(C>T)/, {beta}N/{beta}c.316-290delinsAGGGCAATAATTT and {beta}3.5 kb deletion/{beta}N ) and resolved ten trans and three cis configurations within the globin gene allele. Clinically, these technical advantages translated to a 9.3% (5/54) increase in the detection rate of high-risk prenatal couples, effectively preventing one birth affected by moderate-to-severe thalassemia. Additionally, SMITH corrected a diagnostic discrepancy in one case (HK vs. -3.7), sparing the couple from an unnecessary invasive procedure. Interpretation Our findings demonstrate that SMITH provides a powerful platform for resolving globin gene rearrangements, detecting rare variants, and enabling direct haplotype phasing. By effectively eliminating diagnostic blind spots, SMITH is expected to become an optimal method for thalassemia prevention programs. Funding This study was supported by Chinese National Natural Science Foundation Projects 81760037 and 82271894.

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: 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: Genome-wide association studies (GWAS) have identified numerous lung cancer susceptibility loci based on single nucleotide polymorphisms (SNPs), yet a substantial proportion of heritability remains unexplained. We therefore evaluated germline copy number variants (CNVs) as an underexplored source of genetic susceptibility and potential contributors to genomic instability in lung cancer. Methods: We conducted a genome-wide analysis of germline CNVs using 19,342 cases and 15,917 controls from the Transdisciplinary Research in Cancer of the Lung (TRICL) consortium, with replication in two independent cohorts. High-confidence CNVs were identified by integrating two CNV callers including PennCNV and modSaRa2. Association analyses were performed using both gene-based and CNV region-based approaches. Polygenic risk scores (PRS) were constructed from top loci, and functional validation was conducted using siRNA-mediated knockdown in lung fibroblast cells. Results: We identified CNVs in four genomic regions (1p36.22, 2q31.2, 6p21.32, and 19q13.32) significantly associated with lung cancer risk. Two loci (1p36.22 and 2q31.2) were consistently supported across both analytical strategies. A CNV-based PRS constructed from key genes (CLCN6, NFE2L2, OPA3, and PSMB8) was significantly associated with lung cancer risk and replicated across independent datasets. Functional assays demonstrated that knockdown of NFE2L2 and OPA3 increased endogenous DNA damage, supporting a role in genomic stability. Conclusions: Germline CNVs contribute to lung cancer susceptibility and may influence carcinogenesis through mechanisms related to genomic instability. Impact: These findings expand the genetic architecture of lung cancer and highlight CNVs as potential biomarkers for improving risk stratification and informing precision prevention strategies.

Background: Modern therapy for childhood and adolescent leukemia requires accurate risk classification of genomic subtype. Although short-read next-generation sequencing (NGS)- based approaches provide comprehensive clinical diagnostics in limited, highly resourced settings, they remain expensive, slow, and inaccessible to most children worldwide. Transformative approaches are needed to improve diagnostic classification for leukemia globally. Methods: We simultaneously continued to develop an analytical pipeline NASVar (Nanopore variant calling for adaptive sampling), and conducted a multicenter, type-two hybrid clinical validation study of an Oxford Nanopore Technologies (ONT) adaptive-sampling whole-genome sequencing (asWGS) assay across hospitals with varying diagnostic resources. In preparation for implementation, a global panel developed a leukemia-based standardized gene set and consensus laboratory-developed test (LDT) validation guidelines. Measures of assay effectiveness compared to both conventional and orthogonal NGS methods, where available, were simultaneously collected with data to measure the implementation outcomes of feasibility, fidelity, appropriateness, and cost. Results: All four centers successfully completed the LDT validation, with minimal adaptations required for regulatory compliance. A total of 457 specimens were sequenced (331 B-ALL, 83 AML, 43 T-ALL). For the 210 B-ALL cases with locally resolved genomic subtypes defined by DNA alterations, asWGS was 100% concordant (210/210). Cases locally defined as B-other were resolved via asWGS with disease-defining DNA alterations in 47% (49/105) of cases. An additional 41% (43/105) of locally defined B-other cases were classified by incorporation of DNA methylation, and all 16 B-ALL patient-derived xenograft controls were correct, for a total of 96% (318/331) of all B-ALL cases in the cohort resolved with single assay asWGS. For AML, 97% (56/58) of cases with locally resolved genomic subtypes were identified by automated asWGS analysis, while an additional two cases were identified after targeted manual review. At Indus Hospital in Pakistan, the B-ALL and AML diagnostic genomic subtype yield increased from 28% with local standard of care diagnostic testing, to 84% with asWGS. The cost of reagents and consumables in the United States, assuming pooled three-plexing, was $343/sample. Based on the combined hybrid validation results, all centers are independently preparing for clinical return of results. Conclusions: ONT asWGS was successfully validated as a clinical assay in four diverse hospital settings. As a single, multi-omic platform that delivers value across the continuum of high-resource to resource-limited contexts, the approach offers a disruptive solution to address the global equity gap in cancer diagnostics.

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.

Background: Population-relevant BRCA1/BRCA2 data from Bangladesh are scarce, creating challenges for hereditary breast and ovarian cancer variant interpretation, counseling, and follow-up testing. We examined a clinically referred Bangladeshi cohort to characterize assay-derived BRCA1/BRCA2 short variants, sequencing-depth performance, and copy-number findings in a conservative pilot framework. Methods: Twenty-three de-identified blood-derived DNA samples were assessed using a targeted BRCA1/BRCA2 next-generation sequencing workflow. Downstream analysis used assay-generated short-variant, coverage, and CNV outputs, with coordinates reported on hg19/GRCh37. Short variants were evaluated from high-confidence PASS/VCC-H calls, and CNV review incorporated both target-region and amplicon-level copy-number patterns. Results: After removal of four low-VAF review observations, the primary germline-compatible dataset comprised 304 short-variant observations representing 34 unique variants. Both BRCA1 and BRCA2 contributed comparable variant burdens, while the overall profile was mainly composed of missense and synonymous changes. Six sample-specific heterozygous BRCA1 truncating candidates were observed, including five frameshift variants and one stop-gain variant. Protein-level mapping placed these events across the central-to-C-terminal portion of BRCA1. Sequencing depth was consistently high across the targeted regions, with all 4,255 amplicon-sample measurements exceeding 280x and 99.91% reaching at least 500x. Copy-number analysis highlighted one candidate BRCA1 multi-exon deletion-like event involving exons 15-20 in BCSIR-BRCA-21, with unresolved partial exon 14 involvement. Conclusions: This study provides an initial Bangladesh-focused targeted BRCA1/BRCA2 dataset and identifies candidate short-variant and CNV findings for validation. These findings should be interpreted as analytical candidates only and require confirmatory testing and expert clinical curation before any clinical application. The cohort is referral-enriched and should not be used to infer population prevalence.

Rapid, portable detection of multiple nucleic acid targets is essential for infectious disease surveillance and precision oncology. CRISPR-based diagnostics have set a high bar for sensitivity and single-base specificity, yet their reliance on collateral nuclease activity complicates multiplexing, integration with amplification, and point-of-care deployment. Here we present TIMBER (Templated Incision Mediated By Endonucleases of Repair), a non-CRISPR, isothermal platform that achieves comparable performance without nonspecific nuclease activity. TIMBER uses a repair endonuclease to cleave probes containing abasic sites only when they are hybridized to a matched target. The system exhibits strong specificity, enabling single-nucleotide polymorphism discrimination. TIMBER provides an analytical limit of detection 12 pM without preamplification or 1 copy per {micro}L (1.6 aM) with preamplification through RT-PCR or RT-LAMP, with results observable by visual fluorescence or on lateral flow. We deploy TIMBER to detect SARS-CoV-2 in clinical saliva samples and further demonstrate multiplex detection of four targets enabling identification of EGFR mutations in lung cancer samples. This approach offers a flexible, rapid, and low-instrument ready solution for diverse nucleic acid diagnostics, from viral detection to cancer genotyping.

BackgroundSickle cell disease (SCD) is the most common recessive genetic disorder, caused by pathogenic variants of the HBB gene. SCD is associated with a range of clinical manifestations, including vaso-occlusive crises, infections, and severe anaemia, which contribute to increased morbidity and mortality. The frequency of pathogenic alleles is high in Sub-Saharan African countries, with heterozygous carriers reaching up to 25% of the population. Several methods can be employed for molecular diagnostics, with HBB gene sequencing being the most precise. However, access to DNA analyses and sequencing in Low- and Middle-Income Countries (LMICs), where SCD prevalence is high, is limited. Understanding genetic profiles is crucial at both individual and population levels, as it can guide public health strategies and facilitate accurate genetic counselling. AimThis feasibility study aimed to demonstrate that a portable medical genetic laboratory (in suitcases) can be used to genotype individuals for the HBB A, S, and C alleles and their combinations within a few hours outside of a laboratory setting. Methods and resultsWe established a portable medical genetics laboratory capable of DNA extraction and isothermal DNA amplification using a commercially available kit for the A, S, and C alleles of the HBB gene. During one single study day, this portable lab was set up in a room where the Swiss Association of Patients with SCD was holding its annual meeting. We analysed the samples of 27 participants who were aware of their A, S, or C status. We collected buccal swabs and dried blood samples for genotyping. Genotype results for all participants were obtained within five hours after sample collection. In four cases, we observed discrepancies between the buccal swab and blood genotypes; three were resolved upon repeat testing, and one reflected donor chimerism following hematopoietic stem-cell transplantation. ConclusionsThis study demonstrates the feasibility and efficiency of using a portable medical genetics laboratory for rapid genotyping of HBB SCD alleles in community settings.This approach can improve access to molecular diagnostics in resource-limited environments. Such tools have the potential to significantly enhance local capabilities for genetic screening, counselling, and public health planning in regions heavily affected by SCD.

Flow cytometry can establish T cell clonality by detecting a restricted expression pattern of the T cell receptor (TCR) {beta} constant region (TRBC), expressed in association with CD3. However, T cell neoplasms frequently lose surface expression of the CD3/TCR complex, posing a challenge to demonstrating T cell lineage and clonality. To address this challenge, here we present a 12-color flow cytometry panel, called cytoTCR, to characterize cytoplasmic expression of CD3/TCR complex components. We apply cytoTCR to 38 patient specimens with immunophenotypically abnormal T cell populations, demonstrating this approach can efficiently establish T cell lineage and clonality in challenging T cell neoplasms that have lost surface CD3 expression. While we show that natural killer (NK)-lineage neoplasms can express cytoplasmic CD3 at similar levels to T cells, we show that absent expression of cytoplasmic TCR components by mature lymphocytes can help confirm NK cell lineage. We demonstrate that cytoTCR can detect cytoplasmic TRBC-restriction in challenging cases of null-phenotype anaplastic large cell lymphoma, which lack surface expression of pan-T cell antigens. In cases of T-lymphoblastic leukemia, cytoTCR shows that cytoplasmic TRBC expression matches the expected developmental stage of the leukemia. Finally, we use cytoTCR to characterize atypical cCD3-CD7- T cells in a patient with a history of T-lymphoblastic leukemia as well as recent CAR-T therapy, showing that this atypical population is polytypic and represents CAR-T product rather than residual disease. Our study presents a broadly applicable flow cytometric approach to simultaneously assess T cell lineage and clonality in suspected T lineage populations with absent surface CD3 expression.

Importance: Non-medullary thyroid cancer (NMTC) and melanoma are associated with inherited long telomeres due to germline pathogenic/likely pathogenic variants (PV/LPV) in POT1, TINF2, and ACD resulting in long-telomere syndrome (LTS) and they commonly have somatic TERT promoter mutations. The genetic relationship between these variants and their clinical associations are defined incompletely and may inform clinical practice. Objective: To test the hypothesis that germline LTS-associated PV/LPV are exclusive from functional somatic TERT variants and assess clinical/genetic associations. Design: Retrospective observational cohort study with/without germline LTS variants, that have somatic sequencing and pathology data. Setting: Participants were enrolled through 18 cancer centers participating in the Oncology Research Information Exchange Network (ORIEN). Participants: 995 adults with NMTC and 993 with melanoma between 2013 and 2025. All adult patients at an ORIEN center were offered enrollment Exposures: All patients with NMTC or melanoma are included. There are no required exposures. Main Outcomes and Measures: The presence/absence of a germline or somatic long-telomere variant; secondary outcomes are associations with tumor stage, telomerase expression, and oncogenes. Results: Germline and somatic variants in POT1/TINF2/ACD, somatic TERT promoter variants, TERT fusions, oncogenes, and telomerase mRNA expression were evaluated in 995 NMTC and 993 melanoma patients. In NMTC, 13 (1.5%) had a germline LTS variant while 0/12 with tumor sequencing had somatic TERT promoter variants/fusions. In melanoma, 7 (0.7%) had a LTS variant; 0/2 with tumor sequencing had a TERT promoter variant/ fusion. Meta-analysis including NMTC and melanoma in the current study, a recent thyroid cancer study, and thyroid TCGA, germline LTS-associated PV/LPV and somatic TERT variants/fusions were mutually exclusive (p=0.036). High telomerase mRNA levels were associated with TERT promoter variants/fusions (p<4e-11) and larger NMTC/distant metastases (p=0.016), but not germline LTS variants. NMTCs with somatic TERT promoter variants/fusions had higher tumor mutation burden (p<0.02) versus tumors from patients with a germline LTS variant. TERT promoter mutant variant allele frequency was lower in smaller and non-metastatic vs larger/metastatic NMTC. Conclusion and Relevance: Germline LTS-associated variants appear to be exclusive from somatic TERT promoter variants/fusions but are not associated with aggressive NMTC, suggesting common roles in tumorigenesis but different biological impacts.