Cytomegalovirus, Epstein-Barr virus, BK virus, and adenovirus cause significant morbidity and mortality in immunocompromised individuals, especially those undergoing solid organ and hematopoietic stem cell transplant. Qu...Cytomegalovirus, Epstein-Barr virus, BK virus, and adenovirus cause significant morbidity and mortality in immunocompromised individuals, especially those undergoing solid organ and hematopoietic stem cell transplant. Quantitative viral load testing is essential to the monitoring and treatment of disease associated with these viruses in the post-transplant period. In this review, the current guidelines for viral load monitoring are described, highlighting the differences in testing recommendations by virus and transplant type. The state of commercially available, Food and Drug Administration-cleared quantitative viral load assays are also reviewed and the ongoing challenges associated with quantitative viral load testing in the clinical laboratory are described.
In November 2023, our Canadian tertiary care facility implemented optical genome mapping (OGM) as a first-line diagnostic test for adults with newly diagnosed acute leukemias. Here, the analytical performance and clinica...In November 2023, our Canadian tertiary care facility implemented optical genome mapping (OGM) as a first-line diagnostic test for adults with newly diagnosed acute leukemias. Here, the analytical performance and clinical utility of OGM alongside karyotype, fluorescence in situ hybridization (FISH), and next-generation sequencing are reported. During test validation, OGM demonstrated robust analytical performance, reproducibility, and limits of detection, revealing 100% specificity, 96.1% sensitivity, and 98.0% accuracy. After implementation, clinical reports from the entire cytogenetic and molecular genetic workflow were prospectively compiled from the first 200 cases to evaluate concordance with parallel karyotype/FISH and added yield of OGM. A total of 640 reportable variants were detected by OGM and stratified on the basis of clinical significance, classified as tier 1A (25%), tier 1B (3%), tier 2 (2%), or tier 3A (70%). Of these, 64 variants were missed by karyotype and FISH, 3 KMT2A partial tandem duplications were missed by next-generation sequencing, and 9 cases with failed karyotype were rescued by OGM: overall impacting 35 cases (18%) by altering diagnostic classification (n = 12) and/or risk stratification (n = 31). Despite comprehensive pre-existing diagnostic workflows, implementation of OGM has revealed diagnostically and prognostically significant alterations in cases otherwise cryptic or failed by karyotype, demonstrating strong clinical utility and supporting its use as a first-tier diagnostic test for hematolymphoid malignancies.
Barberán-Martínez P, Balanzá M, García-Bohórquez B
… +7 more, Escobar-Parra S, García-Gil R, Feliciano-Sánchez A, Jaijo T, Aller E, García-García G, Millán JM
Inherited retinal dystrophies (IRDs) represent a diverse group of rare pathologies affecting vision, with significant genetic and clinical variability. Clinical exome sequencing was performed on 143 families clinically d...Inherited retinal dystrophies (IRDs) represent a diverse group of rare pathologies affecting vision, with significant genetic and clinical variability. Clinical exome sequencing was performed on 143 families clinically diagnosed with IRDs. The obtained variants were filtered and classified according to the American College of Medical Genetics and Genomics guidelines. Overall, a genetic diagnosis was achieved for 68.53% of the families in the cohort; 35 causative genes were identified, predominantly ABCA4 and USH2A. A total of 170 clinically relevant variants were identified, 45 (26.47%) of which were novel, with missense variants being the most common type (40.59%). This study reported aberrant splicing generated by the ABCA4 (NM_000350.2): c.1299A>G mutation through the functional assay of a minigene. Furthermore, the genes FAM161A and GUCY2D were associated with IRDs that are not typically linked to these genes. Consequently, this study expands the current understanding of IRDs and supports the use of clinical exome sequencing as an effective strategy for the genetic diagnosis of these pathologies.
Pathogenic POLE mutations (pPOLE) undermine mismatch error correction by polymerase ε during DNA replication, and the resulting somatic ultramutation predicts response to immunotherapy. Beyond frequently recurrent allele...Pathogenic POLE mutations (pPOLE) undermine mismatch error correction by polymerase ε during DNA replication, and the resulting somatic ultramutation predicts response to immunotherapy. Beyond frequently recurrent alleles, historical pPOLE classification has been largely based on exonuclease domain localization. A POLE-specific phenotypic classification model was developed, encompassing tumor mutational burden (TMB), mutational signatures, germline frequency, and consideration of comutation with other POLE mutations to identify pPOLE. This model was applied to >490,000 samples and identified 29 predicted pPOLE, including 16 not previously reported. A total of 748 tumors (0.2%) had one or more pPOLE, most commonly in endometrial and colorectal cancers, although pPOLE were observed in many additional cancer types. pPOLE were associated with ultramutation [median TMB, 186.3 mutations per megabase (mut/Mb)] across tumor types. Concurrent pPOLE and microsatellite instability were more common than previously appreciated and produced a synergistic TMB impact, with medians of 135.7 mut/Mb for pPOLE/microsatellite stable samples compared with 325.6 mut/Mb for pPOLE/microsatellite instability-high samples. Comutation analysis in endometrial and colorectal cancers highlighted associations with homologous recombination pathway gene mutations that were predominantly monoallelic passengers that are unlikely to predict response to therapies targeting DNA repair deficiencies. pPOLE have been incorporated into treatment guidelines for several malignancies and are an important predictor of immunotherapy response. This study provides biological insight to guide classification and clinical management of patients with tumors harboring pPOLE.
In this multilaboratory validation study of 145 ovarian cancer samples, the SOPHiA DDM HRD Solution was compared with the regulatory-approved Myriad myChoice HRD assay to assess clinical comparability for class 3 in-hous...In this multilaboratory validation study of 145 ovarian cancer samples, the SOPHiA DDM HRD Solution was compared with the regulatory-approved Myriad myChoice HRD assay to assess clinical comparability for class 3 in-house in vitro diagnostic medical device companion diagnostic use. BRCA (BRCA1 and BRCA2) mutation status showed 100% concordance, and genomic instability (GI) measurements demonstrated strong linear agreement, absence of bias, and high analytical precision. Receiver operating characteristic analysis suggested a threshold adjustment from 0 to -1.5, improving overall accuracy to 91.2% when combined with BRCA mutation status to assign homologous recombination deficiency (HRD) status. Approximately 6% of samples were excluded because of inconclusive results, whereas GI classification discordance was concentrated near the clinical threshold. Neither inconclusiveness nor discordance was associated with sample-related factors. These findings indicate that the SOPHiA HRD assay can provide results broadly interchangeable with Myriad myChoice, although caution is warranted when assigning HRD status to borderline GI values.
Large genomic rearrangements (LGRs) account for at least 10% of the mutations in BRCA1 and 5% of BRCA2 mutations in outbred families with hereditary breast and ovarian cancer. A total of 21 probands with breast cancer wh...Large genomic rearrangements (LGRs) account for at least 10% of the mutations in BRCA1 and 5% of BRCA2 mutations in outbred families with hereditary breast and ovarian cancer. A total of 21 probands with breast cancer who carried BRCA1 or BRCA2 LGRs were identified from a cohort of 4678 Chinese patients. There was a total of 13 BRCA1 LGR carriers and 8 BRCA2 LGR carriers, including 12 large genomic deletions and 1 duplication. Ten and three specific breakpoints from BRCA1 and BRCA2, respectively, were identified by either whole-genome sequencing by nanopore sequencing or long-range PCR. Five of these LGRs were recurrent LGRs. Three LGRs were novel founder LGRs in the southeast Chinese population. Chinese LGR carriers exhibited clinical phenotypes that were generally similar to those of non-LGR mutation carriers. However, there was a notable tendency for triple-negative breast cancer to be more prevalent among Chinese LGR carriers (P = 0.007), largely because of the predominance of BRCA1 mutations. This suggests a potential association that warrants further investigation.
Lundie B, Chai SY, Byrne AB
… +9 more, Azmanov D, Christodoulou J, Haas MA, Kassahn KS, Lunke S, Stott A, Thompson BA, Badrick T, Bennetts B
J Mol Diagn
· 2026 Mar · PMID 41421497
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Few external quality assurance programs adequately address the complexity of largescale human genome or exome analysis. To bridge this gap, Australian Genomics and the Royal College of Pathologists of Australasia Quality...Few external quality assurance programs adequately address the complexity of largescale human genome or exome analysis. To bridge this gap, Australian Genomics and the Royal College of Pathologists of Australasia Quality Assurance Programs (QAP) developed a pilot interpretive module focused on genomic testing for childhood syndromes and intellectual disabilities. The program assessed laboratories' proficiency in interpreting complex genomic data for pediatric disorders. Six clinically accredited laboratories analyzed standardized genomic, phenotypic, and referral data. Reports were evaluated using a rubric adapted from the European Molecular Genetics Quality Network model, covering genotyping, variant classification, interpretation, and report content. Feedback included comparative performance results and individual recommendations. All laboratories correctly identified and classified target variants, but variation was observed in report structure, inclusion of genetic counseling advice, and application of the American College of Medical Genetics and Genomics/Association for Molecular Pathology classification framework. Participants noted that data-sharing limitations and differences in local reporting practices contributed to scoring inconsistencies. The pilot demonstrated the feasibility of a disease-specific interpretive QAP for complex pediatric genomic testing. Future rounds will address logistical challenges, refine scoring criteria, and strengthen standardization, supporting broader implementation. This initiative lays the groundwork for integrating specialized QAP modules into routine practice to improve diagnostic accuracy and consistency across laboratories.
Gastrointestinal stromal tumors (GISTs) are predominantly characterized by mutations in KIT or PDGFRA. Mutation detection is important for optimal therapy. Next-generation sequencing (NGS) panels are useful in GIST asses...Gastrointestinal stromal tumors (GISTs) are predominantly characterized by mutations in KIT or PDGFRA. Mutation detection is important for optimal therapy. Next-generation sequencing (NGS) panels are useful in GIST assessment as they allow for simultaneous evaluation of multiple genes. However, inherent to use of NGS with short-read sequences on formalin-fixed specimens is the potential to miss larger insertion or deletion variants. Over 9 years, GIST testing was performed on specimens from 55 patients by amplicon-based, semiconductor NGS using the Ion AmpliSeq Cancer Hotspot Panel version 2, a Cancer Hotspot Panel version 2-based GIST panel, or the Oncomine Precision Assay. Negative cases were evaluated by dideoxy sequencing for detection of mutations in KIT and PDGFRA, as per the clinical protocol. Before reflexive sequencing, of 55 completed analyses, 47 (85%) were positive for KIT or PDGFRA mutations by NGS. Three cases were attributed to positivity for pathogenic variants in other genes. Of the five cases evaluated by dideoxy sequencing, all five (9% of all specimens) were positive for pathogenic or likely pathogenic KIT mutations. A total of 12% of KIT mutations required dideoxy sequencing for identification. Mutations were 12 to 39 nucleotide deletion or duplication variants. The results suggest that short-read, amplicon-based NGS assays may miss a significant number of clinically actionable KIT mutations and that follow-up of KIT and PDGFRA NGS-negative cases by alternative testing modalities should be considered.
This large-scale retrospective study investigated the allele, diplotype, and phenotype frequencies of CYP2B6, CYP2C19, and CYP2D6 in a Han Chinese population (N > 10,000), using real-world genetic data from The First Hos...This large-scale retrospective study investigated the allele, diplotype, and phenotype frequencies of CYP2B6, CYP2C19, and CYP2D6 in a Han Chinese population (N > 10,000), using real-world genetic data from The First Hospital of Hebei Medical University, assessed between March 2021 and April 2025. Single-nucleotide polymorphism genotyping was performed using the Agena MassARRAY assay. CYP2D6 copy number variation (CNV) was analyzed by TaqMan real-time quantitative PCR. The sample sizes were 9729 for CYP2B6, 11,479 for CYP2C19, and 11,315 for CYP2D6, all from the Han Chinese population. The high frequencies of alleles were CYP2B6∗6 (16.19%), CYP2C19∗2 (30.36%), and CYP2D6∗10 (41.67%), with the most common diplotypes being CYP2B6 ∗1/∗6 (23.65%), CYP2C19 ∗1/∗2 (37.62%), and CYP2D6 ∗1/∗10 (16.05%), respectively. At the phenotype level, normal metabolizer was most common for CYP2B6 (57.86%) and CYP2D6 (60.50%), whereas the intermediate metabolizer phenotype was noted for CYP2C19 (44.77%). CYP2D6 CNVs included zero copies (0.43%), one copy (13.64%), two copies (83.79%), three copies (2.11%), and more than three copies (0.03%). This large-scale, real-world study of a Northern Han Chinese cohort provides a valuable pharmacogenomic reference and demonstrates the necessity of integrating CNV analysis with single-nucleotide polymorphism genotyping to ensure accurate clinical phenotyping of CYP2D6.
Large-scale tumor molecular profiling has enabled the discovery of diagnostic, prognostic, and therapeutic biomarkers, and expanded the clinical utility of alterations such as gene amplifications (GAMPs), homozygous dele...Large-scale tumor molecular profiling has enabled the discovery of diagnostic, prognostic, and therapeutic biomarkers, and expanded the clinical utility of alterations such as gene amplifications (GAMPs), homozygous deletions (HMZ-Dels), and biallelic inactivation (BI) of tumor suppressor genes. Comprehensive clinical detection of these events is essential for optimal patient management. Illumina's TruSight Oncology 500 (TSO500) kit detects multiple biomarkers, including GAMPs for select genes, but does not assess HMZ-Del or BI events. To address this gap, OncCNV, a genome-wide copy number analysis and visualization pipeline that integrates both on-target and off-target probe data from TSO500 sequencing, was developed. Performance optimization evaluated copy number calling tools, on-target and off-target probe selection strategies, off-target bin sizes, and panel-of-normal configurations. Clinical validation was conducted using 132 unique solid tumors characterized by a clinically validated microarray assay. OncCNV showed >96% positive percentage agreement, >99% negative percentage agreement, and >99% accuracy at the assay's established limit of detection (40 ng DNA at 40% tumor content). Sensitivity for HMZ-Del and BI detection decreased to 62%-70% at tumor content of 20%-39% in in silico dilution experiments; however, intrarun, interrun, and interanalyst precision remained >99%. OncCNV extends the analytical capabilities of TSO500 by enabling robust and precise detection of GAMP, HMZ-Del, and BI events, enhancing solid tumor comprehensive molecular profiling.
Wallander K, Lin Y, Ivanchuk V
… +8 more, Difilippo V, Chellappa V, Murugan SK, Öfverholm I, Bränström R, Nord KH, Carlson J, Haglund de Flon F
J Mol Diagn
· 2026 Feb · PMID 41320126
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Optical genome mapping (OGM) enables high-resolution detection of structural variants (SVs) and copy number aberrations (CNAs) using ultralong DNA molecules and minimal bioinformatics processing. Its diagnostic utility i...Optical genome mapping (OGM) enables high-resolution detection of structural variants (SVs) and copy number aberrations (CNAs) using ultralong DNA molecules and minimal bioinformatics processing. Its diagnostic utility in solid tumors remains underexplored. Whole-genome sequencing (WGS) offers comprehensive variant detection but is resource intensive. This study presents a technical benchmarking of OGM versus WGS for mesenchymal tumors of the gynecologic tract. Twenty-five uterine mesenchymal tumors were prospectively analyzed using matched WGS, transcriptome sequencing, and OGM. Detected SVs, CNAs, and fusion genes were compared across platforms. OGM identified structural driver events in 80% of cases and demonstrated high concordance with WGS for major CNAs and translocations. In select cases, OGM resolved complex rearrangements not clearly defined by WGS, including a PLAG1::RERE fusion and an embedded inversion in a RAD51B::HMGA2 event. Conversely, WGS uniquely detected a truncating NF1 translocation and a TSC2::SENP3 fusion, both clinically significant. OGM is a technically robust platform for SV and CNA detection in mesenchymal tumors, and it may serve as an efficient alternative to sequencing-based cytogenomic approaches in selected clinical contexts, especially in tumors known to be driven by gross chromosomal rearrangements. WGS provides a comprehensive view of the cancer genome, suitable for tumors driven by single-nucleotide variants, SVs, and CNAs. The choice between platforms should be guided by clinical context, diagnostic needs, and available resources.
Epidermal growth factor receptor variant (EGFRv)-III, a common oncogenic variant in glioblastoma and other solid tumors, results from an in-frame deletion of exons 2 to 7 from the EGFR gene. Detection of EGFRvIII is cruc...Epidermal growth factor receptor variant (EGFRv)-III, a common oncogenic variant in glioblastoma and other solid tumors, results from an in-frame deletion of exons 2 to 7 from the EGFR gene. Detection of EGFRvIII is crucial for understanding tumor biology, guiding targeted therapies, and developing personalized treatment strategies. In this study, two detection approaches based on DNA next-generation sequencing-read depth (RD)-based and split read (SR)-based detection-were compared to evaluate their sensitivity and accuracy in identifying EGFRvIII. Thirty-one tumor samples, including glioblastoma and pancreatic adenocarcinoma, were analyzed using both methods. The SR-based method detected EGFRvIII in 20 of 31 samples, while the RD-based method identified it in only 12 samples (P < 0.001), demonstrating that the SR-based method had significantly higher sensitivity. RNA next-generation sequencing confirmed EGFRvIII expression in most SR-positive cases. Additionally, the SR-based method identified multiple breakpoints in several tumors, revealing intratumor heterogeneity and the subclonal origins of EGFRvIII. The RD-based method was prone to false negatives, particularly in cases with high EGFR amplification or low tumor cell percentage, where copy number variations could be masked by background noise. The findings highlight the sensitivity and accuracy of SR-based detection in identifying EGFRvIII and capturing intratumor heterogeneity. SR-based analysis is recommended as the method for EGFRvIII detection in both clinical and research settings.
Donor chimerism analysis is used for monitoring engraftment status and risk of disease relapse following allogeneic stem cell transplantation. Recently developed assays using next-generation sequencing have demonstrated...Donor chimerism analysis is used for monitoring engraftment status and risk of disease relapse following allogeneic stem cell transplantation. Recently developed assays using next-generation sequencing have demonstrated enhanced sensitivity and accuracy compared with standard capillary electrophoresis methods. This work presents validation results using One Lambda Devyser Chimerism assay, a next-generation sequencing-based test for monitoring donor chimerism. A total of 270 samples, including clinical and cell-line DNA, were tested. There was a high correlation between chimerism results obtained with One Lambda Devyser and short tandem repeat assays (R = 0.998). Determination of the limit of blank, limit of detection, and limit of quantitation indicated that One Lambda Devyser Chimerism assay can reliably detect recipient DNA fractions as low as 0.1%. Analytical specificity was >99.9%. Reproducibility, linearity, DNA library characteristics, and sequencing metrics are presented. The suitability of DNA markers was verified in a population predominantly African American and Hispanic, comprising 30 recipient/donor pairs. The average number of informative markers per pair was seven, with a lower representation (five markers) in related pairs. In conclusion, the results show that One Lambda Devyser Chimerism assay is a highly sensitive test for detecting donor chimerism in a diverse patient population. The assay performed remarkably well at low recipient concentrations, having the potential to detect early changes associated with disease relapse.
Breast cancer is the most common cancer among women, and metastasis to the lung is associated with poor prognosis. Reliable biomarkers for predicting lung metastasis are urgently needed to improve early detection and cli...Breast cancer is the most common cancer among women, and metastasis to the lung is associated with poor prognosis. Reliable biomarkers for predicting lung metastasis are urgently needed to improve early detection and clinical decision-making. This study used microarray data sets comprising gene expression profiles and clinical data from primary breast cancer patients who were followed up for lung metastasis outcomes. High-throughput screening combined with Venn diagram analysis was used to identify common candidate probes, and the least absolute shrinkage and selection operator method were used to select 11 genes for model development. Logistic regression was used to construct predictive models, and the final risk signature consisted of 10 candidate genes (CDK19, GLUD1, GTPBP4, HLCS, HYI, KCND3, MAP2K1, NMUR1, PRKD3, and SLC16A3). The model achieved strong performance in training and validation cohorts (areas under the curve >0.87) and generalized to the independent METABRIC data set (area under the curve = 0.706). Subset analyses restricted to patients with early-stage disease confirmed that the signature retained predictive value. Kaplan-Meier analyses showed that patients with high-risk scores had shorter lung metastasis-free survival, recurrence-free survival, and overall survival. Multivariate Cox analysis confirmed that the risk signature provided independent predictive information from clinical variables. In conclusion, the risk signature accurately identifies patients with breast cancer at risk of lung metastasis, enabling clinicians to better assess risk and tailor effective treatment strategies.
Splice-site variants within the ABO gene have the potential to impair ABO glycosyltransferase biosynthesis, leading to decreased expression of A or B antigens on the surface of red blood cells. This study characterized h...Splice-site variants within the ABO gene have the potential to impair ABO glycosyltransferase biosynthesis, leading to decreased expression of A or B antigens on the surface of red blood cells. This study characterized how four intron 6 splice-site variants-three in the A1 allele (c.374+4A>T, c.374+4A>G, and c.374+5G>A) and one in the ABO∗B.01 allele (c.374+2_374+3insT)-affect ABO pre-mRNA splicing. A combination of serologic, molecular genetic, bioinformatic, and minigene assays established a genotype-splicing-phenotype association model. Bioinformatics predictions showed that all variants impaired the recognition of the 5' splice site. Specifically, the A allele variant c.374+5G>A induced approximately 2.8% exon 6 retention, whereas the c.374+4A>G (A/A) and c.374+4A>T (A) variants preserved approximately 6.9% and 10.2% functional transcripts, respectively. The novel B allele insertion variant c.374+2_374+3insT (B subtype) retained approximately 4.7% exon 6-containing transcripts, sufficient for trace B glycosyltransferase expression. Quantitative real-time PCR analysis confirmed that the transcriptional levels of the ABO gene in the AB subtype individual carrying the c.374+2_374+3insT variant were approximately 51.7% of those in the ABO∗A1.02/ABO∗B.01 control. This study demonstrated that splice-site variants in the ABO gene reduce the abundance of functional transcripts via altered transcriptional regulation, which, in turn, leads to decreased ABO glycosyltransferase expression, ultimately resulting in weak antigen phenotypes of varying intensity.
Cameroon is an epicenter of diverse HIV-1 strains, with diagnostic and management challenges. The objective herein was to update HIV-1 non-M prevalence and compare diagnostic performance of two- versus three-test algorit...Cameroon is an epicenter of diverse HIV-1 strains, with diagnostic and management challenges. The objective herein was to update HIV-1 non-M prevalence and compare diagnostic performance of two- versus three-test algorithms. A facility-based study was conducted in February 2024 on 2207 HIV-1 clinical samples at the Chantal Biya International Reference Centre (Yaoundé, Cameroon). HIV-1 non-M were identified by molecular phylogeny. Rapid diagnostic tests (RDTs) used in the two-test (Determine and KHB colloidal gold) versus three-test (First Response, One Step, and KHB) algorithms were evaluated on non-M, with ACRO (HIV1/2 and p24) as independent RDT. No group N (0%) nor P (0%) was found, whereas nine group O were identified (0.4%; 95% CI, 0.2%-0.8%). For individuals harboring group O (mean age, 43 ± 12 years; 50% female), median (IQR): duration since HIV diagnosis was 627 (423 to 775) weeks; viremia, 12,385 (5340 to 72,682) copies/mL; and CD4 count, 52 (39 to 228) cells/mm. One Step, KHB, and ACRO detected eight of eight group O (100%); First Response HIV1-2.0, seven of eight (87.5%); and Determine HIV1/2, six of eight (75%), P = 1.00. In this Cameroonian setting, HIV-1 group N and P are scarce, whereas group O remains low (<1%). Transitioning from the two-test (75% performance) to the three-test algorithm (87.5% performance) could lead to improved diagnostic performance on currently circulating HIV-1 group O, calling for updates in RDTs to adapt to viral dynamics.
Conventional methods for spinal muscular atrophy (SMA) screening have been challenging in detecting SMN1/2 single-nucleotide variants (SNVs) and small insertions and deletions, SMN1 2 + 0 silent carrier, and the copy num...Conventional methods for spinal muscular atrophy (SMA) screening have been challenging in detecting SMN1/2 single-nucleotide variants (SNVs) and small insertions and deletions, SMN1 2 + 0 silent carrier, and the copy number (CN) of SMN2. To address these limitations, a long-read sequencing (LRS)-based approach termed comprehensive analysis of SMA 2 (CASMA2) was developed. CASMA2 was used to perform CN analysis by integrating Poisson distribution with an endogenous reference gene, the first such method developed for LRS platforms. The performance and clinical feasibility of CASMA2 were evaluated by using 414 retrospective peripheral blood samples and 303 prospective dried blood spot samples. CASMA2 displayed 100% accuracy in SMN1/2 CN analysis and identified the SNVs/insertions and deletions in SMN1/2. CASMA2 also showed the capability of screening for the SMN1 2 + 0 silent carrier with family-trio haplotype analysis. It achieved a 99.0% (410 of 414) first-attempt success rate for long-term peripheral blood samples and a 98.7% (299 of 303) rate for dried blood spot samples. CASMA2 offers a clinically feasible, precise, and efficient method for SMA carrier and newborn screening.
Despite the availability of various in silico prediction tools, accurately assessing the pathogenicity of splice-region variants remains limited. In this study, 18 splice-region variants of uncertain significance (VUSs)...Despite the availability of various in silico prediction tools, accurately assessing the pathogenicity of splice-region variants remains limited. In this study, 18 splice-region variants of uncertain significance (VUSs) were functionally characterized using either in vitro (minigene) or in vivo (RT-PCR) experiments. The impacts of in-frame mutants on the indicated protein were further analyzed by modeling the three-dimensional protein structures. The accuracy of different in silico prediction tools (SpliceAI, varSEAK, and Splicing Prediction Pipeline) was also compared. The 18 VUSs included 3 canonical and 15 noncanonical splicing variants. Splicing abnormalities were observed in 16 variants (88.9%), with exon skipping (33.3%, 6/18) being the most prevalent event. Among them, 11 variants were predicted to generate premature termination codons, 3 caused in-frame alterations, and 2 resulted in both outcomes. Structural modeling revealed significant disruption of protein secondary structure in four of five in-frame alterations. Integrating functional and structural evidence, 15 VUSs (83.3%) were reclassified as pathogenic/likely pathogenic, enabling definitive diagnoses and informed reproductive decisions for affected families. Among the in silico tools, SpliceAI demonstrated the highest accuracy in predicting splicing anomalies; however, all tools showed decreased accuracy in analyzing specific splicing patterns. This study establishes an integrated workflow for assessing the pathogenicity of splicing VUSs in clinical diagnostics, offering a practical approach to overcome this critical diagnostic challenge.
Kim J, Venkataraman S, Chavan SN
… +7 more, Bokka R, Tange JI, Leger RR, Pruthi RK, Chen D, Seheult JN, Pandey A
J Mol Diagn
· 2026 Jan · PMID 41176274
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Factor XIII (FXIII) is a heterotetramer that plays a crucial role in the coagulation cascade, facilitating fibrin crosslinking to stabilize clots. Congenital or acquired deficiency of the FXIII A or B subunits can lead t...Factor XIII (FXIII) is a heterotetramer that plays a crucial role in the coagulation cascade, facilitating fibrin crosslinking to stabilize clots. Congenital or acquired deficiency of the FXIII A or B subunits can lead to prolonged bleeding. Existing enzyme-linked immunosorbent assay-based and latex agglutination assays for measuring FXIII are limited to detecting only the FXIIIA subunit of the complex. In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated to accurately quantify both FXIIIA and FXIIIB subunits in plasma. Peptides measured by this targeted assay were rigorously selected based on analytical robustness, with the method demonstrating excellent sensitivity (limit of detection, 1.56 to 3.67 mU/mL), linearity (r > 0.998), and precision (CV, <10%). Comparison with the conventional assay based on ammonia release demonstrated a strong correlation (r = 0.983) and agreement (Cohen κ = 0.846) for FXIIIA. On analysis of 98 clinical plasma samples, the assay accurately identified cases with FXIII deficiency based on significantly reduced FXIIIA with varying alterations in FXIIIB levels. These results establish the clinical utility of the LC-MS/MS assay, highlight its potential for improving diagnostic accuracy for FXIII deficiency, and lay the foundation for future research on FXIII dynamics in pathologic states.