Journal of Molecular Diagnostics最新文献

Inherited retinal dystrophies (IRDs) represent a diverse group of rare pathologies affecting vision with significant genetic and clinical variability. Clinical exome sequencing (CES) was performed on 143 families clinically diagnosed with IRDs. The obtained variants were filtered and classified according to the American College of Medical Genetics guidelines. Overall, a genetic diagnosis was achieved for 68.53% of the families in the cohort, with 35 causative genes identified, predominantly ABCA4 and USH2A. A total of 170 clinically relevant variants were identified, 45 of which (26.47%) were novel, with missense variants being the most common type (40.59%). This study demonstrated 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 CES 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 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-house in vitro diagnostic medical device companion diagnostic use. BRCA1/2 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.

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.

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 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.

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.

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 (R² = 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.

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.