Journal of Molecular Diagnostics最新文献

The DPYD gene encodes dihydropyrimidine dehydrogenase (DPD), which is involved in the catalysis of uracil and thymine, as well as 5-fluorouracil (5-FU), which is used to treat solid tumors. Patients with decreased DPD activity are at risk of serious, sometimes fatal, adverse drug reactions to this important cancer drug. Pharmacogenetic testing for DPYD is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for clinical DPYD testing. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention–based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 33 DNA samples derived from Coriell cell lines for DPYD. Samples were distributed to four volunteer laboratories for genetic testing using a variety of commercially available and laboratory-developed tests. Sanger sequencing was used by one laboratory and publicly available whole-genome sequence data from the 1000 Genomes Project were used by another to inform genotype. Thirty-three distinct DPYD variants were identified among the 33 samples characterized. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.

Next-generation sequencing–based genomic testing is standard of care for tumor workflows. However, its application across different institutions continues to be challenging given the diversity of needs and resource availability among different institutions globally. Moreover, the use of a variety of different panels, including those from a few individual genes to those involving hundreds of genes, results in a relatively skewed distribution of care for patients. It is imperative to obtain a higher level of standardization without having to be restricted to specific kits or requiring repeated validations, which are generally expensive. We show the validation and clinical implementation of the DH-CancerSeq assay, a tumor-only whole-exome–based sequencing assay with integrated informatics, while providing similar input requirements, sensitivity, and specificity to a previously validated targeted gene panel and maintaining similar turnaround times for patient care.

Polygenic risk scores (PRSs) for breast cancer have a clear clinical utility in risk prediction. PRS transferability across populations and ancestry groups is hampered by population-specific factors, ultimately leading to differences in variant effects, such as linkage disequilibrium and differences in variant frequency (allele frequency differences). Thus, locally sourced population-based phenotypic and genomic data sets are essential to assess the validity of PRSs derived from signals detected across populations. This study assesses the transferability of a breast cancer PRS composed of 313 risk variants (313-PRS) in a Brazilian trihybrid admixed ancestries (European, African, and Native American) whole-genome sequenced cohort, the Rare Genomes Project. 313-PRS was computed in the Rare Genomes Project (n = 853) using the UK Biobank (UKBB; n = 264,307) as reference. The Brazilian cohorts have a high European ancestry (EA) component, with allele frequency differences and to a lesser extent linkage disequilibrium patterns similar to those found in EA populations. The 313-PRS distribution was found to be inflated when compared with that of the UKBB, leading to potential overestimation of PRS-based risk if EA is taken as a standard. However, case controls lead to equivalent predictive power when compared with UKBB-EA samples with area under the receiver operating characteristic curve values of 0.66 to 0.62 compared with 0.63 for UKBB.

Timely detection of Aspergillus infection is crucial given the high mortality rate of pulmonary aspergillosis (PA). Here, the diagnostic performances for PA of mycological culture, Aspergillus real-time PCR, and metagenomic next-generation sequencing (mNGS) assay from bronchoalveolar lavage fluid, were evaluated. In total, 139 patients with suspected fungal pneumonia were enrolled between December 2021 and July 2023, collecting 139 bronchoalveolar lavage fluid samples for real-time PCR and culture, with 87 undergoing mNGS assay. The sensitivity, specificity, positive predictive value, negative predictive value, and area under the curve with 95% CIs of these assays for PA were as follows: 35.3% (14.2%–61.7%), 100.0% (94.0%–100.0%), 100.0% (54.1%–100.0%), 84.5% (79.3%–88.6%), and 0.676 (0.560–0.779), respectively, for culture; 82.4% (56.6%–96.2%), 98.3% (91.1%–100.0%), 93.3% (66.4%–99.0%), 95.2% (87.6%–98.2%), and 0.903 (0.815–0.959), respectively, for same diagnostic performance of real-time PCR and mNGS; and 94.1% (71.3%–99.9%), 96.7% (88.5%–99.6%), 88.9% (67.1%–96.9%), 98.3% (89.6%–99.7%), and 0.954 (0.880–0.989), respectively, for real-time PCR combining mNGS; real-time PCR, mNGS, and their combination significantly improved in area under the curve values over culture (P < 0.001), but real-time PCR testing and mNGS had no significant difference with each other and their combination. Overall, the performance of culture was limited by low sensitivity; both real-time PCR and mNGS assays as single diagnostic tests are promising compared with culture and combined tests.

The value of human papillomavirus (HPV) testing for cervical cancer screening is well established; however, its use as a primary screening option or as a reflex test after atypical cytology results is now gaining wider acceptance. The importance of full genotyping and viral load determination has been demonstrated to enhance the clinical understanding of the viral infection progression during follow-up or after treatment, thereby providing clinicians with supplementary tools for optimized patient management. We developed a new analysis method for the RIATOL quantitative PCR assay, and validated and implemented it in the laboratory of clinical molecular pathology at Algemeen Medisch Laboratorium (AML), under national accreditation and following the International Organization for Standardization guidelines. This study presents the successful validation of a high-throughput, multitarget HPV analysis method, with enhanced accuracy on both qualitative and quantitative end results. This is achieved by software standardization and automation of PCR curve analysis and interpretation, using data science and artificial intelligence. Moreover, the user-centric functionality of the platform was demonstrated to enhance both staff training and routine analysis workflows, thereby saving time and laboratory personnel resources. Overall, the integration of the FastFinder plugin semi-automatic analysis algorithm with the RIATOL real-time quantitative PCR assay proved to be a remarkable advancement in high-throughput HPV quantification, with demonstrated capability to provide highly accurate clinical-grade results and to reduce manual variability and analysis time.

Mastocytosis is a heterogeneous disorder characterized by abnormal mast cell accumulation, in which the clinical severity may be explained by distinct molecular mechanisms. This study aimed to explore plasma protein biomarkers associated with systemic mastocytosis subtypes, as well as the cellular origin of the identified proteins. Plasma samples from patients with mastocytosis, including cutaneous mastocytosis (CM), indolent systemic mastocytosis (ISM), and advanced systemic mastocytosis (AdvSM), and a reference group of patients with polycythemia vera, were analyzed by Proximity Extension Assay technology targeting 275 proteins. Furthermore, potential cellular origin was explored using an available single-cell RNA-sequencing data set generated from patients with ISM. The study cohort included 16 patients with CM, 92 patients with systemic mastocytosis (ISM, n = 80; AdvSM, n = 12), and 60 patients with polycythemia vera. A principal component analysis based on 275 plasma proteins revealed one cluster of patients with CM and ISM that was separated from patients with AdvSM. Up to 29 proteins were associated with distinct severe activity in patients with systemic mastocytosis (ISM versus AdvSM), including IL-1 receptor type 1 (IL-1RT1) and tumor necrosis factor ligand superfamily member 13B (TNFSF13B) (q < 0.01). Furthermore, single-cell RNA-sequencing analysis from ISM-derived bone marrow cells revealed that the mRNA for the identified proteins was not exclusive of mast cells. Distinct plasma protein profiles show potential to refine ISM and AdvSM diagnoses, possibly reflecting differences in pathogenic mechanisms and diverse clinical manifestations.

Genetic analysis of congenital adrenal hyperplasia (CAH) has been challenging because of high homology between CYP21A2 and its pseudogene CYP21A1P. This study aimed to evaluate the clinical utility of long-read sequencing (LRS) in diagnosis of CAH attributable to 21-hydroxylase deficiency by comparing with multiplex ligation-dependent probe amplification plus Sanger sequencing. In this retrospective study, 69 samples, including 49 probands from 47 families with high-risk of CAH, were enrolled and blindly subjected to detection of CAH by LRS. The genotype results were compared with control methods, and discordant samples were validated by additional Sanger sequencing. LRS successfully identified biallelic variants of CYP21A2 in the 39 probands diagnosed as having CAH. The remaining 10 probands were not patients with CAH. Additionally, LRS directly identified two pathogenic single-nucleotide variations (SNVs; c.293-13C/A>G and c.955C>T) in the presence of interference caused by nearby insertions/deletions (indels). The cis-trans configuration of two or more SNVs and indels identified in 18 samples was directly determined by LRS without family analysis. Eight CYP21A1P/A2 or TNXA/B deletion chimeras, composed of five subtypes, were identified; and the junction sites were precisely determined. Moreover, LRS determined the exact genotype in two probands who had three heterozygous SNVs/indels and duplication, which could not be clarified by control methods. These findings highlight that LRS could assist in more accurate genotype imputation and more precise CAH diagnosis.

Bloodstream infection is a major cause of morbidity and death worldwide. Timely and appropriate treatment can reduce mortality among critically ill patients. Current diagnostic methods are too slow to inform precise antibiotic choice, leading to the prescription of empirical antibiotics, which may fail to cover the resistance profile of the pathogen, risking poor patient outcomes. Additionally, overuse of broad-spectrum antibiotics may lead to more resistant organisms, putting further pressure on the dwindling pipeline of antibiotics, and risk transmission of these resistant organisms in the health care environment. Therefore, rapid diagnostics are urgently required to better inform antibiotic choice early in the course of treatment. Sequencing offers great promise in reducing time to microbiological diagnosis; however, the amount of host DNA compared with the pathogen in patient samples presents a significant obstacle. Various host-depletion and bacterial-enrichment strategies have been used in samples, such as saliva, urine, or tissue. However, these methods have yet to be collectively integrated and/or extensively explored for rapid bloodstream infection diagnosis. Although most of these workflows possess individual strengths, their lack of analytical/clinical sensitivity and/or comprehensiveness demands additional improvements or synergistic application. This review provides a distinctive classification system for various methods based on their working principles to guide future research, and discusses their strengths and limitations and explores potential avenues for improvement to assist the reader in workflow selection.

Replication-coupled gene editing using locked nucleic acid–modified single-stranded DNA oligonucleotides (LMOs) can genetically engineer mammalian cells with high precision at single nucleotide resolution. Based on this method, oligonucleotide-directed mutation screening (ODMS) was developed to determine whether variants of uncertain clinical significance of DNA mismatch repair (MMR) genes can cause Lynch syndrome. In ODMS, the appearance of 6-thioguanine–resistant colonies upon introduction of the variant is indicative for defective MMR and hence pathogenicity. Whereas mouse embryonic stem cells (mESCs) hemizygous for MMR genes were used previously, we now show that ODMS can also be applied in wild-type mESCs carrying two functional alleles of each MMR gene. 6-Thioguanine resistance can result from two possible events: first, the mutation is present in only one allele, which is indicative for dominant-negative activity of the variant; and second, both alleles contain the planned modification, which is indicative for a regular loss-of-function variant. Thus, ODMS in wild-type mESCs can discriminate fully disruptive and dominant-negative MMR variants. The feasibility of biallelic targeting suggests that the efficiency of LMO-mediated gene targeting at a nonselectable locus may be enriched in cells that had undergone a simultaneous selectable LMO targeting event. This turned out to be the case and provided a protocol to improve recovery of LMO-mediated gene modification events.

PMS2 is one of the DNA-mismatch repair genes included in routine genetic testing for Lynch syndrome and colorectal, ovarian, and endometrial cancers. PMS2 is also included in the American College of Medical Genetics and Genomics' List of Secondary Findings Genes in the context of clinical exome and genome sequencing. However, sequencing of PMS2 by short-read–based next-generation sequencing technologies is complicated by the presence of the pseudogene PMS2CL, and is often supplemented by long-range–based approaches, such as long-range PCR or long-read–based next-generation sequencing, which increases the complexity and cost. This article describes a bioinformatics homology triage workflow that can eliminate the need for long-read–based testing for PMS2 in the vast majority of patients undergoing exome sequencing, thus simplifying PMS2 testing and reducing the associated cost.