Journal of Molecular Diagnostics : JMD

High-throughput sequencing of the T-cell receptor beta (TRB) and gamma (TRG) loci is increasingly utilized due to its high sensitivity, specificity, and versatility in the diagnosis of various T-cell malignancies. Application of these technologies for tracking disease burden can be valuable in detecting recurrence, determining response to therapy, guiding future management of patients, and establishing endpoints for clinical trials. In this study, the performance of the commercially available LymphoTrack high-throughput sequencing assay was assessed for determining residual disease burden in patients with various T-cell malignancies seen at our institution...

Phenylalanine hydroxylase (PAH) deficiency or phenylketonuria (PKU) is the most common cause of hyperphenylalaninemia (HPA), and approximately 5% of patients remain genetically unsolved. Identifying deep intronic PAH variants may help improve their molecular diagnostic rate. Next-generation sequencing was utilized to detect the whole PAH gene in 96 genetically unsolved HPA patients from 2013 to 2022. The effects of deep intronic variants on pre-mRNA splicing were investigated by minigene-based assay. The allelic phenotype values of recurrent deep intronic variants were calculated...

Despite the rapid application of next-generation sequencing (NGS) technologies, target sequencing in regions of the genome is often required to diagnose many genetic diseases. Target enrichment can be a very effective factor in reducing the cost of sequencing and the duration of sequencing. Recently, several CRISPR-based methods (amplification-free sequencing) have been developed to target enrichment in combination with one of the NGS platforms. CRISPR-based target enrichment strategies act as an auxiliary tool to improve NGS analytical performance, thereby indirectly facilitating nucleic acid detection...

Identification of somatic variants in cancer by high-throughput sequencing has become common clinical practice largely because many of these variants may be predictive biomarkers for targeted therapies. However, there can be high sample quality control (QC) failure rates for some tests preventing the return of results. SLIMamp is a patented technology that has been incorporated into commercially available cancer NGS testing kits with the claimed advantage that these kits can interrogate challenging formalin-fixed paraffin-embedded tissue (FFPET) samples with low tumor purity, poor DNA quality, and/or low input DNA, resulting in a high sample QC pass rate...

Current guidelines for patients with Chronic Lymphocytic Leukemia (CLL) recommend mutation status determination of the clonotypic IGHV gene prior to treatment initiation in order to guide the choice of the first-line therapy. Currently, commercially available next-generation sequencing (NGS) solutions have technical constraints, as they necessitate at least a 2x300 bp sequencing, which restricts their use for routine practice. The cost of the commercial kits also represents an important drawback. We present a new method called Next-CLL, a ready-to-use strategy to evaluate IGHV gene mutation status using any NGS device (including 2 x 150 bp sequencers) in routine diagnostic laboratories...

The standard-of-care diagnostic prenatal testing includes a combination of cytogenetic methods, such as karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarray (CMA), using either direct or cultured amniocytes or chorionic villi sampling. However, each technology has its limitations: karyotyping has a low resolution (>5 Mb), FISH is targeted, and CMA does not detect balanced structural variations (SVs). These limitations necessitate the use of multiple tests, either simultaneously or sequentially, to reach a genetic diagnosis...

The COVID-19 pandemic has provided a stage to illustrate that there is considerable value in obtaining rapid, whole genomic information about pathogens. Herein we describe the utility of a commercially available, automated SARS-CoV-2 library preparation, genomic sequencing and a bioinformatic analysis pipeline to provide rapid, near- "real-time" SARS-CoV-2 variant description. We evaluated the turnaround time, accuracy and other quality parameters obtained from Clear Labs Dx automated sequencing instrumentation from analysis of continuous clinical samples from January 1, 2021 to October 6, 2021...

Δ4 -3-oxosteroid 5β-reductase (AKR1D1) deficiency presents with neonatal cholestasis and liver failure in early infancy and features high levels of 3-oxo- Δ4 - bile acids in urine. Genetic analysis is needed for definitive diagnosis, because in the neonatal period it can be difficult to distinguish a primary from secondary enzyme deficiency. By re-analysis of the gene sequencing data, one AKR1D1 non-canonical splice-site variant (NM_005989.4: c.580-13T>A) with controversial pathogenicity was discovered to be enriched in 8 families with clinical and biochemically confirmed AKR1D1 deficiency...

Epithelial Ovarian Cancers (EOCs) harboring germline or somatic pathogenic variants in BRCA1 and BRCA2 genes show sensitivity to poly(ADP-ribose) polymerase (PARP) inhibition. It has been suggested that BRCA1 promoter methylation is perhaps a better determinant of therapy response, due to its intrinsic dynamic feature, with respect to genomic scars or gene mutation. Conflicting evidence was reported so far and the lack of a validated assay to measure promoter methylation was considered a main confounding factor in data interpretation...

Hypnotizability is a stable trait that moderates the benefit of hypnosis for treating pain, but limited availability of hypnotizability testing deters widespread use of hypnosis. Inexpensive genotyping of 4 single nucleotide polymorphisms in the catechol-o-methyltransferase (COMT) gene was performed using giant magnetoresistive biosensors to determine if hypnotizable individuals can be identified for targeted hypnosis referrals. For individuals with the proposed 'optimal' COMT diplotypes, 89.5% score highly on the Hypnotic Induction Profile (OR = 6...

Prophylactic human papillomavirus (HPV) vaccines are recommended for prevention of HPV-associated cancers. Type-specific detection of HPV in formalin-fixed paraffin-embedded (FFPE) tissues retrieved from diagnostic pathology laboratories is important in monitoring the impact of HPV vaccines. However, few typing assays have been validated for testing FFPE samples. We compared results of the Novaplex II HPV28 Detection (Novaplex) assay with those from our reference assay (Linear Array with reflex Line Probe Assay) on 708 FFPE samples from cervical lesions...

CANTRK is a ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next generation sequencing (NGS) for NTRK1, NTRK2 or NTRK3 fusions. Each laboratory received 12 formalin fixed, paraffin embedded (FFPE) tumor samples with unique NTRK fusions, and 2 control non-NTRK fusion samples (1 ALK and 1 ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus, Oncomine Precision, AmpliSeq Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung and QIAseq Multimodal Lung...

The use of standard next-generation sequencing technologies to detect key mutations in IDH genes for glioma diagnosis imposes several challenges, including high capital cost and turnaround delays associated with the need for batch testing. For both glioma testing and testing in other tumor types where highly specific mutation identification is required, the high-throughput nature of next-generation sequencing limits the feasibility of using it as a primary approach in clinical laboratories. We hypothesized that third-generation nanopore sequencing by Oxford Nanopore Technologies has the capability to overcome these limitations...

No abstract text is available yet for this article.

This study compares optical genome mapping (OGM) performed at multiple sites with current standard-of-care (SOC) methods used in clinical cytogenetics. This study included 50 negative controls and 359 samples from individuals (patients) with suspected genetic conditions referred for cytogenetic testing. OGM was performed using the Saphyr system and Bionano Access software version 1.7. Structural variants, including copy number variants, aneuploidy, and regions of homozygosity, were detected and classified according to American College of Medical Genetics and Genomics guidelines...

The Blood Profiling Atlas in Cancer (BLOODPAC) Consortium is a collaborative effort involving stakeholders from the public, industry, academia, and regulatory agencies focused on developing shared best practices on liquid biopsy. This report describes the results from the JFDI (Just Freaking Do It) study, a BLOODPAC initiative to develop standards on the use of contrived materials mimicking cell-free circulating tumor DNA, to comparatively evaluate clinical laboratory testing procedures. Nine independent laboratories tested the concordance, sensitivity, and specificity of commercially available contrived materials with known variant-allele frequencies (VAFs) ranging from 0...

Sarcomas are a diverse group of tumors, with >70 subtypes in the current World Health Organization classification, each with distinct biological behavior requiring specific clinical management. A significant portion of sarcomas are molecularly defined by expression of a driver fusion gene; identification of such fusions is the basis of molecular diagnostics in sarcomas, which is of increasing complexity due to the ongoing discovery of new gene fusions. Recently, a multiplex NanoString platform-based assay was developed and clinically implemented, with fusion junction-spanning probes that detect the majority of sarcoma fusion types, with high sensitivity and specificity, and with lower cost and shorter turnaround time than those of targeted next-generation sequencing-based alternatives...

No abstract text is available yet for this article.

Nearly 14% of disease-causing germline variants result from disruption of mRNA splicing. Most (67%) DNA variants predicted in silico to disrupt splicing end up classified as variants of uncertain significance (VUS). We developed and validated an analytic workflow - Splice Effect Event Resolver (SPEER) - that uses mRNA sequencing to reveal significant deviations in splicing, pinpoints the DNA variants potentially responsible, and measures the deleterious effect of the altered splicing on mRNA transcripts, providing evidence to assess the pathogenicity of the variant...

No abstract text is available yet for this article.