Recessive variants in GJB2 are the most important genetic cause of sensorineural hearing impairment (SNHI) worldwide. Phenotypes vary significantly in GJB2-related SNHI, even in patients with identical variants. For instance, patients homozygous for the GJB2 p.V37I variant, which is highly prevalent in the Asian populations, usually present with mild-to-moderate SNHI; yet severe-to-profound SNHI is occasionally observed in approximately 10% of p.V37I homozygotes. To investigate the genomic underpinnings of the phenotypic variability, we performed next-generation sequencing of GJB2 and other deafness genes in 63 p.V37I homozygotes with extreme phenotypic severities. We identified additional pathogenic variants of other deafness genes in 5 of the 35 patients with severe-to-profound SNHI. Furthermore, we conducted case-control association analyses for 30 unrelated p.V37I homozygotes with severe-to-profound SNHI against 28 p.V37I homozygotes with mild-to-moderate SNHI, and 120 population controls from the Taiwan Biobank. We found that the severe-to-profound group had a higher frequency of the crystallin lambda 1 (CRYL1) variant (rs14236), located upstream of GJB2, than the mild-to-moderate and Taiwan Biobank groups. Our results demonstrated that pathogenic variants in other deafness genes and a possible modifier, the CRYL1 rs14236 variant, may contribute to phenotypic variability in GJB2-realted SNHI, highlighting the importance of comprehensive genomic surveys to delineate the genotype-phenotype correlations.

Bacterial commensals of the human genitourinary tract, Mycoplasma hominis and Ureaplasma species (parvum and urealyticum) can be sexually transmitted, with the potential to cause nongonococcal urethritis, pelvic inflammatory disease, and infertility. Mycoplasma hominis and Ureaplasma species may also cause severe invasive infections in immunocompromised patients. Current culture-based methods for Mycoplasma/Ureaplasma identification are costly and laborious, with a turnaround time between 1 and 2 weeks. We developed a high-throughput, real-time multiplex PCR assay for the rapid detection of M. hominis and Ureaplasma species in urine, genital swab, body fluid, and tissue. In total, 282 specimens were tested by PCR and compared with historic culture results; a molecular reference method was used to moderate discrepancies. Overall result agreement was 99% for M. hominis (97% positive percentage agreement and 100% negative percentage agreement) and 96% for Ureaplasma species (96% positive percentage agreement and 97% negative percentage agreement). Specimen stability was validated for up to 7 days at room temperature. This multiplex molecular assay was designed for implementation in a high-complexity clinical microbiology laboratory. With this method, >90 samples can be tested in one run, with a turnaround time of 4 to 5 hours from specimen extraction to reporting of results. This PCR test is also more labor effective and cost-effective than the conventional culture-based test, thus improving laboratory efficiency and alleviating strains because of labor shortages.

The current advances and success of next-generation sequencing hold the potential for the transition of cancer cytogenetics toward comprehensive cytogenomics. However, the conventional use of short reads impedes the resolution of chromosomal aberrations with current next-generation sequencing modalities. Thus, this study evaluated the detection and reproducibility of extensive copy number alterations and chromosomal translocations using long-read Oxford Nanopore Technologies whole-genome sequencing compared with short-read Illumina sequencing. On the basis of the mantle cell lymphoma cell line Granta-519, almost 99% copy number reproducibility at the 100-kilobase resolution between replicates was demonstrated, with 98% concordance to Illumina. Collectively, the performance of copy number calling from 1.5 million to 7.5 million long reads was comparable to 1 billion Illumina-based reads (50× coverage). Expectedly, the long-read resolution of canonical translocation t(11; 14) (q13; q32) was superior, with a sequence similarity of 89% to the already published CCND1/IGH junction (9× coverage), spanning up to 69 kilobases. The cytogenetic profile of Granta-519 was in general agreement with the literature and karyotype, although several differences remained unresolved. In conclusion, contemporary long-read sequencing is primed for future cytogenomics or sequencing-guided cytogenetics. The combined strength of long- and short-read sequencing is apparent, where the high-precision junctional mapping complements and splits paired-end reads. The potential is emphasized by the flexible single-sample genomic data acquisition of Oxford Nanopore Technologies with the high resolution of allelic imbalances using Illumina short-read sequencing.

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 four 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 (odds ratio, 6.12; 95% CI, 1.26-28.75), which identified 40.5% of the treatable population. Mean hypnotizability scores of the optimal group were significantly higher than the total population (P = 0.015; effect size = 0.60), an effect that was present in women (P = 0.0015; effect size = 0.83), but not in men (P = 0.28). In an exploratory cohort, optimal individuals also reported significantly higher postoperative pain scores (P = 0.00030; effect size = 1.93), indicating a greater need for treatment.

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 the reference assay (Linear Array with reflex Line Probe Assay) on 708 FFPE samples from cervical lesions. Novaplex showed high type-specific concordance with the reference method for HPV16/18, 9 types targeted by the Gardasil 9 vaccine, 14 high-risk types, and 21 types covered by comparison assays. The rate of inadequate samples was low in both approaches (reference, 3.4%; Novaplex, 1.7%). The proportion of discrepant types was less than 3.5% and positive concordance was greater than 75.0%. Furthermore, the type-specific positive agreement (92.0% to 98.0%), negative agreement (96.0% to 99.0%), and accuracy (97.0% to 99.0%) was high. Cohen's κ ranged from 0.86 to 0.89, indicating excellent agreement between Novaplex and reference assays. The results show that Novaplex is a suitable method for detection of HPV in FFPE tissues.

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. The aim of this study was to substantiate that claim using Pillar's oncoReveal Solid Tumor Panel. 48 samples that had failed one or more pre-analytical QC sample parameters for whole exome sequencing (WES) from ATGC's ISO15189 accredited diagnostic genomics laboratory were acquired. XING Genomic Services (XGS) performed an exploratory data analysis to characterize the samples and then tested the samples in their ISO15189 accredited laboratory. Clinical reports could be generated for 37 samples (77%), of which 29 (60%) contained clinically actionable or significant variants that would not have otherwise been identified. 11 samples were deemed to be unreportable and the sequencing data were likely dominated by artefacts. A novel post-sequencing QC metric was developed which can discriminate between clinically reportable and unreportable samples.

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. We validated the performance of Next-CLL on genomic DNA and complementary DNA obtained from 80 CLL patients at diagnosis. Next-CLL identified a productive clone in 100% of cases, whereas PCR with Sanger sequencing led to a 12.5% failure rate. Next-CLL had 100% concordance with the reference technique for IGHV gene identification and allowed assessment of the IGHV mutation status from the leader sequence, following international guidelines. By comparing a large retrospective series of samples, analyzed using Sanger sequencing (n=773) or Next-CLL (n=352), we showed no bias in IGHV usage or mutational status, further validating our strategy in the real-life setting. Next-CLL represents a straight-forward workflow for IGHV analysis in routine practice to assess clonal architecture and prognosis of CLL patients.

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. Optical genome mapping (OGM) is an emerging technology that can detect several classes of SVs in a single assay, but it has not been evaluated in the prenatal setting. This validation study analyzed 114 samples that were received in our laboratory for traditional cytogenetic analysis with karyotyping, FISH, and/or CMA. OGM was 100% concordant in identifying the 101 aberrations that included 29 interstitial/terminal deletions, 28 duplications, 26 aneuploidies, 6 absence of heterozygosity regions, 3 triploid genomes, 4 isochromosomes, and 1 translocation; and the method revealed the identity of 3 marker chromosomes and 1 chromosome with additional material not determined by karyotyping. In addition, OGM detected 64 additional clinically reportable SVs in 43 samples. OGM has a standardized laboratory workflow and reporting solution that can be adopted in routine clinical laboratories and demonstrates the potential to replace the current standard-of-care methods for prenatal diagnostic testing.

The coronavirus disease 2019 (COVID-19) pandemic has provided a stage to illustrate that there is considerable value in obtaining rapid, whole-genome-based information about pathogens. This article describes the utility of a commercially available, automated severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2 library preparation; genome sequencing; and a bioinformatics analysis pipeline to provide rapid, near-real-time SARS-CoV-2 variant description. This study evaluated the turnaround time, accuracy, and other quality-related parameters obtained from commercially available automated sequencing instrumentation, from analysis of continuous clinical samples obtained from January 1, 2021, to October 6, 2021. This analysis included a base-by-base assessment of sequencing accuracy at every position in the SARS-CoV-2 chromosome using two commercially available methods. Mean turnaround time, from the receipt of a specimen for SARS-CoV-2 testing to the availability of the results, with lineage assignment, was <3 days. Accuracy of sequencing by one method was 100%, although certain sites on the genome were found repeatedly to have been sequenced with varying degrees of read error rate.

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 a secondary enzyme deficiency. By re-analysis of the gene-sequencing data, one AKR1D1 noncanonical splice-site variant (NM_005989.4: c.580-13T>A) with controversial pathogenicity was discovered to be enriched in eight families with clinical and biochemically confirmed AKR1D1 deficiency. Further RNA sequencing of liver tissue suggested this variant causes complete degradation of mRNA. An in vitro minigene experiment indicated that this variant led to partial intron retention or exon jumping, which then leads to coding sequence frameshift and nonsense-mediated mRNA decay. Thus, AKR1D1 variant c.580-13T>A was considered pathogenic and, therefore, should be screened during genetic studies in infants with a suspicion of a congenital bile acid synthetic disorder.

Epithelial ovarian cancers (EOCs) harboring germline or somatic pathogenic variants in BRCA1 and BRCA2 genes show sensitivity to poly(ADP-ribose) polymerase inhibition. It has been suggested that BRCA1 promoter methylation is perhaps a better determinant of therapy response, because of 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. To contribute to the validation process of a pyrosequencing assay for BRCA1 promoter methylation, 109 EOCs from two Italian centers were reciprocally blindly investigated. By comparing two different pyrosequencing assays, addressing a partially overlapping region of BRCA1 promoter, an almost complete concordance of results was obtained. Moreover, the clinical relevance of this approach was also supported by the finding of BRCA1 transcript down-regulation in BRCA1-methylated EOCs. These findings could lead to the development of a simple and cheap pyrosequencing assay for diagnostics, easily applicable to formalin-fixed, paraffin-embedded tissues. This technique may be implemented in routine clinical practice in the near future to identify EOCs sensitive to poly(ADP-ribose) polymerase inhibitor therapy, thus increasing the subset of women affected by EOCs who could benefit from such treatment.

Microsatellite instability (MSI) is an evolving biomarker for cancer detection and treatment. MSI was first used to identify patients with Lynch syndrome, a hereditary form of colorectal cancer (CRC), but has recently become indispensable in predicting patient response to immunotherapy. To address the need for pan-cancer MSI detection, a new multiplex assay was developed that uses novel long mononucleotide repeat (LMR) markers to improve sensitivity. A total of 469 tumor samples from 20 different cancer types, including 319 from patients with Lynch syndrome, were tested for MSI using the new LMR MSI Analysis System. Results were validated by using deficient mismatch repair (dMMR) status according to immunohistochemistry as the reference standard and compared versus the Promega pentaplex MSI panel. The sensitivity of the LMR panel for detection of dMMR status by immunohistochemistry was 99% for CRC and 96% for non-CRC. The overall percent agreement between the LMR and Promega pentaplex panels was 99% for CRC and 89% for non-CRC tumors. An increased number of unstable markers and the larger size shifts observed in dMMR tumors using the LMR panel increased confidence in MSI determinations. The LMR MSI Analysis System expands the spectrum of cancer types in which MSI can be accurately detected.

Patterns of somatic mutations have emerged from the broad sequencing of human cancer genomes. These mutational signatures reflect mechanisms of mutagenesis and DNA repair defects and represent an emerging class of cancer biomarkers. The appropriate interpretation of mutational signatures from sequencing assays holds implications in the reporting of molecular diagnostic results for patients with cancer. This brief review describes the scientific principles, laboratory considerations, and emerging clinical applications of mutational signature analysis from clinical cancer genomes.

The Canadian NTRK (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 tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.

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. This study aimed to develop and validate a nanopore-based IDH mutation detection assay for clinical practice using glioma formalin-fixed, paraffin-embedded (FFPE) tissue. Glioma FFPE (n = 66) samples with confirmed IDH gene mutational status were sequenced on the MinION device using an amplicon-based approach. All cases were concordant when compared with the reference results. Limit of blank and limit of detection for the variant allele fraction were 1.5% and 3.3%, respectively, at 500× read depth per gene. Total sequencing cost per sample was CAD$50 to CAD$134 with results being available in 9 to 15 hours. These findings demonstrate that nanopore-sequencing technology can be leveraged to develop low-cost, high-performance clinical sequencing-based assays with quick turnaround times to support the detection of targeted mutations in FFPE tumor tissue.

Nearly 14% of disease-causing germline variants result from the disruption of mRNA splicing. Most (67%) DNA variants predicted in silico to disrupt splicing are classified as variants of uncertain significance. An analytic workflow-splice effect event resolver (SPEER)-was developed and validated to use mRNA sequencing to reveal significant deviations in splicing, pinpoint the DNA variants potentially involved, and measure the deleterious effects of the altered splicing on mRNA transcripts, providing evidence for assessing the pathogenicity of the variant. SPEER was used to analyze leukocyte RNA encoding 63 hereditary cancer syndrome-related genes in 20,317 patients. Among 3563 patients (17.5%) with at least one DNA variant predicted to affect splicing, 971 (4.8%) had altered splicing with a deleterious effect on the transcript, and 40 had altered splicing due to a DNA variant located outside of the reportable range of the test. Integrating SPEER results into the interpretation of variants allowed variants of uncertain significance to be reclassified as pathogenic or likely pathogenic in 0.4%, and as benign or likely benign in 5.9%, of the 20,317 patients. SPEER-based evidence was associated with a significantly greater effect on classifications of pathogenic or likely pathogenic and benign or likely benign in nonwhite versus non-Hispanic white patients, illustrating that evidence derived from RNA-splicing analysis may help to reduce ethnic/ancestral disparities in genetic testing.

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 an effective factor in reducing the cost of sequencing and the duration of sequencing. Recently, several clustered system regularly interspaced short palindromic repeats (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. The direct DNA cleavage approach by CRISPR-CRISPR-associated (Cas) at genome-specific sites enhances the possibility of separating native large fragments from disease-related genomic regions. The CRISPR-Cas can isolate the target region without any amplification; subsequently, long-read sequencing technologies were also implemented. These methods, as promising tools, have the ability to assess genetic and epigenetic composition for clinical application and treatment responses in cancer precision medicine. By modifying CRISPR-based enrichment protocols, it is possible to identify different types of mutations, including structural variants, short tandem repeats, fusion genes, and mobile elements. The Cas9 can specifically eliminate wild-type sequences, and it also enables the enrichment and detection of small amounts of tumor DNA fragments among the highly heterogeneous fragments of wild-type DNA.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for simple, low-cost, and scalable diagnostics that can be widely deployed for rapid testing. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as a promising technology, but its implementation in clinical laboratories has been limited by the requirement of a separate amplification step prior to CRISPR-associated (Cas) enzyme-based detection. This article reports the discovery of two novel Cas12 enzymes (SLK9 and SLK5-2) that exhibit enzymatic activity at 60°C, which, when combined with loop-mediated isothermal amplification (LAMP), enable a real-time, single-step nucleic acid detection method [real-time SHERLOCK (real-time SLK)]. Real-time SLK was demonstrated to provide accurate results comparable to those from real-time quantitative RT-PCR in clinical samples, with 100% positive and 100% negative percent agreement. The method is further demonstrated to be compatible with direct testing (real-time SLK Direct) of samples from anterior nasal swabs, without the need for standard nucleic acid extraction. Lastly, SLK9 was combined with either Alicyclobacillus acidoterrestris AacCas12b or with SLK5-2 to generate a real-time, multiplexed CRISPR-based diagnostic assay for the simultaneous detection of SARS-CoV-2 and a human-based control in a single reaction, with sensitivity down to 5 copies/μL and a time to result of under 30 minutes.

Clinical laboratory implementation of next-generation sequencing (NGS)-based constitutional genetic testing has been rapid and widespread. In the absence of widely adopted comprehensive guidance, there remains substantial variability among laboratories in the practice of NGS. One issue of sustained discussion in the field is whether and to what extent orthogonal confirmation of genetic variants identified by NGS is necessary or helpful. The Association for Molecular Pathology Clinical Practice Committee convened the NGS Germline Variant Confirmation Working Group to assess current evidence regarding orthogonal confirmation and to establish recommendations for standardizing orthogonal confirmation practices to support quality patient care. On the basis of the results of a survey of the literature, a survey of laboratory practices, and subject expert matter consensus, eight recommendations are presented, providing a common framework for clinical laboratory professionals to develop or refine individualized laboratory policies and procedures regarding orthogonal confirmation of germline variants detected by NGS.

The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations for a minimum panel of variant alleles (tier 1) and an extended panel of variant alleles (tier 2) that will aid clinical laboratories when designing assays for PGx testing. The Association for Molecular Pathology PGx Working Group considered functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, and other technical considerations for PGx testing when developing these recommendations. The goal of this Working Group is to promote standardization of PGx gene/allele testing across clinical laboratories. This document will focus on clinical CYP3A4 and CYP3A5 PGx testing that may be applied to all CYP3A4- and CYP3A5-related medications. These recommendations are not to be interpreted as prescriptive but to provide a reference guide.