Genetic diagnosis of inborn errors of immunity using clinical exome sequencing

Inborn errors of immunity (IEI) include a variety of heterogeneous genetic disorders in which defects in the immune system lead to an increased susceptibility to infections and other complications. Accurate, prompt diagnosis of IEI is crucial for treatment plan and prognostication. In this study, clinical utility of clinical exome sequencing (CES) for diagnosis of IEI was evaluated. For 37 Korean patients with suspected symptoms, signs, or laboratory abnormalities associated with IEI, CES that covers 4,894 genes including genes related to IEI was performed. Their clinical diagnosis, clinical characteristics, family history of infection, and laboratory results, as well as detected variants, were reviewed. With CES, genetic diagnosis of IEI was made in 15 out of 37 patients (40.5%). Seventeen pathogenic variants were detected from IEI-related genes, BTK, UNC13D, STAT3, IL2RG, IL10RA, NRAS, SH2D1A, GATA2, TET2, PRF1, and UBA1, of which four variants were previously unreported. Among them, somatic causative variants were identified from GATA2, TET2, and UBA1. In addition, we identified two patients incidentally diagnosed IEI by CES, which was performed to diagnose other diseases of patients with unrecognized IEI. Taken together, these results demonstrate the utility of CES for the diagnosis of IEI, which contributes to accurate diagnosis and proper treatments

A Comparative Study for Detection of EGFR Mutations in Plasma Cell-Free DNA in Korean Clinical Diagnostic Laboratories

Liquid biopsies to genotype the epidermal growth factor receptor (EGFR) for targeted therapy have been implemented in clinical decision-making in the field of lung cancer, but harmonization of detection methods is still scarce among clinical laboratories. We performed a pilot external quality assurance (EQA) scheme to harmonize circulating tumor DNA testing among laboratories. For EQA, we created materials containing different levels of spiked cell-free DNA (cfDNA) in normal plasma. The limit of detection (LOD) of the cobas® EGFR Mutation Test v2 (Roche Molecular Systems) was also evaluated. From November 2016 to June 2017, seven clinical diagnostic laboratories participated in the EQA program. The majority (98.94%) of results obtained using the cobas assay and next-generation sequencing (NGS) were acceptable. Quantitative results from the cobas assay were positively correlated with allele frequencies derived from digital droplet PCR measurements and showed good reproducibility among laboratories. The LOD of the cobas assay was 5~27 copies/mL for p.E746_A750del (exon 19 deletion), 35~70 copies/mL for p.L858R, 18~36 copies/mL for p.T790M, and 15~31 copies/mL for p.A767_V769dup (exon 20 insertion). Deep sequencing of materials (>100,000X depth of coverage) resulted in detection of low-level targets present at frequencies of 0.06~0.13%. Our results indicate that the cobas assay is a reliable and rapid method for detecting EGFR mutations in plasma cfDNA. Careful interpretation is particularly important for p.T790M detection in the setting of relapse. Individual laboratories should optimize NGS performance to maximize clinical utility

Development and validation of sensitive BCR::ABL1 fusion gene quantitation using next-generation sequencing

Abstract Background BCR::ABL1 fusion has significant prognostic value and is screened for chronic myeloid leukemia (CML) disease monitoring as a part of routine molecular testing. To overcome the limitations of the current standard real-time quantitative polymerase chain reaction (RQ-PCR), we designed and validated a next-generation sequencing (NGS)-based assay to quantify BCR::ABL1 and ABL1 transcript copy numbers. Methods After PCR amplification of the target sequence, deep sequencing was performed using an Illumina Nextseq 550Dx sequencer and in-house–designed bioinformatics pipeline. The Next-generation Quantitative sequencing (NQ-seq) assay was validated for its analytical performance, including precision, linearity, and limit of detection, using serially diluted control materials. A comparison with conventional RQ-PCR was performed with 145 clinical samples from 77 patients. Results The limit of detection of the NQ-seq was the molecular response (MR) 5.6 [BCR::ABL1 0.00028% international scale (IS)]. The NQ-seq exhibited excellent precision and linear range from MR 2.0 to 5.0. The IS value from the NQ-seq was highly correlated with conventional RQ-PCR. Conclusions We conclude that the NQ-seq is an effective tool for monitoring BCR::ABL1 transcripts in CML patients with high sensitivity and reliability. Prospective assessment of the unselected large series is required to validate the clinical impact of this NGS-based monitoring strategy

Emergence of multidrug-resistant Providencia rettgeri isolates co-producing NDM-1 carbapenemase and PER-1 extended-spectrum β-lactamase causing a first outbreak in Korea

Abstract Background Nosocomial outbreak due to carbapenem-resistant Enterobacteriaceae has become serious challenge to patient treatment and infection control. We describe an outbreak due to a multidrug-resistant Providencia rettgeri from January 2016 to January 2017 at a University Hospital in Seoul, Korea. Methods A total of eight non-duplicate P. rettgeri isolates were discovered from urine samples from eight patients having a urinary catheter and admitted in a surgical intensive care unit. The β-lactamase genes were identified using polymerase chain reaction and direct sequencing, and strain typing was done with pulsed-field gel electrophoresis (PFGE). Results All isolates showed high-level resistance to extended-spectrum cephalosporins, aztreonam, meropenem, ertapenem, ciprofloxacin, and amikacin. They harbored the bla NDM-1 carbapenemase and the bla PER-1 type extended-spectrum β-lactamases genes. PFGE revealed that all isolates from eight patients were closely related strains. Conclusions The 13-month outbreak ended following reinforcement of infection control measures, including contact isolation precautions and environmental disinfection. This is the first report of an outbreak of a P. rettgeri clinical isolates co-producing NDM-1 and PER-1 β-lactamase

Precision Medicine through Next-Generation Sequencing in Inherited Eye Diseases in a Korean Cohort

In this study, we investigated medically or surgically actionable genes in inherited eye disease, based on clinical phenotype and genomic data. This retrospective consecutive case series included 149 patients with inherited eye diseases, seen by a single pediatric ophthalmologist, who underwent genetic testing between 1 March 2017 and 28 February 2018. Variants were detected using a target enrichment panel of 429 genes and known deep intronic variants associated with inherited eye disease. Among 149 patients, 38 (25.5%) had a family history, and this cohort includes heterogeneous phenotype including anterior segment dysgenesis, congenital cataract, infantile nystagmus syndrome, optic atrophy, and retinal dystrophy. Overall, 90 patients (60.4%) received a definite molecular diagnosis. Overall, NGS-guided precision care was provided to 8 patients (5.4%). The precision care included cryotherapy to prevent retinal detachment in COL2A1 Stickler syndrome, osteoporosis management in patients with LRP5-associated familial exudative vitreoretinopathy, and avoidance of unnecessary phlebotomy in hyperferritinemia-cataract syndrome. A revision of the initial clinical diagnosis was made in 22 patients (14.8%). Unexpected multi-gene deletions and dual diagnosis were noted in 4 patients (2.7%). We found that precision medical or surgical managements were provided for 8 of 149 patients (5.4%), and multiple locus variants were found in 2.7% of cases. These findings are important because individualized management of inherited eye diseases can be achieved through genetic testing

Identification of cell morphology parameters from automatic hematology analyzers to predict the peripheral blood CD34-positive cell count after mobilization

<div><p>Optimal timing of apheresis initiation is important for maximizing the hematopoietic stem cell (HSC) yield. This study aimed to identify useful parameters from automatic hematology analyzers for predicting the peripheral blood CD34+ cell count after mobilization. We prospectively enrolled 53 healthy donors and 72 patients, and evaluated 43 cell morphology parameters from Unicel DxH800 (Beckman Coulter, USA) and Advia 2120i (Siemens Healthcare Diagnostics, USA). The correlation of each parameter with the CD34+ cell count in pre-apheresis blood samples was analyzed. The delta neutrophil index (DNI) from Advia 2120i, standard deviation of volume of neutrophils and monocytes (SD-V-NE and SD-V-MO), standard deviation of conductivity of neutrophils and monocytes (SD-C-NE and SD-C-MO), mean conductivity of neutrophils and monocytes (MN-C-NE and MN-C-MO), and standard deviation of axial light loss of neutrophils and monocytes (SD-AL2-NE and SD-AL2-MO) from DxH800 showed significant correlations with the CD34+ cell count. SD-V-NE, SD-C-NE, and SD-C-MO showed good or fair area under the curve values for the prediction of the CD34+ cell count. SD-V-NE, SD-C-NE, and SD-C-MO from DxH800 will provide rapid, useful information for the initiation of apheresis after mobilization.</p></div

Correction: A novel association between relaxin receptor polymorphism and hematopoietic stem cell yield after mobilization.

[This corrects the article DOI: 10.1371/journal.pone.0179986.]