Genomic technologies for detecting structural variations in hematologic malignancies.

Genomic structural variations in myeloid, lymphoid, and plasma cell neoplasms can provide key diagnostic, prognostic, and therapeutic information while elucidating the underlying disease biology. Several molecular diagnostic approaches play a central role in evaluating hematological malignancies. Traditional cytogenetic diagnostic assays, such as chromosome banding and fluorescence in situ hybridization, are essential components of the current diagnostic workup that guide clinical care for most hematologic malignancies. However, each assay has inherent limitations, including limited resolution for detecting small structural variations and low coverage, and can only detect alterations in the target regions. Recently, the rapid expansion and increasing availability of novel and comprehensive genomic technologies have led to their use in clinical laboratories for clinical management and translational research. This review aims to describe the clinical relevance of structural variations in hematologic malignancies and introduce genomic technologies that may facilitate personalized tumor characterization and treatment.

Performance evaluation of the SMG HHV-6 Q Real-Time PCR Kit for quantitative detection and differentiation of human herpesvirus 6A and 6B.

The aim of this study was to compare the performance of the newly developed SMG HHV-6 Q Real-Time PCR Kit (SMG assay) with the RealStar HHV-6 PCR Kit (RealStar assay). The analytical sensitivity and specificity, linearity, and precision of the SMG assay were evaluated. The clinical performance of the SMG assay was assessed and compared with that of the RealStar assay using 207 clinical specimens (HHV-6A positive, n = 51; HHV-6B positive, n = 64; HHV-6A/B negative, n = 92). The limit of detection of the SMG assay was 2.92 log10 copies/mL for HHV-6A DNA and 2.88 log10 copies/mL for HHV-6B DNA. The linear range was determined to be 3.40-9.00 log10 copies/mL for both viruses. Intra- and inter-assay variability were below 5% at concentrations ranging from 4 to 9 log10 copies/mL. No cross-reactivity was observed with the 25 microorganisms included in the specificity panel. The clinical sensitivity and specificity of the SMG and RealStar assays compared to in-house polymerase chain reaction and sequencing were as follows: SMG assay, 98.0% and 100% for HHV-6A DNA, respectively, and 96.9% and 100% for HHV-6B DNA, respectively; RealStar assay, 98.0% and 100% for HHV-6A DNA, respectively, and 90.6% and 100% for HHV-6B DNA, respectively. The correlation coefficients between viral loads measured by the two assays were 0.948 and 0.975, with mean differences of 0.62 and 0.32 log10 copies/mL for HHV-6A and HHV-6B DNA, respectively. These results demonstrate that the SMG assay is a sensitive and reliable tool for the quantitative detection and differentiation of HHV-6A and HHV-6B DNA.IMPORTANCEQuantitative real-time PCR (qPCR) that can distinguish between HHV-6A and HHV-6B DNA is recommended for diagnosis of active infection. The SMG HHV-6 Q Real-Time PCR Kit (SMG assay) is a newly developed qPCR assay that can differentiate between HHV-6A and HHV-6B DNA; however, little is known about its performance. In this study, we assessed the performance of the SMG assay and compared it with that of a commercially available qPCR assay, the RealStar HHV-6 PCR Kit (RealStar assay). The SMG assay demonstrated excellent analytical sensitivity and specificity, precision, and linearity. Furthermore, the viral loads measured by the SMG assay were highly correlated with those measured by the RealStar assay. Our results suggest that the SMG assay is a useful diagnostic tool for quantitative detection and differentiation of HHV-6A and HHV-6B DNA.

Communication: Evaluation of the Humasis COVID-19 Antigen Rapid Diagnostic Test for the Diagnosis of SARS-CoV-2 Infection.

OBJECTIVE: We assessed the performance of the Humasis COVID-19 AgHS Test (Humasis, Korea), a novel antigen rapid diagnostic test (Ag-RDT) based on lateral flow immunoassay. METHODS: 85 SARS-CoV-2-positive and 155 SARS-CoV-2-negative nasopharyngeal swab specimens confirmed by rRT-PCR were tested using the Humasis and PBCheck Ag-RDTs. The analytical specificity of the Humasis Ag-RDT was evaluated using 27 strains of human respiratory pathogens. RESULTS: The overall sensitivity and specificity were 72.9% and 99.4% for the Humasis Ag-RDT and 64.7% and 100% for the PBCheck Ag-RDT, respectively. The sensitivity for specimens with Ct≤25 was 100% for both Ag-RDTs. The Humasis Ag-RDT showed no cross-reactivity with other respiratory pathogens. CONCLUSION: Our data suggests that the Humasis Ag-RDT can be a useful diagnostic tool for the detection of SARS-CoV-2 infection.

Enhancing the Reliability of PMP22 Copy Number Variation Detection with an Inherited Peripheral Neuropathy Panel.

The utility of the next-generation sequencing (NGS) panel could be increased in hereditary peripheral neuropathies, given that the duplication of PMP22 is a major abnormality. In the present study, the analytical performance of an algorithm for detecting PMP22 copy number variation (CNV) from the NGS panel data was evaluated. The NGS panel covers 141 genes, including PMP22 and five genes within 1.5-megabase duplicated region at 17p11.2. CNV calling was performed using a laboratory-developed algorithm. Among the 92 cases subjected to targeted NGS panel from March 2018 to January 2021, 26 were suggestive of PMP22 CNV. Multiplex ligation-dependent probe amplification analysis was performed in 58 cases, and the results were 100% concordant with the NGS data (23 duplications, 2 deletions, and 33 negatives). Analytical performance of the pipeline was further validated by another blind data set, including 14 positive and 20 negative samples. Reliable detection of PMP22 CNV was possible by analyzing not only PMP22 but also the adjacent genes within the 1.5-megabase region of 17p11.2. On the basis of the high accuracy of CNV calling for PMP22, the testing strategy for diagnosis of peripheral polyneuropathies could be simplified by reducing the need for multiplex ligation-dependent probe amplification.

CRYAB stop-loss variant causes rare syndromic dilated cardiomyopathy with congenital cataract: expanding the phenotypic and mutational spectrum of alpha-B crystallinopathy.

Missense mutations in the alpha-B crystallin gene (CRYAB) have been reported in desmin-related myopathies with or without cardiomyopathy and have also been reported in families with only a cataract phenotype. Dilated cardiomyopathy (DCM) is a disorder with a highly heterogeneous genetic etiology involving more than 60 causative genes, hindering genetic diagnosis. In this study, we performed whole genome sequencing on 159 unrelated patients with DCM and identified an unusual stop-loss pathogenic variant in NM_001289808.2:c.527A>G of CRYAB in one patient. The mutant alpha-B crystallin protein is predicted to have an extended strand with addition of 19 amino acid residues, p.(Ter176TrpextTer19), which may contribute to aggregation and increased hydrophobicity of alpha-B crystallin. The proband, diagnosed with DCM at age 32, had a history of bilateral congenital cataracts but had no evidence of myopathy or associated symptoms. He also has a 10-year-old child diagnosed with bilateral congenital cataracts with the same CRYAB variant. This study expands the mutational spectrum of CRYAB and deepens our understanding of the complex phenotypes of alpha-B crystallinopathies.

Overcoming challenges associated with identifying FBN1 deep intronic variants through whole-genome sequencing.

BACKGROUND: Marfan syndrome (MFS), caused by pathogenic variants of FBN1 (fibrillin-1), is a systemic connective tissue disorder with variable phenotypes and treatment responsiveness depending on the variant. However, a significant number of individuals with MFS remain genetically unexplained. In this study, we report novel pathogenic intronic variants in FBN1 in two unrelated families with MFS. METHODS: We evaluated subjects with suspected MFS from two unrelated families using Sanger sequencing or multiplex ligation-dependent probe amplification of FBN1 and/or panel-based next-generation sequencing. As no pathogenic variants were identified, whole-genome sequencing was performed. Identified variants were analyzed by reverse transcription-PCR and targeted sequencing of FBN1 mRNA harvested from peripheral blood or skin fibroblasts obtained from affected probands. RESULTS: We found causative deep intronic variants, c.6163+1484A>T and c.5788+36C>A, in FBN1. The splicing analysis revealed an insertion of in-frame or out-of-frame intronic sequences of the FBN1 transcript predicted to alter function of calcium-binding epidermal growth factor protein domain. Family members carrying c.6163+1484A>T had high systemic scores including prominent skeletal features and aortic dissection with lesser aortic dilatation. Family members carrying c.5788+36C>A had more severe aortic root dilatation without aortic dissection. Both families had ectopia lentis. CONCLUSION: Variable penetrance of the phenotype and negative genetic testing in MFS families should raise the possibility of deep intronic FBN1 variants and the need for additional molecular studies. This study expands the mutation spectrum of FBN1 and points out the importance of intronic sequence analysis and the need for integrative functional studies in MFS diagnosis.

WBC YOLO-ViT: 2 Way - 2 stage white blood cell detection and classification with a combination of YOLOv5 and vision transformer.

Accurate detection and classification of white blood cells, otherwise known as leukocytes, play a critical role in diagnosing and monitoring various illnesses. However, conventional methods, such as manual classification by trained professionals, must be revised in terms of accuracy, efficiency, and potential bias. Moreover, applying deep learning techniques to detect and classify white blood cells using microscopic images is challenging owing to limited data, resolution noise, irregular shapes, and varying colors from different sources. This study presents a novel approach integrating object detection and classification for numerous type-white blood cell. We designed a 2-way approach to use two types of images: WBC and nucleus. YOLO (fast object detection) and ViT (powerful image representation capabilities) are effectively integrated into 16 classes. The proposed model demonstrates an exceptional 96.449% accuracy rate in classification.

Optimization of extraction-free protocols for SARS-CoV-2 detection using a commercial rRT-PCR assay.

In the ongoing global fight against coronavirus disease 2019 (COVID-19), the sample preparation process for real-time reverse transcription polymerase chain reaction (rRT-PCR) faces challenges due to time-consuming steps, labor-intensive procedures, contamination risks, resource demands, and environmental implications. However, optimized strategies for sample preparation have been poorly investigated, and the combination of RNase inhibitors and Proteinase K has been rarely considered. Hence, we investigated combinations of several extraction-free protocols incorporating heat treatment, sample dilution, and Proteinase K and RNase inhibitors, and validated the effectiveness using 120 SARS-CoV-2 positive and 62 negative clinical samples. Combining sample dilution and heat treatment with Proteinase K and RNase inhibitors addition exhibited the highest sensitivity (84.26%) with a mean increase in cycle threshold (Ct) value of + 3.8. Meanwhile, combined sample dilution and heat treatment exhibited a sensitivity of 79.63%, accounting for a 38% increase compared to heat treatment alone. Our findings highlight that the incorporation of Proteinase K and RNase inhibitors with sample dilution and heat treatment contributed only marginally to the improvement without yielding statistically significant differences. Sample dilution significantly impacts SARS-CoV-2 detection, and sample conditions play a crucial role in the efficiency of extraction-free methods. Our findings may provide insights for streamlining diagnostic testing, enhancing its accessibility, cost-effectiveness, and sustainability.

Clinical, Mutational, and Transcriptomic Characteristics in Elderly Korean Individuals With Clonal Hematopoiesis Driver Mutations.

Background: Clonal hematopoiesis of indeterminate potential (CHIP), which is defined as the presence of blood cells originating from somatically mutated hematopoietic stem cells, is common among the elderly and is associated with an increased risk of hematologic malignancies. We investigated the clinical, mutational, and transcriptomic characteristics in elderly Korean individuals with CHIP mutations. Methods: We investigated CHIP in 90 elderly individuals aged ≥60 years with normal complete blood counts at a tertiary-care hospital in Korea between June 2021 and February 2022. Clinical and laboratory data were prospectively obtained. Targeted next-generation sequencing of 49 myeloid malignancy driver genes and massively parallel RNA sequencing were performed to explore the molecular spectrum and transcriptomic characteristics of CHIP mutations. Results: We detected 51 mutations in 10 genes in 37 (41%) of the study individuals. CHIP prevalence increased with age. CHIP mutations were observed with high prevalence in DNMT3A (26 individuals) and TET2 (eight individuals) and were also found in various other genes, including KDM6A, SMC3, TP53, BRAF, PPM1D, SRSF2, STAG1, and ZRSR2. Baseline characteristics, including age, confounding diseases, and blood cell parameters, showed no significant differences. Using mRNA sequencing, we characterized the altered gene expression profile, implicating neutrophil degranulation and innate immune system dysregulation. Conclusions: Somatic CHIP driver mutations are common among the elderly in Korea and are detected in various genes, including DNMT3A and TET2. Our study highlights that chronic dysregulation of innate immune signaling is associated with the pathogenesis of various diseases, including hematologic malignancies.

Dikkopf-1 promotes matrix mineralization of osteoblasts by regulating Ca+-CAMK2A- CREB1 pathway.

Dickkopf-1 (DKK1) is a secreted protein that acts as an antagonist of the canonical WNT/ß-catenin pathway, which regulates osteoblast differentiation. However, the role of DKK1 on osteoblast differentiation has not yet been fully clarified. Here, we investigate the functional role of DKK1 on osteoblast differentiation. Primary osteoprogenitor cells were isolated from human spinal bone tissues. To examine the role of DKK1 in osteoblast differentiation, we manipulated the expression of DKK1, and the cells were differentiated into mature osteoblasts. DKK1 overexpression in osteoprogenitor cells promoted matrix mineralization of osteoblast differentiation but did not promote matrix maturation. DKK1 increased Ca+ influx and activation of the Ca+/calmodulin-dependent protein kinase II Alpha (CAMK2A)-cAMP response element-binding protein 1 (CREB1) and increased translocation of p-CREB1 into the nucleus. In contrast, stable DKK1 knockdown in human osteosarcoma cell line SaOS2 exhibited reduced nuclear translocation of p-CREB1 and matrix mineralization. Overall, we suggest that manipulating DKK1 regulates the matrix mineralization of osteoblasts by Ca+-CAMK2A-CREB1, and DKK1 is a crucial gene for bone mineralization of osteoblasts. [BMB Reports 2022; 55(12): 627-632].

Gardnerella vaginalis in Recurrent Urinary Tract Infection Is Associated with Dysbiosis of the Bladder Microbiome.

Recent studies on the urine microbiome have highlighted the importance of the gut-vagina-bladder axis in recurrent urinary tract infection (rUTI). In particular, the role of Gardnerella as a covert pathogen that activates E. coli in animal experiments has been reported. Herein, we conducted a human bladder microbiome study to investigate the effect of Gardnerella on rUTI. Urine 16S ribosomal RNA gene sequencing via transurethral catheterization was conducted in the normal control group (NC) (n = 18) and rUTI group (n = 78). The positive detection rate of Gardnerella species did not differ between the NC and rUTI groups (22.2% vs. 18.0%, p = 0.677). In addition, the Gardnerella-positive NC and Gardnerella-positive rUTI groups showed similar levels of microbiome diversity. The Gardnerella-positive group was categorized into three subgroups: the Escherichia-dominant group, Gardnerella-dominant group, and Lactobacillus-dominant group. All of the Escherichia-dominant groups were associated with rUTI. The Gardnerella-dominant or Lactobacillus-dominant groups expressed rUTI with symptoms when risk factors such as the degree of Gardnerella proliferation or causative agents of bacterial vaginosis were present. The presence of Gardnerella in the urine is considered to be related to rUTI depending on other risk factors. New guideline recommendations regarding antibiotic selection based on a novel method to detect the cause of rUTI may be required to reduce antibiotic resistance.

Clinical Manifestations and Genetic Analysis of 5 Korean Choroideremia Patients Initially Diagnosed With Retinitis Pigmentosa.

BACKGROUND: To investigate the clinical findings of choroideremia patients and perform genetic analysis by whole-exome sequencing (WES). METHODS: A total of 94 patients initially diagnosed with retinitis pigmentosa (RP) at another hospital, and who visited our hospital for genetic analysis by WES, were included in the study, along with 64 family members. All subjects underwent comprehensive ophthalmic evaluation, including best-corrected visual acuity, slit lamp examination, fundus photography, fundus autofluorescence (FAF), fluorescein angiography (FAG), visual field (VF), electroretinogram (ERG), and optical coherence tomography (OCT). RESULTS: In six male patients with suspected choroideremia, extensive retinal pigment epithelium (RPE) and severe loss of choroid were observed in the fundus, but not in the macula. CHM gene mutation was confirmed in five patients. A novel single nucleotide variant at a splice site was observed in one patient. OCT showed marked thinning of the outernuclear layer and choroid, except in the macula. FAF showed a small area of hyperfluorescence in the posterior pole. In addition, characteristic interlaminar bridges were observed in four patients. On FAG, hypofluorescence was seen up to the far-peripheral retina in five patients. CONCLUSION: Of the 94 patients initially diagnosed with RP, CHM mutation was identified in five (5.3%) by WES. Choroideremia should be considered as a differential diagnosis of RP. WES would be useful for identifying the causes of hereditary retinal disease.

WNT16 elevation induced cell senescence of osteoblasts in ankylosing spondylitis.

BACKGROUND: WNT16 is critical for bone homeostasis, but the effect of WNT16 in ankylosing spondylitis (AS) is still unknown. Here, we investigated whether WNT16 influences bone formation and pathophysiological changes of AS in an in vitro model. METHODS: The bone tissue from the facet joints was obtained from seven disease control and seven AS patients. Primary osteoprogenitor cells of the facet joints were isolated using an outgrowth method. Isolated osteoprogenitor cells from both control and AS tissues were analyzed by microarray, RT-qPCR, immunoblotting, and immunohistochemistry. The bone-forming activity of osteoprogenitor cells was assessed by various in vitro assays. ß-galactosidase staining and senescence-associated secretory phenotype (SASP) using RT-qPCR were used to assess cell senescence. RESULTS: In microarray analysis, WNT16 expression was significantly elevated in AS osteoprogenitor cells compared to the control. We also validated that WNT16 expression was elevated in AS-osteoprogenitor cells and human AS-bone tissues. WNT16 treatment inhibited bone formation in AS-osteoprogenitor cells but not in the control. Intriguingly, AS-osteoprogenitor cells were stained markedly with ß-galactosidase for cell senescence in WNT16 treatment. Furthermore, in an H2O2 stress-induced premature senescence condition, WNT16 treatment increased cell senescence in AS-osteoprogenitor cells and WNT16 treatment under the H2O2 stress condition showed an increase in p21 protein and SASP mRNA expression. The WNT16-induced SASP expression in AS-osteoprogenitor cells was reduced in WNT16 knockdown cultures. CONCLUSION: WNT16 is highly expressed in AS and WNT16 treatment facilitated cell senescence in AS-osteoprogenitor cells during osteoblast differentiation accompanied by suppression of bone formation. The identified role of WNT16 in AS could influence bone loss in AS patients.

A Rare Case of Essential Thrombocythemia with Coexisting JAK2 and MPL Driver Mutations.

Philadelphia-negative (Ph-) classical myeloproliferative neoplasms (MPNs) include polycythemia vera, essential thrombocythemia (ET), and primary myelofibrosis. Somatic driver mutations in the JAK2, CALR, and MPL genes serve as major diagnostic criteria of the Ph- MPNs and these mutations occur in a mutually exclusive manner. In this report, we describe the first case of ET harboring double mutations in JAK2 V617F and MPL. For MPL, the patient had multiple clones of MPL mutations: c.1543_1546delinsAGGG (p.Trp515_Gln516delinsArgGlu) and c.1546C>G (p.Gln516Glu). The JAK2 V617F allele burden in our patient is very low (4%) compared to the relatively high (17%-78%) allele frequency of MPL mutations. The low JAK2 mutant burden might be explained by preexisting clonal hematopoiesis before overt signs of MPNs, followed by the acquisition of a second oncogenic mutation of CALR or MPL leading to the MPN phenotype. This highlights that screening for a second driver mutation should be considered in patients with a low JAK2 mutant burden by reporting a 57-year-old Korean man with ET.

Rare Cases of PLOD1-Related Kyphoscoliotic Ehlers-Danlos Syndrome in a Korean Family Identified by Next Generation Sequencing.

Kyphoscoliotic Ehlers-Danlos syndrome (kEDS) is an autosomal recessive connective tissue disorder characterized by muscular hypotonia, hyperextensible skin, skin fragility, joint hypermobility, and progressive kyphoscoliosis. The disorder results from a deficiency of the enzyme collagen lysyl hydroxylase 1 due to mutations in the gene PLOD1. We describe the rare cases of kEDS in Korean siblings with two novel compound heterozygous variants, c.926_934del (p.Leu309_Leu311del) and c.2170_2172del (p.Phe724del) in the PLOD1 gene. They had congenital hypotonia, joint laxity, skin hyperextensibility, Marfanoid habitus, high myopia and atrophic scarring. The younger sibling had an early-onset progressive kyphoscoliosis, while the older sibling showed mild scoliosis during childhood. Intrafamilial variability of the clinical severity and age of kyphoscoliosis onset observed in our cases.