Genetic profiling and diagnostic strategies for patients with ectodermal dysplasias in Korea.

BACKGROUND: Ectodermal dysplasia (ED) is a rare genetic disorder that affects structures derived from the ectodermal germ layer. RESULTS: In this study, we analyzed the genetic profiles of 27 Korean patients with ED. Whole exome sequencing (WES) was performed on 23 patients, and targeted panel sequencing was conducted on the remaining 4 patients. Among the patients in the cohort, 74.1% (20/27) tested positive for ED. Of these positive cases, EDA and EDAR mutations were found in 80% (16/20). Notably, 23.1% (3/13) of EDA-positive cases exhibited copy number variations. Among the 23 patients who underwent WES, we conducted a virtual panel analysis of eight well-known genes, resulting in diagnoses for 56.5% (13/23) of the cases. Additionally, further analysis of approximately 5,000 OMIM genes identified four more cases, increasing the overall positivity rate by approximately 17%. These findings underscore the potential of WES for improving the diagnostic yield of ED. Remarkably, 94.1% of the patients manifesting the complete triad of ED symptoms (hair/skin/dental) displayed detectable EDA/EDAR mutations. In contrast, none of the 7 patients without these three symptoms exhibited EDA/EDAR mutations. CONCLUSIONS: When conducting molecular diagnostics for ED, opting for targeted sequencing of EDA/EDAR mutations is advisable for cases with classical symptoms, while WES is deemed an effective strategy for cases in which these symptoms are absent.

Changes in Daily life due to the COVID-19 Pandemic and Mental Health Status in Korean Adolescents.

This study assessed the relationship between daily life changes and mental health in Korean adolescents during the COVID-19 pandemic. Data from middle and high school students who responded to the relevant survey items the 2021 Korea Youth Risk Behavior Survey were analyzed (n = 53,868). The survey assessed changes in economic status, physical activity, skipping breakfast, drinking, smoking, and mental health, including stress, depression, generalized anxiety disorder, and suicidal ideation, due to the COVID-19 pandemic. The overall prevalence of perceived stress, depression, generalized anxiety disorder, and suicidal ideation was 38.6%, 26.4%, 12.2%, and 12.4%. Adolescents whose economic status worsened due to COVID-19 had a higher risk of perceived stress, depression, generalized anxiety disorder, and suicidal ideation. Changes in physical activity and breakfast consumption due to the COVID-19 pandemic were significantly associated with increases in perceived stress, depression, generalized anxiety disorder, and suicidal ideation. Adolescents who increased their alcohol consumption had the highest risk of perceived stress, depression, generalized anxiety disorder, and suicidal ideation. Changes in economic status and health behaviors caused by the COVID-19 pandemic had a negative effect on the mental health of Korean adolescents. These results can be used to identify adolescents at high risk of developing mental health problems.

Harnessing Institutionally Developed Clinical Targeted Sequencing to Improve Patient Survival in Breast Cancer: A Seven-Year Experience.

Purpose: Considering the high disease burden and unique features of Asian patients with breast cancer (BC), it is essential to have a comprehensive view of genetic characteristics in this population. An institutional targeted sequencing platform was developed through the Korea Research-Driven Hospitals project and was incorporated into clinical practice. This study explores the use of targeted next-generation sequencing (NGS) and its outcomes in patients with advanced/metastatic BC in the real world. Materials and Methods: We reviewed the results of NGS tests administered to BC patients using a customized sequencing platform - FiRST Cancer Panel (FCP) - over seven years. We systematically described clinical translation of FCP for precise diagnostics, personalized therapeutic strategies, and unraveling disease pathogenesis. Results: NGS tests were conducted on 548 samples from 522 patients with BC. 97.6% of tested samples harbored at least one pathogenic alteration. The common alterations included mutations in TP53(56.2%), PIK3CA(31.2%), GATA3(13.8%), BRCA2(10.2%), and amplifications of CCND1(10.8%), FGF19(10.0%), and ERBB2(9.5%). NGS analysis of ERBB2 amplification correlated well with HER2 immunohistochemistry and in situ hybridization. RNA panel analyses found potentially actionable and prognostic fusion genes. FCP effectively screened for potentially germline pathogenic/likely pathogenic mutation. 10.3% of BC patients received matched therapy guided by NGS, resulting in a significant overall survival advantage (p=0.022), especially for metastatic BCs. . Conclusion: Clinical NGS provided multifaceted benefits, deepening our understanding of the disease, improving diagnostic precision, and paving the way for targeted therapies. The concrete advantages of FCP highlight the importance of multi-gene testing for BC, especially for metastatic conditions.

Utility of Next-Generation Sequencing-Based Chimerism Analysis for Early Relapse Prediction following Allogenic Hematopoietic Cell Transplantation.

Chimerism monitoring following allogeneic hematopoietic cell transplantation (HCT) plays a pivotal role in evaluating engraftment status and identifying early indicators of relapse. Recent advancements in next-generation sequencing (NGS) technology have introduced AlloSeq HCT as a more sensitive alternative to short tandem repeat (STR) analysis. This study aimed to compare AlloSeq HCT with STR, focusing on the prediction of early relapse post-allogeneic HCT. Chimerism levels in 29 HCT recipients were assessed using both STR and NGS, employing a total of 125 whole blood or bone marrow aspirate samples (68 post-HCT and 57 pre-HCT samples from recipients or donors). AlloSeq HCT exhibited high concordance with STR and demonstrated the potential for early detection of chimeric changes, particularly at extremely low levels. The combined advantages of high sensitivity and automated data analysis offered by AlloSeq HCT substantiate its clinical adoption for effective chimerism monitoring.

Comparison of Measurable Residual Disease in Pediatric B-Lymphoblastic Leukemia Using Multiparametric Flow Cytometry and Next-Generation Sequencing.

Measurable residual disease (MRD) testing, a standard procedure in B-lymphoblastic leukemia (B-ALL) diagnostics, is assessed using multiparametric flow cytometry (MFC) and next-generation sequencing (NGS) analysis of immunoglobulin gene rearrangements. We evaluated the concordance between eight-color, two-tube MFC-MRD the LymphoTrack NGS-MRD assays using 139 follow-up samples from 54 pediatric patients with B-ALL. We also assessed the effect of hemodilution in MFC-MRD assays. The MRD-concordance rate was 79.9% (N=111), with 25 (18.0%) and 3 (2.2%) samples testing positive only by NGS-MRD (MFC-NGS+MRD) and MFC-MRD (MFC+NGS-MRD), respectively. We found a significant correlation in MRD values from total nucleated cells between the two methods (r=0.736 [0.647-0.806], P<0.001). The median MRD value of MFC-NGS+MRD samples was estimated to be 0.0012% (0.0001%-0.0263%) using the NGS-MRD assays. Notably, 14.3% of MFC-NGS+MRD samples showed NGS-MRD values below the limit of detection in the MFC-MRD assays. The percentages of hematogones detected in MFC-MRD assays significantly differed between the discordant and concordant cases (P<0.001). MFC and NGS-MRD assays showed relatively high concordance and correlation in MRD assessment, whereas the NGS-MRD assay detected MRD more frequently than the MFC-MRD assay in pediatric B-ALL. Evaluating the hematogone percentages can aid in assessing the impact of sample hemodilution.

Clinical Practice Guideline for Blood-based Circulating Tumor DNA Assays.

Circulating tumor DNA (ctDNA) has emerged as a promising tool for various clinical applications, including early diagnosis, therapeutic target identification, treatment response monitoring, prognosis evaluation, and minimal residual disease detection. Consequently, ctDNA assays have been incorporated into clinical practice. In this review, we offer an in-depth exploration of the clinical implementation of ctDNA assays. Notably, we examined existing evidence related to pre-analytical procedures, analytical components in current technologies, and result interpretation and reporting processes. The primary objective of this guidelines is to provide recommendations for the clinical utilization of ctDNA assays.

Acral Pigmentation in Peutz-Jeghers Syndrome: Dermoscopic Findings and Treatment with the Q-Switched Nd:YAG Laser.

Peutz-Jeghers syndrome (PJS; MIM 175200) is an autosomal dominant multiple-organ cancer syndrome. It is characterized by brown macules distributed in the perioral skin, oral mucosa, hands and feet, and hamartomatous gastrointestinal polyps that can eventually lead to intestinal obstruction, abdominal pain, bleeding, and anemia. Patients with PJS are at a higher risk of ovarian, testicular, breast, lung, and pancreatic cancers. This predisposition is due to the pathogenic variant in serine/threonine kinase 11 (STK11) gene located on chromosome 19p13.3. Here, we present the dermoscopic findings, histopathologic features of acral pigmentation, and DNA sequencing results of the patient with PJS. We also report a successful removal of acral pigmentation using the Q-switched Nd:YAG laser (QSNYL) treatment. Our results suggest that QSNYL therapy could be a treatment option for acral pigmentation in patients with PJS.

Distinct mutational pattern of T-cell large granular lymphocyte leukemia combined with pure red cell aplasia: low mutational burden of STAT3.

T-cell large granular lymphocyte leukemia (T-LGL) is often accompanied by pure red cell aplasia (PRCA). A high depth of next generation sequencing (NGS) was used for detection of the mutational profiles in T-LGL alone (n = 25) and T-LGL combined with PRCA (n = 16). Beside STAT3 mutation (41.5%), the frequently mutated genes included KMT2D (17.1%), TERT (12.2%), SUZ12 (9.8%), BCOR (7.3%), DNMT3A (7.3%), and RUNX1 (7.3%). Mutations of the TERT promoter showed a good response to treatment. 3 of 41 (7.3%) T-LGL patients with diverse gene mutations were revealed as T-LGL combined with myelodysplastic syndrome (MDS) after review of bone marrow slide. T-LGL combined with PRCA showed unique features (low VAF level of STAT3 mutation, low lymphocyte count, old age). Low ANC was detected in a STAT3 mutant with a low level of VAF, suggesting that even the low mutational burden of STAT3 is sufficient for reduction of ANC. In retrospective analysis of 591 patients without T-LGL, one MDS patient with STAT3 mutation was revealed to have subclinical T-LGL. T-LGL combined with PRCA may be classified as unique subtype of T-LGL. High depth NGS can enable sensitive detection of concomitant MDS in T-LGL. Mutation of the TERT promoter may indicate good response to treatment of T-LGL, thus, its addition to an NGS panel may be recommended.

Parallel Analysis of Pre- and Postoperative Circulating Tumor DNA and Matched Tumor Tissues in Resectable Pancreatic Ductal Adenocarcinoma: A Prospective Cohort Study.

BACKGROUND: Circulating tumor DNA (ctDNA) is a promising biomarker for early tumor detection and minimal residual disease (MRD) assessment in early-stage cancer, but quantifying minute amounts of ctDNA is challenging and well-designed studies on ctDNA in early-stage cancer are still lacking. Here, we adapted a sensitive next-generation sequencing (NGS) technology and performed parallel analysis of pre- and postoperative ctDNA and matched tumor tissues in a prospective cohort of patients with resectable pancreatic ductal adenocarcinoma (PDAC). METHODS: In total, 70 consecutive patients undergoing curative resection for resectable PDAC were enrolled. We performed integrated digital error suppression-enhanced cancer personalized profiling by deep sequencing NGS of triple-matched samples (pre/postoperative plasma cell-free DNA [cfDNA], tumor tissue, and genomic DNA) targeting 77 genes. RESULTS: Preoperative ctDNA was detected in 37.7% of the evaluable patients, with a median variant allele frequency of 0.09%. Twelve additional oncogenic mutations were detected exclusively in preoperative ctDNA but not in tissue. When quantitative concentrations of ctDNA were estimated in haploid genome equivalents per milliliter (hGE/mL), the risk of early recurrence was high in patients with postoperative ctDNA >1 hGE/mL. cfDNA variants from 24.5% of patients had features compatible with clonal hematopoiesis. CONCLUSIONS: An optimized NGS approach might add value beyond tissue analysis through the highly sensitive detection of minute amounts of ctDNA in resectable PDAC. Postoperative ctDNA concentration could be a tool for MRD assessment. Moreover, parallel analyses of matched tissues and leukocytes might be required to accurately detect clinically relevant ctDNA.

Clinical staging and genetic profiling of Korean patients with primary lymphedema using targeted gene sequencing.

Lymphedema is a progressive disease caused by lymphatic flow blockage in the lymphatic pathway. Primary (hereditary) lymphedema is caused by genetic mutations without secondary causes. We performed clinical profiling on Korean primary lymphedema patients based on their phenotypes using lymphoscintigraphy and made genetic diagnoses using a next-generation sequencing panel consisting of 60 genes known to be related to primary lymphedema and vascular anomalies. Of 27 patients included in this study, 14.8% of the patients had lymphedema of the upper extremities, 77.8% had lymphedema of the lower extremities and 7.4% had 4-limbs lymphedema. Based on the International Society of Lymphology staging, 14, 10, and 3 patients had stage 3, 2, and 1 lymphedema, respectively. Only one family was genetically confirmed to harbor likely pathogenic variants in CELSR1. The proband was carrying two likely pathogenic variants in CELSR1, while her symptomatic mother was confirmed to carry only one of the variants. Furthermore, two other variants of uncertain significance in CELSR1 were detected in other patients, making CELSR1 the most commonly altered gene in our study. The clinical and genetic profile of hereditary lymphedema reported here is the first such data series reported for South Korea.

Immunohistochemical Staining to Identify Concomitant Systemic Mastocytosis in Acute Myeloid Leukemia with RUNX1::RUNX1T1.

Systemic mastocytosis with associated hematological neoplasm (SM-AHN) poses diagnostic challenges because of the coexistence of atypical mast cell proliferation and hematological neoplasms. We assessed the presence of SM-AHN in patients with acute myeloid leukemia (AML) with RUNX1::RUNX1T1 from 2014 to 2020. Bone marrow (BM) samples were evaluated for mast cell aggregates using CD117 and CD25 immunohistochemical (IHC) staining. The KIT D816V variant burden at diagnosis and post induction was assessed using droplet digital PCR. Among 23 patients diagnosed as having AML with RUNX1::RUNX1T1, four (17.4%) were also diagnosed as having SM-AHN. No significant differences in clinical characteristics or overall survival (P=0.565) were observed between patients with or without SM-AHN, except for the presence of KIT variants (P=0.040). After induction therapy, IHC staining revealed the presence of mast cell aggregates in the BM, and the KIT D816V variant burden decreased with decreasing blast count and was similar in BM aspirates, smear slides, and sections. Concomitant SM-AHN was not infrequent in AML patients with RUNX1::RUNX1T1. This study showed the importance of CD117 and CD25 IHC staining after induction chemotherapy for SM-AHN screening, especially in patients with KIT variants.

Two wrongs sometimes do make a right: errors in aortic valve stenosis assessment by same-day Doppler echocardiography and 4D flow MRI.

This study aims to systematically verify if the simplified geometry and flow profile of the left ventricular outflow tract (LVOT) assumed in 2D echocardiography is appropriate while examining the utility of 4D flow MRI to assess valvular disease. This prospective study obtained same-day Doppler echocardiography and 4D flow MRI in 37 healthy volunteers (age: 51.9 ± 18.2, 20 females) and 7 aortic stenosis (AS) patients (age: 64.2 ± 9.6, 1 female). Two critical assumptions made in echocardiography for aortic valve area assessment were examined, i.e. the assumption of (1) a circular LVOT shape and (2) a flat velocity profile through the LVOT. 3D velocity and shape information obtained with 4D flow MRI was used as comparison. It was found that the LVOT area was lower (by 26.5% and 24.5%) and the velocity time integral (VTI) was higher (by 28.5% and 30.2%) with echo in the healthy and AS group, respectively. These competing errors largely cancelled out when examining individual and cohort averaged LVOT stroke volume. The LVOT area, VTI and stroke volume measured by echo and 4D flow MRI were 3.6 ± 0.7 vs. 4.9 ± 1.0 cm2 (p < 0.001), 21.2 ± 3.0 vs 15.2 ± 2.8 cm (p < 0.001), and 75.6 ± 15.6 vs 72.8 ± 14.1 ml (p = 0.3376), respectively. In the ensemble average of LVOT area and VTI, under- and over-estimation seem to compensate each other to result in a 'realistic' stroke volume. However, it is important to understand that this compensation may fail. 4D flow MRI provides a unique insight into this phenomenon.

Intronic LINE-1 insertion in SLCO1B3 as a highly prevalent cause of rotor syndrome in East Asian population.

Rotor syndrome is caused by digenic loss-of-function variants in SLCO1B1 and SLCO1B3 but only a few studies have reported co-occurring inactivating variants from both genes. A rotor syndrome-causing long interspersed element-1 (LINE-1) insertion in SLCO1B3 had been reported to be highly prevalent in the Japanese population but there has been no additional report. In spite of its known association with various human diseases, LINE-1 is hard to detect with current sequencing technologies. In this study, we aimed to devise a method to screen the LINE-1 insertion variant and investigate the frequency of this variant in various populations. A chimeric sequence, that was generated by concatenating the reference sequence at the junction and a part of inserted LINE-1 sequence, was searched from 725 raw sequencing data files. In cases containing the chimeric sequence, confirmatory long-range PCR and gap-PCR were performed. In total, 95 (13.1%) of 725 patients were positive for the chimeric sequence, and all were confirmed to have the SLCO1B3 LINE-1 insertion by PCR-based tests. The same chimeric sequence was searched from the 1000 Genomes Project data repository and the carrier frequency was remarkably high in the East Asian populations (10.1%), especially in Southern Han Chinese (18.5%), but almost absent in other populations. This SLCO1B3 LINE-1 insertion should be screened in a population-specific manner under suspicion of Rotor syndrome and the methods proposed in this study would enable this in a simple way.

SnackVar: An Open-Source Software for Sanger Sequencing Analysis Optimized for Clinical Use.

Despite the wide application of next-generation sequencing, Sanger sequencing still plays a necessary role in clinical laboratories. However, recent developments in the field of bioinformatics have focused mostly on next-generation sequencing, while tools for Sanger sequencing have shown little progress. In this study, SnackVar (https://github.com/Young-gonKim/SnackVar, last accessed June 22, 2020), a novel graphical user interface-based software for Sanger sequencing, was developed. All types of variants, including heterozygous insertion/deletion variants, can be identified by SnackVar with minimal user effort. The featured reference sequences of all of the genes are prestored in SnackVar, allowing for detected variants to be precisely described based on coding DNA references according to the nomenclature of the Human Genome Variation Society. Among 88 previously reported variants from four insertion/deletion-rich genes (BRCA1, APC, CALR, and CEBPA), the result of SnackVar agreed with reported results in 87 variants [98.9% (93.0%; 99.9%)]. The cause of one incorrect variant calling was proven to be erroneous base callings from poor-quality trace files. Compared with commercial software, SnackVar required less than one-half of the time taken for the analysis of a selected set of test cases. We expect SnackVar to be a cost-effective option for clinical laboratories performing Sanger sequencing.