In the era of precision medicine, pharmacogenetics has substantial potential for addressing inter-individual variability in drug responses. Although pharmacogenetics has been a research focus for many years, resulting in the establishment of several formal guidelines, its clinical implementation remains limited to several gene-drug combinations in most countries, including Korea. The main causes of delayed implementation are technical challenges in genotyping and knowledge gaps among healthcare providers; therefore, clinical laboratories play a critical role in the timely implementation of pharmacogenetics. This paper presents an update of the Clinical Pharmacogenetic Testing and Application guidelines issued by the Korean Society for Laboratory Medicine and aims to provide the necessary information for clinical laboratories planning to implement or expand their pharmacogenetic testing. Current knowledge regarding nomenclature, gene-drug relationships, genotyping technologies, testing strategies, methods for clinically relevant information delivery, QC, and reimbursements has been curated and described in this guideline.

Background. TP53 mutations are associated with poor prognosis in myelodysplastic neoplasm (MDS) and AML. The updated 5th WHO classification and International Consensus Classification (ICC) categorize TP53-mutated MDS and AML as unique entities. We conducted a multicenter study in Korea to investigate the characteristics of TP53-mutated MDS and AML, focusing on diagnostic aspects based on updated classifications. Methods. This study included patients aged >= 18 yrs who were diagnosed as having MDS (N=1,244) or AML (N=2,115) at six institutions. The results of bone marrow examination, cytogenetic studies, and targeted next-generation sequencing, including TP53, were collected and analyzed. Results. TP53 mutations were detected in 9.3% and 9.2% of patients with MDS and AML, respectively. Missense mutation was the most common, with hotspot codons R248/R273/G245/Y220/R175/C238 accounting for 25.4% of TP53 mutations. Ten percent of patients had multiple TP53 mutations, and 78.4% had a complex karyotype. The median variant allele frequency (VAF) of TP53 mutations was 41.5%, with a notable difference according to the presence of a complex karyotype. According to the 5th WHO classification and ICC, the multi-hit TP53 mutation criteria were met in 58.6% and 75% of MDS patients, respectively, and the primary determinants were a TP53 VAF >50% for the 5th WHO classification and the presence of a complex karyotype for the ICC. Conclusions. Collectively, we elucidated the molecular genetic characteristics of patients with TP53-mutated MDS and AML, highlighting key factors in applying TP53 mutation-related criteria in updated classifications, which will aid in establishing diagnostic strategies.

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 7 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. Ninety-seven point six percentage 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 human epidermal growth factor receptor 2 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. Ten point three percent 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.

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.

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.

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 indepth 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.

Jisoo G. Kim, M.D., Sun Mi Choi, M.D., Hobin Sung, B.S., Yongsook Park, M.S., Sung Im Cho, M.S., Jee-Soo Lee, M.D., Man Jin Kim, M.D., Sung Sup Park, M.D., Moon-Woo Seong, M.D.. Lab Med Online 2024;14:127-32. https://doi.org/10.47429/lmo.2024.14.2.127

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.

Background Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors in multiple endocrine organs, caused by variants in the MEN1 gene. This study analyzed the clinical and genetic features of MEN1 in a Korean cohort, identifying prevalent manifestations and genetic variants, including novel variants. Methods This multicenter retrospective study reviewed the medical records of 117 MEN1 patients treated at three tertiary centers in Korea between January 2012 and September 2022. Patient demographics, tumor manifestations, outcomes, and MEN1 genetic testing results were collected. Variants were classified using American College of Medical Genetics and Genomics (ACMG) and French Oncogenetics Network of Neuroendocrine Tumors propositions (TENGEN) guidelines. Results A total of 117 patients were enrolled, including 55 familial cases, with a mean age at diagnosis of 37.4±15.3 years. Primary hyperparathyroidism was identified as the most common presentation (84.6%). The prevalence of gastroenteropancreatic neuroendocrine tumor and pituitary neuroendocrine tumor (PitNET) was 77.8% (n=91) and 56.4% (n=66), respectively. Genetic testing revealed 61 distinct MEN1 variants in 101 patients, with 18 being novel. Four variants were reclassified according to the TENGEN guidelines. Patients with truncating variants (n=72) exhibited a higher prevalence of PitNETs compared to those with non-truncating variants (n=25) (59.7% vs. 36.0%, P=0.040). Conclusion The association between truncating variants and an increased prevalence of PitNETs in MEN1 underscores the importance of genetic characterization in guiding the clinical management of this disease. Our study sheds light on the clinical and genetic characteristics of MEN1 among the Korean population.

BACKGROUND:Although the epidemiology of Huntington's disease (HD) in Korea differs notably from that in Western countries, the genetic disparities between these regions remain unclear. OBJECTIVE:To investigate the characteristics and clinical significance of cytosine-adenine-guanine (CAG) repeat size associated with HD in the Korean population. METHODS:We analyzed the CAG repeat lengths of the HTT gene in 941 healthy individuals (1,882 alleles) and 954 patients with chorea (1,908 alleles) from two referral hospitals in Korea. We presented normative CAG repeat length data for the Korean population and computed the reduced penetrance (36-39 CAG) and intermediate allele (27-35 CAG) frequencies in the two groups. Furthermore, we investigated the relationship between intermediate alleles and chorea development using logistic regression models in individuals aged ≥55 years. RESULTS:The mean (±standard deviation) CAG repeat length in healthy individuals was 17.5 ± 2.0, with a reduced penetrance allele frequency of 0.05 % (1/1882) and intermediate allele frequency of 0.69 % (13/1882). We identified 213 patients with genetically confirmed HD whose CAG repeat length ranged from 39 to 140, with a mean of 45.2 ± 7.9 in the longer allele. Compared with normal CAG repeat alleles, intermediate CAG repeat alleles were significantly related to a higher risk of developing chorea (age of onset range, 63-84 years) in individuals aged ≥55 years. CONCLUSIONS:This study provides insights into the specific characteristics of CAG repeat lengths in the HTT gene in the Korean population. The reduced penetrance and intermediate allele frequencies in the Korean general population seem to be lower than those reported in Western populations. The presence of intermediate alleles may increase the risk of chorea in the Korean elderly population, which requires further large-scale investigations.

Heerah Lee, M.D., Jung Ae Lee, M.T., M.S., Hyesu Lee, B.S., Jee-Soo Lee, M.D., Ph.D., Jung Min Ko, M.D., Ph.D., Man Jin Kim, M.D., Ph.D., and Moon-Woo Seong, M.D., Ph.D.. Ann Lab Med 2023;43:638-41. https://doi.org/10.3343/alm.2023.43.6.638

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder affecting ciliary structure and function. PCD exhibiting dynein regulatory complex subunit 1 (DRC1) exon 1–4 deletion has been reported in several Japanese PCD patients; however, no large scale studies have been performed. Here, we aimed to determine the prevalence and founder effect of this variant in the Korean population. Using an in-house copy number variation tool, we screened for DRC1 exon 1–4 deletion in 20 patients with PCD and exome data of 1435 patients in the Seoul National University Hospital repository. In cases of suspected DRC1 deletion, confirmatory gap-PCR was performed. In a PCD cohort, three of 20 (15%) patients were positive for DRC1 exon 1–4 deletion (NM_145038.5(DRC1): c.1‐3952_540 + 1331del27748‐bp) while pathogenic variants were found in CCDC39 (N = 1), DNAAF6 (N = 1), DNAH9 (N = 1). In the 1,435-sample exome data, seven patients (0.49%) were confirmed to have DRC1 exon 1–4 deletion. A chimeric sequence including the junction was searched from the 1000 Genomes Project data repository. One Japanese patient (0.96%) was found to have the same DRC1 exon 1–4 deletion, which was absent in other populations. This study demonstrated that the DRC1 exon 1–4 deletion is a founder mutation based on haplotype analysis. In summary, the prevalence of PCD based on DRC1 exon 1–4 deletion is particularly high in Korean and Japanese populations, which is attributed to the founder effect. Genetic testing for DRC1 exon 1–4 deletion should be considered as an initial screening tool for Korean and Japanese patients with PCD.

The Korean Genetic Diagnosis Program for Rare Disease (KGDP) enrolled 1890 patients with rare diseases between March 2017 and October 2022. Children and adolescents accounted for the majority of the patients, and systemic disease was the most common presenting symptom. The exome-based virtual disease-specific multigene panel was the most frequently used analytical method, with an overall diagnostic yield of 33.3%. A total of 629 positive cases were diagnosed, involving 297 genes. All 297 genes identified in these cases were confirmed to be known genes listed in the OMIM database. The nationwide KGDP network and its cooperation with the Korean Undiagnosed Diseases Program (KUDP) provide a more comprehensive genetic analysis of undiagnosed cases. The partnership between the KGDP and KUDP has the potential to improve the diagnosis and treatment options for patients. In conclusion, KGDP serves as the primary access point or gateway to KUDP.

Boram Kim, M.D., Yo Han Ahn, M.D., Ph.D., Jae Hyeon Park, M.D., Han Sol Lim, M.S., Seung Won Chae, M.S., Jee-Soo Lee, M.D., Ph.D., Hee Gyung Kang, M.D., Ph.D., Man Jin Kim, M.D., Ph.D., and Moon-Woo Seong, M.D., Ph.D.. Ann Lab Med -0001;0:. https://doi.org/10.3343/alm.2023.0361

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.

: A 19-month-old Korean boy with a history of recurrent apnea and desaturation presented with multiple erythematous hard plaques on his trunk and upper extremities (Fig. 1A~C). Skin lesions spontaneously developed after birth without trauma and gradually increased in size and number. On physical examination, he was obese with a body weight of 17.5 kg, which was above the 97th percentile. He had a round moon face and mild brachydactyly in the fifth fingers (Fig. 1D). Laboratory work-up showed elevated parathyroid hormone (113 pg/ml, reference 8~76 pg/ml), hyperphosphatemia (7.9 mg/dl, reference 2.5~4.5 mg/dl), and a calcium level close to the upper

Topic: 12. Bone marrow failure syndromes incl. PNH - Clinical Background: T-cell large granular lymphocyte leukemia (T-LGL) is a mature neoplasm of T cells. Autoimmune diseases are often associated with T-LGL in Western countries, while pure red cell aplasia (PRCA) is more often reported in Asian countries. Development of PRCA occurs along with various underlying diseases such as thymoma, T-LGL, and autoimmune diseases. It is also a bone marrow failure syndrome (BMFS) along with aplastic anemia and myelodysplastic syndrome (MDS). The discovery of somatic mutations of STAT3 has led to a more optimistic outlook regarding the genetic basis of T-LGL pathogenesis. Activation of STAT3 induced by somatic mutation is thought to facilitate proliferation of T cells. Because mutation of clonal hematopoiesis-related genes in PRCA, aplastic anemia and a subset of T-LGL occurs concomitantly with BMFS, mutational patterns of T-LGL may differ according to the concomitant disorders. Aims: Conduct of the study demonstrated the difference in mutational pattern according to the concomitant PRCA, STAT3 mutation, or treatment response and the association of mutational extent of STAT3 mutation and clinical features. We also examined the frequency of STAT3 and STAT5B mutations in hematologic diseases without T-LGL to diagnose the presence of T-LGL. Methods: High depth of next generation sequencing (NGS) of 84 candidate genes including all genes detected in PRCA or aplastic anemia was performed. Patients diagnosed with T-LGL (n=41) or isolated PRCA (n=3) were enrolled. Patients with T-LGL were divided into two groups; T-LGL alone (n=25) and T-LGL+PRCA (n=16). A review of 591 patients who were requested for an NGS panel of hematologic malignancies was conducted for identification of STAT3 and STAT5B mutation frequencies in hematologic diseases without T-LGL. Two custom targeted NGS panels were used. For T-LGL and/or PRCA, 84 genes reported to show mutation in PRCA or aplastic anemia, STAT3 and STAT5B were used. Sequencing was performed using NovaSeq6000. The median depth of coverage was 1502x. For hematologic diseases without T-LGL, a panel of 103 hematologic malignancy-related genes was used. Sequencing was performed using NextSeq550Dx. The average depths of coverage were >200 x and the cut-off value was set as ≥2% VAF for all samples. Results: No mutation was detected in PRCA (n=3). Of 41 patients with T-LGL or T-LGL+PRCA, STAT3 mutation was detected in 17 patients (41.5%), nine (36.0%) of 25 patients with T-LGL, and eight (50.0%) of 16 patients with T-LGL+PRCA. When aligning with the level of STAT3 VAF, a low level of VAF (<10.0%) was detected in only two patients with T-LGL (22.2%) compared to those with T-LGL+PRCA (87.5%, P=0.015). STAT5B mutations were detected in two patients with STAT3 wild type. Excluding the three patients with a concomitant MDS clone, 65 mutations were detected in 28 genes (33.3%) of all 84 targeted genes in 32 patients with T-LGL or T-LGL+PRCA. After excluding STAT3 mutations, the genes showing frequent mutation included KMT2D (17.1%), TERT (12.2%), SUZ12 (9.8%). Conduct of a retrospective analysis of 591 hematologic patients without T-LGL resulted in identification of three patients with STAT3 (n=1) or STAT5B (n=2) mutations. Summary/Conclusion: The findings of our study suggest that T-LGL combined with PRCA is a unique disease entity that can be classified as a subtype of T-LGL. Routine utilization of high depth NGS testing can provide sensitive detection of concomitant MDS in patients with T-LGL. STAT3 mutations suggest the presence of subclinical T-LGL in hematologic diseases.Keywords: Bone marrow failure, Pure red cell aplasia, Large granular lymphocytic leukaemia, STAT3

in contrast, refers to the presence of genetic variants in different cells of the body but not in the germline (Fig. 1) [2].Replicating human DNA, consisting of 3 billion base pairs per haploid, is a complex process that is susceptible to errors, despite various proofreading mechanisms in cells [3].The process of differentiation, which involves rapid DNA replication, is especially prone to induction of mutations.In the scenario where a mutation arises during the differentiation process following fertilization, the process results in somatic mosaicism.This phenomenon impacts speci c tissues or cells within the organism, rather than its entirety.This localized mosaicism can result in genetic disorders with symptoms manifesting in speci c tissues or organs.With the advent of high-throughput next-generation sequencing (NGS) in 2005, genome analysis has signi cantly progressed, enabling the detection of mosaicism and enhancing our comprehension of mosaic disorders.However, even healthy donors can exhibit mosaic variation, with mutant allele fractions ranging from 1.0% to 29.7% within organ samples [4].Therefore, there is an increasing demand for effective strategies to detect relevant mosaicism in clinical laboratories.This review aims to provide a comprehensive overview of the most effective methods for detecting clinically relevant mosaic sequence variants, focusing on target diseases, analytical techniques, and sample types.

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.