Background: As nationwide government-led whole-genome sequencing (WGS) projects progress, optimizing the clinical integration of large-scale WGS results is crucial. We explored how the initial analysis from Korea’s First WGS Pilot Study for Rare Diseases was applied in clinical practice, and then we reanalyzed the data comprehensively at Samsung Medical Center (SMC) Seoul, Korea. Methods: A prospective cohort study designed to collect WGS data under a Korean national initiative was conducted from August 2020 to December 2021. We focused on patients with rare diseases recruited from 16 university hospitals. The participants included 5,000 individuals (2,200 probands and 2,800 family members). The initial WGS data and diagnostic reference reports (from 682 probands and 484 family members), generated based on the First Korean WGS Pilot Study for Rare Diseases, were subsequently reanalyzed by SMC. Results: The initial analysis of the First Korean WGS Pilot Study data revealed a diagnostic rate of 17%. Upon receiving these results, the SMC conducted two rounds of reanalysis, increasing the diagnostic rate from 15% in the first analysis, to 18% in the second, and finally to 24% in the third (P = 1.6 × 10 −5 ). Key factors in improving the genetic diagnosis included increased detection of novel (likely) pathogenic variants (P = 1.0 × 10 −4 ), improved diagnostic rates with larger family recruitment (P = 0.004), and refined clinical information for more precise genotype–phenotype correlation analysis (40%). Conclusions Although national WGS projects lay a foundation for rare disease diagnosis, hospital-level reanalysis and multidisciplinary collaborations are crucial for optimizing diagnostic outcomes.

Although venous invasion (VI) is associated with distant metastasis and observed in >50% of pT2–4 colorectal cancers (CRCs), the role of VI in pT1 CRCs is not well-defined. Methods: Thirty-four surgically resected pT1 CRCs were reevaluated for 2-dimensional (2D) VI using hematoxylin and eosin (H&E)–stained slides with additional elastic and desmin immunohistochemical staining (cohort A). Additionally, 27 pT1 CRCs without knowing VI status were selected for 3-dimensional (3D) VI evaluation only (cohort B). All 61 cases (cohorts A and B) were studied in 3D using tissue clearing. Results: VI was detected more commonly in 3D (17/34, 50.0%) than in 2D H&E slide evaluation (9/34, 26.5%, p = .047). When VI was identified in 3D (27/61, 44.3%), the most common phase was that of intraluminal growth (22/27, 81.5%), followed by intravasation (7/27, 25.9%) and extravasation (5/27, 18.5%). E-cadherin expression was characterized in 3D in foci of VI and varied in each phase of invasion. Conclusions: All three phases were observed in VI of pT1 CRCs. The extravasation of neoplastic cells from foci of VI in pT1 CRC suggests that VI could be a route of intratumoral spreading in a subset of pT1 CRCs.

Purpose: While TP53 mutations are well known to be associated with adverse prognosis in hematological diseases, their functional impact remains incompletely understood. This study examines the spectrum of TP53 mutations across various hematologic malignancies and evaluates their functional impact. Materials and Methods: Using targeted sequencing panels, we analyzed TP53 mutations in the bone marrow aspiration samples of a retrospective cohort of 856 patients diagnosed with hematologic malignancies. To assess the impact of TP53 mutations, we applied the evolutionary action (EAp53) score and the relative fitness score (RFS), previously proposed functional scoring methods. The effects of variant allele frequency (VAF), disruptive mutations, EAp53 score, and RFS on overall survival (OS) were evaluated. Results: TP53 mutations were associated with inferior OS compared with wildtype TP53 (median OS 10.0 months versus not estimable; hazard ratio (HR) 4.6; p<0.001). In the acute myeloid leukemia, multiple myeloma, and myelodysplastic syndrome subgroups, TP53 mutations had a significant adverse impact on OS. (HRs 3.8, 4.2, 6.0, respectively; p<0.001, p=0.005, p<0.001, respectively). Patients with VAF >50% had significantly poorer OS compared to those with VAF ≤50% (median OS 7.5 months versus 22.8 months; HR 2.2, p=0.016). Moreover, patients in the high-risk RFS group (RFS >0.22) had significantly worse OS compared to those in the low-risk RFS group (RFS ≤0.22) (median OS 5.6 months versus 16.3 months; HR 2.2, p=0.041). However, no significant survival difference was observed between the EAp53 high-risk (>75) and low-risk (≤75) groups, or between patients with disruptive and non-disruptive mutations. Conclusion Our findings highlight VAF and RFS as valuable tools for stratifying TP53-mutant patients into high-risk and low-risk groups.

Introduction: Severe hypertriglyceridemia, though rare in pregnancy, is a clinically significant condition that poses substantial maternal and perinatal risks. Case: We present a case of a 42-year-old pregnant female with class I obesity and pre-existing type 2 diabetes mellitus, with a booking hemoglobin A1c 12.1%. Her poorly controlled diabetes was complicated by nephrotic syndrome, with a 24-hour urine protein 4.9 g/day. She also had bilateral diabetic retinopathies requiring multiple sessions of panretinal photocoagulation. Throughout the pregnancy, her triglycerides rose progressively, peaking at 30 mmol/L by 33 weeks. Investigations revealed normal thyroid function, urea 4.8 mmol/L, creatinine 66 umol/L, and albumin 26 g/dL. The patient was hospitalized at 33 weeks of gestation and started on an intravenous insulin infusion along with a very low-fat, low-carbohydrate diet prescribed by a dietitian. Pulmonary embolism was diagnosed during evaluation for maternal tachycardia, and subcutaneous enoxaparin was initiated. One week after admission in 35 weeks of gestation, the baby was delivered via emergency Caesarean section due to fetal distress. The baby was small for gestational age, with a birth weight of 1.9 kg, below the 10th percentile for gestational age, and required admission to the neonatal intensive care unit with oxygen support for pneumonia-related respiratory distress. Post-delivery, intravenous insulin infusion and dietary control were continued, reducing her triglyceride level to the lowest level of 8.3 mmol/L. Both mother and baby were discharged well. Lipid-lowering agents such as rosuvastatin and fenofibrate were started after breastfeeding stopped, aiming to normalize her triglyceride levels. Conclusion Hypertriglyceridemia in this patient was the result of uncontrolled diabetes mellitus with nephrotic-range proteinuria. Severe hypertriglyceridemia can cause acute pancreatitis and/or hyperviscosity syndrome, both of which are life-threatening to the mother and baby. Thus, rapid reduction of triglyceride levels is crucial. This case highlights the importance of achieving good diabetes control prior to pregnancy, with strict adherence to treatment and enhanced patient education.
Female ; Pregnancy ; Glycemic Control ; Hypertriglyceridemia

Introduction: Diabetic ketoacidosis (DKA) and thyroid storm are individually rare but potentially fatal endocrine crises in pregnancy. Each carries significant maternal and fetal morbidity, with mortality risk compounded when they occur concomitantly. Physiological and pharmacokinetic changes of pregnancy, combined with overlapping symptoms, necessitate urgent treatment strategies. Case: A 29-year-old G2P2 female at 29 weeks’ gestation, with poorly controlled type 2 diabetes mellitus (hemoglobin A1c 8.1%) on a basal–bolus insulin regimen and Graves’ disease managed with carbimazole, non-adherent to medications, presented with fever, vomiting, and dyspnea. On examination, she was tachycardic (HR 138 bpm), hypotensive (BP 94/60 mmHg), and hypoxic. Laboratory investigations revealed hyperglycemia (glucose 27.1 mmol/L), severe metabolic acidosis (pH 7.02, bicarbonate 4.9 mmol/L), and elevated serum ketones (4.6 mmol/L), consistent with DKA. Thyroid function tests showed suppressed thyroid-stimulating hormone (<0.005 mIU/L) and elevated free T4 (28.2 pmol/L), with a Burch–Wartofsky score of 70. Unfortunately, intrauterine fetal demise was confirmed upon the patient’s presentation to the emergency department. She was intubated and admitted to the intensive care unit, receiving fluid resuscitation judiciously according to the DKA regimen, with frequent assessment of volume status. Intravenous insulin and potassium supplements were commenced concurrently. Metabolic stabilization was achieved within 24 hours. Carbimazole, propranolol, Lugol’s iodine, and intravenous hydrocortisone were started for treatment of thyroid storm. A breech-assisted vaginal delivery was performed, and her postpartum course was uneventful. Conclusion The case reveals the catastrophic potential of concurrent DKA and thyroid storm in pregnancy, where rapid maternal deterioration and poor fetal outcomes can occur despite timely intervention. High clinical suspicion, early biochemical confirmation, and coordinated multidisciplinary management are vital. Precipitating factors, particularly medication non-adherence, must be addressed through intensive patient education and structured follow-up to prevent recurrence.
Female ; Pregnancy ; Diabetic Ketoacidosis ; Thyroid Crisis

Introduction: Incidental hyperparathyroidism with hypercalcemia is not uncommon. However, parathyroid carcinoma is an extremely rare endocrine malignancy, accounting for less than 1% of cases of primary hyperparathyroidism. Case: A 57-year-old Malay female with well-controlled type 2 diabetes was incidentally found to have hypercalcemia during hospitalization for pyelonephritis. She was asymptomatic, with no history of calcium supplementation or family history of endocrine disorders. Physical examination revealed no obvious neck swelling, and other systemic examinations were unremarkable. Investigations showed elevated serum calcium (2.87–3.4 mmol/L), low phosphate (0.34–0.76 mmol/L), and elevated intact parathyroid hormone (iPTH) (6.81 pmol/L). Her 25-OH vitamin D was deficient (45.58 nmol/L). Ultrasound of the neck revealed a solid lesion (1.6 × 2.0 × 2.8 cm) posterior to the right thyroid lobe with bilateral thyroid nodules, with the highest TR4, and normal cervical lymph nodes. Sestamibi scan showed multinodular goiter with cold nodules in bilateral thyroid lobes and a soft tissue lesion posterior to the right thyroid lobe. Subsequently, she underwent right hemithyroidectomy with intraoperative nerve monitoring and right inferior parathyroidectomy with intraoperative iPTH monitoring; the intraoperative iPTH level dropped appropriately from 24 to 2.4 pmol/L. Histopathological examination of the excised right parathyroid gland revealed parathyroid carcinoma, while the right thyroid lobe showed nodular hyperplasia. Postoperatively, calcium and iPTH levels normalized, and she remained well under follow-up. Conclusion Parathyroid carcinoma typically presents with markedly elevated calcium and iPTH levels, often alongside a palpable neck mass. However, in this patient, serum iPTH was only slightly above the upper limit of normal, with tumor size less than 3 cm, no lymph node or surrounding structures involvement from pre-operative evaluation imaging. This highlights the variability in both physical and biochemical presentations of parathyroid carcinoma and the challenges in distinguishing it from benign parathyroid tumors preoperatively.
Parathyroid Neoplasms

Acanthamoeba is an opportunistic pathogen responsible for granulomatous amoebic encephalitis and amoebic keratitis. Despite its clinical significance, effective treatments remain challenging due to a limited understanding of its pathogenic mechanism. This study developed a genetic manipulation system in Acanthamoeba to facilitate gene function and drug screening studies. We applied the Cre/loxP system to integrate the gene encoding the tdTomato fluorescent protein into the genome of Acanthamoeba castellanii via homologous recombination. The polyubiquitin gene and its untranslated regions were identified and verified, after which the tdTomato gene was cloned between the untranslated regions of the polyubiquitin gene. The construct was then introduced into the Acanthamoeba genome using a modified pLPBLP vector containing loxP sites. Cre recombinase was utilized to remove the neomycin resistance cassette flanked by loxP sites, and genetically modified cells were selected by clonal dilution. The integration of the tdTomato gene, confirmed through PCR and fluorescence microscopy, showed stable expression in both trophozoites and cysts without the need for antibiotic selection. We demonstrated the feasibility of antibiotic-free reporter gene expression in Acanthamoeba. The system provides a valuable tool for functional genomics, allowing us to explore gene functions in Acanthamoeba and develop reliable drug screening models. Furthermore, the ability to express genes without the continuous use of selection markers opens up new possibilities for studying the pathobiology of this pathogen and advancing the development of novel therapeutic strategies against Acanthamoeba infections.

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.

Acanthamoeba is an opportunistic pathogen responsible for granulomatous amoebic encephalitis and amoebic keratitis. Despite its clinical significance, effective treatments remain challenging due to a limited understanding of its pathogenic mechanism. This study developed a genetic manipulation system in Acanthamoeba to facilitate gene function and drug screening studies. We applied the Cre/loxP system to integrate the gene encoding the tdTomato fluorescent protein into the genome of Acanthamoeba castellanii via homologous recombination. The polyubiquitin gene and its untranslated regions were identified and verified, after which the tdTomato gene was cloned between the untranslated regions of the polyubiquitin gene. The construct was then introduced into the Acanthamoeba genome using a modified pLPBLP vector containing loxP sites. Cre recombinase was utilized to remove the neomycin resistance cassette flanked by loxP sites, and genetically modified cells were selected by clonal dilution. The integration of the tdTomato gene, confirmed through PCR and fluorescence microscopy, showed stable expression in both trophozoites and cysts without the need for antibiotic selection. We demonstrated the feasibility of antibiotic-free reporter gene expression in Acanthamoeba. The system provides a valuable tool for functional genomics, allowing us to explore gene functions in Acanthamoeba and develop reliable drug screening models. Furthermore, the ability to express genes without the continuous use of selection markers opens up new possibilities for studying the pathobiology of this pathogen and advancing the development of novel therapeutic strategies against Acanthamoeba infections.

Acanthamoeba is an opportunistic pathogen responsible for granulomatous amoebic encephalitis and amoebic keratitis. Despite its clinical significance, effective treatments remain challenging due to a limited understanding of its pathogenic mechanism. This study developed a genetic manipulation system in Acanthamoeba to facilitate gene function and drug screening studies. We applied the Cre/loxP system to integrate the gene encoding the tdTomato fluorescent protein into the genome of Acanthamoeba castellanii via homologous recombination. The polyubiquitin gene and its untranslated regions were identified and verified, after which the tdTomato gene was cloned between the untranslated regions of the polyubiquitin gene. The construct was then introduced into the Acanthamoeba genome using a modified pLPBLP vector containing loxP sites. Cre recombinase was utilized to remove the neomycin resistance cassette flanked by loxP sites, and genetically modified cells were selected by clonal dilution. The integration of the tdTomato gene, confirmed through PCR and fluorescence microscopy, showed stable expression in both trophozoites and cysts without the need for antibiotic selection. We demonstrated the feasibility of antibiotic-free reporter gene expression in Acanthamoeba. The system provides a valuable tool for functional genomics, allowing us to explore gene functions in Acanthamoeba and develop reliable drug screening models. Furthermore, the ability to express genes without the continuous use of selection markers opens up new possibilities for studying the pathobiology of this pathogen and advancing the development of novel therapeutic strategies against Acanthamoeba infections.