Medical University of Gdańsk

Skrobisz K., Miszewski K., Miszewska L., Bieńkowski M., Matuszewski M., Studniarek M.

Background and Clinical Significance: Prostate cancer (PCa) is among the most commonly diagnosed malignancies in men worldwide. While bone and lymph nodes are the most frequent metastatic sites, prostate cancer cells have the potential to spread to virtually any organ, including the pleura, which is an exceedingly rare initial site of presentation that can mimic mesothelioma or primary lung cancer. Case Presentation: We describe a 77-year-old man who presented with exertional dyspnea and intermittent cough, initially suggesting a cardiopulmonary etiology. Imaging revealed multiple pleural nodules and an extensive right-sided pleural effusion. Despite a borderline serum prostate-specific antigen (PSA) level of 2.91 ng/mL, histopathology and immunohistochemistry of pleural biopsies confirmed metastatic prostate adenocarcinoma. Subsequent imaging identified a PIRADS 5 lesion in the prostate, and a biopsy confirmed ISUP Grade Group 5 disease (Gleason score 4 + 5 = 9). A bone scan showed no skeletal metastases, and a contrast-enhanced CT of the abdomen found no additional metastatic lesions. The patient was started on androgen deprivation therapy followed by abiraterone. This case underscores the diagnostic challenge posed by atypical metastatic presentations of prostate cancer. Low or moderately elevated PSA can obscure suspicion of prostate origin, especially with pleural-based lesions suggestive of mesothelioma. Immunohistochemical markers, including androgen receptors, AMACR, and Prostein, are critical for accurate diagnosis. Conclusions: Clinicians must maintain a high index of suspicion for prostate cancer in older men with unexplained pleural effusions, nodules, or masses, even with low-normal PSA levels. Early recognition and prompt treatment can improve outcomes, despite the rarity and aggressiveness of pleural metastases.

Balewski Ł., Inkielewicz-Stępniak I., Gdaniec M., Turecka K., Hering A., Ordyszewska A., Kornicka A.

Background/Objectives: Recently, there has been great interest in metallopharmaceuticals as potential anticancer agents. In this context, presented studies aim to synthesize and evaluate of two copper(II) complexes derived from phthalazine- and imidazoline-based ligands against on three human cancer cell lines: cervix epithelial cell line (HeLa), breast epithelial-like adenocarcinoma (MCF-7), and triple–negative breast epithelial cancer cell line (MDA-MB-231), as well as non-tumorigenic cell line (HDFa). Moreover their antimicrobial, and antioxidant properties were assessed. Methods: The synthetized compounds—both free ligands L1, L2, L3 and copper(II) complexes C1 and C2—were characterized by elemental analysis, infrared spectroscopy. Additionally, a single-crystal X-ray diffraction studies we performed for free ligand L3 and its copper(II) complex C2. The stability of Cu(II)-complexes C1 and C2 was evaluated by UV-Vis spectroscopy. The cytotoxic potency of free ligands and their copper(II) complexes was estimated on HeLa, MCF-7, MDA-MB-231, as well as non-cancerous HDFa by use of an MTT assay after 48 h of incubation. Moreover, the antimicrobial activity of ligands L1 and L3 and their copper(II) complexes C1 and C2 was evaluated using reference strains of the following bacteria and yeasts: Staphylococcus aureus, Escherichia coli, and Candida albicans. The free radical scavenging properties of free ligands L1, L3 and the corresponding copper(II) complexes C1, C2 was tested with two colorimetric methods—ABTS, DPPH, and reduction ability assay (FRAP). Additionally, the ADME webtool was used to assess the drug-likeness of the synthesized compounds, as well as their physicochemical and pharmacokinetic properties. Results: Copper(II) complex C2 exhibited antitumor properties towards MDA-MB-231 compared with Cisplatin (cancer cell viability rate of 23.6% vs. 22.5%). At a concentration of 200 μg/mL, complexes C1 and C2 were less cytotoxic than the reference Cisplatin against a normal, non-cancerous skin fibroblast cell line (HDFa). According to in vitro tests, C2 reduced the viability of HeLa, MCF-7, and MDA-MB-231 cells by about 57.5–81.2%. It was evident that all compounds were devoid of antibacterial or antifungal activity. In vitro assays revealed that a moderate antiradical effect was observed for free ligand L1 containing phthalazin-1(2H)-imine in the ABTS radical scavenging assay (IC50 = 23.63 µg/mL). Conclusions: The anticancer studies revealed that the most potent compound was copper(II) complex C2 bearing a phthalazin-1(2H)-one scaffold. None of the tested compounds showed antimicrobial or antifungal activity. This feature seems to be beneficial in terms of their potential uses as anticancer agents in the future. In vitro antiradical assays revealed that a moderate antioxidant effect was observed only for free ligand L1 containing phthalazin-1(2H)-imine.

Moya-Mendez M.E., Bidzimou M., Muralidharan P., Zhang Z., Ezekian J.E., Perelli R.M., Parker L.E., Prange L., Boggs A., Kim J.J., Howard T.S., Word T.A., Wehrens X.H., Reyes Valenzuela G., Caraballo R., et. al.

ImportanceAlternating hemiplegia of childhood (AHC) is a disorder that can result from pathogenic variants in ATP1A3-encoded sodium-potassium adenosine triphosphatase alpha 3 (ATP1A3). While AHC is primarily a neurologic disease, some individuals experience sudden unexplained death (SUD) potentially associated with cardiac arrhythmias.ObjectiveTo determine the impact of ATP1A3 variants on cardiac electrophysiology and whether lethal ventricular arrhythmias are associated with SUD in patients with AHC.Design, Setting, and ParticipantsIn this international, multicenter case-control study from 12 centers across 10 countries, patients with AHC were grouped by ATP1A3 variant status (positive vs negative) and into subgroups with the most common AHC variants (D801N, E815K, G947R, and other). A healthy control cohort was established for comparison. Blinded, manual measurements of QT intervals and corrected QT interval (QTc) were performed independently by 2 pediatric cardiac electrophysiologists. Induced pluripotent stem cell cardiomyocytes were derived from patients with AHC who were positive for the D801N variant of ATP1A3 (iPSC-CMD801N cells). Data analysis was performed from April to June 2022.ExposurePresence of ATP1A3 variant.Main Outcomes and MeasuresThe primary outcome was QTc. Outcomes, including survival, were abstracted and variants were mapped on cryogenic electron microscopy structure maps. iPSC-CMD801N cells were used to validate ventricular repolarization and arrhythmic susceptibility in vitro.ResultsAmong the 222 individuals included (148 with AHC and 74 control), the mean (SD) age at diagnostic electrocardiography was 11.0 (9.4) years and 119 (54%) were female. The cohort with AHC consisted of 148 largely unrelated probands (mean [SD] age at diagnostic electrocardiography, 11.5 [10.5] years). Of these, 123 individuals were ATP1A3 genotype positive, including 35 (28%) with the D801N variant, 21 (17%) with the E815K variant, 8 (7%) with the G947R variant, and 8 (7%) with a loss-of-function variant. Probands with the D801N variant had shorter mean (SD) QTcs (381.8 [36.6] milliseconds; 24 [69%] with QTc <370 milliseconds) compared with those who had the E815K variant (393.6 [43.1] milliseconds; P = .001; 4 [19%] with QTC <370 milliseconds), the G947R variant (388.4 [26.5] milliseconds; P = .02; 1 [13%] with QTc <370 milliseconds), a loss-of-function variant (403.0 [33.5] milliseconds; P < .001; 1 [13%] with QTc <370 milliseconds), all other variants (387.8 [37.1] milliseconds; P < .001; 44 [86%] with QTc <370 milliseconds), and healthy controls (415.4 [21.0] milliseconds; P < .001; 0 with QTc <370 milliseconds). Three D801N-positive individuals had a major cardiac event, compared with 0 major cardiac events in all other individuals (P = .02). The D801N variant and 4 rare variants (D805N, P323S, S772R, and C333F) found in individuals with the shortest QTcs localized to the potassium-binding domain of ATP1A3. IPSC-CMD801N lines demonstrated shortened action potential duration, higher mean diastolic potential, and delayed afterdepolarizations compared with controls.Conclusions and RelevanceNearly 70% of individuals with D801N variants of ATP1A3 had short QTcs (<370 milliseconds), with an association between ventricular arrhythmias and cardiac arrest. This may underlie the SUD etiology in AHC.

Piechowicz L., Kosznik-Kwaśnicka K., Kaźmierczak N., Grzenkowicz M., Stasiłojć M., Necel A., Werbowy O., Pałubicka A.

Background/Objectives: The viral pandemic caused by the SARS-CoV-2 virus has affected millions of people. However, it was noticed that high mortality was often a result of bacterial co-infections. One of the main pathogens responsible for secondary infections in patients with viral respiratory tract infections, including COVID-19, is Staphylococcus aureus. In recent years, the number of infections caused by drug-resistant strains of S. aureus has been growing rapidly, often exceeding the number of infections caused by antibiotic-sensitive strains. In addition, biofilm-related infections are more difficult to treat due to the lower sensitivity of biofilm structure to antibiotics. Bacteriophages are seen as alternative treatment of bacterial infections. Therefore, in our work, we have analyzed the efficacy of three Kayviruses against S. aureus strains isolated from COVID-19 patients. Methods: We analyzed the ability of tested phages to remove S. aureus biofilm both from polystyrene plates as well as from the surface of pulmonary epithelial cells. Results: We have observed that tested Kayviruses had a broad host range. Furthermore, phages were able to effectively reduce biofilm biomass and number of viable cells in pure culture. During our research, none of the tested phages was shown to have a negative effect on cell viability and were able to inhibit the negative effect S. aureus had on cell condition. Conclusions: Our results show tested phages were effective in reducing the biofilm of S. aureus strains isolated from COVID-19 patients, had no adverse effect on lung epithelial cell viability. Therefore, it should be recognized that the properties of three studied Kayviruses give them an advantage in the selection of phages for treatment of staphylococcal infections.

Kaniewska M., Lewandowski K., Krogulecki M., Filipiuk A., Gonciarz M., Pietrzak A., Janiak M., Adrych K., Klufczynska A., Piotrowicz G., Kopertowska-Majchrzak M., Panasiuk A., Mahadea D., Eder P., Tarasiuk A., et. al.

Background: Upadacitinib (UPA) is a new oral selective Janus Kinase (JAK) inhibitor that has shown high efficacy in the treatment of ulcerative colitis (UC). We present data from a multicenter real-world study. Methods: To assess efficacy of UPA, Total Mayo Score (TMS), fecal calprotectin (FC), endoscopy, and intestinal ultrasonography (IUS) were performed. Results: The study population included 76 patients. An amount of 26.3% of the patients were biologics and small molecules-naive, while 73.7% were exposed. By Week 8, 93.4% of the patients had achieved a clinical response (94.7% naive vs. 92.9% exposed), 72.4% achieved endoscopic improvement (78.9% vs. 71.4%), and 57.9% had clinical remission (78.9% vs. 51.8%). Endoscopic remission was achieved in 31.6% of patients (35.0% vs. 30.4%) and biochemical remission in 82.1% (53.3% vs. 68.3%). All of the results were not significantly different apart from the steroid-free clinical remission—36.8% (68% vs. 26.8%, p = 0.002) after 8 weeks of follow-up. IUS was performed in 33 patients. Bowel wall thickness (BWT), inflammatory fat (iFAT), color Doppler signal (CDS), loss of bowel wall stratification (BWS), and Milano Ultrasound Criteria (MUC) had decreased significantly by Weeks 4 and 8 (p < 0.005 for all). Correlation between the IUS results and TMS, FC and endoscopic remission in Week 8 was confirmed (p < 0.001). UPA was well tolerated, and no new safety signals were registered in our group. Conclusions: In this study, UPA was confirmed to be safe and highly effective in inducing remission in UC patients in both the naive group and the biologically exposed patients. The correlation between the IUS results and TMS, FC, and endoscopic remission provides valuable information for clinicians.

Flis D.J., Bialobrodzka E.G., Rodziewicz-Flis E.A., Jost Z., Borkowska A., Ziolkowski W., Kaczor J.J.

Due to the lack of enzyme 17-α hydroxylase, rats and mice cannot produce appreciable cortisol; however, low cortisol concentrations are detectable in these rodents’ blood [...]

Bielińska-Wąż D., Wąż P., Błaczkowska A.

Objective: The objective of this research is to demonstrate that alignment-free bioinformatics approaches are effective tools for analyzing the similarity and dissimilarity of protein sequences. All numerical parameters representing sequences are expressed analytically, ensuring precision, clarity, and efficient processing, even for large datasets and long sequences. Additionally, a novel approach for identifying previously unknown virus strains is introduced. Methods: A novel approach is proposed, integrating the unique features of our newly developed method, the 20D-Dynamic Representation of Protein Sequences, with the K-means clustering algorithm. The sequences are represented as clouds of material points in a 20-dimensional space (20D-dynamic graphs), with their spatial distribution being unique to each protein sequence. The numerical parameters, referred to as descriptors in molecular similarity theory, represent quantities characteristic of dynamic systems and serve as input data for the K-means clustering algorithm. Results: Examples of the application of the approach are presented, including projections of the 20D-dynamic graphs onto 3D spaces, which serve as a visual tool for comparing sequences. Additionally, cluster plots for the analyzed sequences are provided using the proposed method. Conclusion: It has been demonstrated that the 20D-Dynamic Representation of Protein Sequences, combined with the K-means clustering algorithm, successfully classifies subtypes of influenza A virus strains.

Jopek M.A., Sieczczyński M., Pastuszak K., Łapińska-Szumczyk S., Jassem J., Żaczek A.J., Rondina M., Supernat A.

Abstract Ovarian cancer (OC) presents a diagnostic challenge, often resulting in poor patient outcomes. Platelet RNA sequencing, which reflects host response to disease, shows promise for earlier OC detection. This study examines the impact of sex, age, platelet count, and the training on cancer types other than OC on classification accuracy achieved in the previous platelet-alone training data set. A total of 339 samples from healthy donors and 1396 samples from patients with cancer, spanning 18 cancer types (including 135 OC cases) were analyzed. Logistic regression was applied to verify our classifiers’ performance and interpretability. Models were tested at 100% specificity and 100% sensitivity levels. Incorporating patient age as an additional feature along with gene expression increased sensitivity from 68.6% to 72.6%. Models trained on data from both sexes and on female-only data achieved a sensitivity of 68.6% and 74.5%, respectively. Training solely on OC data reduced late-stage sensitivity from 69.1% to 44.1% but increased early-stage sensitivity from 66.7% to 69.7%. This study highlights the potential of platelet RNA profiling for OC detection and the importance of clinical variables in refining classification accuracy. Incorporating age with gene expression data may enhance OC diagnostic accuracy. The inclusion of male samples deteriorates classifier performance. Data from diverse cancer types improves advanced cancer detection but negatively affects early-stage diagnosis.

Szekalska M., Kasparavičienė G., Bernatonienė J., Wolska E., Misiak P., Markiewicz K.H., Wilczewska A.Z., Czajkowska-Kośnik A., Winnicka K.

Background/Objectives: Posaconazole is an antifungal agent from triazoles with variable bioavailability. To avoid its irregular absorption caused by gastric conditions and ensure more repeatable pharmacokinetic enabling the maximization of its absorption regardless of food intake without the need to administer multiple doses, can be provided by the technology of enteric drug preparations. The cross-linking of polysaccharide polymers with divalent and trivalent cations enables multi-unit formulations to be obtained that prevent drug absorption in the stomach. Microcapsules, as an example of multi-unit drug dosage forms, provide more predictable gastric emptying, depending on nutritional status, and spread extensively throughout the gastrointestinal tract. Methods: Therefore, the utilization of zinc acetate for the cross-linking of the alginate and pectin mixture was evaluated. The obtained formulations were evaluated for the impact of cross-linking process and pectin’s presence on their pharmaceutical, mucoadhesive, physicochemical and antifungal properties. Results: It was shown that cross-linked microcapsules by zinc acetate provided delayed posaconazole release. Additionally, the cross-linking process with Zn2+ ions significantly enhanced antifungal activity against the analyzed Candida strains. It was observed that pectin content in the formulation enhanced the swelling ability in an intestinal condition and increased the mucoadhesive properties of drug-loaded formulations to the intestinal mucosa.

Kaliszewska A., Struczyński P., Bączek T., Niedźwiecki M., Konieczna L.

This study aimed to develop and optimize an analytical method for profiling 21 amino acids in cerebrospinal fluid and plasma, addressing the need for improved diagnostic tools in leukemia research. Using high-performance liquid chromatography coupled with electrospray ionization mass spectrometry, the method achieved enhanced resolution, sensitivity, and specificity. Rigorous sample preparation, including liquid–liquid extraction, ensured high recovery rates, while validation confirmed the method’s accuracy and reproducibility. Clinical application in pediatric leukemia patients revealed significant variations in amino acid concentrations across treatment stages, providing insights into disease progression and therapeutic response. Statistical analysis with IBM SPSS Statistics 25 compared amino acid levels in patients to healthy controls, identifying distinct patterns on day 1, day 15, and day 33 of treatment. Correlation analysis highlighted relationships between amino acid levels and factors such as treatment duration, sex, age, and blood test results. Key amino acids, including proline, leucine, and hydroxyproline, emerged as significant predictors of white blood cell count, effectively distinguishing between patient and control groups. This method demonstrates robust potential for broader leukemia research applications, pending further validation on larger cohorts.

Greber K.E., Topka Kłończyński K., Nicman J., Judzińska B., Jarzyńska K., Singh Y.R., Sawicki W., Puzyn T., Jagiello K., Ciura K.

Biomimetic chromatography is a powerful tool used in the pharmaceutical industry to characterize the physicochemical properties of molecules during early drug discovery. Some studies have indicated that biomimetic chromatography may also be useful for the evaluation of toxicologically relevant molecules. In this study, we evaluated the usefulness of the biomimetic chromatography approach for determining the lipophilicity, affinity to phospholipids, and bind to plasma proteins of selected organophosphate pesticides. Quantitative structure–retention relationship (QSRR) models were proposed to understand the structural features that influence the experimentally determined properties. ACD/labs, Chemicalize, and alvaDesc software were used to calculate theoretical descriptors. Multilinear regression was used as the regression type, and feature selection was supported by a genetic algorithm. The obtained QSRR models were validated internally and externally, and they demonstrated satisfactory performance with key statistical parameters ranged from 0.844 to 0.914 for R2 and 0.696–0.898 for R2ext, respectively, indicating good predictive ability.

Kwiecień-Jaguś K., Mędrzycka-Dąbrowska W., Kopeć M.

Background and Objectives: A medication error can occur at any stage of medication administration at the ward, from the moment the medication is prescribed through the preparation to the administration to the patient. The statistics indicate that the scale of the problem, which has a significant impact on the safety and health of patients, is still poorly known. The purpose of the systematic review was to synthesise the published research about the number of medication errors in operating room theatres and intensive care units. Materials and Methods: The literature review was conducted in the third quarter of 2023. The overview included papers found in Science Direct, EBSCO, PubMed, Ovid, Scopus, and original research papers published in English meeting the PICOS criteria. Original articles published between 2017 and 2023 that meet the inclusion criteria were included for further analysis. Results: The review included 13 articles and original studies, which met the PICOS-based criteria. The analyses confirmed that the operating theatre’s medication error rate was 7.3% to 12%. In the case of intensive care units, the medication error rate was from 1.32 to 31.7%. Conclusions: Medication errors in the operating room and intensive care are high. However, the values presented herein do not differ from the general Medication Error Index for medical centres, as calculated by the World Health Organization.

Szarmach A., Sabiniewicz-Ziajka D., Grzywińska M., Gać P., Piskunowicz M., Wszędybył-Winklewska M.

Background/Objectives: The increasing use of computed tomography (CT) scans significantly contributes to population exposure to ionizing radiation. Traditional dose metrics, such as dose–length product (DLP) and effective dose (ED), lack precision in reflecting individual radiation exposure. This study introduces a novel parameters such as size-specific effective dose (EDss) and the size-specific dose–length product (DLPss), to improve patient-specific dose estimation. The aim of this study is to enhance dose calculation accuracy, optimize CT protocols, and guide the development of next-generation CT technologies. Methods: A retrospective analysis of 247 abdominal and pelvic CT scans (113 women, 134 men) was conducted. Anthropometric parameters, including body mass index (BMI), cross-sectional dimensions, and dose indices, were measured. EDss and DLPss were calculated using size-specific correction factors, and statistical correlations between these parameters were assessed. Results: The mean BMI was 25.92 ± 5.34. DLPss values ranged from 261.63 to 1217.70 mGy·cm (mean: 627.83 ± 145.32) and were roughly 21% higher than traditional DLP values, with men showing slightly higher mean values than women. EDss values ranged from 6.65 to 15.45 mSv (mean: 9.42 ± 2.18 mSv), approximately 22% higher than traditional ED values, demonstrating improved individualization. Significant correlations were observed between BMI and effective diameter (r = 0.78), with stronger correlations in men (r = 0.85). The mean CTDIvol was 11.37 ± 3.50 mGy, and SSDE averaged 13.91 ± 2.39 mGy. Scan length reductions were observed in 53.8% of cases, with statistically significant differences by gender. Conclusions: EDss and DLPss offer improved accuracy in radiation dose estimation, addressing the limitations of traditional methods. Their adoption into clinical protocols, supported by AI-driven automation, could optimize diagnostic safety and significantly reduce radiation risk for patients. Further multicenter studies and technological advancements are recommended to validate these metrics and facilitate their integration into daily practice.

Mazur N.K., Fercho J.M., Kałas M., Szaruta-Raflesz K., Grzybowska M.E., Siemiński M., Wydra D.G.

Intracranial hemorrhage is a rare yet potentially devastating event during pregnancy with a significant risk of maternal and fetal mortality and morbidity. The risk of intracranial hemorrhage increases during the third trimester of pregnancy and is greatest during labor and the postpartum period. Interdisciplinary diagnosis and treatment of the pregnant population often begins in the emergency department setting and is key to increasing patient survival rates through immediate and adequate treatment, including emergency medicine, neurosurgical and obstetrical procedures. A unique case report with a diagnostic pathway for intracranial hemorrhage due to eclampsia in a primipara at 24 weeks of gestation is presented, illustrating potential diagnostic dilemmas as the patient rapidly progresses into hemolysis, elevated liver enzymes and low platelets syndrome. A literature review was conducted to uncover the etiology of intracranial hemorrhage during pregnancy, as well as its diagnostic challenges and treatment. Pregnancy should not be viewed as a barrier to performing angiography or endovascular treatment for vascular causes of intracranial hemorrhage. Patient transport to a tertiary reference center and the interdisciplinary cooperation of specialists are key to achieving correct and rapid treatment. Continuous prevention of preeclampsia and patient education are necessary to decrease the incidence of eclampsia and its complications. Key message: Intracranial hemorrhage and eclampsia in pregnant patients are rare yet may result in high rates of maternal and fetal morbidity and mortality. The diagnostic process is difficult and requires interdisciplinary cooperation to start the correct treatment immediately.

De Wachter M., Millevert C., Nicolai J., Cats E., Kluger G., Milh M., Cloarec R., Syrbe S., Arts K., Jansen K., Krygier M., Smigiel R., Auvin S., Olofson K., Gjerulfsen C.E., et. al.

AbstractObjectiveHeterozygous gain‐of‐function (GOF) variants in KCNQ2 and KCNQ3, encoding the voltage‐gated potassium channel subunits Kv7.2 and Kv7.3, lead to neurodevelopmental disorders for which no established treatments are available. Amitriptyline, an antidepressant, blocks Kv7.2/Kv7.3 and has previously been reported to be effective in a single individual with a KCNQ2 GOF variant. We designed a retrospective, single‐arm, multicenter study to investigate the effects of amitriptyline in a real‐world setting.MethodsWe used a 7‐point Likert scale to measure seizure frequency, clinical examination, motor function, alertness, skill acquisition, communication, mood, behavior, self‐care, sleep, tiredness, and electroencephalogram at baseline, after a minimum of 6 weeks of intervention, and, if applicable, after discontinuation. Adverse events were assessed in all participants, and the effectiveness of the treatment was evaluated in 11 individuals who received a minimum dosage of .5 mg/kg/day for at least 6 weeks. Data were collected from October 2023 to August 2024.ResultsThirteen individuals, eight with a pathogenic KCNQ2 GOF variant and five with a pathogenic KCNQ3 GOF variant, were included. Nine were female, and the median age at start of amitriptyline was 7.1 years (range = 1.5–20 years). Eleven individuals received a minimum dosage of .5 mg/kg/day for at least 6 weeks. The median dosage of amitriptyline administered was 1 mg/kg/day, with a median treatment duration of 29 weeks. Although amitriptyline was ineffective in two individuals (18%), eight (72%) demonstrated at least minimal improvement in two or more domains, with improvements in alertness and communication being the most frequently reported. In those with reported improvements, amitriptyline was discontinued in four individuals, but continued improvements were seen, to the same or greater extent compared to treatment. The remaining five individuals are on continued treatment because of perceived benefits.SignificanceOverall, the effect of amitriptyline remains unclear, and formal n‐of‐1 trials are needed to investigate the precise effects of amitriptyline in KCNQ GOF‐related neurodevelopmental disorders.