Neuropsychological adverse drug reactions of Remdesivir: analysis using VigiBase, the WHO global database of individual case safety reports.

OBJECTIVE: Although remdesivir (GS-5734) has recently demonstrated clinical benefits against the pandemic outbreak of coronavirus disease 2019 (COVID-19), neuropsychological adverse reactions (ADRs) remain to be examined in real-world settings. Therefore, we aimed to identify and characterize the neuropsychological ADRs associated with remdesivir use. MATERIALS AND METHODS: We obtained data for this international pharmacovigilance cohort study from individual case safety reports (ICSRs) in a World Health Organization database (VigiBase) from the first report on remdesivir on February 17, 2020, until August 30, 2020 (n=1,403,532). ADRs reported to be relevant to remdesivir were compared with the full database by using a Bayesian neural network method to calculate the information component (IC). RESULTS: A total of 2,107 reported cases of neuropsychological ADRs suspected to be associated with remdesivir were identified from among all ICSRs in the database during the observation period. Although 108 neuropsychological ADRs (64 neurologic events and 44 psychologic events) were reported in association with the medication, no statistically significant pharmacovigilance signal could be detected; the IC025 value was negative for all of the neuropsychological dysfunctions (anxiety [n=13, 0.62%], seizures [n=12, 0.57%], lethargy [n=6, 0.28%], agitation [n=5, 0.25%], cerebral infarction [n=3, 0.14%], ischemic stroke [n=3, 0.14%], and hemiparesis [n=3, 0.14%]). CONCLUSIONS: Our study demonstrates that remdesivir, a novel drug applied to the treatment of COVID-19, does not have a significant association with adverse neurologic or psychiatric reactions in the real-world setting.

Lactobacillus intestinalis YT2 restores the gut microbiota and improves menopausal symptoms in ovariectomized rats.

There are many studies focusing on the alleviation of menopausal symptoms; however, little is known about the role of gut microorganisms in menopausal symptoms. Ovariectomized (OVX) rats were administered a novel strain (YT2) of Lactobacillus intestinalis (a species with significantly reduced abundance in OVX rats) and the potential probiotic effect on the improvement of menopausal symptoms was evaluated. Of note, the gut microbial composition completely shifted after ovariectomy in rats. Treatment with L. intestinalis YT2 significantly alleviated menopausal symptoms, such as increased fat mass, decreased bone mineral density, increased pain sensitivity, depression-like behaviour, and cognitive impairment. Additionally, the administration of L. intestinalis YT2 restored the intestinal microbial composition, including an increased Firmicutes/Bacteroides ratio. L. intestinalis YT2 also promoted gut barrier integrity by increasing the mRNA levels of tight junction-related markers. In conclusion, L. intestinalis YT2 treatment alleviated menopausal symptoms via the modulation of the gut microbiota. Importantly, these results suggest that L. intestinalis YT2 should be considered as a therapeutic probiotic agent for menopausal women.

Tipping the immunostimulatory and inhibitory DAMP balance to harness immunogenic cell death.

Induction of tumor cell death is the therapeutic goal for most anticancer drugs. Yet, a mode of drug-induced cell death, known as immunogenic cell death (ICD), can propagate antitumoral immunity to augment therapeutic efficacy. Currently, the molecular hallmark of ICD features the release of damage-associated molecular patterns (DAMPs) by dying cancer cells. Here, we show that gemcitabine, a standard chemotherapy for various solid tumors, triggers hallmark immunostimualtory DAMP release (e.g., calreticulin, HSP70, and HMGB1); however, is unable to induce ICD. Mechanistic studies reveal gemcitabine concurrently triggers prostaglandin E2 release as an inhibitory DAMP to counterpoise the adjuvanticity of immunostimulatory DAMPs. Pharmacological blockade of prostaglandin E2 biosythesis favors CD103+ dendritic cell activation that primes a Tc1-polarized CD8+ T cell response to bolster tumor rejection. Herein, we postulate that an intricate balance between immunostimulatory and inhibitory DAMPs could determine the outcome of drug-induced ICD and pose COX-2/prostaglandin E2 blockade as a strategy to harness ICD.

Anatomical analysis to establish the optimal positioning of an osteotomy for genioglossal advancement: a trial in cadavers.

Genioglossal advancement, which is one of the treatments for obstructive sleep apnoea, can be effective only if it contains enough genial tubercle for an osteotomy. The aim of this study was to establish the position of the genial tubercle and of the optimal osteotomy during genioglossal advancement. Twenty-four adult cadavers with intact bony mandibular structures were included. Five variables were measured: the width and height of the genial tubercle (GTW); the distance from its inferior border to the inferior border of the mandible (IGT-IBM); the distance from the superior border of the genial tubercle to the inferior border of the mandible (SGT-IBM); and the width of the intermental foramen (IMFW). The following mean (SD) (range) measurements were obtained: GTW 7.38 (1.43) (4.5-10.0); GTH 7.94 (1.45) (5.0-10.0); IGT-IBM 7.96 (2.29) (4.0-12.0); SGT-IBM 15.90 (2.29) (12.0-20.0); and IMFW 56.65 (6.44) (43.0-67.0) mm. Of the 24 cadavers, 22 showed evidence of optimal positioning when the osteotomy was placed 2mm higher than the SGT-IBM measured on the inner table. This suggests that an optimal osteotomy, which includes the genial tubercle, may be possible in most patients when the osteotomy is positioned 2mm higher at the SGT-IBM.

Astrocytic water channel aquaporin-4 modulates brain plasticity in both mice and humans: a potential gliogenetic mechanism underlying language-associated learning.

The role of astrocytes in brain plasticity has not been extensively studied compared with that of neurons. Here we adopted integrative translational and reverse-translational approaches to explore the role of an astrocyte-specific major water channel in the brain, aquaporin-4 (AQP4), in brain plasticity and learning. We initially identified the most prevalent genetic variant of AQP4 (single nucleotide polymorphism of rs162008 with C or T variation, which has a minor allele frequency of 0.21) from a human database (n=60 706) and examined its functionality in modulating the expression level of AQP4 in an in vitro luciferase reporter assay. In the following experiments, AQP4 knock-down in mice not only impaired hippocampal volumetric plasticity after exposure to enriched environment but also caused loss of long-term potentiation after theta-burst stimulation. In humans, there was a cross-sectional association of rs162008 with gray matter (GM) volume variation in cortices, including the vicinity of the Perisylvian heteromodal language area (Sample 1, n=650). GM volume variation in these brain regions was positively associated with the semantic verbal fluency. In a prospective follow-up study (Sample 2, n=45), the effects of an intensive 5-week foreign language (English) learning experience on regional GM volume increase were modulated by this AQP4 variant, which was also associated with verbal learning capacity change. We then delineated in mice mechanisms that included AQP4-dependent transient astrocytic volume changes and astrocytic structural elaboration. We believe our study provides the first integrative evidence for a gliogenetic basis that involves AQP4, underlying language-associated brain plasticity.

Impaired development and dysfunction of endothelial progenitor cells in type 2 diabetic mice.

AIM: Dysfunction of circulating endothelial progenitor cells (EPCs) has been shown to affect the development of microvascular diseases in diabetes patients. The aim of this study was to elucidate the development and mechanical dysfunction of EPCs in type 2 diabetes (T2D). METHODS: The colony-forming capacity of EPCs and differentiation potential of bone marrow (BM) c-Kit(+)/Sca-I(+) lineage-negative mononuclear cells (KSL) were examined in T2D mice, db/db mice and KKAy mice, using EPC colony-forming assay (EPC-CFA). RESULTS: T2D mice had fewer BM stem/progenitor cells, and proliferation of KSL was lowest in the BM of db/db mice. In T2D mice, the frequency of large colony-forming units (CFUs) derived from BM-KSL was highly reduced, indicating dysfunction of differentiation into mature EPCs. Only a small number of BM-derived progenitors [CD34(+) KSL cells], which contribute to the supply of EPCs for postnatal neovascularization, was also found. Furthermore, in terms of their plasticity to transdifferentiate into various cell types, BM-KSL exhibited a greater potential to differentiate into granulocyte macrophages (GMs) than into other cell types. CONCLUSION: T2D affected EPC colony formation and differentiation of stem cells to mature EPCs or haematopoietic cells. These data suggest opposing regulatory mechanisms for differentiation into mature EPCs and GMs in T2D mice.

Identifying the potential long-term survivors among breast cancer patients with distant metastasis.

BACKGROUND: We aimed to develop a prediction model to identify long-term survivors after developing distant metastasis from breast cancer. PATIENTS AND METHODS: From the institution's database, we collected data of 547 patients who developed distant metastasis during their follow-ups. We developed a model that predicts the post-metastasis overall survival (PMOS) based on the clinicopathologic factors of the primary tumors and the characteristics of the distant metastasis. For validation, the survival data of 254 patients from four independent institutions were used. RESULTS: The median duration of the PMOS was 31.0 months. The characteristics of the initial primary tumor, such as tumor stage, hormone receptor status, and Ki-67 expression level, and the characteristics of the distant metastasis presentation including the duration of disease-free interval, the site of metastasis, and the presence of metastasis-related symptoms were independent prognostic factors determining the PMOS. The association between tumor stage and the PMOS was only seen in tumors with early relapses. The PMOS score, which was developed based on the above six factors, successfully identified patients with superior survival after metastasis. The median PMOS for patients with a PMOS score of <2 and for patients with a PMOS score of >5 were 71.0 and 12 months, respectively. The clinical significance of the PMOS score was further validated using independent multicenter datasets. CONCLUSIONS: We have developed a novel prediction model that can classify breast cancer patients with distant metastasis according to their survival after metastasis. Our model can be a valuable tool to identify long-term survivors who can be potential candidates for more intensive multidisciplinary approaches. Furthermore, our model can provide a more reliable survival information for both physicians and patients during their informed decision-making process.

A Large National Cohort Study of the Association between Bisphosphonates and Osteonecrosis of the Jaw in Patients with Osteoporosis: A Nested Case-control Study.

The purpose of this study was to examine the association between bisphosphonate exposure and osteonecrosis of the jaw (ONJ) in Korean patients with osteoporosis. A nested case-control study was performed using the claims database during 2002 to 2010 provided by the National Health Insurance Service. We identified a cohort of individuals with diagnosis of osteoporosis during 2002 to 2010. Cases and controls were identified during 2004 to 2010, and the date of potential cases of ONJ was defined as the index date. Bisphosphonate exposure was evaluated during 2 y prior to the index date. The association between bisphosphonate exposure and ONJ was tested by performing a conditional logistic regression analysis for matched data, and odds ratios (ORs) with 95% confidence intervals (CIs) were presented. Subjects were classified as nonuser, recent user, past user, or continuous user, depending on the prescription of bisphosphonates in 2 periods (1 to 2 y and 0 to 1 y prior to the index date). Continuous users were defined as patients who were exposed to bisphosphonate in both periods. We also examined the impact of bisphosphonate medication compliance by measuring the cumulative duration of exposure (CDE) on the risk of ONJ. A total of 212 cases with ONJ and 2,120 controls matched by sex, age, income level, and insurance type were identified among 109,787 patients with osteoporosis out of 1,025,340 enrollees in the sample cohort. The odds of having ONJ after adjusting for patient comorbidities significantly increased in continuous users of bisphosphonates (OR, 3.9; 95% CI, 2.4 to 6.2) compared to nonusers. Increased odds of ONJ were observed as CDE increased. The adjusted OR in patients with 1.5 y < CDE ≤ 2 y prior to the index date was 7.8 (95% CI, 4.0 to 15.5) versus nonusers. Our study results support significantly increased occurrences of potential ONJ in patients with osteoporosis who were exposed to bisphosphonates compared to those without exposure.

Effects of Palonosetron on Perioperative Cardiovascular Complications in Patients Undergoing Noncardiac Surgery With General Anesthesia: A Retrospective Cohort Study.

We retrospectively investigated whether palonosetron administered during the induction of general anesthesia is associated with an increased risk of perioperative cardiovascular complications in a single tertiary center cohort consisting of 4,517 palonosetron-exposed patients and 4,517 propensity score-matched patients without palonosetron exposure. The primary endpoint was a composite of perioperative cardiovascular complications, including intraoperative cardiac arrhythmia, intraoperative cardiac death, and myocardial injury within the first postoperative week, and there was no significant difference between the groups (odds ratio [OR] = 1.04; 95% confidence interval [CI] = 0.92-1.19). As secondary endpoints, intraoperative cardioversion, cardiac compression, use of cardiovascular drugs, postoperative hospital stay, and in-hospital mortality showed no differences between the groups. However, the palonosetron group showed decreased intraoperative hypotension (OR = 0.88; 95% CI = 0.79-0.97) and length of postoperative intensive care unit (ICU) stay (4.26 ± 9.86 vs. 6.14 ± 16.75; P = 0.026). Palonosetron did not increase the rate of perioperative cardiovascular complications, and can therefore be used safely during anesthetic induction.

Modulation of dendritic cell function by the radiation-mediated secretory protein γ-synuclein.

Recently, γ-synuclein (SNCG), which is also known as breast cancer-specific gene-1, has been demonstrated to be an adverse and aggressive marker in breast cancer. In our previous study, SNCG was significantly upregulated in irradiated human breast cancer cells. The aim of this study was to investigate whether radiation-induced, tumor-derived SNCG can influence dendritic cell (DC) function in immune systems. The phenotypical and functional changes of DCs in the presence or absence of SNCG were investigated by FACS analysis, ELISA, and real-time PCR. The ability of SNCG-treated DCs to influence T cells was also examined by coculturing with T cells. The treatment of DCs with SNCG protein inhibited the surface expression of the co-stimulatory molecules CD40 and CD86, and decreased the mRNA levels of pro-inflammatory cytokines. The SNCG-treated DCs inhibited T-cell proliferation slightly, but distinctively increased the population of regulatory T cells. In addition, the production of TGF-ß from T cells was significantly increased when they were cocultured with SNCG-treated DCs. Taken together, these results demonstrate that tumor-derived SNCG contributes to immunosuppressive effects via the inhibition of DC differentiation and activation, thus making it a potential target for cancer treatment.

Steroid plus antiviral treatment for Bell's palsy.

OBJECTIVES: The effectiveness of antiviral agents for the treatment of Bell's palsy is uncertain. We evaluated whether a steroid with an antiviral agent (S + A group) provided better recovery outcomes than a steroid alone (S group) in patients with Bell's palsy. SUBJECTS AND DESIGN: A total of 1342 patients diagnosed with Bell's palsy who visited the Kyung Hee Medical Center in Seoul, Korea, from 2002 to 2012 were included in this study. Patients in the S + A group were treated with prednisolone and antiviral agents (n = 569) and those in the S group with prednisolone alone (n = 773). Outcomes were measured using the House-Brackmann (HB) scale according to age, initial disease severity, electroneurography (ENoG) findings and underlying comorbidities. RESULTS: The rate of recovery (HB grades I and II) with initially severe Bell's palsy (HB grades V and VI) was higher in the S + A than in the S group (P = 0.001). However, the rates of recovery were similar with initially moderate palsy (HB grades II-IV) (P = 0.502). In patients classified according to age and ENoG-determined severity of palsy, the overall recovery rate was higher in the S + A than in the S group, but the differences were not statistically significant (P > 0.05 for both). The recovery rate without diabetes mellitus (DM) and hypertension (HTN) was higher in the S + A group than in the S group (P = 0.031). But in the patients with HTN and DM, the difference in recovery rates between the S + A and S groups was not statistically significant (P = 0.805). CONCLUSIONS: Treatment with a steroid plus antiviral agent resulted in significantly higher recovery rates than steroid therapy alone in patients with initially severe Bell's palsy and without either HTN or DM, and a nonsignificant trend towards higher recovery rates in all patients with Bell's palsy in this study. Antiviral agents may therefore help in the treatment of Bell's palsy.

Comparison of steroid administration methods in patients with idiopathic sudden sensorineural hearing loss: a retrospective observational study.

OBJECTIVES: To compare the recovery rates of patients with idiopathic sudden sensorineural hearing loss (ISSHL) treated with oral systemic steroids (PO) or intratympanic steroid injection (IT) or both. DESIGN: A retrospective observational study. SETTING: Tertiary referral centre. PARTICIPANTS: Eight hundred and forty-four patients diagnosed with ISSHL within 14 days of the onset of symptoms. The patients were divided into three groups by treatment modality. MAIN OUTCOME MEASURES: Threshold of pure-tone tests, age, accompanying symptoms and underlying diseases were compared. The level of final hearing recovery was evaluated by the application of the results of the pure-tone test that was performed at least 3 months after the completion of each treatment. RESULTS: Final hearing recovery rate differed significantly by the type of treatment (P = 0.031). Recovery rates in the PO and combined groups were significantly higher in patients with mild (85.1% and 88.6%, respectively) than with profound (52.8% and 69.0%, respectively) hearing loss (P < 0.05). In contrast, severity and recovery rate were not significantly correlated in the IT group (P > 0.05). Combined treatment yielded significantly higher recovery rates than other treatment modalities in patients without hypertension (HTN) and diabetes mellitus (DM) (P = 0.021). CONCLUSION: In the group treated with combined therapy, better hearing improvement was obtained than in the groups treated with systemic steroid only or with intratympanic steroid injection only without complications. These findings suggest that the combination of systemic administration and intratympanic injection may improve patient prognosis.

Neuritin can normalize neural deficits of Alzheimer's disease.

Reductions in hippocampal neurite complexity and synaptic plasticity are believed to contribute to the progressive impairment in episodic memory and the mild cognitive decline that occur particularly in the early stages of Alzheimer's disease (AD). Despite the functional and therapeutic importance for patients with AD, intervention to rescue or normalize dendritic elaboration and synaptic plasticity is scarcely provided. Here we show that overexpression of neuritin, an activity-dependent protein, promoted neurite outgrowth and maturation of synapses in parallel with enhanced basal synaptic transmission in cultured hippocampal neurons. Importantly, exogenous application of recombinant neuritin fully restored dendritic complexity as well as spine density in hippocampal neurons prepared from Tg2576 mice, whereas it did not affect neurite branching of neurons from their wild-type littermates. We also showed that soluble recombinant neuritin, when chronically infused into the brains of Tg2576 mice, normalized synaptic plasticity in acute hippocampal slices, leading to intact long-term potentiation. By revealing the protective actions of soluble neuritin against AD-related neural defects, we provide a potential therapeutic approach for patients with AD.

CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110).

Checkpoint kinase 2 (CHK2) kinase is a key mediator in many cellular responses to genotoxic stresses, including ionizing radiation (IR) and topoisomerase inhibitors. Upon IR, CHK2 is activated by ataxia telangiectasia mutated kinase and regulates the S-phase and G1-S checkpoints, apoptosis and DNA repair by phosphorylating downstream target proteins, such as p53 and Brca1. In addition, CHK2 is thought to be a multi-organ cancer susceptibility gene. In this study, we used a tandem affinity purification strategy to identify proteins that interact with CHK2 kinase. Cyclin-dependent kinase 11 (CDK11)(p110) kinase, implicated in pre-mRNA splicing and transcription, was identified as a CHK2-interacting protein. CHK2 kinase phosphorylated CDK11(p110) on serine 737 in vitro. Unexpectedly, CHK2 kinase constitutively phosphorylated CDK11(p110) in a DNA damage-independent manner. At a molecular level, CDK11(p110) phosphorylation was required for homodimerization without affecting its kinase activity. Overexpression of CHK2 promoted pre-mRNA splicing. Conversely, CHK2 depletion decreased endogenous splicing activity. Mutation of the phosphorylation site in CDK11(p110) to alanine abrogated its splicing-activating activity. These results provide the first evidence that CHK2 kinase promotes pre-mRNA splicing via phosphorylating CDK11(p110).

Establishment of isolation and expansion protocols for human cardiac C-kit-positive progenitor cells for stem cell therapy.

Although cardiac stem cells (CSCs) have emerged in regeneration research, the number of isolated CSCs is low, making a sufficient supply of functional elements an important consideration in cardiovascular research. In this study, we established an efficient method for CSC isolation. We directly compared cultures of single cells to human cardiac-derived c-kit-positive progenitor cells (hCPCs(c-kit+)). The two protocols employed enzymatically digested hCPCs(c-kit+) (ED-hCPCs) with tissue-expanded hCPC(c-kit+) (TE-hCPCs). Using fluorescence-activated cell sorting, we showed the concentration of c-kit in TE-hCPCs to be higher than in ED-hCPCs, although the total number of c-kit positive cells resulting from ED-hCPCs was similar to that resulting from TE-hCPCs. The cardiomyocyte-associated proteins, GATA4 and Nkx2-5, which were expressed during hCPCs expansion, did not differ between the isolation methods. Importantly, the expression of the CSC stem cell marker, c-kit, was more efficiently preserved using the ED-hCPCs versus the TE-hCPCs method. In a cell proliferation assay, the ED-hCPCs method produced a significantly greater number of cells. Finally, hCPCs derived using both protocols differentiated into endothelial, smooth muscle, and cardiomyocyte lineages. In conclusion, the single-cell culture protocol using an enzymatic digestion method may be more useful to isolate human cardiac-derived c-kit-positive elements compared with the tissue expansion method.

Polyethylene glycol bowel preparation does not eliminate the risk of acute renal failure: a population-based case-crossover study.

BACKGROUND AND STUDY AIMS: Polyethylene glycol (PEG) bowel preparations are regarded as effective and safe for colonoscopy; however, recent reports have indicated a risk of acute renal failure (ARF). This population-based case-crossover study evaluated the association between PEG and ARF in screening colonoscopy patients aged ≥ 50 years. PATIENTS AND METHODS: Korean Health Insurance Review and Assessment Service (HIRA) claims data from 1 January 2005 to 31 December 2009 were used in the study. The study population consisted of patients aged ≥ 50 years who were first hospitalized for ARF following colonoscopy involving PEG bowel preparation. For each patient, PEG use in a 1-, 2-, or 4-week period prior to the first hospital admission date for ARF (hazard period) was compared with PEG use in four earlier 1-, 2-, or 4-week control periods. Conditional logistic regression analysis was used to estimate odds ratios (ORs) and 95 % confidence intervals (CIs), adjusting for concomitant medications that could induce ARF. RESULTS: The total number of study patients was 1064 (59 % were male). A greater proportion of patients used PEG during the hazard period than during the control periods (for 4-week time window: 8.8 % vs. 3.2 %). The adjusted ORs for ARF incidence when applying the 1-, 2-, and 4-week periods were 3.1 (95 %CI 2.06 - 4.73), 2.5 (95 %CI 1.76 - 3.53), and 2.1 (95 %CI 1.61 - 4.85), respectively. CONCLUSIONS: The use of PEG was associated with the risk of ARF. Adequate hydration and renal function monitoring should be assured before and after colonoscopy, regardless of the bowel preparation regimen used.

Long-term clinical outcomes and risk factors for the occurrence of post-operative complications after cardiovascular surgery in patients with Behçet's disease.

OBJECTIVES: Cardiovascular surgery in patients with Behçet's disease (BD) frequently leads to postoperative complications such as anastomotic leakage, occlusion or pseudoaneurysm. We evaluated the clinical outcomes and related risk factors of postoperative complications in BD patients undergoing cardiovascular surgeries, as well as the long-term efficiency of postoperative immunosuppressive treatment. METHODS: Forty-one patients with BD who had undergone cardiovascular surgery between 1990 and 2009 were studied. We evaluated the patients' clinical data, postoperative complications, and survival rate. Risk factors related to the occurrence of postoperative complications were identified by univariate analysis using the Kaplan-Meier method with the log-rank test and multivariate analysis using the Cox proportional hazards regression model. RESULTS: Fifty-nine operations were performed in 41 patients. During the mean follow-up period of 65.3±48.1 months, complications such as paravalvular leakage, dehiscence, fistula, graft occlusion, or pseudoaneurysm occurred in 29 operations (49.2%). The cumulative occurrence rate of postoperative complication was 10.2% at three months, 32.8% at 12 months, and 43.8% at 24 months. Upon univariate analysis, young age, high Creactive protein levels, lack of postoperative immunosuppression, and short disease duration were identified as significant factors responsible for the occurrence of postoperative complications. In multivariate analysis, postoperative immunosuppression was found to independently lower the risk of complications. The 5-year survival rate was significantly higher in patients with postoperative immunosup immunosuppression than in those without (84.5% vs. 45.0%, p=0.011). CONCLUSIONS: The present study suggests that postoperative immunosuppressive therapy after cardiovascular surgeries in BD patients is important for reducing the development of serious postoperative complications.

Mutation spectrum and inhibitor risk in 100 Korean patients with severe haemophilia A.

Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by defects in the F8 gene encoding the coagulation factor VIII. Mutation analysis in HA is important to confirm the diagnosis, genotype-phenotype correlations and for genetic counselling and family study. The aim of this study was to detect causative mutations of F8 in severe HA patients in Korea and to correlate the mutation type with the risk of inhibitor development. A total of 100 unrelated Korean patients with severe HA were enrolled for this study. The Nijeman modification of the Bethesda assay was used to determine the presence of inhibitor. Molecular analysis of F8 was performed using a combination of molecular techniques, including long-distance polymerase chain reaction, direct sequencing and multiplex ligation-dependent probe amplification (MLPA). We identified causative mutations in 98% of severe HA patients (98/100). Inv22 and Inv1 mutations were detected in 30 patients and one patient, respectively. A total of 59 unique mutations were identified in 69 non-inversion patients, including 24 novel mutations. The overall prevalence of inhibitor was 26%. Inhibitor risk was highest in patients with large deletion mutations identified using MLPA (100%). Among those with point mutations, the prevalence of inhibitor was highest when the mutation occurred in the A3 and C2 domains (60% and 50%, respectively). The molecular diagnostic strategy involving multiplex PCR, sequencing and dosage analyses identified causative mutations in most cases of severe HA. The high inhibitor risk was associated with large deletion mutations and point mutations in A3 and C2 domains.