Opposite Effects of Apoptotic and Necroptotic Cellular Pathways on Rotavirus Replication.

Group A rotavirus (RVA), one of the leading pathogens causing severe acute gastroenteritis in children and a wide variety of young animals worldwide, induces apoptosis upon infecting cells. Though RVA-induced apoptosis mediated via the dual modulation of its NSP4 and NSP1 proteins is relatively well studied, the nature and signaling pathway(s) involved in RVA-induced necroptosis are yet to be fully elucidated. Here, we demonstrate the nature of RVA-induced necroptosis, the signaling cascade involved, and correlation with RVA-induced apoptosis. Infection with the bovine NCDV and human DS-1 RVA strains was shown to activate receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL), the key necroptosis molecules in virus-infected cells. Using an immunoprecipitation assay, RIPK1 was found to bind phosphorylated RIPK3 (pRIPK3) and pMLKL. pMLKL, the major executioner molecule in the necroptotic pathway, was translocated to the plasma membrane of RVA-infected cells to puncture the cell membrane. Interestingly, transfection of RVA NSP4 also induced necroptosis through the RIPK1/RIPK3/MLKL necroptosis pathway. Blockage of each key necroptosis molecule in the RVA-infected or NSP4-transfected cells resulted in decreased necroptosis but increased cell viability and apoptosis, thereby resulting in decreased viral yields in the RVA-infected cells. In contrast, suppression of RVA-induced apoptosis increased necroptosis and virus yields. Our findings suggest that RVA NSP4 also induces necroptosis via the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, necroptosis and apoptosis-which have proviral and antiviral effects, respectively-exhibited cross talk in RVA-infected cells. These findings significantly increase our understanding of the nature of RVA-induced necroptosis and the cross talk between RVA-induced necroptosis and apoptosis. IMPORTANCE Viral infection usually culminates in cell death through apoptosis, necroptosis, and, rarely, pyroptosis. Necroptosis is a form of programmed necrosis that is mediated by signaling complexes of the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Although apoptosis induction by rotavirus and its NSP4 protein is well known, rotavirus-induced necroptosis is not fully understood. Here, we demonstrate that rotavirus and also its NSP4 protein can induce necroptosis in cultured cells through activation of the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, rotavirus-induced necroptosis and apoptosis have opposite effects on viral yield, i.e., they function as proviral and antiviral processes, respectively, and counterbalance each other in rotavirus-infected cells. Our findings provide important insights for understanding the nature of rotavirus-induced necroptosis and the development of novel therapeutic strategies against infection with rotavirus and other RNA viruses.

Printable organometallic perovskite enables large-area, low-dose X-ray imaging.

Medical X-ray imaging procedures require digital flat detectors operating at low doses to reduce radiation health risks. Solution-processed organic-inorganic hybrid perovskites have characteristics that make them good candidates for the photoconductive layer of such sensitive detectors. However, such detectors have not yet been built on thin-film transistor arrays because it has been difficult to prepare thick perovskite films (more than a few hundred micrometres) over large areas (a detector is typically 50 centimetres by 50 centimetres). We report here an all-solution-based (in contrast to conventional vacuum processing) synthetic route to producing printable polycrystalline perovskites with sharply faceted large grains having morphologies and optoelectronic properties comparable to those of single crystals. High sensitivities of up to 11 microcoulombs per air KERMA of milligray per square centimetre (µC mGyair-1 cm-2) are achieved under irradiation with a 100-kilovolt bremsstrahlung source, which are at least one order of magnitude higher than the sensitivities achieved with currently used amorphous selenium or thallium-doped cesium iodide detectors. We demonstrate X-ray imaging in a conventional thin-film transistor substrate by embedding an 830-micrometre-thick perovskite film and an additional two interlayers of polymer/perovskite composites to provide conformal interfaces between perovskite films and electrodes that control dark currents and temporal charge carrier transportation. Such an all-solution-based perovskite detector could enable low-dose X-ray imaging, and could also be used in photoconductive devices for radiation imaging, sensing and energy harvesting.

Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating.

The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections.

Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens.

Some viruses within the Caliciviridae family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Ley HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked fucose and α1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines, and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored.IMPORTANCE Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples, and bovine duodenal sections. We further discovered that α1,2-linked fucose and α1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus.

Evaluation of global laboratory methods and establishing on-therapy ranges for monitoring apixaban and rivaroxaban: Experience at a single institution.

BACKGROUND: Apixaban and rivaroxaban are approved for the prevention and treatment of deep vein thrombosis (DVT), pulmonary embolism (PE), and embolic stroke in atrial fibrillation (AF) patients. The aim of this study was to find appropriate methods of monitoring the anticoagulant effects of are direct oral anticoagulants (DOACs) and establish on-therapy ranges using conventional tests. METHODS: A total of 184 samples were collected from 91 patients receiving DOACs. Concentrations of apixaban and rivaroxaban in plasma were accessed by an anti-factor Xa chromogenic assay. PT, APTT, antithrombin, D-dimer, dRVVT screen/confirm, FDP, and fibrinogen levels were measured. On-therapy ranges were calculated by substituting previously reported trough plasma concentrations of DOACs. RESULTS: Anti-factor Xa chromogenic assay-based DOACs levels were 26.0-279.5 (115.9 ± 56.5) ng/mL for apixaban at 2.5 mg BID, 19.9-565.1 (205.3 ± 162.4) ng/mL for apixaban at 5 mg BID, 2.3-395.3 (205.3 ± 162.4) ng/mL for rivaroxaban at 15 mg OD, 3.6-494.8 (119.6 ± 95.1) ng/mL for rivaroxaban at 20 mg OD, and 9.6-431.4 (140.8 ± 113.6) ng/mL for rivaroxaban at 15 mg BID. PT (%), antithrombin, and dRVVT confirm tests showed good correlation with plasma apixaban levels. Plasma rivaroxaban concentrations were correlated well with PT (sec), PT (%),and dRVVT confirm results. On-therapy ranges established for dRVVT confirm test by linear regression were as follows: 1.32-1.52 for apixaban 2.5 mg BID, 1.12-1.75 for apixaban 5 mg BID, 1.11-1.78 for rivaroxaban 15 mg OD, 1.09-1.64 for rivaroxaban 20 mg OD, and 1.22-1.81 for rivaroxaban 20 mg BID. CONCLUSIONS: Apixaban concentrations were well correlated with PT (%), antithrombin, and dRVVT confirm test. Rivaroxaban concentrations showed good correlation with PT (sec), PT (%), and dRVVT confirm test.

How useful is bone marrow study as an initial investigative tool without lymph node biopsy in malignant lymphoma?: Eleven years of experience at a single institution.

BACKGROUND: Bone marrow (BM) study plays an important role as initial investigation specimen of lymphoma as well as staging lymphoma. This study aimed to investigate the utility of BM studies for classification of lymphoma and evaluate features of BM involvement by lymphoma over a period of 11 years. METHODS: A total of 1162 cases of BM studies for lymphoma evaluation were reviewed for the incidence of lymphoma subtypes, the percentage of marrow involvement, the pattern of involvement and discordance with histopathologic diagnoses of lymph nodes and other tissues. RESULTS: A total of 255 of 1162 cases underwent BM study without pathologic information, and 108 cases show lymphoma involvement. Lymph node biopsy underwent in 66 cases, and 10 cases show discordant result between BM and lymph node biopsy. Seven discordant cases were due to insufficient further studies. Lymphoma was diagnosed only by BM study in 38 cases. Abnormal lymphocytes were found in BM aspiration in 34 cases. Also, abnormal clonal lymphocytes were detected by flow cytometry in 26 cases. Four cases showed disease-related chromosomal abnormalities. FISH analysis detected abnormal findings in two cases, however, discordant with other additional studies. CONCLUSIONS: Discrepancies between the BM study and lymph node biopsy were due to insufficient further study and discordance of immunohistochemical stain result. BM study can be utilized as initial diagnosis of lymphoma by the combination of morphological feature, involvement pattern, and additional tests such as flow cytometry, chromosomal analysis, and FISH analysis. Thus, BM study with further analysis is an essential choice when lymph node biopsies are unavailable.

Detection and genomic analysis of genital group B streptococcus in pregnant Korean women.

AIM: Group B streptococcus (GBS) is a leading cause of life-threatening bacterial infections among newborns, and neonates born to heavily colonized women may be subject to vertical transmission. We sought to determine an appropriate detection method for genital GBS in pregnant women by comparing culture-based methods and real-time polymerase chain reaction (PCR). In addition, we performed molecular serotyping and multilocus sequence typing (MLST) on isolates. METHODS: A total of 150 pregnant women were enrolled and underwent vaginal-rectal swabbing at 16-40 weeks of gestation. GBS was identified by conventional culture and real-time PCR with or without enrichment. Molecular serotyping and MLST were performed on isolates. RESULTS: Overall genital GBS positive rate among the 150 study subjects was 17.3%. Direct culture identified 18 (12.0%) positive specimens, enrichment culture 22 (14.6%), direct PCR 24 (16.0%) and enrichment PCR 25 (16.6%). The sensitivity and specificity by direct and enrichment PCR were as follows: for direct PCR, 90.9% and 96.9%, respectively; and for enrichment PCR, 95.5% and 96.9%, respectively. Resistance rates to clindamycin and erythromycin were 33.3% and 19.1%, respectively. Serotype III-1 was the most common (26.3%), followed by serotype Ib (21.1%), III-3 (15.8%), V (15.8%), II (10.5%), IV (5.3%) and VI (5.3%). Most common sequence types (ST) were ST-1, ST-19 and ST-862 (15.8%), followed by ST-2 and ST-654 (10.5%). CONCLUSION: Direct real-time PCR using vaginal-rectal specimen could be used for detecting GBS in emergent conditions. Molecular serotypes III, Ib and V were most common. MLST analysis frequently presented ST-1, ST-19 and ST-862.

Universal Approach toward Hysteresis-Free Perovskite Solar Cell via Defect Engineering.

Organic-inorganic halide perovskite is believed to be a potential candidate for high efficiency solar cells because power conversion efficiency (PCE) was certified to be more than 22%. Nevertheless, mismatch of PCE due to current density (J)-voltage (V) hysteresis in perovskite solar cells is an obstacle to overcome. There has been much lively debate on the origin of J-V hysteresis; however, effective methodology to solve the hysteric problem has not been developed. Here we report a universal approach for hysteresis-free perovskite solar cells via defect engineering. A severe hysteresis observed from the normal mesoscopic structure employing TiO2 and spiro-MeOTAD is almost removed or does not exist upon doping the pure perovskites, CH3NH3PbI3 and HC(NH2)2PbI3, and the mixed cation/anion perovskites, FA0.85MA0.15PbI2.55Br0.45 and FA0.85MA0.1Cs0.05PbI2.7Br0.3, with potassium iodide. Substantial reductions in low-frequency capacitance and bulk trap density are measured from the KI-doped perovskite, which is indicative of trap-hysteresis correlation. A series of experiments with alkali metal iodides of LiI, NaI, KI, RbI and CsI reveals that potassium ion is the right element for hysteresis-free perovskite. Theoretical studies suggest that the atomistic origin of the hysteresis of perovskite solar cells is not the migration of iodide vacancy but results from the formation of iodide Frenkel defect. Potassium ion is able to prevent the formation of Frenkel defect since K+ energetically prefers the interstitial site. A complete removal of hysteresis is more pronounced at mixed perovskite system as compared to pure perovskites, which is explained by lower formation energy of K interstitial (-0.65 V for CH3NH3PbI3 vs -1.17 V for mixed perovskite). The developed KI doping methodology is universally adapted for hysteresis-free perovskite regardless of perovskite composition and device structure.

Activation of COX-2/PGE2 Promotes Sapovirus Replication via the Inhibition of Nitric Oxide Production.

Enteric caliciviruses in the genera Norovirus and Sapovirus are important pathogens that cause severe acute gastroenteritis in both humans and animals. Cyclooxygenases (COXs) and their final product, prostaglandin E2 (PGE2), are known to play important roles in the modulation of both the host response to infection and the replicative cycles of several viruses. However, the precise mechanism(s) by which the COX/PGE2 pathway regulates sapovirus replication remains largely unknown. In this study, infection with porcine sapovirus (PSaV) strain Cowden, the only cultivable virus within the genus Sapovirus, markedly increased COX-2 mRNA and protein levels at 24 and 36 h postinfection (hpi), with only a transient increase in COX-1 levels seen at 24 hpi. The treatment of cells with pharmacological inhibitors, such as nonsteroidal anti-inflammatory drugs or small interfering RNAs (siRNAs) against COX-1 and COX-2, significantly reduced PGE2 production, as well as PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We observed that pharmacological inhibition of COX-2 dramatically increased NO production, causing a reduction in PSaV replication that could be restored by inhibition of nitric oxide synthase via the inhibitor N-nitro-l-methyl-arginine ester. This study identified a pivotal role for the COX/PGE2 pathway in the regulation of NO production during the sapovirus life cycle, providing new insights into the life cycle of this poorly characterized family of viruses. Our findings also reveal potential new targets for treatment of sapovirus infection. IMPORTANCE: Sapoviruses are among the major etiological agents of acute gastroenteritis in both humans and animals, but little is known about sapovirus host factor requirements. Here, using only cultivable porcine sapovirus (PSaV) strain Cowden, we demonstrate that PSaV induced the vitalization of the cyclooxygenase (COX) and prostaglandin E2 (PGE2) pathway. Targeting of COX-1/2 using nonsteroidal anti-inflammatory drugs (NSAIDs) such as the COX-1/2 inhibitor indomethacin and the COX-2-specific inhibitors NS-398 and celecoxib or siRNAs targeting COXs, inhibited PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE2 pathway. We further demonstrate that the production of PGE2 provides a protective effect against the antiviral effector mechanism of nitric oxide. Our findings uncover a new mechanism by which PSaV manipulates the host cell to provide an environment suitable for efficient viral growth, which in turn can be a new target for treatment of sapovirus infection.

Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II.

Caliciviruses in the genus Sapovirus are a significant cause of viral gastroenteritis in humans and animals. However, the mechanism of their entry into cells is not well characterized. Here, we determined the entry mechanism of porcine sapovirus (PSaV) strain Cowden into permissive LLC-PK cells. The inhibition of clathrin-mediated endocytosis using chlorpromazine, siRNAs, and a dominant negative (DN) mutant blocked entry and infection of PSaV Cowden strain, confirming a role for clathrin-mediated internalization. Entry and infection were also inhibited by the cholesterol-sequestering drug methyl-ß-cyclodextrin and was restored by the addition of soluble cholesterol, indicating that cholesterol also contributes to entry and infection of this strain. Furthermore, the inhibition of dynamin GTPase activity by dynasore, siRNA depletion of dynamin II, or overexpression of a DN mutant of dynamin II reduced the entry and infection, suggesting that dynamin mediates the fission and detachment of clathrin- and cholesterol-pits for entry of this strain. In contrast, the inhibition of caveolae-mediated endocytosis using nystatin, siRNAs, or a DN mutant had no inhibitory effect on entry and infection of this strain. It was further determined that cell entry of PSaV Cowden strain required actin rearrangements for vesicle internalization, endosomal trafficking from early to late endosomes through microtubules, and late endosomal acidification for uncoating. We conclude that PSaV strain Cowden is internalized into LLC-PK cells by clathrin- and cholesterol-mediated endocytosis that requires dynamin II and actin rearrangement, and that the uncoating occurs in the acidified late endosomes after trafficking from the early endosomes through microtubules.

Laboratory diagnosis of Clostridium difficile infection: Comparison of Techlab C. diff Quik Chek Complete, Xpert C. difficile, and multistep algorithmic approach.

BACKGROUND: Clostridium difficile is a major pathogen responsible for nosocomial infectious diarrhea. We explored optimal laboratory strategies for diagnosis of C. difficile infection (CDI) in our clinical settings, a 1400-bed tertiary care hospital. METHODS: Using 191 fresh stool samples from adult patients, we evaluated the performance of Xpert C. difficile (Xpert CD), C. diff Quik Chek Complete (which simultaneously detects glutamate dehydrogenase [GDH] and C. difficile toxins [CDT]), toxigenic culture, and a two-step algorithm composed of GDH/CDT as a screening test and Xpert CD as a confirmatory test. RESULTS: Clostridium difficile was detected in 35 samples (18.3%), and all isolates were toxigenic strains. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value of each assay for detecting CDI were as follows: Quik Chek Complete CDT (45.7%, 100%, 100%, 89.1%), Quik Chek Complete GDH (97.1%, 99.4%, 97.1%, 99.4%), Xpert CD (94.3%, 100%, 100%, 98.7%), and toxigenic culture (91.4%, 100%, 100%, 98.1%). A two-step algorithm performed identically with Xpert CD assay. CONCLUSION: Our data showed that most C. difficile isolates from adult patients were toxigenic. We demonstrated that a two-step algorithm based on GDH/CDT assay followed by Xpert CD assay as a confirmatory test was rapid, reliable, and cost effective for diagnosis of CDI in an adult patient setting with high prevalence of toxigenic C. difficile.

Performance evaluation of Mindray CAL 8000(BC-6800 and SC-120) hematology analyzer and slidemaker/stainer.

INTRODUCTION: The Mindray CAL 8000 is a cellular analysis line that consists of the BC-6800, an automated hematology analyzer, and the SC-120, an automated slidemaker/stainer. We evaluated the performances of the BC-6800 and the SC-120. METHODS: Four hundred and eight normal and abnormal samples were analyzed. The performance of the BC-6800 and Sysmex XE-2100 were compared, and blood films by the SC-120 and manual method were compared according to the CLSI guideline H26-A2 and H20-A2. RESULTS: Most parameters measured by the BC-6800 matched well with the XE-2100 and manual differential. The flag efficiency of the BC-6800 for blasts (95.3%) and atypical lymphocytes (92.6%) were higher while immature granulocytes (89.7%) and NRBCs (94.1%) were lower than that of the XE-2100. Additionally, the BC-6800 detected four of five samples infected with plasmodium parasites. The SC-120 showed no carry-over and expected repeatability. There was good agreement on the five-part differential including abnormal cells between blood films by the SC-120 and manually prepared blood films. The shape of the RBC was also comparable between blood films. CONCLUSION: The CAL-8000 analysis line is beneficial for precise, fast hematology work, and even more useful in malaria endemic areas.

Pathogenesis of Korean SapelovirusA in piglets and chicks.

Sapelovirus A (SV-A), formerly known as porcine sapelovirus as a member of a new genus Sapelovirus, is known to cause enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. We have recently identified α2,3-linked sialic acid on GD1a ganglioside as a functional SV-A receptor rich in the cells of pigs and chickens. However, the role of GD1a in viral pathogenesis remains elusive. Here, we demonstrated that a Korean SV-A strain could induce diarrhoea and intestinal pathology in piglets but not in chicks. Moreover, this Korean SV-A strain had mild extra-intestinal tropisms appearing as mild, non-suppurative myelitis, encephalitis and pneumonia in piglets, but not in chicks. By real-time reverse transcription (RT) PCR, higher viral RNA levels were detected in faecal samples than in sera or extra-intestinal organs from virus-inoculated piglets. Immunohistochemistry confirmed that high viral antigens were detected in the epithelial cells of intestines from virus-inoculated piglets but not from chicks. This Korean SV-A strain could bind the cultured cell lines originated from various species, but replication occurred only in cells of porcine origin. These data indicated that this Korean SV-A strain could replicate and induce pathology in piglets but not in chicks, suggesting that additional porcine-specific factors are required for virus entry and replication. In addition, this Korean SV-A strain is enteropathogenic, but could spread to the bloodstream from the gut and disseminate to extra-intestinal organs and tissues. These results will contribute to our understanding of SV-A pathogenesis so that efficient anti-sapelovirus drugs and vaccines could be developed in the future.

Whole genomic characterization of Korean porcine G8P[7] reassortant rotaviruses.

This study analyzed eleven genomic segments of three Korean porcine G8P[7] group A rotavirus (RVA) strains. Phylogenetically, these strains contained two bovine-like and nine porcine-like genomic segments. Eight genes (VP1, VP2, VP6 and NSP1-NSP5) of strains 156-1 and 42-1 and seven genes (VP1, VP2, VP6 and NSP2-NSP5) of strain C-1 clustered closely with porcine and porcine-like animal strains and distantly from typical human Wa-like strains. The VP3-M2 genotype of these strains clustered closely with bovine-like strains, but distantly with typical human DS-1-like strains. These data indicate that multiple reassortments involving porcine and bovine RVA strains in Korea must have occurred.

Distinct frequencies and mutation spectrums of genetic thrombophilia in Korea in comparison with other Asian countries both in patients with thromboembolism and in the general population.

Hereditary natural anticoagulant deficiencies are the major cause of genetic thrombophilia in Asia. Given the growing acknowledgment of the risk of venous thromboembolism in Asian populations, we investigated the frequency and mutation spectrums of natural anticoagulant deficiency in Korea. The group of patients consisted of consecutive patients with venous thromboembolism screened for thrombophilia. Genetic tests were performed on suspicion of natural anticoagulant deficiency. For the population group, >3,000 individuals were screened from routine check-ups, and those with a low level (<1(st) percentile) of natural anticoagulant underwent genetic tests. Mutations were detected by direct sequencing of PROC, PROS1, and SERPINC1, followed by additional multiplex ligation-dependent probe amplification for PROS1 and SERPINC1 for dosage mutations. Among 500 patients screened, 127 were suspected of having a natural anticoagulant deficiency, and this was genetically confirmed in 71: protein C deficiency in 36 (50.7%), antithrombin deficiency in 21 (29.6%), and protein S deficiency in 14 (19.7%). Among 3,129 individuals from the population who were screened, the frequency of natural anticoagulant deficiency was ~1.0%: antithrombin deficiency 0.49%, protein C deficiency 0.35%, and protein S deficiency 0.16%. Two PROC mutations causing type I protein C deficiency were prevalent (Arg211Trp and Met406Ile in patients and Arg211Trp in the population). Two SERPINC1 mutations causing type II antithrombin deficiency, Arg79Cys and Ser158Pro, were prevalent in the population group. This is the first study on the genetic epidemiology of natural anticoagulant deficiencies in Korea. The results demonstrated that the frequencies and spectrum of mutations underlying genetic thrombophilia in Korea are different not only from those in Caucasians but also those in other Asian populations.

Hereditary gene mutations in Korean patients with isolated erythrocytosis.

Most cases of erythrocytosis occur secondary to chronic tissue hypoxia or as a clonal disease such as polycythemia vera with somatic mutations in the Janus kinase 2 (JAK2) gene. Rarely, erythrocytosis is caused by hereditary gene mutations. This study investigated hereditary gene mutations in 38 unrelated Korean patients with isolated erythrocytosis without (1) JAK2 mutation and (2) secondary causes of erythrocytosis other than smoking history. Direct sequencing analyses were performed on six genes associated with hereditary erythrocytosis [HBB, exon 2 and exon 3 of HBA2, VHL, EGLN1 (previously PHD2), exon 12 of EPAS1 (previously HIF2A), and exons 5-8 of EPOR]. As a result, mutations were detected in five patients (three never smokers and two current smokers) out of 38 patients (13.2 %). The mutations detected in those five patients were EPOR:p.W439*, EPOR:p.G212C, HBB:p.H98Q (or conventionally H97Q, Hb Malmö [ß 97(FG4) His > Gln]), HBB:p.V138M (V137M), and EGLN1:p.L279Tfs43*, all in heterozygous state. No patient had mutations in HBA2, VHL, or in EPAS1. This study indicates that workup for hereditary gene mutations is needed for isolated erythrocytosis with or without smoking history.

Clinical significance of SF3B1 mutations in Korean patients with myelodysplastic syndromes and myelodysplasia/myeloproliferative neoplasms with ring sideroblasts.

Somatic mutations in the SF3B1 gene, a gene encoding the splicing factor 3B subunit 1, were recently reported in myelodysplastic syndromes (MDS), particularly in the presence of ring sideroblasts (RS). The authors investigated the prevalence and clinical significance of SF3B1 mutations in Korean patients with myeloid neoplasms with RS. The study subjects were 43 Korean patients with myeloid neoplasms. Twenty-nine patients (67 %) had 15 % or more RS (high-count RS [HC-RS]), and 14 (33 %) had RS less than 15 % (low-count RS [LC-RS]). Molecular genetic tests were performed to detect SF3B1 mutations by direct sequencing on bone marrow samples of the patients. SF3B1 mutations were detected in 55 % (16/29) of the HC-RS group: 3 RARS (3/3), 8 RCMD (8/16), 3 RARS-t (3/4), 1 RAEB (1/4), and 1 MDS-U (1/1). All mutations were previously reported mutations with K700E being the most common (63 % of mutation-positive cases). On the other hand, none (0 %) of the LC-RS group had SF3B1 mutation. The patients with SF3B1 mutations had higher platelet counts (p = 0.023), higher proportions of RS (p = 0.003), and lower proportions of bone marrow blasts (p = 0.026) than those without SF3B1 mutations. SF3B1 mutations showed a favorable survival implication (p = 0.025), but not in multivariate analysis (p = 0.178). This study confirmed that SF3B1 mutation is highly specific to the HC-RS phenotype in Korean patients with myeloid neoplasms with similar frequencies and distributions in previous findings and is associated with distinct hematologic features.

Steroid profiling for congenital adrenal hyperplasia by tandem mass spectrometry as a second-tier test reduces follow-up burdens in a tertiary care hospital: a retrospective and prospective evaluation.

BACKGROUND: Newborn screening for congenital adrenal hyperplasia (CAH) based on measuring 17-hydroxyprogesterone (17-OHP) by immunoassay generates a number of false-positive results, especially in preterm neonates. We applied steroid profiling by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a second-tier test in newborns with positive CAH screening and evaluated its clinical utility in a tertiary care hospital setting. METHODS: By performing a 4-year retrospective data review, we were able to test 121 dried blood spots from newborns with positive CAH screening for 17-OHP, androstenedione and cortisol levels by LC-MS/MS. We prospectively evaluated the clinical utility of steroid profiling after the implementation of steroid profiling as a second-tier test in our routine clinical practice. During the 2-year prospective study period, 104 cases with positive initial screening by FIA were tested by LC-MS/MS. Clinical and laboratory follow-up were performed for at least 6 months. RESULTS: The preterm neonates accounted for 50.7% (76/150) and 70.4% (88/125) of screening-positive cases in retrospective and prospective cohorts, respectively. By applying steroid profiling as a second-tier test for positive CAH screening, we eliminated all false-positive results and decreased the median follow-up time from 75 to 8 days. CONCLUSIONS: Our data showed that steroid profiling reduced the burden of follow-up exams by improving the positive predictive value of the CAH screening program. The use of steroid profiling as a second-tier test for positive CAH screening will improve clinical practice particularly in a tertiary care hospital setting where positive CAH screening from preterm neonates is frequently encountered.

Founder effects in two predominant intronic mutations of UNC13D, c.118-308C>T and c.754-1G>C underlie the unusual predominance of type 3 familial hemophagocytic lymphohistiocytosis (FHL3) in Korea.

Familial hemophagocytic lymphohistiocytosis (familial HLH or FHL) is a potentially fatal autosomal recessive disorder. Our previous study demonstrated that UNC13D mutations (FHL3) account for ~90 % of FHL in Korea with recurrent splicing mutation c.754-1G>C (IVS9-1G>C). Notably, half of the FHL3 patients had a monoallelic mutation of UNC13D. Deep intronic mutations in UNC13D were recently reported in patients of European descent. In this study, we performed targeted mutation analyses for deep intronic mutations and investigated on the founder effect in FHL3 in Korean patients. The study patients were 72 children with HLH including those with FHL3 previously reported to have a monoallelic UNC13D mutation. All patients were recruited from the Korean Registry of Hemophagocytic Lymphohistiocytosis. In addition to conventional sequencing of FHL2-4, targeted tests for c.118-308C>T and large intronic rearrangement mutations of UNC13D were performed. Haplotype analysis was performed for founder effects using polymorphic markers in the FHL3 locus. FHL mutations were detected in 20 patients (28 %). Seventeen patients had UNC13D mutations (FHL3, 85 %) and three had PRF1 mutations (FHL2, 15 %). UNC13D:c.118-308C>T was detected in ten patients, accounting for 38 % of all mutant alleles of UNC13D, followed by c.754-1G>C (26 %). Haplotype analyses revealed significantly shared haplotypes in both c.118-308C>T and c.754-1G>C, indicating the presence of founder effects. The deep intronic mutation UNC13D:c.118-308C>T accounts for the majority of previously missing mutations and is the most frequent mutation in FHL3 in Korea. Founder effects of two recurrent intronic mutations of UNC13D explain the unusual predominance of FHL3 in Korea.

A case of maturity-onset diabetes of the young type 4 in Korea.

Maturity-onset diabetes of the young (MODY) is a rare, autosomal dominant disease characterized by non-ketogenic diabetes mellitus (DM). MODY type 4, caused by PDX1 mutation, is a very rare subtype of MODY, especially in Korea. We report a case of a 10-year-old, nonobese girl with a family history of type 2 DM. After diagnosis, the patient's serum glucose level was well controlled using metformin monotherapy; however, the glycated hemoglobin level increased to 9.0% approximately 2 years after treatment. No obesity or lifestyle problems were observed, and serum fasting C-peptide level was within the normal range. Furthermore, no islet-related autoantibodies were detected. A genetic screening for MODY using a next-generation sequencing panel was performed, and a likely heterozygous pathogenic PDX1 mutation (p.Gly246ArgfsTer21) was identified. The PDX1 variant was not detected in her mother, implying that the mutation had arisen de novo in the proband. She was prescribed insulin degludec in addition to metformin therapy, which improved her hyperglycemia. This report presents a novel MODY type 4 phenotype and highlights the importance of genetic screening in patients with MODY characteristics.