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Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency has been a target of expanded newborn screening (ENBS) using tandem mass spectrometry in Japan. Since the implementation of ENBS, a number of novel ACADVL variants responsible for VLCAD deficiency have been identified. In this study, genotypic differences in Japanese patients with VLCAD deficiency were investigated before and after ENBS. The ACADVL variants in 61 subjects identified through ENBS (ENBS group) and in 40 patients who subsequently developed clinical symptoms without undergoing ENBS (pre-ENBS group) were compared. Subjects in the ENBS group underwent genetic testing and/or VLCAD enzyme activity measurements. Patients in the pre-ENBS group were stratified into three clinical phenotypes and underwent genetic testing. This study revealed that the variants p.K264E, p.K382Q and c.996dupT were found in both groups, but their frequencies were lower in the ENBS group (5.2%, 3.1% and 4.2%, respectively) than in the pre-ENBS group (16.5%, 12.7% and 10.1%, respectively). In addition, p.C607S, p.T409M, p.M478I, p.G289R, p.C237R, p.T260M, and p.R229* were exclusively identified in the ENBS group. Among these variants, p.C607S exhibited the highest frequency (18.8%). The patients who were heterozygous for p.C607S demonstrated 7-42% of control enzyme activity. p.C607S is suspected to be unique to Japanese individuals. According to a comparison of enzyme activity, patients with the p.C607S variant may exhibit higher enzyme activity than those with the p.A416T, p.A180T, p.R450H, and p.K264E variants, which are responsible for the myopathic form of the disease. The VLCAD deficiency genotypes have changed since the initiation of ENBS in Japan.

CXCR4 antagonists sensitize FLT3/ITD

Very long‐chain acyl‐CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive mitochondrial fatty acid oxidation disorder that manifests in three clinical forms: (a) severe, (b) milder, and (c) myopathic. Patients with the myopathic form present intermittent muscular symptoms such as myalgia, muscle weakness, and rhabdomyolysis during adolescence or adulthood. Here, the clinical symptoms and serum creatine kinase (CK) levels of a pregnant 31‐year‐old woman with the myopathic form of VLCAD deficiency were reduced during pregnancy. Clinical symptoms rarely appeared during pregnancy, although she had sometimes suffered from muscular symptoms before pregnancy. When ritodrine was administered for threatened premature labor at 35 weeks of gestation, her CK level was elevated to over 3900 IU/L. She delivered a full‐term baby via cesarean section but suffered from muscle weakness with elevated CK levels soon after delivery. It has been reported that an unaffected placenta and fetus can improve maternal β‐oxidation during pregnancy. However, in our case, the baby was also affected by VLCAD deficiency. These suggest that the clinical symptoms of a woman with VLCAD deficiency might be reduced during pregnancy even if the fetus is affected with VLCAD deficiency.

Multiple acyl-CoA dehydrogenase deficiency (MADD), also known as glutaric acidemia type II, is classically caused by a congenital defect in electron transfer flavoprotein (ETF) or ETF dehydrogenase (ETFDH). Flavin adenine dinucleotide synthase (FADS) deficiency caused by mutations in FLAD1 was recently reported as a novel riboflavin metabolism disorder resembling MADD. Here, we describe a Japanese boy with FADS deficiency due to a novel mutation (p.R249*) in FLAD1. In the asymptomatic male infant born at full term, newborn screening showed positive results with elevated C5 and C14:1 acylcarnitine levels and an increased C14:1/C2 ratio. Biochemical studies were unremarkable except for lactic acidosis (pH 7.197, lactate 61 mg/dL). A diagnosis of MADD was suspected because of mild abnormalities of the acylcarnitine profile and apparent abnormalities of urinary organic acids, although mutations in the ETFA, ETFB, ETFDH, and riboflavin transporter genes (SLC52A1, SLC52A2, and SLC52A3) were not detected. Administration of riboflavin and L-carnitine was initiated at one month of age based on the diagnosis of “biochemical MADD” despite a lack of symptoms. Nevertheless, the acylcarnitine profile was not normalized. Symptoms resembling bulbar palsy, such as vocal cord paralysis and dyspnea with stridor, were present from 3 months of age. At 4 months of age, he became bedridden because of hypoxic-ischemic encephalopathy due to fulminant respiratory failure with aspiration pneumonia. At 2 years and 5 months of age, a homozygous c.745C > T (p.R249*) mutation in the FLAD1 gene was identified, confirming the diagnosis of FADS deficiency. His severe clinical course may be caused by this nonsense mutation associated with poor responsiveness to riboflavin. Persistent lactic acidosis and neuropathy, such as bulbar palsy, may be important for diagnosing FADS deficiency. Although the biochemical findings in FADS deficiency are similar to those in MADD, their clinical symptoms and severity may not be identical.

Background While providing various benefits, concerns about the potential risks of kangaroo mother care, or skin-to-skin contact (SSC), between mother and her preterm infant hinder its widespread implementation in some resource- rich countries. In neonates, salivary chromogranin A (s-CgA) is elevated upon exposure to stress, whereas the perfusion index (PI) is associated with hemodynamics and peripheral perfusion. Here, we investigated the effects of SSC on s-CgA and the PI in preterm infants. Methods Twelve infants were enrolled in the study. Factors associated with baseline s-CgA were analyzed. Baseline s-CgA and the level after SSC were compared. Secreted IgA in the saliva was compared as the control. The PI before, throughout, and after SSC were compared. Results Baseline s-CgA was significantly lower in infants who were supplemented with baby formula milk in addition to breast milk before SSC (n = 2) compared with those fed with their mother's breast milk alone (n = 10, P = 0.03). SSC significantly decreased s-CgA in babies who were fed breast milk only before SSC (n = 10, P = 0.01) but not in those supplemented with formula milk before SSC (n = 2). Secreted IgA in saliva was not affected by SSC. The PI was significantly elevated during SSC (P = .01). Conclusion Our data indicate that SSC can reduce s-CgA levels when combined with mother's breast milk and increase the PI in preterm infants, thereby providing additional evidence of the benefit of SSC.

To explore the additional medical costs (AMCs) due to hospital-acquired falls (falls), as well as their impact on clinical services within hospitals under the nationally uniform universal health insurance system in Japan.With the use of administrative profiling data based on accounting systems linked with the Japanese social insurance medical fee schedule, we analyzed data from 2 teaching hospitals: Shimane University Hospital (SUH) and St. Mary's Hospital (SMH). We extracted 588 fall cases from 4669 incident reports in SUH and 1168 fall cases from 7717 incident reports in SMH that potentially incurred AMCs.Additional medical costs were 364 ± 2129 USD for minor injuries and 4336 ± 3645 USD for major injuries at SUH (P0.001) and 114 ± 124 USD for minor injuries and 2267 ± 2811 USD for major injuries at SMH (P0.001). Among the clinical services provided, imaging services were the most frequently used, with 89.9% (n = 205) of 228 minor injuries at SUH and 86.7% (n = 339) of 391 minor injuries at SMH; imaging services were used in all major injury cases at both hospitals. Although the number of cases using additional procedure/surgery services was lower than those using imaging services at both hospitals, AMCs for procedure/surgery services accounted for the highest proportions of total AMCs in both hospitals.Although falls with minor injuries outnumbered falls with major injuries, fall-related AMCs for the latter were higher at both teaching hospitals because procedure/surgery services were required for cases with major injuries such as femoral neck and trochanteric fractures. The findings suggest that hospital administrators and policy makers have to take appropriate measures to prevent major injuries inpatients due to hospital-acquired falls.

Background: Expanded newborn screening (ENBS) utilizing tandem mass spectrometry (MS/MS) for inborn metabolic diseases (IMDs), such as organic acidemias (OAs), fatty acid oxidation disorders, (FAODs), and amino acid disorders (AAs), is increasingly popular but has not yet been introduced in many Asian countries. This study aimed to determine the incidence rates of OAs, FAODs, and AAs in Asian countries and Germany using selective screening and ENBS records. Materials and methods: Selective screening for IMDs using gas chromatography–mass spectrometry and MS/MS was performed among patients suspected to be afflicted in Asian countries (including Japan, Vietnam, China, and India) between 2000 and 2015, and the results from different countries were compared. Similarly, ENBS results from Japan, South Korea, Taiwan, and Germany were compared. Additionally, the results of selective screening and ENBS in Japan were compared. Results: Among 39,270 patients who underwent selective screening, IMDs were detected in 1170. Methylmalonic acidemia was frequently identified in several countries, including Japan (81/377 diagnosed IMDs), China (94/216 IMDs), and India (72/293 IMDs). In Vietnam, however, β-ketothiolase deficiency was particularly frequent (33/250 IMDs). ENBS yielded differences in overall IMD rates by country: 1:8557 in Japan, 1:7030 in Taiwan, 1:13,205 in South Korea, and 1:2200 in Germany. Frequently discovered diseases included propionic acidemia (PPA) and phenylketonuria (PKU) in Japan, 3-methylcrotonyl-CoA carboxylase deficiency (MCCD) and PKU in Taiwan, MCCD and citrullinemia type I in South Korea, and PKU and medium-chain acyl-CoA dehydrogenase deficiency in Germany. Furthermore, in Japan, selective screening and ENBS yielded respective PPA frequencies of 14.7% and 49.4% among all organic acidemias. Conclusion: The incidence rates of IMDs vary by country. Moreover, the disease spectra of IMDs detected via selective screening differ from those detected via ENBS. Keywords: Organic acidemia, Fatty acid oxidation disorder, Amino acid disorder, Inherited metabolic disease, Expanded newborn screening, Incidence rate

RUNX1 generally functions as a tumor suppressor in the hematopoietic system. However, RUNX1 expression is significantly elevated in human AML cells with FLT3/ITD mutations, promotes leukemogenesis induced by FLT3/ITD (Behrens et al. JEM 2017) and enhances the resistance of FLT3/ITD + cells to type-II FLT3 inhibitor quizartinib (Hirade et al IJH 2016). We previously reported that RUNX1 expression is higher in CXCR4-low FLT3/ITD + cells compared to Cxcr4-high FLT3/ITD + cells, even though Cxcr4 expression is trans-activated by RUNX1. This difference in RUNX1 expression level was associated with divergent response to CXCL12 in FLT3/ITD + cells harboring different CXCR4 expression levels that were exposed to quizartinib (Fukuda S. et al. ASH 2019). Our data also demonstrated that RUNX1 expression is down-regulated following withdrawal of quizartinib in FLT3/ITD + cells that became refractory to quizartinib (Hirade et al. IJH 2016), suggesting that RUNX1 expression may be up-regulated by quizartinib in FLT3/ITD + cells. Since RUNX1 regulates proliferation of FLT3/ITD + AML cells, the present study investigated association between RUNX1 expression levels and proliferation of quizartinib resistant FLT3/ITD + cells that are exposed to quizartinib. In the sensitive FLT3/ITD + Ba/F3 cells, RUNX1 protein expression was transiently up-regulated but eventually down-regulated by 5 nM quizartinib, coincident with decline in the viable cells. In contrast, RUNX1 expression was up-regulated by quizartinib and remained elevated in the resistant FLT3/ITD + Ba/F3 cells. Since RUNX1 enhances proliferation of FLT3/ITD + cells, we next examined whether proliferation FLT3/ITD + cells that acquired resistance to quizartinib is facilitated by quizaritinib as a result from quizartinib-mediated up-regulation of RUNX1, using the Cxcr4-low and Cxcr4-high FLT3/ITD + cells that acquired resistance to quizartinib. Although CXCL12 barely enhanced the proliferation of refractory FLT3/ITD + Ba/F3 cells, 5 nM quizartinib significantly increased the proliferation of both Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells that acquired resistance to quizartinib compared to those without quizartinib. This increase in the proliferation of Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells coincided with the elevation in RUNX1 and CXCR4 protein expression. Moreover, the resistant Cxcr4-low FLT3/ITD + Ba/F3 cells proliferated significantly faster than Cxcr4-high FLT3/ITD + cells, with concomitant higher expression of RUNX1 in Cxcr4-low FLT3/ITD + cells than in Cxcr4-high FLT3/ITD + cells. Likewise, type-I FLT3 inhibitor gilteritinib significantly enhanced proliferation of Cxcr4-low and Cxcr4-high FLT3/ITD + Ba/F3 cells that acquired resistance to gilteritinib. Knocking down Runx1 using shRNAs significantly decreased the enhanced proliferation induced by quizartinib in refractory FLT3/ITD + Ba/F3 cells, coincident with reduction in CXCR4 expression. Since CXCR4 expression level was elevated by quizartinib in the FLT3/ITD + cells refractory to quizartinib, we next examined CXCL12-induced migration in quizartinib-resistant FLT3/ITD + cells following exposure to quzartinib. Pre-incubating the quizartinib resistant Cxcr4-low or Cxcr4-high FLT3/ITD + Ba/F3 cells with 5 nM quizartinib for 72 hours significantly enhanced their migration to 100 ng/ml of Cxcl12 compared to those without quizartinib, coincident with elevation in RUNX1 levels. Surprisingly, migration of CXCR4-low FLT3/ITD + cells to CXCL12 was significantly elevated compared to CXCR4-high cells, with concomitant higher expression of RUNX1 in Cxcr4-low FLT3/ITD + cells than in Cxcr4-high FLT3/ITD + cells. Silencing Runx1 using shRNAs significantly decreased migration to CXCL12 in refractory Cxcr4-low FLT3/ITD + Ba/F3 cells. These data indicate that the FLT3 inhibitor itself can facilitate the proliferation and migration to CXCL12 in FLT3/ITD + cells that are refractory to FLT3 inhibitors by up-regulating RUNX1. The results implicate that FLT3 inhibitors may worsen the disease progression in the patients that became refractory to FLT3 inhibitors by facilitating proliferation and migration to CXCL12 of the resistant FLT3/ITD + AML cells. In this regard, targeting RUNX1 may represent additional strategy to eradicate resistant FLT3/ITD + AML cells, in which their proliferation and migration are supported by FLT3 inhibitors. Disclosures No relevant conflicts of interest to declare.

Mitochondrial trifunctional protein (TFP) deficiency is an inherited metabolic disorder of mitochondrial fatty-acid oxidation. Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency is often reported in Caucasian countries due to a common mutation. However, the molecular and clinical basis of complete TFP deficiency has not been extensively reported. In this study, 14 Japanese cases (13 families) with complete TFP deficiency, including 9 previously reported cases, were analyzed to clarify the clinical and molecular characteristics of TFP deficiency. The clinical types of the 14 patients were as follows: 12 cases of neonatal (n=7) or myopathic (n=5) types and 2 cases of intermediate type. Peripheral neuropathy was found in four cases and hypocalcemia due to hypoparathyroidism, which is rarely reported in Caucasian patients, had developed in four cases. Maternal hemolysis, elevated liver enzymes and low platelet count syndrome and acute fatty liver of pregnancy were noted in two and one mothers, respectively. Fourteen mutations were identified in 26 alleles in Japanese patients, including two novel mutations (HADHA: c.361C>T, and HADHA-HADHB: g.26233880_ 26248855del), although no common mutations were found. This study suggests that the molecular and clinical aspects of Japanese patients with TFP deficiencies differ from those of Caucasian patients.

Introduction We evaluated the effects of bezafibrate (BEZ) on β-oxidation in fibroblasts obtained from patients with glutaric acidemia type II (GA2) of various clinical severities using an in vitro probe (IVP) assay. Methods Cultured fibroblasts from 12 patients with GA2, including cases of the neonatal-onset type both with and without congenital anomalies (the prenatal- and neonatal-onset forms, respectively), the infantile-onset, and the myopathic forms, were studied. The IVP assay was performed by measuring acylcarnitines (ACs) in the cell culture medium of fibroblasts incubated with palmitic acid for 96 h in the presence of 0–800 μM BEZ using tandem mass spectrometry. Results The IVP assay showed that 100 μM BEZ markedly reduced the level of palmitoylcarnitine (C16) in the neonatal-onset, infantile-onset, and myopathic forms of GA2, either increasing or maintaining a high level of acetylcarnitine (C2), which serves as an index of energy production via β-oxidation. In the prenatal-onset form, although a small reduction of C16 was also observed in the presence of 100 μM BEZ, the level of C2 remained low. At concentrations higher than 100 μM, BEZ further decreased the level of ACs including C16, but a concentration over 400 μM decreased the level of C2 in most cases. Discussion BEZ at 100 μM was effective for all GA2 phenotypes except for the prenatal-onset form, as a reduction of C16 without deterioration of C2 is considered to indicate improvement of β-oxidation. The effects of higher doses BEZ could not be estimated by the IVP assay but might be small or nonexistent.

Introduction: Various markers, such as C14:1 and the C14:1/C2 ratio, are used as diagnostic markers of very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). However, the levels of these markers in patients with VLCADD overlap with those in heterozygous carriers and even healthy subjects. Materials and methods: In twenty-three affected patients and 15 heterozygous carriers with VLCADD, the accuracies of C14:1, C14:1/C12:1, C14:1/C2, and C14:1/C16 in dried blood spots (DBS) and serum were statistically estimated. Results: Among the serum markers, the sensitivity, specificity, positive predictive value, negative predictive value, false-positive rate, false-negative rate, and validity of C14:1/C12:1 were superior to those of C14:1, C14:1/C2, and C14:1/C16, but C14:1/C2 demonstrated a statistical advantage compared with only C14:1 and C14:1/C16. Elevation in serum C14:1/C12:1 was observed in only one heterozygous carrier, whereas almost half of the carriers displayed false positive results for the other markers. Among the DBS markers, although the accuracy of C14:1/C2 was ostensibly the best, no statistical significance was observed. Discussion: Serum C14:1/C12:1 might be useful for differentiating patients with VLCADD from heterozygous carriers. Although serum C14:1/C2 was significantly useful for the detection of VLCADD, this marker could not distinguish the affected patients from carriers. C14:1/C12:1 might be optimal compared with the other markers. Keywords: Very long-chain acyl-CoA dehydrogenase deficiency, Tetradecenoyl carnitine, Dodecenoyl carnitine, Heterozygous carrier, Newborn screening, Diagnostic markers

To examine the clinical features and risk factors of secondary carnitine deficiency due to long-term use of pivalate-conjugated antibiotics (PCAs).We retrospectively investigated the age, clinical manifestations, PCA administration period, and background of 22 patients who showed a decrease in free carnitine (C0; ≤20 μmol/L) concomitant with an increase in pivaloyl carnitine (detected as C5-acylcarnitine) on acylcarnitine analysis with tandem mass spectrometry. Administration of PCAs was confirmed in all cases.The patients ranged in age from 2 months to 42 years (median, 1 year, 11 months). One patient was aged1 year, 10 patients were aged 1 year, 1 patient was aged 2 years, and 10 patients were aged ≥3 years. Nine patients had known underlying disease. Fourteen patients developed acute encephalopathy, 13 with accompanying hypoglycemia. Four patients presented with hypoglycemia without signs of encephalopathy. C0 values ranged from 0.25 to 19.66 μmol/L (median, 1.31 μmol/L); C5-acylcarnitine values, from 0.43 to 11.92 μmol/L (median, 3.23 μmol/L). There was no correlation between the PCA administration period and C0 level. Ten patients developed the symptoms after PCA administration for ≥14 days, whereas 6 patients showed symptoms after PCA administration for14 days.Carnitine deficiency resulting from PCA treatment was most frequently observed in 1-year-old infants. Most patients manifested acute encephalopathy and/or hypoglycemia. Some patients developed carnitine deficiency after PCA administration for14 days.

Introduction An increasing number of adult patients have been diagnosed with fatty acid β-oxidation disorders with the rising use of diagnostic technologies. In this study, clinical, biochemical, and molecular characteristics of 2 Japanese patients with adult-onset glutaric acidemia type II (GA2) were investigated and compared with those of pediatric cases. Methods The patients were a 58-year-old male and a 31-year-old male. In both cases, episodes of myopathic symptoms, including myalgia, muscle weakness, and liver dysfunction of unknown cause, had been noted for the past several years. Muscle biopsy, urinary organic acid analysis (OA), acylcarnitine (AC) analysis in dried blood spots (DBS) and serum, immunoblotting, genetic analysis, and an in vitro probe acylcarnitine (IVP) assay were used for diagnosis and investigation. Results In both cases, there was no obvious abnormality of AC in DBS or urinary OA, although there was a increase in medium- and long-chain ACs in serum; also, fat deposits were observed in the muscle biopsy. Immunoblotting and gene analysis revealed that both patients had GA2 due to a defect in electron transfer flavoprotein dehydrogenase (ETFDH). The IVP assay indicated no special abnormalities in either case. Conclusion Late-onset GA2 is separated into the intermediate and myopathic forms. In the myopathic form, episodic muscular symptoms or liver dysfunction are primarily exhibited after later childhood. Muscle biopsy and serum (or plasma) AC analysis allow accurate diagnosis in contrast with other biochemical tests, such as analysis of AC in DBS, urinary OA, or the IVP assay, which show fewer abnormalities in the myopathic form compared to intermediate form.

Carnitine palmitoyltransferase II (CPT-2) deficiency, an autosomal recessive disorder of fatty acid oxidation, can be detected by newborn screening using tandem mass spectrometry (TMS). Our case was a boy born at 38 weeks and 6 days of gestation via normal vaginal delivery; his elder sister was affected with CPT-2 deficiency. Acylcarnitine (AC) was analyzed in both dried blood spots (DBS) and serum 2 h after birth to determine whether the boy was also affected. His C16 and C18:1 AC levels in DBS were in the normal range, while his serum long-chain AC levels were marginally increased but lower than those of his sister. After the samples were taken, he was treated with glucose infusion to prevent any catabolism for 2 days. On day 4, the long-chain AC levels in both DBS and serum obtained were higher than those on day 0 and were equivalent to those of his sister. Genetic testing confirmed the presence of the same mutation found in his sister, a homozygous F383Y mutation in the CPT2 gene, thus leading to the diagnosis of CPT-2 deficiency. The sample for TMS should be taken between days 1 and 7. If the sample is not obtained at an appropriate time, correct diagnosis may not be made, as in our case. Although early diagnosis is required, samples taken within 24 h after birth should not be used for TMS.

When Japanese National Railways was privatized and separated in 1987, the government established a new freight train operation scheme where rail tracks were owned by six JR Passenger companies, and JR-Freight was allowed access to the JR Passenger company's tracks by paying an access charge under the avoidable cost rule, regulated by the national government. The aim of this scheme was to reduce the track costs for JR-Freight. We found that rail freight infrastructure investment had not been high enough because it was only conducted by JR-Freight, and it only included investment in a few facilities owned by JR-Freight but excluded investment in the rail tracks owned by the JR passenger companies. Additionally, the track access charges, calculated by the avoidable cost rule, could not compensate for the track maintenance costs and would induce an undersupply of rail tracks for rail freight traffic. In conclusion, the track access charges paid from JR-Freight to the JR Passenger companies were too low, causing difficulties in the promotion of a modal shift to rail freight.

Mucopolysaccharidoses (MPSs) are rare lysosomal storage diseases caused by the accumulation of undegraded glycosaminoglycans in cells and tissues. The effectiveness of early intervention for MPS has been reported. Multiple-assay formats using tandem mass spectrometry have been developed. Here, we developed a method for simultaneous preparation and better measurement of the activities of five enzymes involved in MPSs, i.e., MPS I, MPS II, MPS IIIB, MPS IVA, and MPS VI, which were validated using 672 dried blood spot samples obtained from healthy newborns and 23 patients with MPS. The mean values of the enzyme activities and standard deviations in controls were as follows: &alpha, iduronidase (IDUA), 4.19 &plusmn, 1.53 &micro, M/h, iduronate-2-sulfatase (I2S), 8.39 &plusmn, 2.82 &micro, N-acetyl-&alpha, glucosaminidase (NAGLU), 1.96 &plusmn, 0.57 &micro, N-acetylgalactosamine-6-sulfatase (GALNS), 0.50 &plusmn, 0.20 &micro, and N-acetylgalactosamine-4-sulfatase (ARSB), 2.64 &plusmn, 1.01 &micro, M/h. All patients displayed absent or low enzyme activity. In MPS I, IIIB, and VI, each patient group was clearly separated from controls, whereas there was some overlap between the control and patient groups in MPS II and IVA, suggesting the occurrence of pseudo-deficiencies. Thus, we established a multiplex assay for newborn screening using liquid chromatography tandem mass spectrometry, allowing simultaneous pretreatment and measurement of five enzymes relevant to MPSs.

In order to determine the associations between sudden unexpected death in infancy (SUDI) or acute life-threatening events (ALTE) and inborn errors of metabolism, particularly organic acidemia and fatty acid oxidation disorders, we evaluated clinical features in patients with SUDI or ALTE. The subjects were infants between the ages of 7 days and 3 years who developed SUDI or ALTE between January 2004 and December 2013. They were then diagnosed as having inborn errors of metabolism on gas chromatography-mass spectrometry (GC/MS) and/or tandem mass spectrometry (MS/MS). The age distribution, onset forms, and clinical findings were evaluated during the acute phase. Inborn errors of metabolism were detected in three of 196 patients with SUDI, and in seven of 167 patients with ALTE. Of these 10 patients, nine had a history of poor feeding and somnolence during the neonatal period, and symptoms of infection such as cough, fever or vomiting during infancy. Routine laboratory tests during an acute phase indicated hyperammonemia, liver dysfunction, increased blood creatine kinase, acidosis, positive ketone bodies in urine or blood, or hypoglycemia. When SUDI or ALTE are encountered in the emergency unit, it is essential that a detailed medical history is taken, particularly with regard to the neonatal period, and that specific abnormalities are investigated on routine laboratory tests. Moreover, samples such as urine, serum, and filter paper blood specimens should be collected for GC/MS and/or MS/MS of organic acids and acylcarnitines, to identify inborn metabolic disorders.

We report the case of an infant girl with incontinentia pigmenti (IP) complicated by fatal pulmonary arterial hypertension (PAH). She was diagnosed with IP, based on the presence of specific skin lesions, neonatal seizures, hypereosinophilia and a maternal family history of IP. At the age of 2 months, she was diagnosed with PAH on systolic heart murmur due to tricuspid valve regurgitation. Despite several treatments for PAH but not including epoprostenol, severe PAH persisted and she died of pulmonary hypertensive crisis at the age of 5 months. On postmortem histopathology the pulmonary artery had severe intimal thickening, with occlusion or stenosis of the vascular lumen of the small pulmonary arteries as well as partial plexiform lesions, all of which were compatible with PAH. Modulation of nuclear factor-κB signaling may be involved in the development of PAH in IP.

The Runx1 transcriptional factor is significantly elevated in human AML cells with FLT3/ITD mutations (Behrens et al. JEM 2017) and enhances the resistance of FLT3/ITD+ 32D cells to the type II FLT3 inhibitor quizartinib (Hirade et al. IJH 2016). While Cxcr4 levels are significantly downregulated in human FLT3/ITD+ AML cells and FLT3/ITD+ Ba/F3 cells compared to FLT3/ITD- cells (Onishi et al. JBC 2014), Runx1 transactivates Cxcr4 expression (Jacob et al. Blood 2010), indicating that Cxcr4 is inversely regulated by FLT3/ITD and Runx1. Studies have indicated that Cxcl12 in the microenvironment protects FLT3/ITD+AML cells from chemotherapeutic insults through Cxcr4 (Zheng et al. Blood 2009). Herein, we investigated the interaction between Cxcl12/Cxcr4 signaling and Runx1 on the refractory activity of FLT3/ITD+ cells against quizartinib. Runx1 shRNAs downregulated Cxcr4 levels in FLT3/ITD+ Ba/F3 cells, indicating that Runx1 enhances the expression of Cxcr4. However, a comparison of the intracellular Runx1 levels in FLT3/ITD+ Ba/F3 cells expressing different surface Cxcr4 levels that were sorted by FACS based on Cxcr4 expression demonstrated that the Runx1 protein levels were the highest in FLT3/ITD+ Ba/F3 cells harboring the lowest Cxcr4 compared to those expressing higher Cxcr4 levels, and were inversely correlated with the Cxcr4 levels (r=-0.78, P Studies have indicated that Cxcr4 levels are downregulated and Runx1 levels are upregulated by FLT3/ITD, even though Runx1 enhances Cxcr4 expression. Our data indicate that Cxcr4 dim FLT3/ITD+ cells are more refractory to quizartinib in the presence or absence of Cxcl12 concomitant with higher Runx1 levels, compared to Cxcr4 high cells. Antagonizing Runx1 partially abrogated the refractory against quizartinib in Cxcr4 dim cells in the presence or absence of Cxcl12. These data implicate that Cxcr4 dim FLT3/ITD+ cells display increased FLT3/ITD activity compared to Cxcr4 high cells as a consequence of elevated Runx1 expression. Runx1 also enhanced chemotaxis of quizartinib resistant FLT3/ITD+ cells to Cxcl12. Targeting Runx1 represents an additional strategy to overcome FLT3 inhibitor refractory in FLT3/ITD+AML cells that are protected in the microenvironment. Disclosures No relevant conflicts of interest to declare.

Rotavirus and norovirus are common pathogens associated with gastroenteritis in children. Although rotavirus occasionally induces central nervous system disease, only 3 cases with rotavirus-induced acute myositis have been reported in the English literature. We recently treated a female patient with acute myositis associated with gastroenteritis induced by concurrent infection with rotavirus and norovirus. Having suffered from gastroenteritis for 3 days, she suddenly developed myositis affecting her lower extremities with concomitant creatine kinase elevation. Herein, we present our patient and review the previous cases including those reported in the Japanese literature.

Although mesenchymal stromal cells (MSCs) have significant potential in cell-based therapies, little is known about the factors that regulate their functions. While exploring regulatory molecules potentially involved in MSC activities, we found that the endogenous multifunctional factor Survivin is essential for MSC survival, expansion, lineage commitment, and migration. Pharmacological or genetic blockade of Survivin expression in mouse and human bone marrow MSC enhances caspase 3 and 7 expression and reduces proliferation resulting in fewer MSC and clonogenic colony-forming unit-fibroblasts (CFU-F), whereas ectopic Survivin overexpression in MSC results in their expansion. Survivin is also required for the MSC proliferative responses to basic fibroblast growth factor and platelet derived growth factor. In a wound healing model, Survivin inhibition results in suppression of MSC migration to the wound site. In addition, loss of Survivin in MSCs compromises their hematopoiesis-supporting capacity. These results demonstrate that Survivin is a key regulator of mouse and human MSC function, and suggest that targeted modulation of Survivin in MSCs may have clinical utility to enhance MSC recovery and activity following insult or stress.

Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROβ, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.

Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression.

Internal tandem duplication in the FLT3 gene (FLT3/ITD), which is found in patients with acute myeloid leukemia (AML), causes resistance to FLT3 inhibitors. We found that RUNX1, a transcription factor that regulates normal hematopoiesis, is up-regulated in patients with FLT3/ITD(+) AML. While RUNX1 can function as a tumor suppressor, recent data have shown that RUNX1 is required for AML cell survival. In the present study, we investigated the functional role of RUNX1 in FLT3/ITD signaling. FLT3/ITD induced growth factor-independent proliferation and impaired G-CSF mediated myeloid differentiation in 32D hematopoietic cells, coincident with up-regulation of RUNX1 expression. Silencing of RUNX1 expression significantly decreased proliferation and secondary colony formation, and partially abrogated the impaired myeloid differentiation of FLT3/ITD(+) 32D cells. Although the number of FLT3/ITD(+) 32D cells declined after incubation with the FLT3/ITD inhibitor AC220, the cells became refractory to AC220, concomitant with up-regulation of RUNX1. Silencing of RUNX1 abrogated the emergence and proliferation of AC220-resistant FLT3/ITD(+) 32D cells in the presence of AC220. Our data indicate that FLT3/ITD deregulates cell proliferation and differentiation and confers resistance to AC220 by up-regulating RUNX1 expression. These findings suggest an oncogenic role for RUNX1 in FLT3/ITD(+) cells and that inhibition of RUNX1 function represents a potential therapeutic strategy in patients with refractory FLT3/ITD(+) AML.

A 52-year-old male visited a local clinic with a subjective complaint of pain in the left side of his abdomen. Abdominal CT revealed the presence of a pancreatic body tumor. On EUS, the tumor presented hypoechoic signals with an obscure boundary, which continued from the pancreatic parenchyma to the inside of the main pancreatic duct. Abdominal contrast CT revealed a hypervascular tumor with densely stained pancreatic parenchyma. ERP findings revealed that main pancreatic duct invasion was suspected based on partial radiolucency in the duct. Distal pancreatectomy was performed, and a definitive diagnosis of pancreatic neuroendocrine tumor (WHO class G1) was made histopathologically.

Mesenchymal stemcells (MSCs) are found throughout adult organisms and are involved in tissue maintenance and repair, as well as in the regulation of hematopoiesis and immunologic responses. Several clinical trials are currently under way to use allogeneic MSCs for enhancement of hematopoietic stem cell transplantation and treatment of graft-versus-host disease, spinal cord injury, cartilage and meniscus repair, and stroke. However, the therapeutic uses of MSCs can be limited by insufficient MSC number, their survival, and their ability to differentiate into multiple lineages, pointing to an important need to identify factors regulating MSC survival, proliferation, and differentiation. In this study, we explored the role of the anti-apototic protein Survivin in MSC functions. Survivin is an intracellular member of the inhibitor-of-apoptosis protein (IAP) family and is implicated in regulation of apoptosis, cell division, and cell cycle control of many cell types including hematopoietic stem cells, leukemic cells, and endothelial cells. However, whether Survivin is involved in MSC activity or function is unknown. Using flow cytometry and function assays, we found that adult mouse and human MSCs, identified as CD45-Ter119-CD51+PDGFR+Nestin+ cells, express high levels of Survivin, which regulates MSCs survival and expansion under normal physiological and stressful conditions. Flow cytometry analysis revealed that approximately 40.5±3.6% of mouse bone marrow MSCs, 33.6±4.2% of adult human bone marrow MSCs, and 43.6±5.3% human cord blood MSCs express Survivin. Treatment of mice in vivo with YM155 (10 mg/kg), a small molecule Survivin inhibitor, for 6 days decreased bone marrow CD45-Ter119-CD51+PDGFR+Nestin+ MSC by 2.1 fold and functional fibroblast colony formation (CFU-F) by 2.8 fold. Survivin gene deletion using Survivin-specific SiRNA decreased mouse and human bone marrow derived MSCs number and CFU-F ability by 3.2 fold and 2.8 fold respectively. Retroviral overexpression of Survivin in mouse MSCs enhanced CFU-F formation by 4 fold. In an in vitro wound healing assay YM155-treated MSCs recovered more slowly compared to control cultures. To determine underlying mechanisms involved in Survivin dependent regulation of MSC function, we measured the survival and proliferation of YM155 treated MSCs. Treatment of mouse or human bone marrow-derived MSCs with YM155 (50 nmol/l) for 48 hours enhanced the caspase 3 and 7 expression by 42.8% and 63.9% respectively, while BrdU incorporation was similar in control and YM155-treated MSCs, suggesting that Survivin is primarily involved in MSC survival. To explore whether Survivin is also involved in growth factor-mediated MSC survival and expansion, mouse bone marrow derived MSCs were cultured in the presence of platelet derived growth factor (PDGF) or basic fibroblast growth factor (b-FGF) with or without the Survivin inhibitor YM155. PDGF enhanced the Survivin expression by 52.8±2.2% and b-FGF enhanced Survivin expression by 43.8±4.5% and increased the CFU-F counts by 5.6 fold and 4.2 fold, respectively. In contrast, YM155 treated MSCs did not show any enhancement in Survivin expression, and CFU-F were substantially lower than the corresponding controls (PDGF or b-FGF treated cultures). To determine whether Survivin affects the Survival of stressed MSCs, we exposed mouse bone marrow MSCs to 4 Gy irradiation dose and followed by culture for 3 days with or without the Survivin inhibitor YM155. Irradiation alone reduced the survival of MSCs by 53.5%, however the viability of irradiated MSCs treated with YM155 was reduced by 82.8%. The hallmark of MSC function is the capacity to differentiate into several mesodermal lineages. We found that the exposure of MSC cultures to YM155 during the lineage differentiation process inhibits MSC osteogenic and adipogenic differentiation while sparing chondrogenic cell differentiation. In conclusion, our study demonstrates that Survivin controls basal and growth factor dependent survival and expansion of mouse and human MSCs, and protects them from irradiation induced cell death. Furthermore, our data suggest the regulating Survivin expression in MSCs would be beneficial to enhance MSC recovery for clinical utility. Disclosures No relevant conflicts of interest to declare.

FMS-like tyrosine kinase 3 (FLT3) is a membrane type tyrosine kinase and has important roles for the proliferation and differentiation of the hematopoietic cells. The Internal Tandem Duplications of FLT3 (FLT3/ITD) is detected in approximately 30 % of patients with acute myeloid leukemia (AML) and the prognoses of FLT3/ITD+ AML are very poor. While a number of inhibitors targeting FLT3 tyrosine kinase have been developed, few drugs are effective for the FLT3/ITD+ AML because of emergence of resistant cells against the drugs. Recently, AC220 (Quizartinib), a second generation class III tyrosine kinase inhibitor (TKI) for FLT3/ITD+ AML was developed and used in clinical trials. Although AC220 is a more potent and specific inhibitor for FLT3/ITD compared to the other TKIs, report demonstrates that prolonged exposure to AC220 can generate resistant clones to AC220 in FLT3/ITD+ cells (Smith et al. Nature 2012). These findings underscore the need to develop additional therapeutic strategies to overcome the resistance of FLT3/ITD+ AML to TKIs. However, the mechanism responsible for drug resistance of FLT3/ITD+ AML cells remains to be investigated. We previously reported that mRNA expression of RUNX1, a core-binding transcription factor that regulates the differentiation and proliferation of hematopoietic stem cells, is significantly higher in FLT3/ITD+AML cells compared to FLT3/ITD-AML cells (Hirade et al. ASH 2013). Although loss of RUNX1 function (i.e. RUNX1/ETO fusion gene) contributes to the development of AML, RUNX1 also promotes survival of AML cells (Goyama et al. JCI 2013) and can function as an oncogene in cancer cells (Kilbey et al. Cancer Research 2010). These findings lead us hypothesize that RUNX1 may confer resistance of AML cells to TKIs. In the present study, we investigated if Runx1 is involved in the refractory phenotype of Flt3/ITD+cells to AC220. Transduction of Flt3/ITD into IL3-dependent mouse 32D cells allowed the cells proliferate in a growth factor independent fashion, concomitant with up-regulation of Runx1 mRNA level, similar to the patients’ samples with FLT3/ITD+AML. Silencing Runx1 expression using shRNA resulted in 70% reduction of Flt3/ITD+32D cells that proliferated in the absence of growth factors. Similarly, incubating the Flt3/ITD+32D cells with 0.5nM AC220 inhibited their factor independent proliferation by 95%, which was further accentuated up to 99% by the combination with shRNA mediated silencing of Runx1. Although the number of factor independent Flt3/ITD+32D cells cultured in the presence of 2nM AC220 rapidly declined within 96 hours, the residual cells subsequently re-proliferate within 14 days and became no longer sensitive to AC220. Surprisingly, the expression of Runx1 mRNA in the resistant cells to AC220 was 5.0±0.2 fold higher (P Our data indicates that knocking down Runx1 expression enhances the cytotoxic effect of AC220 on Flt3/ITD+32D cells and that Runx1 expression is significantly up-regulated by the AC220 resistance cells. Moreover, Runx1 knockdown recovered the cytotoxicity of AC220 in the refractory Flt3/ITD+32D cells, demonstrating that Flt3/ITD confers resistance to AC220 by up-regulating the expression of Runx1. These findings demonstrate that antagonizing RUNX1 may represent potential therapeutic strategy in the patients with FLT3/ITD+ AML that become refractory to AC220. Disclosures No relevant conflicts of interest to declare.

Introduction: Clinical features and genetic backgrounds of acute myeloid Leukemia (AML) are heterogenous and the prognosis is poor. Cooperating alterations of several genes including oncogenes or tumor suppressor genes lead to development of Leukemia. AML leukemogenesis is thought to require at least two different types of genetic change: class I mutations, which confer a proliferative or survival advantage; and class II mutations, which inhibit myeloid differentiation and apoptotic activity. In hematological malignancies with 11p15 translocations, the NUP98 gene is reportedly fused to various partner genes, often including homeobox genes, such as HOXA9, A11, A13, C11, C13, D11, D13 and PMX. We previously revealed that high frequencies of FLT3 internal tandem duplication (FLT3-ITD), NRAS mutations (NRASMT), and WT1 mutations (WT1MT) in myeloid malignancies with NUP98-HOX fusions were found and their concurrent mutations with NUP98-HOX fusions were associated with clinical prognosis of the patients. However, biological outcome of the combination of NUP98-HOX fusions with FLT3-ITD, NRASMT or WT1MT remains to be investigated. To elucidate the mechanism resposible for the leukemogenesis, we examined the function of NUP98-HOX fusions and the concomitant somatic mutations in vitro. Methods: Full length of NUP98-HOXA9 (NHA9), FLT3-ITD (51 bp-duplication), NRASMT (G13V), and WT1MT (Arg250trp) was cloned into a plasmid that has a mammalian replication initiation region and a nuclear matrix attachment region, which possesses great ability to persist gene amplification for long periods. Each plasmid or the combination of the two plasmids (NHA9/FLT3-ITD, NHA9/NRASMT, NHA9/WT1MT) were transfected into IL3-dependent mouse myeloid 32D cell line. Cell proliferation was determined in medium without IL3. Cellular differentiation was examined in the medium containing G-CSF using May-Giemsa staining and fluorescence-activated cell sorting (FACS). Apoptosis was quantitated using FACS. Colony forming assay was performed in methyl cellulose medium with stem cell factor and IL3. Moreover, autophagy induced by tamoxifen was checked. Results: Compared to the empty vector, IL3-independent cellular growth was significantly increased in the cells transfected with NHA9 and FLT3-ITD alone, but not in NRASMT and WT1MT alone. In contrast, co-transfection of NRASMT or WT1MT with NHA9 significantly increased the IL3 independent proliferation of 32D cells. Similarly, cells transfected with NHA9/FLT3-ITD showed the enhanced IL3-independent proliferation. CD11b and Gr1, of which represent myeloid differentiation marker, decreased in all the transfected cells except for NRASMT. While transfection with FLT3-ITD, NRASMT and WT1MT increased the morphological nuclear segmentation of the cells, NHA9/FLT3-ITD, NHA9/NRASMT, and NHA9/WT1MT abrogated the nuclear segmentation. Both Annexin V positive/propidium iodide negative cells and active caspase 3-positive cells declined in all transfected cells. Transfection of NHA9/FLT3-ITD, NHA9/NRASMT, NHA9/WT1MT tended to increase the number of colony forming unit compared to the single gene-transfected cells. After treatment with tamoxifen, monodansylcadaverine, which was used to reveal autophagic vacuoles, was incorporated into empty vector-transfected cells, not into all transfected cells. Conclusions: NHA9 acquired proliferation capacity, blockage of differentiation, and anti-apoptosis activity, FLT3-ITD did proliferation capacity and anti-apoptosis activity, NRASMT and WT1MT did anti-apoptosis activity. By contrast, NHA9 with FLT3-ITD, NRASMT, or WT1MT conferred growth advantage, inhibited differentiation and apoptosis, augmented self-renewality. In vivo study demonstrated that NHA9 or each genetic mutation (FLT3-ITD and NRASMT) alone was insufficient to achieve myeloid leukemia. These suggested that leukemogenesis of NUP98-HOX fusion genes with simultaneous gene mutations might be induced by not only harmonization between class I and class II mutations but also self-renewality and anti-autophagic cell death. Disclosures No relevant conflicts of interest to declare.