The magnitude of CXCR4 signaling regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1.

CXCR4 antagonists sensitize FLT3/ITD+ AML cells to FLT3 inhibitors; however, CXCR4 signaling can induce apoptosis in AML cells, raising the question of whether CXCR4 signaling exerts divergent effects on FLT3/ITD+ cells. The present study investigated the paradoxical function of CXCR4 in resistance to FLT3 inhibitors. The FLT3 inhibitor quizartinib significantly decreased the number of FLT3/ITD+ Ba/F3 cells, whereas 1 ng/ml CXCL12 showed a significant protective effect against quizartinib. In contrast, CXCL12 over 100 ng/ml significantly decreased FLT3/ITD+ cell viability with concomitant downregulation of Runx1. Moreover, the survival of FLT3/ITD+ Ba/F3 or MOLM13 cells with low surface CXCR4 expression incubated with quizartinib was significantly enhanced by 100 ng/ml CXCL12; however, this protective effect of CXCL12 against quizartinib was barely detected in cells with high surface CXCR4 expression. Although silencing Runx1 downregulated CXCR4 expression, RUNX1 expression levels were significantly higher in CXCR4LOW FLT3/ITD+ Ba/F3 cells incubated with 100 ng/ml CXCL12 than in CXCR4HIGH cells, coincident with an increase in FLT3 phosphorylation. Silencing RUNX1 partially abrogated resistance to quizartinib in CXCR4LOW cells incubated with CXCL12, whereas ectopic RUNX1 significantly restored resistance in CXCR4HIGH cells. These results indicate that CXCR4 signaling of different magnitudes paradoxically regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1.

The frequencies of very long-chain acyl-CoA dehydrogenase deficiency genetic variants in Japan have changed since the implementation of expanded newborn screening.

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.

Kangaroo mother care alters chromogranin A and perfusion index in preterm babies.

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.

Validation of Liquid Chromatography-Tandem Mass Spectrometry-Based 5-Plex Assay for Mucopolysaccharidoses.

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: α-iduronidase (IDUA), 4.19 ± 1.53 µM/h; iduronate-2-sulfatase (I2S), 8.39 ± 2.82 µM/h; N-acetyl-α-glucosaminidase (NAGLU), 1.96 ± 0.57 µM/h; N-acetylgalactosamine-6-sulfatase (GALNS), 0.50 ± 0.20 µM/h; and N-acetylgalactosamine-4-sulfatase (ARSB), 2.64 ± 1.01 µM/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.

Serum C14:1/C12:1 ratio is a useful marker for differentiating affected patients with very long-chain acyl-CoA dehydrogenase deficiency from heterozygous carriers.

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.

Clinical course in a patient with myopathic VLCAD deficiency during pregnancy with an affected baby.

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 ß-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.

Flavin adenine dinucleotide synthase deficiency due to FLAD1 mutation presenting as multiple acyl-CoA dehydrogenation deficiency-like disease: A case report.

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.

Diversity in the incidence and spectrum of organic acidemias, fatty acid oxidation disorders, and amino acid disorders in Asian countries: Selective screening vs. expanded newborn screening.

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, ß-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.

Additional Medical Costs Due to Hospital-Acquired Falls.

OBJECTIVES: 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. METHODS: 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. RESULTS: Additional medical costs were 364 ± 2129 USD for minor injuries and 4336 ± 3645 USD for major injuries at SUH (P < 0.001) and 114 ± 124 USD for minor injuries and 2267 ± 2811 USD for major injuries at SMH (P < 0.001). 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. CONCLUSIONS: 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.

Survivin Is Required for Mouse and Human Bone Marrow Mesenchymal Stromal Cell Function.

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. Stem Cells 2018;36:123-129.

Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell.

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, GROß, 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.

Clinical and molecular investigation of 14 Japanese patients with complete TFP deficiency: a comparison with Caucasian cases.

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.

A newborn case with carnitine palmitoyltransferase II deficiency initially judged as unaffected by acylcarnitine analysis soon after birth.

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.

Efficacy of bezafibrate on fibroblasts of glutaric acidemia type II patients evaluated using an in vitro probe acylcarnitine assay.

INTRODUCTION: We evaluated the effects of bezafibrate (BEZ) on ß-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 96h in the presence of 0-800µM BEZ using tandem mass spectrometry. RESULTS: The IVP assay showed that 100µM 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 ß-oxidation. In the prenatal-onset form, although a small reduction of C16 was also observed in the presence of 100µM BEZ, the level of C2 remained low. At concentrations higher than 100µM, BEZ further decreased the level of ACs including C16, but a concentration over 400µM decreased the level of C2 in most cases. DISCUSSION: BEZ at 100µM 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 ß-oxidation. The effects of higher doses BEZ could not be estimated by the IVP assay but might be small or nonexistent.

Internal Tandem Duplication in FLT3 Attenuates Proliferation and Regulates Resistance to the FLT3 Inhibitor AC220 by Modulating p21Cdkn1a and Pbx1 in Hematopoietic Cells.

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.

Fatal pulmonary arterial hypertension in an infant girl with incontinentia pigmenti.

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.

Clinical Features of Carnitine Deficiency Secondary to Pivalate-Conjugated Antibiotic Therapy.

OBJECTIVE: To examine the clinical features and risk factors of secondary carnitine deficiency due to long-term use of pivalate-conjugated antibiotics (PCAs). STUDY DESIGN: 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. RESULTS: The patients ranged in age from 2 months to 42 years (median, 1 year, 11 months). One patient was aged <1 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 for <14 days. CONCLUSION: 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 for <14 days.

A fetus with mitochondrial trifunctional protein deficiency: Elevation of 3-OH-acylcarnitines in amniotic fluid functionally assured the genetic diagnosis.

Mitochondrial trifunctional protein (TFP) is a multienzyme complex that catalyzes the last three steps of the ß-oxidation cycle of long-chain fatty acids. In the prenatal diagnosis of TFP deficiency, acylcarnitine (AC) analysis has been considered difficult because of limited excretion of long-chain ACs into the fetal urine and hence into the amniotic fluid. Here, we report our experience with prenatally diagnosing TFP deficiency using AC analysis of amniotic fluid. The index case was a boy born at 38 weeks gestation and weighing 2588 g. He suddenly became unconscious and hypoglycemic and died on day 6 of life. Postmortem blood AC analysis and gene sequencing revealed TFP deficiency. Therefore, the parents underwent prenatal diagnoses for their subsequent 2 pregnancies. Mutation analysis suggested that one (Case 1) was affected and the other (Case 2) was not. AC analysis also demonstrated identical results, with significantly elevated 3-hydroxy-AC levels in the amniotic fluid of the affected pregnancy compared with those of heterozygotes and normal controls (n = 2 for heterozygotes and n = 8 for normal controls). Our findings suggest that AC analysis can functionally confirm results even in families with unidentified mutations, without raising issues related to maternal cell contamination. During prenatal diagnosis, misdiagnosis has to be avoided, and combining AC analysis with gene sequencing may result in more accurate prenatal diagnosis of TFP deficiency.

Internal tandem duplication of FLT3 deregulates proliferation and differentiation and confers resistance to the FLT3 inhibitor AC220 by Up-regulating RUNX1 expression in hematopoietic cells.

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.

Clinical, biochemical and molecular investigation of adult-onset glutaric acidemia type II: Characteristics in comparison with pediatric cases.

INTRODUCTION: An increasing number of adult patients have been diagnosed with fatty acid ß-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.