Hirosuke Inoue

SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7

Adrenal hypoplasia is a rare, life-threatening congenital disorder. Here we define a new form of syndromic adrenal hypoplasia, which we propose to term MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) syndrome. By exome sequencing and follow-up studies, we identified 11 patients with adrenal hypoplasia and common extra-adrenal features harboring mutations in SAMD9. Expression of the wild-type SAMD9 protein, a facilitator of endosome fusion, caused mild growth restriction in cultured cells, whereas expression of mutants caused profound growth inhibition. Patient-derived fibroblasts had restricted growth, decreased plasma membrane EGFR expression, increased size of early endosomes, and intracellular accumulation of giant vesicles carrying a late endosome marker. Of interest, two patients developed myelodysplasitc syndrome (MDS) that was accompanied by loss of the chromosome 7 carrying the SAMD9 mutation. Considering the potent growth-restricting activity of the SAMD9 mutants, the loss of chromosome 7 presumably occurred as an adaptation to the growth-restricting condition.

Moyamoya disease susceptibility gene RNF213 links inflammatory and angiogenic signals in endothelial cells

Moyamoya disease (MMD) is a cerebrovascular disorder characterized by occlusive lesions of the circle of Willis. To date, both environmental and genetic factors have been implicated for pathogenesis of MMD. Allelic variations in RNF213 are known to confer the risk of MMD; however, functional roles of RNF213 remain to be largely elusive. We herein report that pro-inflammatory cytokines, IFNG and TNFA, synergistically activated transcription of RNF213 both in vitro and in vivo. Using various chemical inhibitors, we found that AKT and PKR pathways contributed to the transcriptional activation of RNF213. Transcriptome-wide analysis and subsequent validation with quantitative PCR supported that endogenous expression of cell cycle-promoting genes were significantly decreased with knockdown of RNF213 in cultured endothelial cells. Consistently, these cells showed less proliferative and less angiogenic profiles. Chemical inhibitors for AKT (LY294002) and PKR (C16) disrupted their angiogenic potentials, suggesting that RNF213 and its upstream pathways cooperatively organize the process of angiogenesis. Furthermore, RNF213 down-regulated expressions of matrix metalloproteases in endothelial cells, but not in fibroblasts or other cell types. Altogether, our data illustrate that RNF213 plays unique roles in endothelial cells for proper gene expressions in response to inflammatory signals from environments.

Serum neutrophil gelatinase-associated lipocalin as a predictor of the development of bronchopulmonary dysplasia in preterm infants.

BACKGROUND Bronchopulmonary dysplasia (BPD) is a chronic lung disease mostly occurring in preterm infants. The pathogenesis of BPD involves early inflammation and remodeling of the premature lung. AIM To search for the novel predictive marker of BPD development, we studied serum levels of neutrophil gelatinase-associated lipocalin (NGAL), an innate immune mediator, in preterm infants. METHODS Serum NGAL concentrations at birth were measured by enzyme-linked immunosorbent assay. The reference levels were determined in 52 infants having no anomalies or inherited diseases. The levels and clinical variables were assessed in association with BPD. RESULTS Geometric means (95%CI) of serum NGAL levels at birth of infants having no underlying diseases were 32.4 (22.1-47.5), 58.6 (47.9-71.8), and 126.2 (99.0-168.7) ng/mL for <31, 31-36 and >36 gestational weeks (GW), respectively (p<0.001). These levels positively correlated with neutrophil (p<0.0001) or monocyte counts (p<0.0001). The median NGAL levels (307.8 ng/mL) and neutrophil counts (4141/μL) at birth of 16 preterm infants (<31 GW) who developed BPD were higher than those (42.9 ng/mL and 1357/μL) of 20 infants (<31 GW) who did not (p<0.0001 and p=0.012), respectively. In multivariable analysis for 36 infants born less than 31 GW, higher NGAL levels (≥ 82 ng/mL) but not neutrophil counts at birth had a significant association with developing BPD (gestational-age adjusted odds ratio [OR]=37.45 [3.08-455.49], p<0.01). CONCLUSIONS High serum levels of NGAL at birth could be an early sensitive marker for BPD in preterm infants, because their levels were physiologically low.

Differential Transmission and Postnatal Outcomes in Triplets with Intrauterine Cytomegalovirus Infection

We present a case of triplets with intrauterine cytomegalovirus (CMV) infection, each of whom showed differential transmission, placental pathology, and postnatal outcome. The first- and second-born infants were both vigorous and asymptomatic at birth, although the first-, but not the second-born, triplet had a high copy number of CMV DNA in the peripheral blood (1.2 × 103 copy/mL). The third-born triplet suffered from symptomatic CMV infection with a high viral load (6.0 × 106 copy/mL). The triamniotic-trichorionic placentas were not fused to each other. The histopathologic analysis showed that CMV-positive cells were frequently found in the decidua, villi, and amnion of the third-born triplets placenta but were limited and scattered in the decidua or villi but not amnion of the other 2 placentas. The third-born triplet underwent ganciclovir therapy. None of the infants had physical or auditory problems at 4 years of age, whereas the third-born triplet had been diagnosed with an autistic disorder. This observation exemplifies the preventive roles of the individual placentas of triplets with regard to virus infection, thus suggesting that developing CMV disease largely depends on the placental function.

A novel SCN5A mutation associated with the linker between III and IV domains of Nav1.5 in a neonate with fatal long QT syndrome

A male newborn weighing 2334 g was delivered at 37 weeks of gestation by caesarean section because of prenatal ultrasound findings of fetal hydrops with atrioventricualr block, ventriucular tachycardia (VT), and impaired ventricular function. In spite of the intravenous administration of lidocaine, VT continued. He developed poor perfusion and systemic hypotension. After the intravenous administration of amiodarone, VT was terminated. The electrocardiogram revealed an extremely prolonged corrected QT interval (860 ms) with 2:1 atrioventricular block. Unfortunately, he died 18 h after birth in spite of the administration of lidocaine, beta-blocker and magnesium. Mutational analysis identified a novel heterozygous de novo mutation (F1486del) in SCN5A. This mutation is associated with the IFM motif in the linker between III and IV domains of Na(v)1.5, which serves as an inactivation particle binding within the pore of sodium channels. This report demonstrates an interesting relationship between the clinical phenotype and the location of the mutation in long QT syndrome.

Successful treatment of a patient with tumor necrosis factor receptor-associated periodic syndrome using a half-dose of etanercept

TNF receptor‐associated periodic syndrome (TRAPS) is caused by mutations of TNFRSF1A gene and characterized by recurrent febrile episodes of prolonged duration and initial good response to steroids. Etanercept, a TNF blocker, has been used as a putative molecular‐targeted agent for TRAPS, with some patients showing limited efficacy. Here, we report a patient with TRAPS who recovered from steroid dependency by etanercept and kept remission with a reduced dose of etanercept. The pathophysiology of TRAPS still remains to be elucidated and several hypotheses have been proposed. In the most recent hypothesis, the concerted action of wild‐type and mutant TNF receptors plays an important role in provoking enhanced inflammation in TRAPS. The excellent response to etanercept in our patient suggested that there is heterogeneity in TRAPS patients in terms of the contribution of normal TNF signaling to autoinflammation.

Early Mortality and Morbidity in Infants with Birth Weight of 500?Grams or Less in Japan.

Objective To assess the short‐term prognosis of Japanese infants with a birth weight (BW) of ≤500 g. Study design Demographic and clinical data were reviewed for 1473 live born infants with a BW ≤500 g at gestational age ≥22 weeks who were treated in the 204 affiliated hospitals of the Neonatal Research Network of Japan between 2003 and 2012. Results Survival to hospital discharge occurred in 811 of 1473 infants (55%; 95% CI 53%‐58%). The survival rates of BW ≤300 g, 301‐400 g, and 401‐500 g were 18% (95% CI 10%‐31%), 41% (95% CI 36%‐47%), and 60% (95% CI 57%‐63%), respectively. In a multivariable Cox proportional hazards analysis, antenatal corticosteroid use (adjusted hazard ratio: 0.68; 95% CI 0.58‐0.81; P < .01), cesarean delivery (0.69; 95% CI 0.56‐0.85; P < .01), advanced gestational age per week (0.94; 95% CI 0.89‐0.99; P = .02), BW per 100‐g increase (0.55; 95% CI 0.49‐0.64; P < .01), Apgar score ≥4 at 5 minutes (0.51; 95% CI 0.43‐0.61; P < .01), and no major congenital abnormalities (0.38; 95% CI 0.29‐0.51; P < .01) were associated with survival to discharge. Despite the improved survival rate over the 10‐year study period (from 40% in 2003 [95% CI 30%‐51%] to 68% in 2012 [95% CI 61%‐75%]), at least 1 severe morbidity was present in 81%‐89% of the survivors. Conclusions Improvements in perinatal‐neonatal medicine have improved the survival, but not the rate of major morbidities, of infants with a BW ≤500 g in Japan.

Age-specific onset and distribution of the natural anticoagulant deficiency in pediatric thromboembolism

Background:The early diagnosis of inherited thrombophilia in children is challenging because of the rarity and hemostatic maturation.Methods:We explored protein C (PC), protein S (PS), and antithrombin (AT) deficiencies in 306 thromboembolic patients aged ≤20 y using the screening of plasma activity and genetic analysis.Results:Reduced activities were determined in 122 patients (40%). Low PC patients were most frequently found in the lowest age group (0–2 y, 45%), while low PS or low AT patients were found in the highest age group (16–20 y; PS: 30% and AT: 20%). Genetic study was completed in 62 patients having no other causes of thromboembolism. Mutations were determined in 18 patients (8 PC, 8 PS, and 2 AT genes). Six of eight patients with PC gene mutation were found in age 0–2 y (75%), while six of eight patients with PS gene mutation were in 7–20 y. Two AT gene–mutated patients were older than 4 y. Four PC-deficient and two PS-deficient patients carried compound heterozygous mutations. All but one PC gene–mutated patient suffered from intracranial thromboembolism, while PS/AT gene–mutated patients mostly developed extracranial venous thromboembolism.Conclusion:Stroke in low PC infants and deep vein thrombosis in low PS/AT school age children could be targeted for genetic screening of pediatric thrombophilias.

Activation of Nod1 Signaling Induces Fetal Growth Restriction and Death through Fetal and Maternal Vasculopathy

Intrauterine fetal growth restriction (IUGR) and death (IUFD) are both serious problems in the perinatal medicine. Fetal vasculopathy is currently considered to account for a pathogenic mechanism of IUGR and IUFD. We previously demonstrated that an innate immune receptor, the nucleotide-binding oligomerization domain-1 (Nod1), contributed to the development of vascular inflammations in mice at postnatal stages. However, little is known about the deleterious effects of activated Nod1 signaling on embryonic growth and development. We report that administration of FK565, one of the Nod1 ligands, to pregnant C57BL/6 mice induced IUGR and IUFD. Mass spectrometry analysis revealed that maternally injected FK565 was distributed to the fetal tissues across placenta. In addition, maternal injection of FK565 induced robust increases in the amounts of CCL2, IL-6, and TNF proteins as well as NO in maternal, placental and fetal tissues. Nod1 was highly expressed in fetal vascular tissues, where significantly higher levels of CCL2 and IL-6 mRNAs were induced with maternal injection of FK565 than those in other tissues. Using Nod1-knockout mice, we verified that both maternal and fetal tissues were involved in the development of IUGR and IUFD. Furthermore, FK565 induced upregulation of genes associated with immune response, inflammation, and apoptosis in fetal vascular tissues. Our data thus provided new evidence for the pathogenic role of Nod1 in the development of IUGR and IUFD at the maternal-fetal interface.

Tracheal aspirate gene expression in preterm newborns and development of bronchopulmonary dysplasia.

Background:  Bronchopulmonary dysplasia (BPD) occurs in association with prenatal conditions predisposing infants to inflammation and remodeling of the premature lungs. Because of the lack of useful biomarkers for BPD, the gene expression of tracheal aspirate fluid (TAF) cells in premature infants was analyzed.