Multiplex Real-Time PCR-Based Newborn Screening for Severe Primary Immunodeficiency and Spinal Muscular Atrophy in Osaka, Japan: Our Results after 3 Years.

In newborn screening (NBS), it is important to consider the availability of multiplex assays or other tests that can be integrated into existing systems when attempting to implement NBS for new target diseases. Recent developments in innovative testing technology have made it possible to simultaneously screen for severe primary immunodeficiency (PID) and spinal muscular atrophy (SMA) using quantitative real-time polymerase chain reaction (qPCR) assays. We describe our experience of optional NBS for severe PID and SMA in Osaka, Japan. A multiplex TaqMan qPCR assay was used for the optional NBS program. The assay was able to quantify the levels of T-cell receptor excision circles and kappa-deleting recombination excision circles, which is useful for severe combined immunodeficiency and B-cell deficiency screening, and can simultaneously detect the homozygous deletion of SMN1 exon 7, which is useful for NBS for SMA. In total, 105,419 newborns were eligible for the optional NBS program between 1 August 2020 and 31 August 2023. A case each of X-linked agammaglobulinemia and SMA were diagnosed through the optional NBS and treated at early stages (before symptoms appeared). Our results show how multiplex PCR-based NBS can benefit large-scale NBS implementation projects for new target diseases.

Postneonatal mortality of severely small for gestational age extremely low birth weight infants.

BACKGROUND: Infants born with weights below the 10th percentile of the expected birth weight for gestational age, defined as small for gestational age (SGA), have an increased risk of neonatal mortality and prematurity-related complications. However, the relationship between SGA and postneonatal (28 days to <1 year) mortality among extremely low birth weight infants (ELBWIs) remains uncertain. Hence, this study aimed to investigate the association between birth weight percentiles and postneonatal mortality in ELBWIs. METHODS: A cohort of ELBWIs with a gestational age greater than 23 weeks who were admitted to Osaka Women's and Children's Hospital between 2008 and 2019 were considered eligible. Infants with major congenital anomalies, those large for their gestational age, or those who died within 28 days of birth were excluded. Baseline characteristics and outcomes of the three groups of ELBWIs-severe SGA (sSGA; birth weight, <3rd percentile), moderate SGA (mSGA; birth weight, 3rd to <10th percentile), and appropriate for gestational age (AGA; birth weight, 10th to <90th percentile)-were compared. Logistic regression analysis was used to identify perinatal factors associated with postneonatal mortality in sSGA infants. RESULTS: sSGA ELBWIs demonstrated higher incidence of meconium obstruction (25% vs. 8.3% vs. 7.6%, P < 0.001), cholestasis (21% vs. 4.2% vs. 9.7%, P < 0.003), and postneonatal mortality (7.3% vs. 0% vs. 0.7%, P < 0.004) than mSGA and AGA ELBWIs. In the logistic regression analysis, cholestasis (odds ratio, 30.1; 95% confidence interval, 2.98-304) and sepsis (odds ratio, 13.5; 95% confidence interval, 1.06-173) were significantly related to postneonatal mortality among ELBWIs with sSGA. The leading cause of postneonatal mortality in sSGA ELBWIs was liver failure (55.5%). CONCLUSION: sSGA ELBWIs exhibited a higher rate of postneonatal mortality compared to mSGA and AGA ELBWIs. Therefore, strategies aimed at preventing liver dysfunction in severely cholestatic ELBWIs with sSGA are necessary.

Newborn screening for spinal muscular atrophy in Osaka -challenges in a Japanese pilot study.

OBJECTIVE: This study aimed to establish an optional newborn screening program for spinal muscular atrophy (SMA-NBS) in Osaka. METHODS: A multiplex TaqMan real-time quantitative polymerase chain reaction assay was used to screen for SMA. Dried blood spot samples obtained for the optional NBS program for severe combined immunodeficiency, which covers about 50% of the newborns in Osaka, were used. To obtain informed consent, participating obstetricians provided information about the optional NBS program to all parents by giving leaflets to prospective parents and uploading the information onto the internet. We prepared a workflow so that babies that were diagnosed with SMA through the NBS could be treated immediately. RESULTS: From 1 February 2021 to 30 September 2021, 22,951 newborns were screened for SMA. All of them tested negative for survival motor neuron (SMN)1 deletion, and there were no false-positives. Based on these results, an SMA-NBS program was established in Osaka and included in the optional NBS programs run in Osaka from 1 October 2021. A positive baby was found by screening, diagnosed with SMA (the baby possessed 3 copies of the SMN2 gene and was pre-symptomatic), and treated immediately. CONCLUSION: The workflow of the Osaka SMA-NBS program was confirmed to be useful for babies with SMA.

Hospital burden and characteristics of pediatric COVID-19 based on a multicenter collaborative retrospective study in Japan.

Objectives: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant emergence preceded a wave of pediatric coronavirus disease 2019 (COVID-19) cases, putting considerable strain on hospitals across Japan. Our study evaluated the pediatric disease burden of COVID-19 in pediatric hospitals. Methods: This retrospective study evaluated all pediatric patients (defined as aged < 21 years) hospitalized with SARS-CoV-2 infection, or as close contacts, at four children's hospitals, between January 1 and May 31, 2022. Clinical characteristics, reasons for admission, and outcome data were analyzed. Results: In total, 492 patients (median age 3.0 years; male 58.7%) were included over the study period. Of these, 232 (47.2%) patients had at least one underlying disease. Asymptomatic and mild diseases were common during the study period (n = 451, 91.7%). Social reasons for hospitalization (including a lack of family support at home) accounted for 36.8% (n = 181) of inpatients. The median length of stay was 4.0 days. Fever was the most common symptom (n = 273, 55.5%), followed by upper respiratory (n = 77, 15.7%) and neurological (n = 60, 12.2%) symptoms. Overall, 34 (6.9%) children required invasive mechanical ventilation, 51 (10.4%) were admitted to the pediatric intensive care unit, and two (0.4%) died. COVID-19 vaccination rate was low (n =14/200, 7.0%). Conclusions: The disease burden during the Omicron-predominant period was attributable to asymptomatic and mild infections, and some patients were hospitalized for social reasons. To maintain a medical care system for critically ill patients, each medical facility must play a role according to its function.

The Association between Early Gram-Negative Bacteria in Tracheal Aspirate Cultures and Severe Bronchopulmonary Dysplasia among Extremely Preterm Infants Requiring Prolonged Ventilation.

OBJECTIVE: The study aimed to evaluate the association between bronchopulmonary dysplasia (BPD) development at 36 weeks' postmenstrual age (PMA) and Gram-negative bacteria in tracheal aspirate cultures among extremely preterm infants. STUDY DESIGN: This study has a retrospective cohort. Patients were 155 infants aged less than or equal to 26 gestational weeks who were admitted to the neonatal intensive care unit of Osaka Women's and Children's Hospital from 2009 to 2018. Primary outcome was respiratory outcomes expressed as BPD development.Multivariable logistic regression analysis was used to identify neonatal and bacterial factors associated with BPD. RESULTS: After adjusting for gestational age, birth weight, sex, chorioamnionitis, Gram-positive cocci (GPC) and Gram-negative rods (GNRs) in tracheal aspirate cultures within 28 days after birth, GNRs were significantly associated with BPD development (odds ratio [OR]: 3.88, 95% confidence interval [CI]: 1.68-8.94). In contrast, GPCs were not associated with BPD development (OR: 0.47, 95% CI: 0.05-1.61). CONCLUSION: Gram-negative bacteria in tracheal cultures within 28 days of birth are associated with BPD development in infants aged less than or equal to 26 gestational weeks. KEY POINTS: · BPD is a factor for morbidity in extremely preterm infants.. · Respiratory infection is an adverse outcome of BPD.. · GNRs in tracheal cultures soon after birth disturb BPD development.. · GPC was not associated with BPD development..

Urine Desmosine as a Novel Biomarker for Bronchopulmonary Dysplasia and Postprematurity Respiratory Disease in Extremely Preterm or Low Birth Weight Infants.

OBJECTIVE: This study aimed to evaluate whether elevated urine desmosine levels at 3 weeks of age were associated with severe radiological findings, bronchopulmonary dysplasia (BPD), and post-prematurity respiratory disease (PRD) in extremely preterm (EP) or extremely low birth weight (ELBW) infants. STUDY DESIGN: This study recruited 37 EP (22-27 completed weeks) or ELBW (<1,000 g) infants. Urine was collected between 21 and 28 postnatal days, and desmosine was measured using an enzyme-linked immunosorbent assay kit; the urine creatinine level was also measured. Bubbly/cystic lungs were characterized by emphysematous chest X-rays on postnatal day 28. Furthermore, provision of supplemental oxygen or positive-pressure respiratory support at 40 weeks' postmenstrual age defined BPD, and increased medical utilization at 18 months of corrected age defined PRD. The desmosine/creatinine threshold was determined by receiver operating characteristic analysis. The adjusted risk and 95% confidence interval (CI) for elevated urine desmosine/creatinine levels were estimated by logistic regression analysis. RESULTS: Elevated urine desmosine/creatinine levels higher than the threshold were significantly associated with bubbly/cystic lungs (8/13 [61.5%] vs. 2/24 [8.3%], p = 0.001), BPD (10/13 [76.9%] vs. 8/24 [33.3%], p = 0.02), and PRD (6/13 [46.2%] vs. 2/24 [8.3%], p = 0.01). After adjusting for gestational age, birth weight, and sex, the urine desmosine/creatinine levels were significantly higher in those who were highly at risk of bubbly/cystic lungs (odds ratio [OR], 13.2; 95% CI, 1.67-105) and PRD (OR, 13.8; 95% CI, 1.31-144). CONCLUSION: Elevated urine desmosine/creatinine levels on the third postnatal week were associated with bubbly/cystic lungs on day 28 and PRD at 18 months of corrected age in EP or ELBW infants. KEY POINTS: · Urine desmosine was prospectively measured in 3-week-old EP/ELBW infants.. · Elevated urine desmosine levels were associated with emphysematous radiological findings on day 28, PRD at 18 months of corrected age.. · Urine desmosine may be a promising biomarker indicating lung damage in EP/ELBW infants..

Genome Analysis in Sick Neonates and Infants: High-yield Phenotypes and Contribution of Small Copy Number Variations.

OBJECTIVE: To delineate the diagnostic efficacy of medical exome, whole exome, and whole genome sequencing according to primary symptoms, the contribution of small copy number variations, and the impact of molecular diagnosis on clinical management. STUDY DESIGN: This was a prospective study of 17 tertiary care centers in Japan, conducted between April 2019 and March 2021. Critically ill neonates and infants less than 6 months of age were recruited in neonatal intensive care units and in outpatient clinics. The patients underwent medical exome, whole exome, or whole genome sequencing as the first tier of testing. Patients with negative results after medical exome or whole exome sequencing subsequently underwent whole genome sequencing. The impact of molecular diagnosis on clinical management was evaluated through contacting primary care physicians. RESULTS: Of the 85 patients, 41 (48%) had positive results. Based on the primary symptoms, patients with metabolic phenotypes had the highest diagnostic yield (67%, 4/6 patients), followed by renal (60%, 3/5 patients), and neurologic phenotypes (58%, 14/24 patients). Among them, 4 patients had pathogenic small copy number variations identified using whole genome sequencing. In the 41 patients with a molecular diagnosis, 20 (49%) had changes in clinical management. CONCLUSIONS: Genome analysis for critically ill neonates and infants had a high diagnostic yield for metabolic, renal, and neurologic phenotypes. Small copy number variations detected using whole genome sequencing contributed to the overall molecular diagnosis in 5% of all the patients. The resulting molecular diagnoses had a significant impact on clinical management.

Feasibility of portable capnometer for mechanically ventilated preterm infants in the delivery room.

This study aimed to determine whether a specific portable capnometer (EMMA™) can facilitate the maintenance of an appropriate partial pressure of arterial carbon dioxide (PaCO2) in intubated preterm infants in the delivery room. This study included preterm infants with a gestational age of 26 + 0 to 31 + 6 weeks who required intubation in the delivery room. We prospectively identified 40 infants who underwent the EMMA™ monitoring intervention group and 43 infants who did not undergo monitoring (historical control group). PaCO2 was evaluated either at admission in the neonatal intensive care unit or at 2 h after birth. The proportion of infants with an appropriate PaCO2 (35-60 mmHg) was greater in the intervention group than in the control group (80% vs. 42%; p = 0.001). There were no significant differences in the rate of accidental extubation (5.0% vs. 7.0%, p = 1.00) or in the proportion of infants with an appropriate PaCO2 among infants whose birth weight was < 1000 g (54% vs. 40%, p = 0.49). However, among infants whose birth weight was ≥ 1000 g, the PaCO2 tended to be more appropriate in the intervention group than in the control group (93% vs. 44%; p < 0.001).Conclusion: The EMMA™ facilitated the maintenance of an appropriate PaCO2 for mechanically ventilated preterm infants, especially infants with birth weight ≥1000 g, in the delivery room. What is Known: • An inappropriate partial pressure of arterial carbon dioxide has been associated with intraventricular hemorrhage in preterm infants. • There is a need to appropriately control the partial pressure of arterial carbon dioxide in preterm infants. What is New: • This is the first report regarding the feasibility of a portable capnometer, the EMMA™, in the delivery room. • The EMMA™ may be considered a feasible monitoring device in the delivery room for intubated preterm infants, especially infants with birth weight ≥1000 g.

Availability of portable capnometers in children with tracheostomy.

BACKGROUND: A capnometer is a noninvasive monitor that is used to assess patients' respiratory status. This study was performed to evaluate the availability of a portable capnometer in children with tracheostomy. METHODS: This retrospective study included children with tracheostomy who were treated at the Osaka Women's and Children's Hospital Osaka, Japan, from 1 September 2018 to 31 October 2019. We assessed the correlation between the partial pressure of venous carbon dioxide (PvCO2 ) and end-tidal carbon dioxide tension (EtCO2 ) using a portable capnometer (EMMA; Masimo, Irvine, CA, USA). RESULTS: Nine infants and 43 simultaneous PvCO2 -EtCO2 pairs were analyzed. The correlation coefficient of these pairs was 0.87 (95% confidence interval, 0.77-0.93; P < 0.001). The Bland-Altman plot showed that EtCO2 was on average 10.0 mmHg lower than its paired PvCO2 value (95% limits of agreement, 1.0-19.1). The difference between PvCO2 and EtCO2 was significantly greater in patients on ventilators. CONCLUSIONS: The portable capnometer evaluated in this study (EMMA) was readily available and useful for assessment of the respiratory condition in children with tracheostomy.

Clostridium tertium Peritonitis and Bacteremia in a Neonate With Congenital Intestinal Atresia: A Case Report.

We report a 1-day-old girl who was affected by peritonitis and bacteremia caused by Clostridium tertium following perforation of congenital intestinal atresia. Splenic infarction was also suspected during C. tertium bacteremia. C. tertium was identified by using mass spectrometry and 16S rRNA sequencing. This patient was successfully treated with emergency laparotomy and broad-spectrum antibiotics.

Severe course with lethal hepatocellular injury and skeletal muscular dysgenesis in a neonate with infantile liver failure syndrome type 1 caused by novel LARS1 mutations.

Infantile liver failure syndrome type 1 (ILFS1) is a recently recognized autosomal recessive disorder caused by deleterious mutations in the leucyl-tRNA synthetase 1 gene (LARS1). The LARS1 enzyme is responsible for incorporation of the amino acid leucine during protein polypeptide synthesis. Individuals with LARS1 mutations typically show liver failure from infancy to early childhood during periods of illness or other physiological stress. While 25 patients from 15 families with ILFS1 have been reported in the literature, histological reports from autopsy findings are limited. We report here a premature male neonate who presented with severe intrauterine growth retardation, microcytic anemia, and fulminant liver failure, and who was a compound heterozygote for two novel deleterious mutations in LARS1. An autopsy showed fulminant hepatitis-like hepatocellular injury and fibrogenesis in the liver and a lack of uniformity in skeletal muscle, accompanied by the disruption of striated muscle fibers. Striking dysgenesis in skeletal muscle detected in the present case indicates the effect of LARS1 functional deficiency on the musculature. Whole-exome sequencing may be useful for neonates with unexplained early liver failure if extensive genetic and metabolic testing is inconclusive.

Association between amount of dopamine and infections in extremely preterm infants.

This study aimed to assess the effect of dopamine on the development of infections after birth in extremely preterm infants. We retrospectively identified 258 extremely preterm infants (born at < 28 gestational weeks) between July 2009 and December 2018 in a tertiary neonatal intensive care unit (NICU). We extracted data on potential risk factors for infection, total amount of dopamine, and infection history during NICU stay for each infant. We compared the infection group with the non-infection group, and used the Cox proportional hazard regression analysis to identify risk factors for infection during NICU stay. After adjustment for all potential risk factors, factors that significantly affected development of infection were gestational age (hazard ratio [HR], 0.70; 95% confidence interval [CI] 0.55-0.89; p = 0.004) and total amount of dopamine (HR, 1.04; 95% CI 1.02-1.07; p = 0.002). The receiver operating characteristic curve of total amount of dopamine for infection suggested that total amount of dopamine greater than 7.271 mg/kg predicted infection development with 80.4% sensitivity and 41.7% specificity.Conclusion: A large amount of dopamine can increase infections in extremely preterm infants. We should avoid using a large amount of dopamine and remain aware of the potential development of infections in extremely preterm infants. What is Known: • Inotropes are often used for extremely preterm infants and dopamine is the most commonly used inotrope. • However, it is suggested that dopamine affects the immune system and related infections. What is New: • This is the first study of the association between the amount of dopamine and infection in extremely preterm infants. • We should avoid using a large amount of dopamine in extremely preterm infants.

Postnatal physiological changes in electrical activity of the diaphragm in extremely preterm infants.

OBJECTIVES: This study aimed to describe postnatal physiological changes in maximum values of peak electrical activity of the diaphragm (Edi) in extremely preterm infants during the preterm period. WORKING HYPOTHESIS: The amplitude and frequency of neural sigh are different at each postmenstrual age in extremely preterm infants. STUDY DESIGN: A retrospective, observational study. PATIENT-SUBJECT SELECTION: Edi values were evaluated in 14 extremely preterm infants with neurally-adjusted ventilatory assist. METHODOLOGY: Data of Edi peak and Edi minimum were collected from a ventilator. Edi-sigh was defined as the Edi peak value that was more than twice as large as the median Edi peak at each postmenstrual week in each patient. The frequency of Edi-sigh, and median values of Edi-sigh, Edi peak, and Edi minimum were evaluated at each postmenstrual week. The Jonckheere-Terpstra test was used to analyze the trend between postmenstrual weeks and Edi values. RESULTS: From 26 to 35 postmenstrual weeks, the number of Edi-sighs per hour significantly increased as postmenstrual weeks increased (P < .001). Furthermore, the median values of Edi-sigh significantly increased as postmenstrual weeks increased (16.9 µV at 26 weeks to 25.4 µV at 35 weeks, P < .001). There were no significant changes in the median values of Edi peak and Edi minimum at each week. CONCLUSIONS: The amplitude and frequency of neural sigh in extremely preterm infants increase with the number of postmenstrual weeks.

Effect of fluctuation of oxygenation on the development of severe retinopathy of prematurity in extremely preterm infants.

OBJECTIVE: To investigate factors associated with development of severe retinopathy of prematurity (ROP) in extremely preterm (EP) infants. STUDY DESIGN: This retrospective cohort study included 213 EP infants (22 + 0 to 27 + 6 weeks gestation) who were admitted to the neonatal intensive care unit of Osaka Women's and Children's Hospital between 2009 and 2017. Multivariable logistic regression analysis was used to identify neonatal factors associated with severe ROP requiring treatment. RESULT: After adjustments for gestational age (GA), birth weight, sex, red blood cell transfusion, average SpO2, and fluctuations of SpO2 from birth to 32 weeks postmenstrual age, fluctuations of SpO2 (odds ratio [OR]: 2.10, 95% confidence interval [CI]: 1.03-4.27), and low GA (OR: 0.95, 95% CI: 0.91-0.98) were significantly associated with severe ROP. CONCLUSIONS: Fluctuations of SpO2 from birth to 32 weeks postmenstrual age and low GA were significantly associated with development of severe ROP requiring treatment in EP infants.

Impact of Time to Neonatal Transport on Outcomes of Transient Tachypnea of the Newborn.

OBJECTIVE: To assess effects of neonatal transport on transient tachypnea of the newborn (TTN) in outborn term neonates. STUDY DESIGN: This retrospective cohort study included 66 term neonates diagnosed with TTN and transported to the Osaka Women's and Children's Hospital neonatal intensive care unit between January 2003 and March 2018. A multivariate logistic regression analysis identified perinatal and neonatal transport factors associated with adverse short-term outcomes defined as mechanical ventilation >48 hours, continuous positive airway pressure >72 hours, pulmonary hemorrhage, and requirement for inhaled nitric oxide, thoracentesis, or surfactant replacement therapy. RESULTS: A lower gestational age (GA) (37.7 [37.2, 38.3] vs. 39.6 [37.8, 40.3] weeks, p = 0.002), longer time to neonatal transport (10.0 [4.3, 25.5] vs. 5.5 [2.7, 9.7] hours, p = 0.01), and higher respiratory rates during transport (70 [60, 85] vs. 60 [55, 78.8] breaths/min, p = 0.04) were significantly associated with adverse short-term outcomes. After adjusting for GA, sex, cesarean section, and time to neonatal transport, GA (odds ratio [OR], 0.37; 95% confidence interval [CI], 0.24-0.87) and time to neonatal transport (OR, 1.07; 95% CI, 1.01-1.13) were significantly associated with adverse outcomes. CONCLUSION: Short-term adverse prognosis of TTN is strongly associated with a lower GA and longer time between birth and neonatal transport.

Ceftriaxone preconditioning confers neuroprotection in neonatal rats through glutamate transporter 1 upregulation.

OBJECTIVE: This study investigated the hypothesis that ceftriaxone preconditioning ameliorates brain damage in neonatal animals through glutamate transporter 1 (GLT-1) upregulation. STUDY DESIGN: Sprague Dawley rats were pretreated with ceftriaxone, erythromycin, minocycline, or saline for 5 consecutive days starting from postnatal day 2 (P2), and GLT-1/glutamate-aspartate transporter (GLAST) messenger RNA (mRNA) and protein levels were examined in the P7 brains. After ceftriaxone or saline preconditioning, the P7 rats underwent hypoxic-ischemic (H-I) procedure or sham operation. One week after the procedure (P14), hematoxylin-eosin staining, microtubule-associated protein 2 (MAP-2) immunostaining, and transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay were used to examine neuronal damage and possible neurotoxicity. RESULTS: Repeated ceftriaxone injections significantly increased GLT-1 mRNA and protein levels but not GLAST. Following such treatment and H-I procedure, the MAP-2-positive area increased and TUNEL-positive cells decreased. CONCLUSION: Antenatal ceftriaxone may help to provide neuroprotection in the immature brain and become a new prophylactic strategy to reduce neonatal encephalopathy in clinical perinatal medicine.

Silica and titanium dioxide nanoparticles cause pregnancy complications in mice.

The increasing use of nanomaterials has raised concerns about their potential risks to human health. Recent studies have shown that nanoparticles can cross the placenta barrier in pregnant mice and cause neurotoxicity in their offspring, but a more detailed understanding of the effects of nanoparticles on pregnant animals remains elusive. Here, we show that silica and titanium dioxide nanoparticles with diameters of 70 nm and 35 nm, respectively, can cause pregnancy complications when injected intravenously into pregnant mice. The silica and titanium dioxide nanoparticles were found in the placenta, fetal liver and fetal brain. Mice treated with these nanoparticles had smaller uteri and smaller fetuses than untreated controls. Fullerene molecules and larger (300 and 1,000 nm) silica particles did not induce these complications. These detrimental effects are linked to structural and functional abnormalities in the placenta on the maternal side, and are abolished when the surfaces of the silica nanoparticles are modified with carboxyl and amine groups.

Regulation of TCR Vγ2 gene rearrangement by the helix-loop-helix protein, E2A.

V(D)J recombination of Ig and TCR genes is strictly regulated by the accessibility of target gene chromatin in a lineage- and stage-specific manner. In the mouse TCRγ locus, rearrangement of the Vγ2 gene predominates over Vγ3 rearrangement in the adult thymus. This preferential rearrangement is likely due to the differential accessibility of the individual Vγ genes, because the levels of germ line transcription and histone acetylation of the Vγ genes are well correlated with the rearrangement frequency in adult thymocytes. However, factors responsible for the differential regulation of the Vγ gene rearrangement have been largely unknown. In this study, we demonstrated that Vγ2 rearrangement in the adult thymus was substantially reduced in mice deficient for the basic helix-loop-helix protein, E2A. The decreased rearrangement is likely caused by the reduced accessibility of Vγ2 chromatin, since germ line transcription and histone acetylation of the Vγ2 gene were reduced in an E2A dosage-dependent manner. We further showed that E2A bound around the Vγ2 gene in vivo and we identified two canonical E-box sites downstream of Vγ2, to which E2A can bind in vitro. Furthermore, these two E-box sites had the ability to activate transcription upon E2A over-expression. These data suggest that E2A directly binds to and increases accessibility of Vγ2 chromatin, thereby facilitating Vγ2 rearrangement in the adult thymus.

Transcriptional regulation of the human establishment of cohesion 1 homolog 2 gene.

Transcriptional regulation of human establishment of cohesion 1 homolog 2 (ESCO2), the causative gene of Roberts syndrome, was investigated. Deletion and mutation analyses of the ESCO2 promoter indicated that the selenocysteine tRNA-activating factor (Staf) binding site (SBS) is an essential element for transcriptional activation of ESCO2. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay revealed that the zinc finger protein 143 (ZNF143), a human homolog of Xenopus Staf, bound to the ESCO2 promoter. The ACTACAN submotif, adjacent to SBS, also contributed to transcriptional activation of ESCO2. EMSA indicated that the ACTACAN submotif was not involved in binding of ZNF143 to SBS. S phase-specific expression of the ESCO2 gene was confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), but EMSA revealed binding of ZNF143 to SBS in G1/S and G2/M phases. These results demonstrated that SBS functioned as the basal transcriptional activator of the S phase-specific gene ESCO2, but other mechanisms are required for cell cycle-dependent expression.