Congenital anaemia associated with loss-of-function variants in DNA polymerase epsilon 1.

DNA polymerase epsilon (Pol ε), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol ε have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE, encoding Pol ε catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole-knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol ε defect in humans, additionally providing unique evidence linking Pol ε to haematopoiesis.

Children's animistic beliefs toward a humanoid robot and other objects.

This study examined children's beliefs about a humanoid robot by examining their behavioral and verbal responses. We investigated whether 3- and 5-year-old children would treat the humanoid robot gently along with other objects and tools with and without a face and whether 3- and 5-year-olds would attribute moral, perceptual, and psychological properties to these targets. Although 3-year-olds did not treat objects gently or rudely, they were likely to affirm that hitting targets was acceptable despite targets having psychological and perceptual properties. Thus, 3-year-olds' perception of the targets was incongruent with their behavior toward them. Most 5-year-olds treated a robot gently and were likely to affirm the robot's psychological characteristics. Behaviors and perceptions of the robot differed between 3- and 5-year-olds. Thus, children may start believing that robots are not alive at age five, and they can distinguish them from other objects even when the latter have faces. Developmental changes in children's animistic beliefs are also discussed.

Epitranscriptomic profiling in human placenta: N6-methyladenosine modification at the 5'-untranslated region is related to fetal growth and preeclampsia.

Intracellular mRNA levels are not always proportional to their respective protein levels, especially in the placenta. This discrepancy may be attributed to various factors including post-transcriptional regulation, such as mRNA methylation (N6-methyladenosine: m6A). Here, we conducted a comprehensive m6A analysis of human placental tissue from neonates with various birth weights to clarify the involvement of m6A in placental biology. The augmented m6A levels at the 5'-untranslated region (UTR) in mRNAs of small-for-date placenta samples were dominant compared to reduction of m6A levels, whereas a decrease in m6A in the vicinity of stop codons was common in heavy-for-date placenta samples. Notably, most of these genes showed similar expression levels between the different birth weight categories. In particular, preeclampsia placenta samples showed consistently upregulated SMPD1 protein levels and increased m6A at 5'-UTR but did not show increased mRNA levels. Mutagenesis of adenosines at 5'-UTR of SMPD1 mRNAs actually decreased protein levels in luciferase assay. Collectively, our findings suggest that m6A both at the 5'-UTR and in the vicinity of stop codon in placental mRNA may play important roles in fetal growth and disease.

Isolation and characterization of fetal nucleated red blood cells from maternal blood as a target for single cell sequencing-based non-invasive genetic testing.

PURPOSE: Although non-invasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) in maternal plasma has been prevailing worldwide, low levels of fetal DNA fraction may lead to false-negative results. Since fetal cells in maternal blood provide a pure source of fetal genomic DNA, we aimed to establish a workflow to isolate and sequence fetal nucleated red blood cells (fNRBCs) individually as a target for NIPT. METHODS: Using male-bearing pregnancy cases, we isolated fNRBCs individually from maternal blood by FACS, and obtained their genomic sequence data through PCR screening with a Y-chromosome marker and whole-genome amplification (WGA)-based whole-genome sequencing. RESULTS: The PCR and WGA efficiencies of fNRBC candidates were consistently lower than those of control cells. Sequencing data analyses revealed that although the majority of the fNRBC candidates were confirmed to be of fetal origin, many of the WGA-based genomic libraries from fNRBCs were considered to have been amplified from a portion of genomic DNA. CONCLUSIONS: We established a workflow to isolate and sequence fNRBCs individually. However, our results demonstrated that, to make cell-based NIPT targeting fNRBCs feasible, cell isolation procedures need to be further refined such that the nuclei of fNRBCs are kept intact.

Task Difficulty Makes 'No' Response Different From 'Yes' Response in Detection of Fragmented Object Contours.

Two-alternative forced choice tasks are often used in object detection, which regards detecting an object as a 'yes' response and detecting no object as a 'no' response. Previous studies have suggested that the processing of yes/no responses arises from identical or similar processing. In this study, we investigated the difference of processing between detecting an object ('yes' response) and not detecting any object ('no' response) by controlling the task difficulty in terms of fragment length and stimulus duration. The results indicated that a 'yes' response depends on accurate and stable decisions through grouping processing, and a 'no' response might involve two distinct processing, including accurate decisions and intuitive decisions. Accurate decisions of 'no' may arise after the rejection of a 'yes' response with grouping processing, which is an accurate but slow response in an easy task. Intuitive decisions of 'no' arise as the result of breaking down the decision process when the received information was insufficient for grouping processing in a difficult task. Therefore, intuitive decisions of 'no' arise quickly but are inaccurate. The different processes associated with yes/no responses were discussed in terms of the hierarchal structure of object recognition, especially with respect to receiving information and grouping.

Placental Development and Nutritional Environment.

The placenta is considered to have developed recently in mammalian evolution. While the fundamental function of the placenta, i.e., providing nutrients and oxygen to the fetus and receiving waste products, is the same in all mammals, the morphology of the placenta varies substantially in a species-dependent manner. Therefore, considerable interest exists in understanding placental development and function in mammals from a molecular biological viewpoint. Numerous recent studies have shown that various environmental factors before and during pregnancy, including nutrition, affect placental formation and function and that alterations in placental formation and function can influence the developing fetus and the offspring after birth. To date, the relationship between nutrition and the placenta has been investigated in several species, various model organisms, and humans. In this chapter, we discuss the current knowledge of the placenta and the epigenome and then highlight the effects of nutrition during pregnancy on the placenta and the fetus and on the offspring after birth.

Essential Role of Lysophosphatidylcholine Acyltransferase 3 in the Induction of Macrophage Polarization in PMA-Treated U937 Cells.

Lysophospholipid acyltransferases (LPLATs) regulate the diversification of fatty acid composition in biological membranes. Lysophosphatidylcholine acyltransferases (LPCATs) are members of the LPLATs that play a role in inflammatory responses. M1 macrophages differentiate in response to lipopolysaccharide (LPS) and are pro-inflammatory, whereas M2 macrophages, which differentiate in response to interleukin-4 (IL-4), are anti-inflammatory and involved in homeostasis and wound healing. In the present study, we showed that LPCATs play an important role in M1/M2-macrophage polarization. LPS changed the shape of PMA-treated U937 cells from rounded to spindle shaped and upregulated the mRNA and protein expression of the M1 macrophage markers CXCL10, TNF-α, and IL-1ß. IL-4 had no effect on the shape of PMA-treated U937 cells and upregulated the M2 macrophage markers CD206, IL-1ra, and TGF-ß in PMA-treated U937 cells. These results suggest that LPS and IL-4 promote the differentiation of PMA-treated U937 cells into M1- and M2-polarized macrophages, respectively. LPS significantly downregulated the mRNA expression of LPCAT3, one of four LPCAT isoforms, and suppressed its enzymatic activity toward linoleoyl-CoA and arachidonoyl-CoA in PMA-treated U937 cells. LPCAT3 knockdown induced a spindle-shaped morphology typical of M1-polarized macrophages, and increased the secretion of CXCL10 and decreased the levels of CD206 in IL-4-activated U937 cells. This indicates that knockdown of LPCAT3 shifts the differentiation of PMA-treated U937 cells to M1-polarized macrophages. Our findings suggest that LPCAT3 plays an important role in M1/M2-macrophage polarization, providing novel potential therapeutic targets for the regulation of immune and inflammatory disorders.

Strict management of a pregnant patient with giant coronary artery aneurysm due to Kawasaki disease.

Coronary artery aneurysms (CAA) may occur in Kawasaki disease (KD). Patients with giant CAA (diameter >8 mm), in particular, have higher risk of myocardial infarction. Previous reports have demonstrated the necessity of anticoagulation therapy in such cases. The management of patients with KD complicated by giant CAA later in life, however, remains controversial. Here, we describe the strict management in the case of a 28-year-old pregnant Japanese woman with KD with giant CAA (diameter, 11 mm). Instead of warfarin, the patient was given low-dose aspirin and i.v. unfractionated heparin during pregnancy to prevent thrombosis in the giant CAA. At 38 weeks of gestation, she had spontaneous delivery of a healthy baby. No thrombotic or bleeding complications were observed. The strict anticoagulation therapy resulted in successful pregnancy and delivery without any adverse events.

Twin anemia-polycythemia sequence after laser surgery for twin-twin transfusion syndrome and maternal morbidity.

Twin anemia-polycythemia sequence (TAPS) is characterized by large inter-twin hemoglobin value differences without inter-twin amniotic fluid discordance. The management of post-laser TAPS remains controversial. Hence, more studies on TAPS, together with the associated maternal complications and outcome of the fetuses and infants are needed. Between 2003 and 2012, we performed 287 cases of fetoscopic laser photocoagulation for twin-twin transfusion syndrome. Among the 114 who were placed under our care until delivery, three cases of TAPS occurred. In one case, we conducted intrauterine intravenous transfusion, while in the other two cases, we adopted expectant management. We performed an emergency caesarean section at 27-30 weeks of gestation in all cases due to a severe condition of anemia in the TAPS donor. Two cases with antenatal TAPS stage 4 had severe maternal complications; one had minute pulmonary embolism, while the other had Mirror syndrome. All three pairs of infants survived. One TAPS donor and one TAPS recipient had neurodevelopmental impairment; bilateral deafness at 9.5 years old and spastic paralysis at 2 years old, respectively. In conclusion, post-laser TAPS in a higher stage can cause severe maternal complications. Close observations for both fetuses and mothers are required for such cases.

Is in vitro fertilization associated with preeclampsia? A propensity score matched study.

BACKGROUND: Although an increased risk of preeclampsia in pregnancies conceived by in vitro fertilization (IVF) has been reported, it remains unknown whether IVF is associated with preeclampsia. In the present study, we sought to investigate whether IVF is associated with preeclampsia in pregnant women using propensity score matching analysis. METHODS: This study included 3,084 pregnant women who visited the National Center for Child Health and Development before 20 weeks of gestation without hypertension or renal disease and delivered a singleton after 22 weeks of gestation between 2009 and 2011. Of the 3084 patients, 474 (15.4%) conceived by IVF (IVF group) and 2,610 (84.6%) conceived without IVF (non-IVF group). The propensity score for receiving IVF was estimated using multiple logistic regression with 27 maternal and paternal variables. This model yielded a c-statistic of 0.852, indicating a strong ability to differentiate between those conceiving with and without IVF. The association between IVF and onset of preeclampsia was assessed by the propensity matched sample (pair of N = 474). RESULTS: There were 46 preeclampsia cases (1.5%) in the total study population, with a higher proportion of cases in the IVF group (15 cases, 3.2%) than the non-IVF group (31 cases, 1.2%). Before propensity score matching, the IVF group was 2.72 (95% confidence intervals [CI]: 1.46-5.08) times more likely to have preeclampsia when unadjusted, and 2.32 (95% CI: 1.08-4.99) times more likely to have preeclampsia when adjusted for maternal and paternal variables by logistic regression. After propensity score matching, the IVF group did not show a significantly greater association with preeclampsia compared to the non-IVF group (odds ratio: 2.50, 95% CI: 0.49-12.89), although point estimates showed a positive direction. CONCLUSIONS: Propensity score matching analysis revealed that the association between IVF and preeclampsia became weaker than when conventional adjustments are made in multivariate logistic regression analysis, suggesting that the association between IVF and preeclampsia might be confounded by residual unmeasured factors.

The infant-doctor relationship: an examination of infants' distress reactions in the presence of a doctor.

Fear of doctors is a common source of distress among infants; however, the underlying sources of this distress are unknown. To investigate the doctor-infant relationship, the behaviors of 61 healthy infants (176-617 days old) were observed in a simulated examination room. Their behaviors and electrocardiograms were recorded. Two groups of infants were analyzed: those who cried and those who did not. When an experimenter dressed in the doctor's attire entered the room, all 9 infants who were crying (14.8% of all infants) stopped crying, all infants gazed at the experimenter, and their mean heart rate (HR) decreased. After the auscultation started, 29.5% of all infants cried, and the HRs of infants who cried were higher than those of infants who did not cry. During the auscultation, 80.0% of infants who cried averted from the experimenter, while 34.4% of infants who did not cry. Within 5 s of gazing at the stethoscope, the number of infants who cried increased from 3 to 12, and their mean HR also increased. Our findings suggest that the fear of doctors is not due to the appearance of doctors but rather to specific actions performed by doctors, such as auscultation. Infants may regard a doctor's appearance as a source of interest. Furthermore, a stethoscope is a possible trigger for infants' crying. These behavioral observations suggest the potential for patient-centered care for infants.

Missense BICD2 variants in fetuses with congenital arthrogryposis and pterygia.

Type 2 spinal muscular atrophy with lower extremity dominance (SMALED2) is caused by bicaudal D cargo adaptor 2 (BICD2) variants. However, the SMALED2 genotype and phenotype correlation have not been thoroughly characterized. We identified de novo heterozygous BICD2 missense variants in two fetuses with severe, prenatally diagnosed multiple arthrogryposis congenita. This report provides further insights into the genetics of this rare disease.

A novel FLNA variant in a fetus with skeletal dysplasia.

Otopalatodigital spectrum disorder (OPDSD) is characterized by variable phenotypes, including skeletal dysplasia, and is caused by pathogenic variants in filamin A-encoding FLNA. FLNA variants associated with lethal OPDSD primarily alter the CH2 subdomain of the ABD of FLNA. Herein, we report a novel FLNA mutation in a fetus with severe skeletal dysplasia in a pregnant multigravida female with a history of repeated miscarriages and terminations.

Pregnancy Outcome and Postnatal Chromosome Analysis of the Cord Blood and Chorionic Villi in Two Cases after Intrauterine Transfer of Mosaic Aneuploid Blastocysts.

The long-term prognosis and genetic mechanism of pregnancy after intrauterine mosaic aneuploid blastocyst transfer remain unknown. We report the case of two babies after the aforementioned procedure and chromosomal analysis of their cord blood and chorionic villi. Case Report 1. A 41-year-old primipara, with two previous spontaneous abortions, was pregnant after intrauterine transfer of a blastocyst carrying 40% mosaicism of long-arm monosomy of chromosome 5. The amniocentesis results were 46,XX. A cesarean section was performed at 39 weeks. The female infant was 3,315 g at birth. Case Report 2. A 44-year-old primipara, with two spontaneous abortions, was pregnant after intrauterine transfer of a blastocyst carrying 40% mosaicism of long-arm monosomy of chromosome 9 and monosomy of chromosome 14. After genetic counselling, she decided not to undergo amniocentesis. No abnormalities were found by ultrasound. A cesarean section was performed at 38 weeks. The male infant was 3,340 g at birth. Chromosome analyses of postnatal cord blood and chorionic villi were performed using SNP arrays. The cord blood and chorionic villi showed no chromosomal structural abnormalities or mosaicism. For both, no disorders were observed at 10 months of age. We experienced the birth of babies after intrauterine transfer of mosaic aneuploid blastocysts.

Glucocorticoids increase the risk of preterm premature rupture of membranes possibly by inducing ITGA8 gene expression in the amnion.

INTRODUCTION: Maternal glucocorticoid exposure increases the risk of preterm delivery; however, the association between glucocorticoids and preterm premature rupture of membranes (pPROM)-a direct cause of preterm delivery-has rarely been investigated. METHODS: To examine this association, we evaluated the clinical data of patients with systemic lupus erythematosus (SLE). Mechanism analysis was performed in both human amnion-derived mesenchymal cells (as a model for fetal membranes) and the amnion from SLE patients. We characterized the effects of glucocorticoids on the amnion in both models through comprehensive gene expression profiling and by electric cell-substrate impedance sensing in the mesenchymal cells. RESULTS: The average glucocorticoid dose in cases with pPROM (13.3 mg/day, n = 10) was significantly higher than in those without pPROM (8.5 mg/day, n = 65; P < 0.01) among pregnant patients with well-controlled SLE (SLEDAI <4, n = 75); however, we did not observe a statistically significant difference in it between cases with or without chorioamnionitis. Glucocorticoid-treated human amnion mesenchymal cells showed decreased electric resistance between cells, indicating increased permeability. Differentially expressed genes upon glucocorticoid treatment were significantly enriched with cell adhesion-related genes. Among them, ITGA8 was strikingly induced in both the amnion mesenchymal cells and in amnion derived from patients with SLE. DISCUSSION: We observed an association between glucocorticoids and pPROM with non-infectious etiology. Our findings indicate that glucocorticoids increase amnion permeability and modulate cell-adhesion related genes. ITGA8 represents a primary molecule that triggers pPROM through fibrotic remodeling and preventing resealing of the rupture site in fetal amnion.

Novel missense COL2A1 variant in a fetus with achondrogenesis type II.

Achondrogenesis type II (ACG2) is a lethal skeletal disorder caused by pathogenic variants in COL2A1. We present a fetus with cystic hygroma and severe shortening of the limbs at 14 weeks of gestation. We performed postnatal genetic analysis of the parents and fetus to diagnose the disease. A novel missense variant of COL2A1 [NM_001844.5: c.2987G>A, (p. Gly996Asp)] was identified, which led to the ACG2 diagnosis.

Collection of 2429 constrained headshots of 277 volunteers for deep learning.

Deep learning has rapidly been filtrating many aspects of human lives. In particular, image recognition by convolutional neural networks has inspired numerous studies in this area. Hardware and software technologies as well as large quantities of data have contributed to the drastic development of the field. However, the application of deep learning is often hindered by the need for big data and the laborious manual annotation thereof. To experience deep learning using the data compiled by us, we collected 2429 constrained headshot images of 277 volunteers. The collection of face photographs is challenging in terms of protecting personal information; we therefore established an online procedure in which both the informed consent and image data could be obtained. We did not collect personal information, but issued agreement numbers to deal with withdrawal requests. Gender and smile labels were manually and subjectively annotated only from the appearances, and final labels were determined by majority among our team members. Rotated, trimmed, resolution-reduced, decolorized, and matrix-formed data were allowed to be publicly released. Moreover, simplified feature vectors for data sciences were released. We performed gender and smile recognition by building convolutional neural networks based on the Inception V3 model with pre-trained ImageNet data to demonstrate the usefulness of our dataset.

Effects of high-intensity interval exercise under hyperoxia on HSP27 and oxidative stress responses.

Hypoxia in working muscles during exercise may be associated with increased oxidative stress. Inhalation of hyperoxic gas diminishes the hypoxia within working muscles during exercise. Exposure to hyperoxia increases the expression of the antioxidant HSP27. We investigated the effects of acute high-intensity interval exercise (HIE) under hyperoxia on HSP27 levels and oxidative stress responses. Eight male subjects participated in two experiments: 1) normoxic HIE (NHIE) and 2) hyperoxic (60 % oxygen) HIE (HHIE). HIE consisted of four 30-s all-out cycling bouts with 4-min rest between bouts. Levels of serum oxidative stress markers (d-ROMs and LPO), HSP27, BAP, IL-6, and TNF-α significantly increased after both trials. The HIE-induced changes in d-ROMs, LPO, and HSP27 levels were significantly lower in the HHIE trial than in the NHIE trial. These findings suggest that inhaling hyperoxic gas during exercise might diminish oxidative stress induced by all-out HIE.

Novel TNFAIP3 microdeletion in a girl with infantile-onset inflammatory bowel disease complicated by a severe perianal lesion.

A20 haploinsufficiency (HA20), a disease caused by loss-of-function TNFAIP3 mutations, manifests various autoinflammatory and/or autoimmune symptoms. Some cases of HA20 were initially diagnosed as very early onset inflammatory bowel disease (VEO-IBD). We performed whole-exome sequencing (WES) for a Japanese girl with infantile-onset IBD and a severe perianal lesion and detected a novel de novo 119 kb microdeletion containing only TNFAIP3 (arr[GRCh37] 6q23.3(138125829_138244816) × 1).

Improved library preparation protocols for amplicon sequencing-based noninvasive fetal genotyping for RHD-positive D antigen-negative alleles.

OBJECTIVE: We aimed to simplify our fetal RHD genotyping protocol by changing the method to attach Illumina's sequencing adaptors to PCR products from the ligation-based method to a PCR-based method, and to improve its reliability and robustness by introducing unique molecular indexes, which allow us to count the numbers of DNA fragments used as PCR templates and to minimize the effects of PCR and sequencing errors. RESULTS: Both of the newly established protocols reduced time and cost compared with our conventional protocol. Removal of PCR duplicates using UMIs reduced the frequencies of erroneously mapped sequences reads likely generated by PCR and sequencing errors. The modified protocols will help us facilitate implementing fetal RHD genotyping for East Asian populations into clinical practice.