Sleep and internalizing problems in primary school children with attention-deficit hyperactivity disorder.

BACKGROUND: Internalizing and externalizing problems have received great attention, and children with ADHD exhibit high rates of comorbid internalizing and externalizing disorders. This study aimed to explore the relationship between sleep and internalizing problems in children with attention-deficit hyperactivity disorder (ADHD) and the probable mediating role of externalizing problems. METHODS: A total of 203 primary school children diagnosed with ADHD for the first time were recruited for this study. Children with ADHD were evaluated by Children's Sleep Habits Questionnaire (CSHQ), Strengths and Difficulties Questionnaire (SDQ). Internalizing problems were represented by emotional symptoms and peer problems of SDQ, and externalizing problems were represented by conduct problems and hyperactivity-inattention problems of SDQ. Multi-step linear regression analysis was used to investigate the mediating effect of externalizing problems on the relationship between sleep and internalizing problems. RESULTS: Sleep in children with ADHD was associated with emotional problems in internalizing problems, and conduct problems in externalizing problems mediated the association between sleep and emotional problems. CONCLUSION: For children with ADHD, when it is difficult to identify internalizing problems, especially emotional problems, we can take sleep and externalizing problems as clues to improve our clinical ability to recognize and deal with emotional problems. IMPACT: 1. We first explored the possible mediating role of conduct problems between sleep and emotional problems in primary school children with ADHD. 2. When it is difficult to identify internalizing problems, especially emotional problems, we can take sleep and externalizing problems as clues to improve our clinical ability to recognize emotional problems for children with ADHD. 3. For children with ADHD with potential internalizing problems, especially emotional problems, interventions for their sleep and externalizing problems may be the possible methods to deal with.

The challenges of screen time in children with typical development and children with developmental disorders during COVID-19 pandemic.

Background: The significant lifestyle changes that occurred during the lockdown period associated with the COVID-19 pandemic may have had many potential adverse effects on children, in particular, sedentary screen exposure among children, including those with developmental disorders. We conducted a cross-sectional study to investigate and compare the screen time and outdoor activity time of children with typically development (TD) and those with developmental disorders during and before the emergence of COVID-19, and identified the risk factors related to screen time during the COVID-19 pandemic. Methods: A total of 496 children were surveyed via online questionnaires. Parents or/and children filled in the online questionnaire, including basic characteristics, screen time, outdoor activity time, and other related factors. The Statistical Product and Service Solutions software was used to analyze all data. Results: Children spent less time outdoors (t=14.774, P<0.001) and more time on electronic screens (t=-14.069, P<0.001) during the lockdown period of COVID-19, compared to the periods before COVID-19. Age (P=0.037), pre-COVID-19 screen time (P=0.005), screen time used for learning/education (P<0.001), screen time of siblings (P=0.007), and use of screen devices as electronic babysitters (P=0.005) were risk factors for screen time during the COVID-19 pandemic, while restrictive use of electronic devices by parents (P<0.05) was a protective factor. The screen time of children with autism spectrum disorder (ASD) or attention deficit hyperactivity disorder (ADHD) was significantly longer than children with TD before COVID-19 pandemic, but there is no statistical difference during the COVID-19 pandemic. Conclusions: During the COVID-19 pandemic, children's screen exposure time increased, and outdoor activities decreased significantly. This represents a significant challenge, and we should focus our efforts on managing children's screen time and promoting healthier lifestyles, including children with typical development, as well as those with developmental disorders.

The association of vitamin A, zinc and copper levels with clinical symptoms in children with autism spectrum disorders in Jilin Province, China.

BACKGROUND: This study evaluated vitamin A (VA), copper (Cu), and zinc (Zn) levels in the population with autism spectrum disorder (ASD) in Jilin Province, China. Furthermore, we examined their links to core symptoms and neurodevelopment, as well as gastrointestinal (GI) comorbidities and sleep disorders. METHODS: This study included 181 children with autism and 205 typically developing (TD) children. The participants had not taken vitamin/mineral supplements in the prior three months. High-performance liquid chromatography was used to measure serum VA levels. By using inductively coupled plasma-mass spectrometry, Zn and Cu concentrations in plasma were determined. Importantly, the Childhood Autism Rating Scale, the Social Responsiveness Scale, and the Autism Behavior Checklist were used to measure core ASD symptoms. However, the Griffith Mental Development Scales-Chinese were used to measure neurodevelopment. GI comorbidities and sleep abnormalities were assessed with the 6 Item-Gastrointestinal Severity Index and Children's Sleep Habits Questionnaire, respectively. Children with ASD with GI issues were grouped according to severity (low GI severity and high GI severity groups). RESULTS: (i) The difference in VA, Zn, Cu levels and the Zn/Cu ratio between ASD and TD children is small. But children with ASD had lower VA levels and Zn/Cu ratio, higher Cu levels than TD children. Cu levels in children with ASD were associated with the severity of core symptoms. (ii) Children with ASD were much more likely than their TD counterparts to suffer from GI comorbidities or sleep problems. Furthermore, it was observed that high GI severity was associated with lower levels of VA, whereas low GI severity was associated with higher levels of VA. (iii) The children with ASD who had both lower VA and lower Zn/Cu ratio had more severe scores on the Autism Behavior Checklist, but not on other measures. CONCLUSION: Children with ASD had lower VA and Zn/Cu ratio, and higher Cu levels. Cu levels in children with ASD were weakly correlated with one subscale on social or self-help. ASD children with lower VA levels may face more serious GI comorbidities. Children with ASD combined VA-Zn/Cu lower had more severe core symptoms. TRIAL REGISTRATION: Registration number: ChiCTR-OPC-17013502. Date of registration: 2017-11-23.

Risk factors for developmental quotients in ASD children: A cross-sectional study.

Objective: To analyze the risk factors for developmental quotients (DQs) of children with autism spectrum disorder (ASD) and to better understand the effects of screen time on neurodevelopment in children with ASD. Methods: We retrospectively analyzed the data of 382 children with ASD, including demographic profiles; socioeconomic status; score on the Chinese parent-child interaction scale (CPCIS); screen time questionnaire; ASD symptom rating scales, including the Autism Behavior Checklist (ABC), Childhood Autism Rating Scale (CARS), and Autism Diagnostic Observation Schedule Second Edition (ADOS-2); and DQs using Griffiths Development Scales-Chinese Edition. Univariate analysis was carried out to analyze the factors related to the DQs of children with ASD, and then the linear regression model was used to identify the independent influencing factors of the DQs of children with ASD. Results: Vitamin D (ß = 0.180, p = 0.002), age (ß = -0.283, p = 0.000) and CARS score (ß = -0.347, p = 0.000) are risk factors related to DQ of locomotor in children with ASD. Vitamin D (ß = 0.108, p = 0.034), CARS score (ß = -0.503, p = 0.000), ADOS-2 severity score (ß = -0.109, p = 0.045) and CPCIS score (ß = 0.198, p = 0.000) are risk factors related to DQ of personal social skill in children with ASD. Vitamin D (ß = 0.130, p = 0.018), CARS score (ß = -0.469, p = 0.000), and CPCIS score (ß = 0.133, p = 0.022) are risk factors related to DQ of hearing-speech in children with ASD. Vitamin D (ß = 0.163, p = 0.003) and CARS score (ß = -0.471, p = 0.000) are risk factors related to DQ of eye-hand coordination in children with ASD. Age (ß = -0.140, p = 0.020), CARS score (ß = -0.342, p = 0.000), ADOS-2 severity score (ß = -0.133, p = 0.034) and CPCIS score (ß = 0.193, p = 0.002) are risk factors related to DQ of performance in children with ASD. Vitamin D (ß = 0.801, p = 0.000) and CPCIS score (ß = 0.394, p = 0.019) are risk factors related to DQ of practical reasoning in children with ASD. Conclusion: Vitamin D status, the severity of autistic symptoms and parent-child interaction are risk factors for developmental quotients in children with ASD. Screen exposure time is negatively associated with DQs in children with ASD but is not an independent risk factor for DQs.

Novel de novo ZNF148 truncating variant causing autism spectrum disorder, attention deficit hyperactivity disorder, and intellectual disability.

ZNF148 gene is a Krüppel-type transcription factor that has transcriptional regulatory function. Heterozygous variant in ZNF148 gene causes an intellectual disability syndrome characterized by global developmental delay, absence, or hypoplasia of corpus callosum, wide intracerebral ventricles, and dysmorphic facial features, while its associations with ASD and ADHD have not been reported. We report a new patient with intellectual disability, autism spectrum disorder (ASD) and attention-deficit hyperactivity disorder (ADHD). The patient had a novel heterozygous truncating variant c.1818dupC (p.Lys607Glnfs*11) in the ZNF148 gene. This variation produces a ZNF148 truncated protein with a deletion of the C-terminal activation domain and may destabilize the protein by affecting the transcriptional activation function. Brain MRI shows normal brain development. Here, we identify a novel ZNF148 heterozygous truncating variant in a patient with distinct phenotypes of ASD and ADHD, which expands the genotype-phenotype spectrum of ZNF148, and indicates ZNF148 is also a potential target gene for ASD.

Analysis of scores of Symptom Checklist 90 (SCL-90) questionnaire of 182 parents of children with spinal muscular atrophy: a cross-sectional study.

Background: Spinal muscular atrophy (SMA) is a hereditary disorder characterized by progressive muscle weakness and atrophy in children. However, less attention is paid to psychiatric symptoms of SMA parents. Attention to the psychiatric symptoms of parents of SMA children can improve the comprehensiveness of family support for SMA children, which is beneficial to the rehabilitation of SMA children. Here, we conducted a survey on the psychiatric symptoms of SMA parents and analyzed its relevant factors, with an attempt to inform the psychological support for SMA parents. Methods: The Symptom Checklist 90 (SCL-90) and a self-designed basic information (such as parent's gender, household area, place of residence, education background, etc.) questionnaire (in electronic questionnaire) were distributed to parents of SMA children aged 0-18 in a charity WeChat group sponsored by the Meier Advocacy & Support Center for SMA during the period from August 1 to August 31, 2021. Parents completed the electronic questionnaires by mobile phone or computer voluntarily. A total of 188 questionnaires were obtained, of which 182 were valid. Comparisons were performed with the SCL-90 adult norms as the standards. The potential correlations between the general data of SMA parents and children and abnormal factors in the SCL-90 for SMA parents were analyzed. Results: The SCL-90 factors somatization (1.56±0.80, P=0.002), depression (1.78±0.98, P<0.001), anxiety (1.58±0.87, P=0.007), fear (1.39±0.74, P=0.003), and sleep and eating problems (1.67±0.87, P=0.014) of SMA parents were significantly higher than the national norms. Place of residence was correlated with sleep and eating problems (r=0.158, P=0.033). Increasing age [odds ratio (OR) =1.012, P=0.014] and continuous home-living status (OR =0.360, P=0.031) of SMA children increased the risk of depression in their parents, and the lack of rehabilitation management in SMA children increased the risk of anxiety of their parents (OR =0.409, P=0.038). Non-urban residence (OR =2.602, P=0.017) and poor physical health (OR =0.163, P=0.031) were the relevant factors for the increased risk of sleep and eating problems in SMA parents. Conclusions: SMA parents have a higher risk of developing psychiatric symptoms problems compared with the general population. Increasing age and the continuous home-living status of SMA children increase the risk of depression in their parents, and the lack of rehabilitation management increase the risk of anxiety in SMA parents.

COVID-19 and mental health disorders in children and adolescents (Review).

The new coronavirus has been present for two years and has had a widespread and sustained impact worldwide. There is growing evidence in the literature that COVID-19 may have negative effects on mental illness in patients and in healthy populations. The unprecedented changes brought about by COVID-19, such as social isolation, school closures, and family stress, negatively affect people's mental health, especially that of children and adolescents. The purpose of this paper is to review the literature and summarize the impact of COVID-19 disorders on children's and adolescents' mental health, the mechanisms and risk factors, screening tools, and intervention and prevention. We hope that the mental dysfunction caused by the pandemic will be mitigated through appropriate and timely prevention and intervention.

Spatial barcoding-enabled highly multiplexed immunoassay with digital microfluidics.

Digital microfluidics (DMF), facilitating independent manipulation of microliter samples, provides an ideal platform for immunoassay detection; however, suffering limited multiplexity. To address the need, herein we described a digital microfluidics (DMF) platform that realizes spatial barcoding on the Teflon-coated indium tin oxide (ITO) glass side to fulfill highly multiplexed immunoassay (10+) with low-volume samples (∼4 µL) in parallel, representing the highest multiplexing recorded to date for DMF-actuated immunoassay. Planar-based spatial immobilization of multiple capture antibodies was realized on a Teflon-coated ITO glass side, which was then used as the top plate of the DMF device. Droplets containing analytes, secondary antibodies, and fluorescent signaling reporters with low volume, which were electrically manipulated by our DMF control system, were shuttled sequentially along the working electrodes to complete the immuno-reaction. Evaluation of platform performance with recombinant proteins showed excellent sensitivity and reproducibility. To test the feasibility of our platform in analyzing multiplex biomarkers of the immune response, we used lipopolysaccharide-stimulated macrophages as a model system for protein secretion dynamics studies. As a result, temporal profiling of pro-inflammatory cytokine secretion dynamics was obtained. The spatial barcoding strategy presented here is easy-to-operate to enable a more comprehensive evaluation of protein abundance from biological samples, paving the way for new opportunities to realize multiplexity-associated applications with the DMF platform.

Treatment Effect of Bumetanide in Children With Autism Spectrum Disorder: A Systematic Review and Meta-Analysis.

Background: The therapeutic effect of bumetanide on autism spectrum disorder (ASD) seems to be controversial. To obtain better evidence on the efficacy of bumetanide, a systematic review and meta-analysis were performed. Methods: Randomized, placebo-controlled trials (RCTs) of bumetanide treatment in children with ASD were identified through systematic review from database inception to January 17, 2021. Subsequently, a meta-analysis was carried out to examine the effect of bumetanide on the severity of symptoms of ASD as assessed by the Childhood Autism Rating Scale (CARS) and Social Responsive Scale (SRS); core symptoms according to criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM)-5 of the American Psychiatric Association [social affect (SA), restricted, repetitive patterns of behavior, interests, or activities (RRB) and sensory symptoms]; and the therapeutic effect as assessed by Clinical Global Impressions-Efficacy (CGI-E). Results: In total, six RCTs involving 496 participants with ASD were identified in our study. The results showed that bumetanide could significantly improve the severity of the ASD symptoms measured by CARS and SRS. There was also evidence that bumetanide had positive effect on the core symptoms of ASD such as the SA and RRB, but there was no statistically significant effect on sensory symptoms. A significant positive effect on CGI-E scores in ASD patients was also observed. Conclusion: Our meta-analysis provided some support that bumetanide could improve the symptoms of children with ASD. However, additional large-scale longitudinal studies that provide clearer information and better control for confounding factors are needed to confirm our findings.

Rapid prototyping of PDMS microdevices viaµPLAT on nonplanar surfaces with flexible hollow-out mask.

A major goal of polydimethylsiloxane (PDMS) microfabrication is to develop a simple and inexpensive method for rapid fabrication. Despite the recent advancements in this field, facile PDMS microfabrication on non-planar surfaces remains elusive. Here we report a facile method for rapid prototyping of PDMS microdevices viaµPLAT (microscale plasma-activated templating) on non-planar surfaces through micropatterning of hydrophilic/hydrophobic (HL/HB) interface by flexible polyvinyl chloride (PVC) hollow-out mask. This mask can be easily prepared with flexible PVC film through a cutting crafter and applied as pattern definer during the plasma treatment for microscale HL/HB interface formation on different substrates. The whole process requires low inputs in terms of time as well as toxic chemicals. Inspired by liquid molding, we demonstrated its use for rapid prototyping of PDMS microstructures. Following the proof-of-concept study, we also demonstrated the use of the flexible hollow-out mask to facilitate cell patterning on curved substrates, which is difficult to realize with conventional methods. Collectively, our work utilizes flexible and foldable PVC film as mask materials for facile microscale HL non-planar surface modification to establish a useful tool for PDMS prototyping and cell patterning.

Fluorescent paper-based sensor based on carbon dots for detection of folic acid.

Paper-based devices have been very much in the foreground of analytical science recently. This work innovatively proposed a fluorescent paper-based sensor (FPS) constructed on a hybrid polydimethylsiloxane (PDMS)/paper platform where cellulose papers functionalized with carbon dots (CDs) as fluorophores by Schiff base chemistry were loaded on the grooves array of a designed PDMS plate. As a proof of concept, the performance of FPS was investigated with folic acid (FA) as the target analyte. Under optimal conditions, FPS enabled a rapid fluorescence quenching response to FA via inner filter effect in a wide range of 1-300 µmol L-1 with the limit of detection of 0.28 µmol L-1. The feasibility of FPS was further verified by the detection of FA in orange juice and urine samples with satisfactory results. The covalent modification of CDs on paper endowed the FPS with good assay reproducibility and stability. Interestingly, FPS achieved a more sensitive assay of FA than the conventional strategy, by which the same CDs were directly used to detect FA in a solution-based system. The FPS illuminated a novel strategy for construction of reliable and sensitive assays based on paper-based devices. It is of paramount importance for its practical application in biosensing and clinical diagnosis. Graphical abstract.

Paper Microfluidics for Cell Analysis.

Paper microfluidics has attracted much attention since its first introduction around one decade ago due to the merits such as low cost, ease of fabrication and operation, portability, and facile integration with other devices. The dominant application for paper microfluidics still lies in point-of-care testing (POCT), which holds great promise to provide diagnostic tools to meet the ASSURED criteria. With micro/nanostructures inside, paper substrates provide a natural 3D scaffold to mimic native cellular microenvironments and create excellent biointerfaces for cell analysis applications, such as long-term 3D cell culture, cell capture/phenotyping, and cell-related biochemical analysis (small molecules, protein DNA, etc.). This review summarizes cell-related applications based on various engineered paper microdevices and provides some perspectives for paper microfluidics-based cell analysis.

Paper-Based 3D Scaffold for Multiplexed Single Cell Secretomic Analysis.

Despite rapid progresses in single-cell analysis technologies, efforts to control the three-dimensional microenvironment for single cell measurements have been lacking. Here, we report a simple method to incorporate three-dimensional scaffolds, including polyvinylidene fluoride (PVDF) membranes and PVDF membrane replicated analog polydimethylsiloxane, into multiplexed single cell secretomic analysis platforms (including a microwell array and a single cell barcode microchip) to mimic the extracellular physical matrix and mechanical support for single cells. Applying this platform to brain tumor cell line U87 to investigate single cell protein secretion behavior on different substrates, we revealed that single cell protein secretions were regulated differently in three-dimensional (3D) microenvironments. This finding was further verified with intracellular cytokine staining, highlighting the significance of 3D single cell microenvironments. This new single cell biomimetic platform can be easily adaptable to other three-dimensional cell culture scaffolds or other single cell assays and may become a broadly applicable three-dimensional single cell analysis system to study the effect of microenvironment conditions on cellular functional heterogeneity in vitro.

RET somatic mutations are underrecognized in Hirschsprung disease.

PURPOSE: We aimed to determine the frequency of RET mosaicism in Hirschsprung disease (HSCR), test whether it has been underestimated, and to assess its contribution to HSCR risk. METHODS: Targeted exome sequencing (n = 83) and RET single-gene screening (n = 69) were performed. Amplicon-based deep sequencing was applied on multiple tissue samples. TA cloning and sequencing were conducted for validation. RESULTS: We identified eight de novo mutations in 152 patients (5.2%), of which six were pathogenic mosaic mutations. Two of these patients were somatic mosaics, with mutations detected in blood, colon, and saliva (mutant allele frequency: 35-44%). In addition, germ-line mosaicism was identified in four clinically unaffected subjects, each with an affected child, in multiple tissues (mutant allele frequency: 1-28%). CONCLUSION: Somatic mutations of the RET gene are underrecognized in HSCR. Molecular investigation of the parents of patients with seemingly sporadic mutations is essential to determine recurrence risk in these families.

Bench to bedside review: Possible role of vitamin D in autism spectrum disorder.

Autism spectrum disorder (ASD) is a group of dysfunctions in social interaction, communication, and behaviors. Studies have demonstrated that vitamin D deficiency during pregnancy and in individuals increased the risk of ASD. A genetic polymorphism study has pinpointed that genotype AA/A-allele of GC rs4588 in children is associated with ASD, which encodes the vitamin D binding protein. Translating the mentioned points into clinical practice, several clinical trials have demonstrated that vitamin D supplementation can improve the core symptoms in children with ASD. One paper also showed that possible prophylactic effect for the reoccurrence of ASD by vitamin D supplement during pregnancy and early childhood. Herein, this review aims to address the recent advances in this field and to clarify the possible role of vitamin D in ASD.

Carboxylated single-walled carbon nanotube-functionalized chiral polymer monoliths for affinity capillary electrochromatography.

Carboxylated single-walled carbon nanotubes (c-SWNTs) were incorporated into poly(glycidylmethacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-co-EDMA)] monoliths to develop a novel monolithic stationary phase for capillary electrochromatography. The prepared monoliths were characterized by scanning electron microscopy and nitrogen adsorption. Additionally, pepsin, which is a chiral selector, was bonded to the c-SWNT-incorporated monoliths via epoxide groups as reactive sites and glutaraldehyde as the spacer. The effects of the c-SWNT concentration on chiral separation were investigated, and the results suggested that the c-SWNTs played a significant role in improving the separation efficiency, although pepsin was the dominant element in determining the chiral recognition ability of the monolith. Moreover, the influences of buffer pH, operating voltage and sample volume were also studied with (±)-nefopam as a model drug. Under the optimized conditions, the pepsin-modified poly(GMA-c-SWNTs-EDMA) monolith exhibited excellent enantioseparation performance for ten pairs of basic chiral drugs and extended the scope of chiral separation of drug enantiomers.

Mutations in the Motile Cilia Gene DNAAF1 Are Associated with Neural Tube Defects in Humans.

Neural tube defects (NTDs) are severe malformations of the central nervous system caused by complex genetic and environmental factors. Among genes involved in NTD, cilia-related genes have been well defined and found to be essential for the completion of neural tube closure (NTC). We have carried out next-generation sequencing on target genes in 373 NTDs and 222 healthy controls, and discovered eight disease-specific rare mutations in cilia-related gene DNAAF1 DNAAF1 plays a central role in cytoplasmic preassembly of distinct dynein-arm complexes, and is expressed in some key tissues involved in neural system development, such as neural tube, floor plate, embryonic node, and brain ependyma epithelial cells in zebrafish and mouse. Therefore, we evaluated the expression and functions of mutations in DNAAF1 in transfected cells to analyze the potential correlation of these mutants to NTDs in humans. One rare frameshift mutation (p.Gln341Argfs*10) resulted in significantly diminished DNAAF1 protein expression, compared to the wild type. Another mutation, p.Lys231Gln, disrupted cytoplasmic preassembly of the dynein-arm complexes in cellular assay. Furthermore, results from NanoString assay on mRNA from NTD samples indicated that DNAAF1 mutants altered the expression level of NTC-related genes. Altogether, these findings suggest that the rare mutations in DNAAF1 may contribute to the susceptibility for NTDs in humans.

Ectopic cross-talk between thyroid and retinoic acid signaling: A possible etiology for spinal neural tube defects.

Previous studies have highlighted the connections between neural tube defects (NTDs) and both thyroid hormones (TH) and vitamin A. However, whether the two hormonal signaling pathways interact in NTDs has remained unclear. We measured the expression levels of TH signaling genes in human fetuses with spinal NTDs associated with maternal hyperthyroidism as well as levels of retinoic acid (RA) signaling genes in mouse fetuses exposed to an overdose of RA using NanoString or real-time PCR on spinal cord tissues. Interactions between the two signaling pathways were detected by ChIP assays. The data revealed attenuated DIO2/DIO3 switching in fetuses with NTDs born to hyperthyroid mothers. The promoters of the RA signaling genes CRABP1 and RARB were ectopically occupied by increased RXRG and RXRB but displayed decreased levels of inhibitory histone modifications, suggesting that elevated TH signaling abnormally stimulates RA signaling genes. Conversely, in the mouse model, the observed decrease in Dio3 expression could be explained by increased levels of inhibitory histone modifications in the Dio3 promoter region, suggesting that overactive RA signaling may ectopically derepress TH signaling. This study thus raises in vivo a possible abnormal cross-promotion between two different hormonal signals through their common RXRs and the subsequent recruitment of histone modifications, prompting further investigation into their involvement in the etiology of spinal NTDs.

Genotyping analysis of 3 RET polymorphisms demonstrates low somatic mutation rate in Chinese Hirschsprung disease patients.

BACKGROUND: Genetic mosaicism has been reported for both coding and non-coding sequences in the RET gene in Hirschsprung disease (HSCR) patients. This study aimed to investigate somatic mutation rate in Chinese population by comparing both homozygous genotype percentage and risk allele frequency of 3 RET single nucleotide polymorphisms (SNPs) among blood and colon samples. METHODS: DNA was extracted from 59 HSCR blood samples, 59 control blood samples and 76 fresh frozen colon tissue samples (grouped into ganglionic, transitional and aganglionic level). Genotype status of rs2435357 and rs2506030 was examined by competitive allele specific hydrolysis probes (Taqman) PCR technology, and rs2506004 was examined by Sanger sequencing. Homozygous genotype percentage and risk allele frequency were calculated for each type of sample and compared by chi-square test. P<0.05 was regarded as being statistically significant. RESULTS: Colon tissue DNA samples showed similar frequency of SNPs as that of the blood DNA samples in HSCR patients, both of which are significantly higher than the control blood group (rs2435357 TT genotype: 71.2%, 74.7% versus 22.0% in HSCR blood, HSCR colon and control blood DNA respectively, P=0.000; rs2506004 AA genotype: 72.4%, 83.1% versus 25.5%, P=0.000; rs2506030 GG genotype: 79.7%, 77.2% versus 54.2%, P=0.000 and 0.004). With respect to DNA extracted from ganglionic, transitional and aganglionic levels, no statistically significant difference was demonstrated in those 3 regions (rs2435357: P=0.897; rs2506004: P=0.740; rs2506030: P=0.901). CONCLUSION: Our data does not support the notion that high frequency of somatic changes as an underlying etiology of Chinese HSCR population.

Different epigenetic alterations are associated with abnormal IGF2/Igf2 upregulation in neural tube defects.

The methylation status of DNA methylation regions (DMRs) of the imprinted gene IGF2/Igf2 is associated with neural tube defects (NTDs), which are caused by a failure of the neural tube to fold and close and are the second-most common birth defect; however, the characterization of the expression level of IGF2/Igf2 in neural tissue from human fetuses affected with NTDs remains elusive. More importantly, whether abnormal chromatin structure also influences IGF2/Igf2 expression in NTDs is unclear. Here, we investigated the transcriptional activity of IGF2/Igf2 in normal and NTD spinal cord tissues, the methylation status of different DMRs, and the chromatin structure of the promoter. Our data indicated that in NTD samples from both human fetuses and retinoic acid (RA)-treated mouse fetuses, the expression level of IGF2/Igf2 was upregulated 6.41-fold and 1.84-fold, respectively, compared to controls. H19 DMR1, but not IGF2 DMR0, was hypermethylated in human NTD samples. In NTD mice, h19 DMR1 was stable, whereas the chromatin structure around the promoter of Igf2 might be loosened, which was displayed by higher H3K4 acetylation and lower H3K27 trimethylation. Therefore, the data revealed that IGF2/Igf2 expression can be ectopically up-regulated by dual epigenetic factors in NTDs. In detail, the upregulation of IGF2/Igf2 is likely controlled by hypermethylation of H19 DMR1 in human NTDs, however, in acute external RA-induced NTD mice it is potentially determined by more open chromatin structure.