Phenotypic and genetic analysis of children with unexplained neurodevelopmental delay and neurodevelopmental comorbidities in a Chinese cohort using trio-based whole-exome sequencing.

BACKGROUND: Trio-based whole-exome sequencing (trio-WES) enables identification of pathogenic variants, including copy-number variants (CNVs), in children with unexplained neurodevelopmental delay (NDD) and neurodevelopmental comorbidities (NDCs), including autism spectrum disorder (ASD), epilepsy, and attention deficit hyperactivity disorder. Further phenotypic and genetic analysis on trio-WES-tested NDD-NDCs cases may help to identify key phenotypic factors related to higher diagnostic yield of using trio-WES and novel risk genes associated with NDCs in clinical settings. METHODS: In this study, we retrospectively performed phenotypic analysis on 163 trio-WES-tested NDD-NDCs children to determine the phenotypic differences between genetically diagnosed and non-genetically diagnosed groups. Additionally, we conducted genetic analysis of ASD genes with the help of Simons Foundation for Autism Research Institute (SFARI) Gene database to identify novel possible ASD-risk genes underlying genetic NDD conditions. RESULTS: Among these 163 patients, pathogenic variants were identified in 82 cases (82/163, 50.3%), including 20 cases with CNVs. By comparing phenotypic variables between genetically diagnosed group (82 cases) and non-genetically diagnosed group (81 cases) with multivariate binary logistic regression analysis, we revealed that NDD-NDCs cases presenting with severe-profound NDD [53/82 vs 17/81, adjusted-OR (95%CI): 4.865 (2.213 - 10.694), adjusted-P < 0.001] or having multiple NDCs [26/82 vs 8/81, adjusted-OR (95%CI): 3.731 (1.399 - 9.950), adjusted-P = 0.009] or accompanying ASD [64/82 vs 35/81, adjusted-OR (95%CI): 3.256 (1.479 - 7.168), adjusted-P = 0.003] and head circumference abnormality [33/82 vs 11/81, adjusted-OR (95%CI): 2.788 (1.148 - 6.774), adjusted-P = 0.024] were more likely to have a genetic diagnosis using trio-WES. Moreover, 37 genes with monogenetic variants were identified in 48 patients genetically diagnosed with NDD-ASD, and 15 dosage-sensitive genes were identified in 16 individuals with NDD-ASD carrying CNVs. Most of those genes had been proven to be ASD-related genes. However, some of them (9 genes) were not proven sufficiently to correlate with ASD. By literature review and constructing protein-protein interaction networks among these 9 candidate ASD-risk genes and 102 established ASD genes obtained from the SFARI Gene database, we identified CUL4B, KCNH1, and PLA2G6 as novel possible ASD-risk genes underlying genetic NDD conditions. CONCLUSIONS: Trio-WES testing is recommended for patients with unexplained NDD-NDCs that have severe-profound NDD or multiple NDCs, particularly those with accompanying ASD and head circumference abnormality, as these independent factors may increase the likelihood of genetic diagnosis using trio-WES. Moreover, NDD patients with pathogenic variants in CUL4B, KCNH1 and PLA2G6 should be aware of potential risks of developing ASD during their disease courses.

Identification of a novel phenotype of external ear deformity related to Coffin-Siris syndrome-9 and literature review.

De novo germline variants of the SRY-related HMG-box 11 gene (SOX11) have been reported to cause Coffin-Siris syndrome-9 (CSS-9), a rare congenital disorder associated with multiple organ malformations, including ear anomalies. Previous clinical and animal studies have found that intragenic pathogenic variant or haploinsufficiency in the SOX11 gene could cause inner ear malformation, but no studies to date have documented the external ear malformation caused by SOX11 deficiency. Here, we reported a Chinese male with unilateral microtia and bilateral sensorineural deafness who showed CSS-like manifestations, including dysmorphic facial features, impaired neurodevelopment, and fingers/toes malformations. Using trio-based whole-exome sequencing, a de novo missense variant in SOX11 (NM_003108.4: c.347A>G, p.Y116C) was identified and classified as pathogenic variant as per American College of Medical Genetics guidelines. Moreover, a systematic search of the literature yielded 12 publications that provided data of 55 SOX11 intragenic variants affecting various protein-coding regions of SOX11 protein. By quantitatively analyzing phenotypic spectrum information related to these 56 SOX11 variants (including our case), we found variants affecting different regions of SOX11 protein (high-mobility group [HMG] domain and non-HMG regions) appear to influence the phenotypic spectrum of organ malformations in CSS-9; variants altering the HMG domain were more likely to cause the widest range of organ anomalies. In summary, this is the first report of CSS with external ear malformation caused by pathogenic variant in SOX11, indicating that the SOX11 gene may be not only essential for the development of the inner ear but also critical for the morphogenesis of the external ear. In addition, thorough clinical examination is recommended for patients who carry pathogenic SOX11 variants that affect the HMG domain, as these variants may cause the widest range of organ anomalies underlying this condition.

An Integrative Pan-Cancer Analysis of the Prognostic and Immunological Role of Casein Kinase 2 Alpha Protein 1 (CSNK2A1) in Human Cancers: A Study Based on Bioinformatics and Immunohistochemical Analysis.

BACKGROUND: Although emerging animal- or cell-based evidence supports the relationship between casein kinase 2 alpha protein 1 (CSNK2A1) and cancers, no pan-cancer analysis is available. Thus, this report aimed to display the prognostic landscape of CSNK2A1 in pan-cancer and investigate the relationship between CSNK2A1 and tumor immunity. METHODS: In the current study, we investigated the expression pattern, genetic alterations and survival analysis of CSNK2A1 in pan-cancer across multiple datasets and online platforms. The correlations between CSNK2A1 expression and tumor immunity were explored and visualized via R language software. Following this, immunohistochemical (IHC) staining and Kaplan-Meier survival analysis were conducted in clinical patients for proving the bioinformatic findings. Analysis of protein-protein interaction and gene functional enrichment was conducted using GeneMANIA platform and gene set enrichment analysis (GSEA), respectively. RESULTS: In TCGA, tumor tissue had a higher expression level of CSNK2A1 compared with that in corresponding normal tissue. An increased expression level of CSNK2A1 was related to poor clinical prognosis in most types of cancer such as LIHC. The following expression and survival analysis in clinical liver hepatocellular carcinoma (LIHC) patients confirmed these TCGA findings. CSNK2A1 expression had significant positive correlations with pro-tumor-infiltrating immune cells (TIICs) like M1-macrophages and fibroblasts, and significant negative correlations with anti-tumor-TIICs like activated CD8+ T cells and NK cells, suggesting specific interactions between CSNK2A1 and certain TIICs subtypes. Furthermore, CSNK2A1 expression had the most significant positive correlations with common markers of immune checkpoint including programmed death ligand-1 (PDL1) in LIHC. These findings were validated by an IHC analysis. GSEA analysis demonstrated that high expression of CSNK2A1 was related to cell signaling pathways and immunity-related activities. CONCLUSION: These findings suggested that CSNK2A1 was not only related to poor clinical prognosis in cancer like LIHC but also a novel immunotherapy-related biomarker in cancers, especially in LIHC, shedding new light on anti-tumor strategy.

Gender roles and behavioral problems in children with 21-hydroxylase deficiency in Southern China.

PURPOSE: To assess the gender roles and behavioral outcomes in children with 21-hydroxylase deficiency (21-OHD) in Southern China. METHODS: A total of 50 individuals with 21-OHD participated in our study, (30 boys and 20 girls), as well as another 19 age-matched non-affected relatives of patients (12 boys and 7 girls). Psychological adjustment was assessed with a preschool activity survey and a Conner parent symptom questionnaire was modified for retrospective reporting. RESULTS: The response rate of the questionnaire in the control group was only 36.5%. All the patients were diagnosed with salt-wasting of 21-OHD. Our study revealed that the masculine score was higher in male patients with 21-OHD than male controls and female patients. Compared with that in the female 21-OHD patient group, the masculine score in the female control group was lower, while comparative masculinization was found in the male controls. Regarding behavioral problems, there was a higher incidence of parent-reported problems among children with 21-OHD than controls, including conduct problems, impulsive hyperactivity, anxiety, and hyperactivity index. CONCLUSION: Parents of 21-OHD patients in Southern China were unwilling to disclose the condition of their children to the society. Masculinization and behavioral problems were prevalent among patients with 21-OHD, which highlighted the importance of psychological and social support for 21-OHD patients and their families.

[Identification of a novel frameshift variant in the SRCAP gene of a child with Floating-Harbor syndrome].

OBJECTIVE: To explore the molecular basis for a child featuring with Floating-Harbor syndrome. METHODS: The 2-year-and-8-month-old child presented with retarded growth and language development. Genomic DNA was extracted from peripheral blood samples from the child and his parents with informed consent and subjected to whole exome sequencing. Suspected variants were verified by Sanger sequencing. Pathogenecity of the variants were predicted by using bioinformatic tools. RESULTS: The child was found to carry a de novo frameshift variant c.7273dupA (p. Thr2425Asnfs*18) in the SRCAP gene. The variant was unreported previously and predicted to be pathogenic by MutationTaster. Analysis using HomoloGene system and MEGA software indicated position 2425 of the SRCAP protein to be highly conserved. Substitution of amino acid (Thr) at this position may cause destruction of three AT-hook domains (Amino acid 2857-2869, 2936-2948 and 3004-3016) and serious damage to the function of SRCAP protein. CONCLUSION: The patient's condition may be attributed to the de novo frameshift variant c.7273dupA (p. Thr2425Asnfs*18) of the SRCAP gene. Above finding can facilitate diagnosis of Floating-Harbor syndrome among Chinese population.

Glycerol's generalized two-dimensional correlation IR/NIR spectroscopy and its principal component analysis.

In this manuscript, the fundamental vibration, the combination vibration and the first overtone vibration of the glycerol hydroxyl were studied by near-infrared and infrared spectroscopy. The composition and variation of hydrogen bond were analyzed by two-dimensional correlation spectroscopy and principal component analysis. The analysis revealed five types of hydroxyl and verified the existence of independent, intramolecular, as well as intermolecular, hydrogen bond hydroxyl. The principal component analysis showed that there were three main forms of glycerol association: the first and second principal components explained the majority of the spectral features, and the third was mainly the independent hydroxyl. The results provided insight into the structure of glycerol and illustrated the potential for using these tools in analyzing bonding in even more complex systems.

Measuring Sharp Waves and Oscillatory Population Activity With the Genetically Encoded Calcium Indicator GCaMP6f.

GCaMP6f is among the most widely used genetically encoded calcium indicators for monitoring neuronal activity. Applications are at both the cellular and population levels. Here, we explore two important and under-explored issues. First, we have tested if GCaMP6f is sensitive enough for the detection of population activity with sparse firing, similar to the sensitivity of the local field potential (LFP). Second, we have tested if GCaMP6f is fast enough for the detection of fast network oscillations critical for the encoding and consolidation of memory. We have focused this study on the activity of the hippocampal network including sharp waves (SWs), carbachol-induced theta oscillations, and interictal-like spikes. We compare simultaneous LFP and optical GCaMP6f fluorescent recordings in Thy1-GCaMP6f mouse hippocampal slices. We observe that SWs produce a clear population GCaMP6f signal above noise with an average magnitude of 0.3% ΔF/F. This population signal is highly correlated with the LFP, albeit with a delay of 40.3 ms (SD 10.8 ms). The population GCaMP6f signal follows the LFP evoked by 20 Hz stimulation with high fidelity, while electrically evoked oscillations up to 40 Hz were detectable with reduced amplitude. GCaMP6f and LFP signals showed a large amplitude discrepancy. The amplitude of GCaMP6f fluorescence increased by a factor of 28.9 (SD 13.5) between spontaneous SWs and carbachol-induced theta bursts, while the LFP amplitude increased by a factor of 2.4 (SD 1.0). Our results suggest that GCaMP6f is a useful tool for applications commonly considered beyond the scope of genetically encoded calcium indicators. In particular, population GCaMP6f signals are sensitive enough for detecting synchronous network events with sparse firing and sub-threshold activity, as well as asynchronous events with only a nominal LFP. In addition, population GCaMP6f signals are fast enough for monitoring theta and beta oscillations (<25 Hz). Faster calcium indicators (e.g., GCaMP7) will further improve the frequency response for the detection of gamma band oscillations. The advantage of population optical over LFP recordings are that they are non-contact and free from stimulation artifacts. These features may be particularly useful for high-throughput recordings and applications sensitive to stimulus artifact, such as monitoring responses during continuous stimulation.

Electroconvulsive Shock Enhances Responsive Motility and Purinergic Currents in Microglia in the Mouse Hippocampus.

Microglia are in a privileged position to both affect and be affected by neuroinflammation, neuronal activity and injury, which are all hallmarks of seizures and the epilepsies. Hippocampal microglia become activated after prolonged, damaging seizures known as status epilepticus (SE). However, since SE causes both hyperactivity and injury of neurons, the mechanisms triggering this activation remain unclear, as does the relevance of the microglial activation to the ensuing epileptogenic processes. In this study, we use electroconvulsive shock (ECS) to study the effect of neuronal hyperactivity without neuronal degeneration on mouse hippocampal microglia. Unlike SE, ECS did not alter hippocampal CA1 microglial density, morphology, or baseline motility. In contrast, both ECS and SE produced a similar increase in ATP-directed microglial process motility in acute slices, and similarly upregulated expression of the chemokine C-C motif chemokine ligand 2 (CCL2). Whole-cell patch-clamp recordings of hippocampal CA1sr microglia showed that ECS enhanced purinergic currents mediated by P2X7 receptors in the absence of changes in passive properties or voltage-gated currents, or changes in receptor expression. This differs from previously described alterations in intrinsic characteristics which coincided with enhanced purinergic currents following SE. These ECS-induced effects point to a "seizure signature" in hippocampal microglia characterized by altered purinergic signaling. These data demonstrate that ictal activity per se can drive alterations in microglial physiology without neuronal injury. These physiological changes, which up until now have been associated with prolonged and damaging seizures, are of added interest as they may be relevant to electroconvulsive therapy (ECT), which remains a gold-standard treatment for depression.

Interleukin-10 inhibits interleukin-1ß production and inflammasome activation of microglia in epileptic seizures.

BACKGROUND: Microglia are important for secreting chemical mediators of inflammatory responses in the central nervous system. Interleukin (IL)-10 and IL-1ß secreted by glial cells support neuronal functions, but the related mechanisms remain vague. Our goal was to demonstrate the efficacy of IL-10 in suppressing IL-1ß and in inflammasome activation in mice with epileptic seizure based on an epileptic-seizure mouse model. METHODS: In this study, mice in which epileptic seizures were induced by administering picrotoxin (PTX) were used as a case group, and mice injected with saline were employed as the control group. The expression of nucleic acids, cytokines, or signaling pathways was detected by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blotting. RESULTS: Our results demonstrated that IL-10 inhibits IL-1ß production through two distinct mechanisms: (1) Treatment with lipopolysaccharides (LPS) results in IL-10 overexpression in microglia and reduced NLRP3 inflammasome activity, thus inhibiting caspase-1-related IL-1ß maturation; (2) next, autocrine IL-10 was found to subsequently promote signal transducer and activator of transcription-3 (STAT-3), reducing amounts of pro-IL-1ß. CONCLUSIONS: Our results indicate that IL-10 is potentially effective in the treatment of inflammation encephalopathy, and suggest the potential usefulness of IL-10 for treating autoimmune or inflammatory ailments.

Establishment of NOD/SCID mouse model of central nervous system leukemia.

In the present study, we successfully established a NOD/SCID mouse model of central nervous system leukemia by injection of acute monocytic leukemia cell line SHI-1 cells into the lateral ventricle. Immunohistochemistry was used to detect human leukocyte common antigen in brain slices. Nested PCR assay was used to detect MLL/AF6 fusion gene expression. After injection, the condition of the mice gradually progressed to cachexia and death (median survival time, 25 days). Leukemic cells were identified in the lung, bone marrow, and lymph node of one mouse. Brain tissue sections showed invasion into the subdural space, pia mater, arachnoid, along the Virchow-Robin space and into the deep brain parenchyma. In summary, a central nervous system leukemia (CNSL) model was established in NOD/SCID mice.

Merosin-deficient congenital muscular dystrophy type 1A: A case report.

The aim of this study was to characterize the clinical and genetic features of a 4-year-old female with merosin-deficient congenital muscular dystrophy type 1A (MDC1A). MDC1A is the most common form of congenital muscular dystrophy. MDC1A is caused by mutation of the laminin α-2 gene (LAMA2), localized to chromosome 6q22-23. Clinical presentation, as well as the results of neuro-imaging, electrophysiology and molecular genetic tests were used to evaluate a patient with MDC1A. The patient exhibited severe hypotonia and marked proximal weakness at 6 months of age, as well as delayed developmental milestones. The serum creatine kinase levels of the patient were elevated at 1,556 IU/l. Magnetic resonance imaging (MRI) showed that the white matter in the frontal, parietal, temporal and occipital lobes was abnormal with low signal intensities on T1-weighted images and high signal intensities on T2-weighted images; however, the cortex was normal. Sequencing of the 65 exons of the LAMA2 revealed a homozygous nonsense mutation in exon 50: a C>T exchange in nucleotide 7147 that resulted in a stop codon (Arg2383X stop). Molecular genetic testing is a reliable method for confirming a diagnosis of MDC1A. When a patient presents with severe congenital hypotonia, muscle weakness, high serum creatine kinase (CK) levels and white matter abnormalities, the evaluation may directly proceed to molecular genetic testing of the LAMA2 gene without performing a muscle biopsy.

Downregulation of miR-383 promotes glioma cell invasion by targeting insulin-like growth factor 1 receptor.

Invasiveness is a major clinical feature of glioma, an aggressive brain tumor with poor prognosis. Although there is emerging evidence that some microRNAs are involved in the glioma cell invasion process, it remains necessary to find functional microRNAs and elucidate the underlying molecular mechanisms. Here, we reported that a microRNA, miR-383, was downregulated in gliomas and inversely correlated with glioma pathological grades. Downregulation of miR-383 enhanced, whereas upregulation of miR-383 inhibited, the glioma cell invasive ability. Furthermore, we found that downregulation of miR-383 activated the AKT signaling following upregulation of MMP2 expression by directly targeting insulin-like growth factor 1 receptor (IGF1R). Importantly, we demonstrated that IGF1R expression is critical for miR-383 downregulation-induced cell invasion. Taken together, these findings uncover a novel regulatory mechanism for constitutive IGF1R signaling activation in glioma cancer and may provide miR-383 as a useful diagnostic marker or therapeutic target.

Human intravenous immunoglobulins suppress seizure activities and inhibit the activation of GFAP-positive astrocytes in the hippocampus of picrotoxin-kindled rats.

We previously showed that human intravenous immunoglobulin (IVIG) can lower seizure severity and prolong seizure latency in picrotoxin-kindled rats. The aim of this study was to further characterize the effects of IVIG on seizure activity and investigate its influence on astrocytes in the hippocampus of picrotoxin-kindled rats. A rat kindling model was established by peritoneal injections of picrotoxin for 21 days in Wistar rats. Seventy-five rats were equally divided into five groups: picrotoxin, IVIG pretreatment, IVIG post-treatment, normal saline control, and IVIG control. Seizure severity was evaluated according to a six-stage classification. The number and morphology of glial fibrillary acidic protein (GFAP)-positive astrocytes were studied by immunohistochemistry using the anti-GFAP antibody. The cross-sectional area and grayscale of GFAP-positive astrocytes were also determined. In picrotoxin-kindled rats, pretreatment with IVIG appeared to inhibit full kindling rates, and it significantly reduced the number of GFAP-positive cells in the hippocampus (p < .001). IVIG also significantly (p < .001) attenuated the increase in the cross-sectional area and grayscale of GFAP-positive astrocytes in the hippocampus. Our results suggest that by suppressing the expression of GFAP, IVIGs may reduce seizure activity and inhibit the activation of GFAP-positive astrocytes in picrotoxin-kindled rats.