ASXL3 gene mutations inhibit cell proliferation and promote cell apoptosis in mouse cardiomyocytes by upregulating lncRNA NONMMUT063967.2.

Congenital heart disease (CHD) is a serious condition with unknown etiology. In a recent study, a compound heterozygous mutation (c.3526C > T [p.Arg1176Trp] and c.4643A > G [p.Asp1548Gly]) in the ASXL3 gene was identified, which is associated with CHD. This mutation was overexpressed in HL-1 mouse cardiomyocyte cells, leading to increased cell apoptosis and decreased cell proliferation. However, whether this effect is mediated by long noncoding RNAs (lncRNAs) is yet to be determined. We identified the differences among lncRNA and mRNA profiles in mouse heart tissues using sequencing to explore this issue. We detected HL-1 cell proliferation and apoptosis through CCK8 and flow cytometry. Fgfr2, lncRNA, and Ras/ERK signaling pathway expressions were evaluated using quantitative real time polymerase chain reaction (qRT-PCR) and western blot (WB) assays. We also conducted functional investigations by silencing lncRNA NONMMUT063967.2. The sequencing revealed significant changes in lncRNA and mRNA profiles, with the expression of lncRNA NONMMUT063967.2 being significantly promoted in the ASXL3 gene mutations group (MT) while the expression of Fgfr2 being downregulated. The in vitro experiments showed that ASXL3 gene mutations inhibited the proliferation of cardiomyocytes and accelerated cell apoptosis by promoting the expression of lncRNAs (NONMMUT063967.2, NONMMUT063918.2, and NONMMUT063891.2), suppressing the formation of FGFR2 transcripts, and inhibiting the Ras/ERK signaling pathway. The decrease in FGFR2 had the same effect on the Ras/ERK signaling pathway, proliferation, and apoptosis in mouse cardiomyocytes as ASXL3 mutations. Further mechanistic studies revealed that suppression of lncRNA NONMMUT063967.2 and overexpression of FGFR2 reversed the effects of the ASXL3 mutations on the Ras/ERK signaling pathway, proliferation, and apoptosis in mouse cardiomyocytes. Therefore, ASXL3 mutation decreases FGFR2 expression by upregulating lncRNA NONMMUT063967.2, inhibiting cell proliferation and promoting cell apoptosis in mouse cardiomyocytes.

Ndufa4 Regulates the Proliferation and Apoptosis of Neurons via miR-145a-5p/Homer1/Ccnd2.

The Dandy-Walker malformation (DWM) is characterized by neuron dysregulation in embryonic development; however, the regulatory mechanisms associated with it are unclear. This study aimed to investigate the role of NADH dehydrogenase 1 alpha subcomplex 4 (NDUFA4) in regulating downstream signaling cascades and neuronal proliferation and apoptosis. Ndufa4 overexpression promoted the proliferation of neurons and inhibited their apoptosis in vitro, which underwent reverse regulation by the Ndufa4 short hairpin RNAs. Ndufa4-knockout (KO) mice showed abnormal histological alterations in the brain tissue, in addition to impaired spatial learning capacity and exploratory activity. Ndufa4 depletion altered the microRNA expressional profiles of the cerebellum: Ndufa4 inhibited miR-145a-5p expression both in the cerebellum and neurons. miR-145a-5p inhibited the proliferation of neurons and promoted their apoptosis. Ndufa4 promoted and miR-145a-5p inhibited the expression of human homer protein homolog 1 and cyclin D2 in neurons. Thus, Ndufa4 promotes the proliferation of neurons and inhibits their apoptosis by inhibiting miR-145a-5p, which directly targets and inhibits the untranslated regions of Homer1 and Ccnd2 expression.

Identification of Alternative Splicing and LncRNA Genes in Brain Tissues of Fetal Mice at Different Developmental Stages.

BACKGROUND: Brain development is an extremely complex and precisely regulated process, with about one-third of genes expressed and precisely regulated during brain development. OBJECTIVE: This study aims to explore the molecular mechanisms involved in brain development. METHODS: We first established the expression profile of long non-coding RNAs (lncRNAs) and mRNAs in brain tissues of fetal mice at 12.5d, 14.5d and 16.5d through high-throughput sequencing. Second, the associated functions, pathways, and networks of the co-differentially expressed lncRNAs and mRNAs were identified via Gene Ontology (GO), pathway analysis, and PPI network. After bioinformatic analysis and screening, 8 differentially expressed lncRNAs and mRNAs with the same genetic origin were verified by RT-qPCR analysis in brain tissues of fetal mice at different developmental stages. RESULTS: The data revealed that there were 972 co-differentially expressed lncRNAs and 992 codifferentially expressed mRNAs in brain tissues of fetal mice at 12.5d, 14.5d and 16.5d. And we discovered 125 differentially expressed lncRNAs and mRNAs, which have the same genetic origin, in brain tissues of fetal mice at 12.5d, 14.5d and 16.5d through sequencing results and bioinformatics analysis. Besides, we proved that 8 lncRNAs, which have had the same genetic origin as differentially expressed mRNAs, were prominently downregulated, while their maternal genes were upregulated during brain development in fetal mice. CONCLUSION: Our results preliminarily illustrated the differentially expressed lncRNAs and mRNAs, both of which were derived from the same parent genes, during brain development in fetal mice, which suggests that alternative splicing of lncRNA exists during brain development. Besides, our study provides a perspective on critical genes for brain development, which might be the underlying therapeutic targets for developmental brain diseases in children.

Identification of differential microRNAs and messenger RNAs resulting from ASXL transcriptional regulator 3 knockdown during during heart development.

Congenital heart disease (CHD) is the most common birth defect. Although ASXL transcriptional regulator 3 (ASXL3) has been reported to cause hereditary CHD, ASXL3-mediated mechanisms in heart development remain unclear. In this study, we used dimethyl sulfoxide (DMSO) to induce differentiation in P19 cells, observed cell morphology using light microscopy after ASXL3 knockdown, and determined the levels of associated myocardial cell markers using reverse transcription-quantitative polymerase chain reaction and western blotting. Subsequently, we used microRNA sequencing, messenger RNA (mRNA) sequencing, and bioinformatics to initially identify the possible mechanisms through which ASXL3-related microRNAs and mRNAs affect heart development. The results indicated that DMSO induced P19 cell differentiation, which could be inhibited by ASXL3 knockdown. We screened 1214 and 1652 differentially expressed microRNAs and mRNAs, respectively, through ASXL3 knockdown and sequencing; these differentially expressed miRNAs were largely enriched in PI3K-Akt, mitogen-activated protein kinase, and Rap1 signaling pathways. Additionally, 11 miRNAs associated with heart development were selected through a literature review. Our analysis indicated the involvement of mmu-miR-323-3p in P19 cell differentiation through the PI3K-Akt pathway. In conclusion, ASXL3 may be involved in the regulation of heart development. This comprehensive study of differentially expressed microRNAs and mRNAs through ASXL3 knockdown in P19 cells provides new insights that may aid the prevention and treatment of CHD.

Prenatal exome sequencing in fetuses with callosal anomalies.

OBJECTIVE: We aimed to investigate the value of exome sequencing (ES) in fetuses with callosal anomalies (CA) with or without other structural anomalies, but with normal findings by karyotyping and chromosome microarray analysis (CMA). METHODS: Cases with CA with or without other structural anomalies were screened for eligibility. Fetuses with abnormal karyotyping or CMA results were excluded. We performed ES on DNA samples from eligible fetus-parental trios and identified diagnostic genetic variants based on the ultrasonographic features. RESULTS: A total of 50 eligible fetus-parental trios were successfully analyzed by ES. We found 17 likely pathogenic or pathogenic variants in 14 genes from 17 fetuses, with a total proportion of diagnostic genetic variants equal to 34.0% (17/50). Of the 17 cases with a diagnosis, 10 (29.4%, 10/35) were isolated and 7 (43.8%, 7/15) were non-isolated. Pregnancy outcome data showed that 70.0% (7/10) of the surviving isolated CA fetuses with negative ES results had a good prognosis in early childhood. CONCLUSIONS: Our study used ES prenatally for CA and showed that ES can be used diagnostically to define the molecular defects that underlie unexplained CA. Most subjects with isolated CA with negative results for genetic causes will have a favorable prognosis in early childhood.

Compound heterozygous mutation of the ASXL3 gene causes autosomal recessive congenital heart disease.

To explore mutations in the additional sex combs-like 3 (ASXL3) gene in two Chinese families with congenital heart disease (CHD). Whole-exome sequencing (WES) was used to reveal a novel compound heterozygous mutation in the ASXL3 gene that was associated with CHD. Sanger sequencing of a further 122 CHD patients was used to determine an additional compound heterozygous mutation in the ASXL3 gene. Cell apoptosis was examined by MTS assay and flow cytometry. The cardiac structure was identified via hematoxylin-eosin (HE), Masson's trichrome, and ultrasound scanning. RNA sequencing was performed to identify a series of differentially expressed mRNAs. The mRNA and protein expressions were identified by quantitative real-time PCR and western blotting, respectively. A compound heterozygous mutation c.2168C > G (p.Pro723Arg) and c.5449C > G (p.Pro1817Ala) in the ASXL3 gene associated with CHD was identified. Overexpression of this compound heterozygous mutation in HL-1 cells resulted in increased apoptosis and reduced cell viability. Moreover, it affected cardiac structure and fibrosis in mice. There were 126 downregulated mRNAs and 117 upregulated mRNAs between the ASXL3 compound heterozygous mutation c.2168C > G (p.Pro723Arg) and c.5449C > G (p.Pro1817Ala) mice and wild-type mice. Ezh2, Slc6a4, and Socs3, which could interact with ASXL3 through proteins, were all upregulated. Another compound heterozygous mutation c.3526C > T (p.Arg1176Trp) and c.4643A > G (p.Asp1548Gly) in the ASXL3 gene was identified by screening a further 122 patients with CHD. The ASXL3 gene is important in cardiac development and may exert this influence by affecting the expression of mRNAs associated with cell apoptosis and cell proliferation.

Whole-exome sequencing in the evaluation of fetal congenital anomalies of the kidney and urinary tract detected by ultrasonography.

OBJECTIVE: We aimed to investigate the value of whole-exome sequencing (WES) in fetuses with congenital anomalies of the kidney and urinary tract (CAKUT) with or without other structural anomalies but with normal findings upon karyotyping and chromosome microarray analysis (CMA). METHODS: Cases with CAKUT with or without other structural anomalies were screened for eligibility. Fetuses with abnormal karyotyping or CMA results were excluded. We performed WES on DNA samples from eligible fetus-parental trios and identified diagnostic genetic variants based on ultrasonographic features. RESULTS: A total of 163 eligible fetus-parental trios were successfully analyzed by WES. We found 26 likely pathogenic or pathogenic variants in 18 genes from 20 fetuses, with a total proportion of diagnostic genetic variants of 12.3% (20/163). Genetic variants were significantly more frequently detected in fetuses with multisystem anomalies (27.0%, 10/37), enlarged kidney/echogenic kidney (20%, 4/20), and multicystic dysplastic kidney (11.1%, 4/36). Pregnancy outcome data showed that 88 (94.6%, 88/93) of the surviving cases with negative WES results had a good prognosis in early childhood. CONCLUSIONS: Our study is the largest to use WES prenatally for CAKUT and shows that WES can be used diagnostically to define the molecular defects that underlie unexplained CAKUT.

All-trans-retinoid acid induces the differentiation of P19 cells into neurons involved in the PI3K/Akt/GSK3ß signaling pathway.

The pluripotent mouse embryonal carcinoma cell line P19 is widely used as a model for research on all-trans-retinoid acid (RA)-induced neuronal differentiation; however, the signaling pathways involved in this process remain unclear. This study aimed to reveal the molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells. Real-time quantitative polymerase chain reaction and Western blot analysis were used to determine the expression of neuronal-specific markers, whereas flow cytometry was used to analyze cell cycle and cell apoptosis. The expression profiles of messenger RNAs (mRNAs) in RA-induced neuronal differentiation of P19 cells were analyzed using high-throughput sequencing, and the functions of differentially expressed mRNAs (DEMs) were determined by bioinformatics analysis. RA induced an increase in both class III ß-tubulin (TUBB3) and neurofilament medium (NEFM) mRNA expression, indicating that RA successfully induces neuronal differentiation of P19 cells. Cell apoptosis was not affected; however, cell proliferation decreased. We found 4117 DEMs, which were enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, Wnt signaling pathway, and cell cycle. Particularly, a few DEMs could be identified in the PI3K/Akt signaling pathway networks, such as PI3K, Akt, glycogen synthase kinase-3ß (GSK3ß), cyclin-dependent kinase 4 (CDK4), P21, and Bax. RA significantly increased the protein expression of PI3K, Akt, phosphorylated Akt, GSK3ß, phosphorylated GSK3ß, CDK4, and P21, but it reduced Bax protein expression. The Akt inhibitor affected the increase of TUBB3 and NEFM mRNA expression in RA-induced P19 cells. The molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells is potentially involved in the PI3K/Akt/GSK3ß signaling pathway. The decreased cell proliferation ability of neuronally differentiated P19 cells could be associated with the expression of cell cycle proteins.

NDUFA4 enhances neuron growth by triggering growth factors and inhibiting neuron apoptosis through Bcl-2 and cytochrome C mediated signaling pathway.

Dandy-Walker malformation (DWM) is the most prevalent congenital malformation in cerebellum, however, pathological mechanism of DWM has not been fully clarified. This study aims to investigate effects of NDUFA4 on growth of neurons. LV5-NDUFA4 and LV3-NDUFA4-RNAi lentivirus were constructed and transfected to neurons. Ciclosporin A, together with the two lentivirus were applied to neurons to observe neuron growth, apoptosis, and related protein expression. MTT assay was used to observe neuron growth. Apoptosis was detected by using flow cytometry assay. Real-time PCR was utilized to examine NDUFA4 mRNA expression. Western blot and immunohistochemistry assay were used to detect nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), brain fibroblast growth factor (bFGF) and cytochrome C (Cyt C) expression. Results indicated that NDUFA4 significantly enhanced neuron activity and inhibited neuron apoptosis (P<0.05). NDUFA4 significantly increased Bcl-2 and decreased cleave caspase-3 expression compared to normal control group (P<0.05). NDUFA4 up-regulated growth factors, including NGF, BDNF, bFGF and Cyt C and inhibited Cyt C expression. NDUFA4 interfere inhibits antagonistic effect of ciclosporin A on apoptosis and decrease up-regulative effect of ciclosporin A on neuron growth. NDUFA4 over-expression enhances antagonistic effect of ciclosporin A on apoptosis and increases up-regulative effect of ciclosporin A on neuron growth. In conclusion, NDUFA4 enhances neuron growth by triggering NGF, BDNF and bFGF expression, inhibits neuron apoptosis by increasing Bcl-2 expression and decreasing cyto C expression. Meanwhile, NDUFA4 regulates the antagonistic effect of ciclosporin A on apoptosis and the up-regulative effect of ciclosporin A on neuron growth.

Clinical application of SNP array analysis in fetuses with ventricular septal defects and normal karyotypes.

PURPOSE: The present study aims to evaluate the utility of high-resolution single-nucleotide polymorphism (SNP) arrays in fetuses with ventricular septal defects (VSDs) with or without other structural anomalies but with normal karyotypes and to investigate the outcomes of cases of prenatal VSDs via clinical follow-up. METHODS: We analyzed 144 fetuses with VSDs and normal karyotypes using Affymetrix CytoScan HD arrays and the analyses were carried out a year after birth. RESULTS: Clinically significant CNVs were detected in 12 fetuses (8.3%). The most common pathogenic CNV was a 22q11.2 deletion with a detection rate of 2.8% (4/144). Well-known microdeletion or microduplication syndromes, including Smith-Magenis, Miller-Dieker, 9q subtelomeric deletion, 1p36 microdeletion, 1q21.1 microduplication, and terminal 4q deletion syndrome, were identified in six cases. Three regions of chromosomal imbalance were also identified: microduplication at 12q24.32q24.33, microdeletion at 16p13.13p13.12 and microdeletion at Xp21.1. The genes TBX1, SKI, GJA5, EHMT1, NOTCH1 were identified as established genes and LZTR1, PRDM26, YWHAE, FAT1, AKAP10, ERCC4, and ULK1 were identified as potential candidate genes of fetal VSDs. There was no significant difference in pathogenic CNVs between isolated VSDs and VSDs with additional structural abnormalities. Ninety-five (74.8%) pregnant women with fetuses with benign CNVs chose to continue the pregnancy and had a favorable prognosis, while nine (75%) pregnant women with fetuses with pathogenic CNVs chose to terminate the pregnancy. CONCLUSIONS: High-resolution SNP arrays are valuable tools for identifying submicroscopic chromosomal abnormalities in the prenatal diagnosis of VSDs. An excellent outcome can be expected for VSD fetuses that are negative for chromosomal anomalies and other severe anatomic abnormalities.

Whole-exome sequencing for prenatal diagnosis of fetuses with congenital anomalies of the kidney and urinary tract.

BACKGROUND: In the absence of cytogenetic abnormality, fetuses with congenital anomalies of the kidney and urinary tract (CAKUT) with/without other structural anomalies show a higher likelihood of monogenic causes; however, defining the underlying pathology can be challenging. Here, we investigate the value of whole-exome sequencing (WES) in fetuses with CAKUT but normal findings upon karyotyping and chromosome microarray analysis. METHODS: WES was performed on DNA from the cord blood of 30 fetuses with unexplained CAKUT with/without other structural anomalies. In the first 23 cases, sequencing was initially performed on fetal DNA only; for the remaining seven cases, the trio of fetus, mother and father was sequenced simultaneously. RESULTS: Of the 30 cases, pathogenic variants were identified in 4 (13%) (UMOD, NEK8, HNF1B and BBS2) and incidental variants in 2 (7%) (HSPD1 and GRIN2B). Furthermore, two of the above four cases had other anomalies in addition to CAKUT. Thus, the detection rate was only 2/22 (9.1%) for isolated CAKUT and 2/8 (25%) for CAKUT with other abnormalities. CONCLUSIONS: Applying WES to the prenatal diagnostic approach in CAKUT fetuses with or without other anomalies allows for an accurate and early etiology-based diagnosis and improved clinical management. To expedite interpretation of the results, trio sequencing should be employed; however, interpretation may nevertheless be compromised by incomplete coverage of all relevant genes.

De Novo 1.77-Mb Microdeletion of 10q22.2q22.3 in a Girl With Developmental Delay, Speech Delay, Congenital Cleft Palate, and Bilateral Hearing Impairment.

Interstitial deletions of chromosome band 10q22.1q22.3 are rare. We here report a 2.5-year-old female patient with developmental delay, speech delay, congenital cleft palate, and bilateral hearing impairment. The girl's karyotype was normal. Chromosome microarray analysis (CMA) revealed a 1.77-Mb de novo interstitial deletion in 10q22.2q22.3. The deletion harbors 9 genes, including KAT6B, DUPD1, DUSP13, SAMD8, VDAC2, COMTD1, ZNF503, NCRNA00245, and C10orf11. This is the first patient with a deletion of the smallest size in 10q22.2q22.3 as detected using single nucleotide polymorphism (SNP) arrays. Comparisons with patients with overlapping deletions and in neighboring regions demonstrate the clinical impact of each deletion and in the context of other deletions within the 10q22q23 region. Additionally, KAT6B and C10orf11 could represent disease-associated genes that contribute to developmental delay, speech and language delay, and congenital cleft palate.

Application of high resolution SNP arrays in patients with congenital oral clefts in south China.

Chromosome microarray analysis (CMA) has proven to be a powerful tool in postnatal patients with intellectual disabilities. However, the diagnostic capability of CMA in patients with congenital oral clefts remain mysterious. Here, we present our clinical experience in implementing whole-genome high-resolution SNP arrays to investigate 33 patients with syndromic and nonsyndromic oral clefts in whom standard karyotyping analyses showed normal karyotypes. We aim to identify the genomic aetiology and candidate genes in patients with congenital oral clefts. CMA revealed copy number variants (CNVs) in every patient, which ranged from 2 to 9 per sample. The size of detected CNVs varied from 100 to 3.2 Mb. In 33 patients, we identified six clinically significant CNVs. The incidence of clinically significant CNVs was 18.2% (6/33). Three of these six CNVs were detected in patients with nonsyndromic clefts, including one who presented with isolated cleft lip with cleft palate (CLP) and two with cleft palate only (CPO). The remaining three CNVs were detected in patients with syndromic clefts. However, no CNV was detected in patients with cleft lip only (CLO). The six clinically significant CNVs were as follows: 8p23.1 microduplication (198 kb); 10q22.2-q22.3 microdeletion (1766 kb); 18q12.3 microduplication (638 kb); 20p12.1 microdeletion (184 kb); 6q26 microdeletion (389 kb); and 22q11.21-q11.23 microdeletion (3163 kb). In addition, two novel candidate genes for oral clefts, KAT6B and MACROD2, were putatively identified. We also found a CNV of unknown clinical significance with a detection rate of 3.0% (1/33). Our results further support the notion that CNVs significantly contributed to the genetic aetiology of oral clefts and emphasize the efficacy of whole-genome high-resolution SNP arrays to detect novel candidate genes in patients with syndromic and nonsyndromic clefts.

Prenatal diagnosis of Smith-Magenis syndrome in two fetuses with increased nuchal translucency, mild lateral ventriculomegaly, and congenital heart defects.

OBJECTIVE: Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation disorder characterized by an interstitial deletion involving chromosome 17p11.2 containing the retinoic acid-induced 1 (RAI1) gene or due to mutation of RAI1. Few cases have been reported in the medical literature regarding prenatal diagnosis of SMS. We report on the prenatal diagnosis of SMS in two fetuses with increased nuchal translucency (NT), mild lateral ventriculomegaly, and congenital heart defects by whole-genome and high-resolution chromosome microarray analysis (CMA). CASE REPORT: The CMA result of Fetus 1, which had increased NT, mild lateral ventriculomegaly, tricuspid regurgitation, and right aortic arch with left ductus arteriosus, revealed a de novo 4.79-Mb deletion at 17p12p11.2. Fetus 2 had increased NT, pulmonary stenosis, and a ventricular septal defect, and showed a de novo 3.68-Mb deletion at 17p11.2. CONCLUSION: The findings further confirm that increased NT is associated with genetic syndromes, and brain imaging is necessary for SMS fetuses. Both deletions encompass the SMS "critical region", which includes many genes including RAI1. However, the precise gene(s) responsible for the heart defects in SMS remain unclear; further efforts should be undertaken to understand the molecular basis of this syndrome.

Prenatal diagnosis of foetuses with congenital abnormalities and duplication of the MECP2 region.

MECP2 duplication results in a well-recognised syndrome in 100% of affected male children; this syndrome is characterised by severe neurodevelopmental disabilities and recurrent infections. However, no sonographic findings have been reported for affected foetuses, and prenatal molecular diagnosis has not been possible for this disease due to lack of prenatal clinical presentation. In this study, we identified a small duplication comprising the MECP2 and L1CAM genes in the Xq28 region in a patient from a family with severe X-linked mental retardation and in a prenatal foetus with brain structural abnormalities. Using high-resolution chromosome microarray analysis (CMA) to screen 108 foetuses with congenital structural abnormalities, we identified additional three foetuses with the MECP2 duplication. Our study indicates that ventriculomegaly, hydrocephalus, agenesis of the corpus callosum, choroid plexus cysts, foetal growth restriction and hydronephrosis might be common ultrasound findings in prenatal foetuses with the MECP2 duplication and provides the first set of prenatal cases with MECP2 duplication, the ultrasonographic phenotype described in these patients will help to recognise the foetuses with possible MECP2 duplication and prompt the appropriate molecular testing.