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Hypospadias refers to the abnormal position of the male urethral orifice, which not only leads to urination disorder but also causes sexual dysfunction in adulthood. However, the complex and diverse pathogenic factors of hypospadias are still unclear. To study the pathogenesis and prognosis of hypospadias, we counted the serological indexes of children with hypospadias, and found that sSBP, TC and LDL increased in children with mild, moderate and severe hypospadias. Subsequently, we used quantitative proteomics to find differential proteins in mild, moderate and severe hypospadias. After bioinformatics analysis and biochemical experiments on the screened DEPs, we found that the expression of proteins related to immune inflammation, coagulation, blood pressure and inflammation, and blood lipid were differential expressed in the prepuce tissue of children with hypospadias. We further confirmed that the proteins FGB, FGG, SERPINA1, and AGT involved in the angiotensin system, cholesterol metabolism, and coagulation were significantly up-regulated by biochemical experiments. In particular, the AGT protein of the angiotensin system involved in blood pressure regulation, we have shown that it increases with the severity of hypospadias. This study suggests that children with hypospadias are more likely to suffer from hyperlipidemia and cardiovascular disease (CVD). Our findings provide a theoretical basis for early monitoring of blood lipids and blood pressure to prevent CVD in children with hypospadias.
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Hirschsprung disease (HSCR) is a heterogeneous group of neurocristopathy characterized by the absence of the enteric ganglia along a variable length of the intestine. Genetic defects play a major role in the pathogenesis of HSCR, whereas family studies of pathogenic variants in all the known genes (loci) only demonstrate incomplete penetrance and variable expressivity for unknown reasons. Here, we applied large-scale, quantitative proteomics of human colon tissues from 21 patients using isobaric tags for relative and absolute quantification. method followed by bioinformatics analysis. Selected findings were confirmed by parallel reaction monitoring verification. At last, the interesting differentially expressed proteins were confirmed by Western blot. A total of 5341 proteins in human colon tissues were identified. Among them, 664 proteins with >1.2-fold difference were identified in six groups: groups A1 and A2 pooled protein from the ganglionic and aganglionic colon of male, long-segment HSCR patients (n = 7); groups B1 and B2 pooled protein from the ganglionic and aganglionic colon of male, short-segment HSCR patients (n = 7); and groups C1 and C2 pooled protein from the ganglionic and aganglionic colon of female, short-segment HSCR patients (n = 7). Based on these analyses, 49 proteins from five pathways were selected for parallel reaction monitoring verification, including ribosome, endocytosis, spliceosome, oxidative phosphorylation, and cell adhesion. The downregulation of three neuron projection development genes ARF4, KIF5B, and RAB8A in the aganglionic part of the colon was verified in 15 paired colon samples using Western blot. The findings of this study will shed new light on the pathogenesis of HSCR and facilitate the development of therapeutic targets.
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Fatores de Ribosilação do ADP/metabolismo , Colo/metabolismo , Doença de Hirschsprung/metabolismo , Cinesinas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Fatores de Ribosilação do ADP/genética , Regulação para Baixo , Feminino , Doença de Hirschsprung/genética , Humanos , Lactente , Cinesinas/genética , Masculino , Neurônios , Proteômica , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Congenital malformations, such as neural tube defects (NTDs) and congenital heart disease (CHD), cause significant fetal mortality and childhood morbidity. NTDs are a common congenital anomaly, and are typically induced by higher maternal homocysteine (Hcy) levels and abnormal folate metabolism. The gene encoding methionine synthase reductase (MTRR) is essential for adequate remethylation of Hcy. Previous studies have focused on the coding region of genes involved in one-carbon metabolism, but recent research demonstrates that an allelic change in a non-coding region of MTRR (rs326119) increases the risk of CHD. We hypothesized that this variant might contribute to the etiology of NTDs as well, based on a common role during early embryogenesis. In the present study, 244 neural tube defect cases and 407 controls from northern China were analyzed to determine any association (by χ (2) test) between rs326119 and disease phenotypes. Significant increased risk of anencephaly was seen in MTRR variant rs326119 heterozygote (het) and homozygote (hom) individuals [odds ratios (OR)het = 1.81; ORhom = 2.05)]. Furthermore, this variant was also a risk factor for congenital malformations of the adrenal gland (OR = 1.85), likely due to multiple systemic malformations in the NTDs case population. Our present data indicate that the rs326119 non-coding variant of MTRR has a pleiotropic effect on the development of multiple tissues, especially during early stages in utero. This suggests the allelic state of MTRR is a significant clinical factor affecting Hcy levels and optimal folic supplementation.
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Anencefalia/genética , Povo Asiático/genética , Ferredoxina-NADP Redutase/genética , Defeitos do Tubo Neural/genética , Polimorfismo de Nucleotídeo Único , China , Estudos de Associação Genética , Predisposição Genética para Doença , Cardiopatias Congênitas/genética , Humanos , ÍntronsRESUMO
OBJECTIVE: To explore the association of polymorphisms in folate metabolism genes, methionine synthase reductase (MTRR) gene and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, with complex congenital abnormalities and to further investigate its association with complex congenital abnormalities derived from three germ layers. METHODS: A total of 250 cases of birth defects (with complex congenital abnormalities including congenital heart disease, neural tube defects, and craniofacial anomalies) in Shanxi Province, China were included in the study. MTRR single nucleotide polymorphism (SNP) (rs1801394) and MTHFR SNP (rs1801133) were genotyped by the SNaPshot method, and the genotyping results were compared with those of controls (n=420). RESULTS: SNPs rs1801394 and rs1801133 were associated with multiple birth defects. For the recessive model, individuals with GG genotype at rs1801394 and CC genotype at rs1801133 had a relatively low risk of developing birth defects, so the two genotypes were protective factors against birth defects. The homozygous recessive genotype at rs1801133, which served as a protective factor, was associated with ectoderm- or endoderm-derived complex congenital abnormalities, while the homozygous recessive genotype at rs1801394, which served as a protective factor, was associated with ectoderm-, mesoderm- or endoderm-derived complex congenital abnormalities. CONCLUSIONS: Among the Chinese population in Shanxi Province, the SNPs in folate metabolism genes (MTRR and MTHFR) are associated with complex congenital abnormalities and related to ectoderm, mesoderm or endoderm development.
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Povo Asiático/genética , Anormalidades Congênitas/genética , Ferredoxina-NADP Redutase/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo de Nucleotídeo Único , China , Genótipo , Camadas Germinativas/embriologia , HumanosRESUMO
DNA damage is associated with hyperhomocysteinemia (HHcy) and neural tube defects (NTDs). Additionally, HHcy is a risk factor for NTDs. Therefore, this study examined whether DNA damage is involved in HHcy-induced NTDs and investigated the underlying pathological mechanisms involved. Embryonic day 9 (E9) mouse neuroectoderm cells (NE4C) and homocysteine-thiolactone (HTL, active metabolite of Hcy)-induced NTD chicken embryos were studied by Western blotting, immunofluorescence. RNA interference or gene overexpression techniques were employed to investigate the impact of Menin expression changes on the DNA damage. Chromatin immunoprecipitation-quantitative polymerase chain reaction was used to investigate the epigenetic regulation of histone modifications. An increase in γH2AX (a DNA damage indicator) was detected in HTL-induced NTD chicken embryos and HTL-treated NE4C, accompanied by dysregulation of phospho-Atr-Chk1-nucleotide excision repair (NER) pathway. Further investigation, based on previous research, revealed that disruption of NER was subject to the epigenetic regulation of low-expressed Menin-H3K4me3. Overexpression of Menin or supplementation with folic acid in HTL-treated NE4C reversed the adverse effects caused by high HTL. Additionally, by overexpressing the Mars gene, we tentatively propose a mechanism whereby HTL regulates Menin expression through H3K79hcy, which subsequently influences H3K4me3 modifications, reflecting an interaction between histone modifications. Finally, in 10 human fetal NTDs with HHcy, we detected a decrease in the expression of Menin-H3K4me3 and disorder in the NER pathway, which to some extent validated our proposed mechanism. The present study demonstrated that the decreased expression of Menin in high HTL downregulated H3K4me3 modifications, further weakening the Atr-Chk1-NER pathway, resulting in the occurrence of NTDs.
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BACKGROUND: Orthostatic intolerance, which includes vasovagal syncope and postural orthostatic tachycardia syndrome, is common in children and adolescents. Elevated plasma homocysteine levels might participate in the pathogenesis of orthostatic intolerance. This study was designed to analyze the plasma metabolomic profile in orthostatic intolerance children with high levels of plasma homocysteine. METHODS: Plasma samples from 34 orthostatic intolerance children with a plasma homocysteine concentration > 9 µmol/L and 10 healthy children were subjected to ultra-high-pressure liquid chromatography and quadrupole-time-of-flight mass spectrometry analysis. RESULTS: A total of 875 metabolites were identified, 105 of which were significantly differential metabolites. Choline, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1-(1Z-octadecenyl)-2-(4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoyl)-sn-glycero-3-phosphocholine, histidine, isocitric acid, and DL-glutamic acid and its downstream metabolites were upregulated, whereas 1-palmitoyl-sn-glycero-3-phosphocholine, 1-stearoyl-sn-glycerol 3-phosphocholine, sphingomyelin (d18:1/18:0), betaine aldehyde, hydroxyproline, and gamma-aminobutyric acid were downregulated in the orthostatic intolerance group compared with the control group. All these metabolites were related to choline and glutamate. Heatmap analysis demonstrated a common metabolic pattern of higher choline, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, and DL-glutamic acid, and lower sphingomyelin (d18:1/18:0), 1-stearoyl-sn-glycerol 3-phosphocholine, and 1-palmitoyl-sn-glycero-3-phosphocholine in patients with certain notable metabolic changes (the special group) than in the other patients (the common group). The maximum upright heart rate, the change in heart rate from the supine to the upright position, and the rate of change in heart rate from the supine to the upright position of vasovagal syncope patients were significantly higher in the special group than in the common group (P < 0.05). Choline, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, and DL-glutamic acid were positively correlated with the rate of change in heart rate from the supine to the upright position in vasovagal syncope patients (P < 0.05). CONCLUSIONS: The levels of choline-related metabolites and glutamate-related metabolites changed significantly in orthostatic intolerance children with high levels of plasma homocysteine, and these changes were associated with the severity of illness. These results provided new light on the pathogenesis of orthostatic intolerance.
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Glicerol/análogos & derivados , Intolerância Ortostática , Fosforilcolina/análogos & derivados , Síncope Vasovagal , Adolescente , Criança , Humanos , Ácido Glutâmico , Glicerilfosforilcolina , Esfingomielinas , Colina , HomocisteínaRESUMO
The mechanism of hypertension in children remains elusive. The objective of this study was to analyze plasma metabolomics characteristics to explore the potential mechanism of hypertension in children. Serum samples from 29 control children, 38 children with normal body mass index and simple hypertension (NBp), 8 children overweight with simple hypertension (OBp), 37 children with normal body mass index and H-type hypertension (NH) and 19 children overweight with H-type hypertension (OH) were analyzed by non-targeted metabolomics. A total of 1235 differential metabolites were identified between children with hypertension and normal controls, of which 193 metabolites including various lipids were significantly expressed. Compared with the control group, 3-dehydroepiandrosterone sulfate, oleic acid and linoleic acid were up-regulated, and gamma-muricholic acid was down-regulated in the NBp group; 3-dehydroepiandrosterone sulfate, 4-acetamidobutanoate and 1-hexadecanoyl-2-octadecadienoyl-sn-glyero-3-phosphocholine were up-regulated in the OBp group, whereas adenosine and 1-myristoyl-sn-glyero-3-phosphocholine were down-regulated; in the NH group, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, phenol and 3-methoxytyramine were up-regulated, while pentadecanoic acid was down-regulated; in the OH group, NG,NG-dimethyl-L-arginine, 1-palmitoyl-sn-glycero-3-phosphocholine and monoethyl phthalate were up-regulated, while phloretin and glycine were down-regulated. The results showed that the children with hypertension had obvious disorders of lipid metabolism (especially in the overweight hypertension group), which led to the occurrence of hypertension. Additionally, the concentration of NO production-related NG, NG-dimethyl-L-arginine, was significantly increased, which may play an important role in H-type hypertension in children.
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Hipertensão , Fosforilcolina , Humanos , Criança , Cromatografia Líquida de Alta Pressão/métodos , Sulfato de Desidroepiandrosterona , Sobrepeso , Metabolômica/métodos , Biomarcadores/metabolismoRESUMO
Introduction: Neural tube defects (NTDs) are serious congenital malformations. The etiology of NTDs involves both genetic and environmental factors. Loss of CECR2 in mice has been shown to result in NTDs. Our previous study indicated that high homocysteine (HHcy) levels could further reduced the expression level of CECR2. This investigation aims to explore the genetic influence of the chromatin remodeling gene, CECR2, in humans and determine if HHcy can have a synergistic effect on protein expression. Methods: We conducted Next-Generation Sequencing (NGS) of the CECR2 gene in 373 NTD cases and 222 healthy controls, followed by functional assay application to select and evaluate CECR2 missense variants and subsequent Western blotting to identify protein expression levels. Results: From the analysis, we identified nine rare, NTD-specific mutations within the CECR2 gene. Significantly, four missense variants (p.E327V, p.T521S, p.G701R, and p.G868R) were selected via functional screening. The E9.5 mouse ectodermal stem cell line NE-4C, transfected with plasmids expressing p.E327V, p.T521S, p.G868R variants or a recombinant harboring all four (named as 4Mut), exhibited notable reductions in CECR2 protein expression. Furthermore, exposure to homocysteine thiolactone (HTL), an extremely reactive homocysteine metabolite, amplified the reduction in CECR2 expression, accompanied by a significant increase in the apoptotic molecule Caspase3 activity, a potential NTD inducer. Importantly, folic acid (FA) supplementation effectively counteracted the CECR2 expression decline induced by CECR2 mutation and HTL treatment, leading to reduced apoptosis. Discussion: Our observations underscore a synergistic relationship between HHcy and genetic variations in CECR2 concerning NTDs, thereby reinforcing the concept of gene-environment interaction phenomena in NTD etiology.
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Introduction: Type 1 diabetes (T1D) is a serious autoimmune disease with high morbidity and mortality. Early diagnosis and treatment remain unsatisfactory. While the potential for development of T1D biomarkers in circulating exosomes has attracted interest, progress has been limited. This study endeavors to explore the molecular dynamics of plasma exosome proteins in pediatric T1D patients and potential mechanisms correlated with T1D progression. Methods: Liquid chromatography-tandem mass spectrometry with tandem mass tag (TMT)6 labeling was used to quantify exosomal protein expression profiles in 12 healthy controls and 24 T1D patients stratified by age (≤ 6 years old and > 6 years old) and glycated hemoglobin (HbA1c) levels (> 7% or > 7%). Integrated bioinformatics analysis was employed to decipher the functions of differentially expressed proteins, and Western blotting was used for validation of selected proteins' expression levels. Results: We identified 1035 differentially expressed proteins (fold change > 1.3) between the T1D patients and healthy controls: 558 in those ≤ 6-year-old and 588 in those > 6-year-old. In those who reached an HbA1c level < 7% following 3 or more months of insulin therapy, the expression levels of most altered proteins in both T1D age groups returned to levels comparable to those in the healthy control group. Bioinformatics analysis revealed that differentially expressed exosome proteins are primarily related to immune function, hemostasis, cellular stress responses, and matrix organization. Western blotting confirmed the alterations in RAB40A, SEMA6D, COL6A5, and TTR proteins. Discussion: This study delivers valuable insights into the fundamental molecular mechanisms contributing to T1D pathology. Moreover, it proposes potential therapeutic targets for improved T1D management.
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Diabetes Mellitus Tipo 1 , Humanos , Criança , Diabetes Mellitus Tipo 1/metabolismo , Hemoglobinas Glicadas , Biomarcadores , Insulina/uso terapêutico , Espectrometria de Massas , Proteínas SanguíneasRESUMO
BACKGROUND: Obesity is a key risk factor of hypertension. Angiotensin-converting enzyme 1 (ACE1) is a key enzyme involved in the renin-angiotensin-aldosterone system (RAAS), which contributes to obesity-related hypertension (OrHTN). Emerging evidence has shown that histone acetylation is also involved in OrHTN. As kidney is an effector organ that activates the RAAS by secreting renin after hypertension occurs, this study aimed to explore the regulatory role of histone acetylation on renal RAAS expression. METHODS: Nineteen male Wistar rats were randomly divided into a control group ( n â=â9, fed normal chow) and a high-fat diet (HFD) group ( n â=â10, fed HFD for 16âweeks). The renal transcriptome and histone acetylation spectrum was analyzed by RNA sequencing and tandem mass spectrometry and was further confirmed by RT-qPCR, western blot, and immunohistochemistry. Then, chromatin immunoprecipitation (ChIP)-qPCR analysis was performed for the detection of DNA-protein interaction. RESULTS: After 16-week HFD, the rats became obese with increased plasma triglyceride and high blood pressure. Increased ACE1 and histone 3 lysine 27 acetylation (H3K27ac) expression levels were found in OrHTN rat kidneys. The following ChIP-qPCR analysis illustrated that the upregulation of ACE1 transcription was mediated by increased H3K27ac. CONCLUSION: H3K27ac could be an important histone acetylation site that activates renal ACE1 in HFD-induced hypertensive rats.
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Dieta Hiperlipídica , Hipertensão , Angiotensinas/metabolismo , Animais , Pressão Sanguínea , Dieta Hiperlipídica/efeitos adversos , Histonas/metabolismo , Rim , Lisina , Masculino , Obesidade/complicações , Obesidade/metabolismo , Peptidil Dipeptidase A/genética , Ratos , Ratos Wistar , Sistema Renina-AngiotensinaRESUMO
BACKGROUND: Neural tube defects (NTDs) are severe congenital malformations. Diabetes during pregnancy is a risk factor for NTDs, but its mechanism remains elusive. Emerging evidence suggests that protein malonylation is involved in diabetes. Here, we report the correlation between histone lysine malonylation in diabetes-induced NTDs. METHODS: Nano-HPLC/MS/MS was used to screen the histone malonylation profile in human embryonic brain tissue. Then, the histone malonylation level was compared between the brains of normal control mice and mice with diabetes-induced NTDs. Finally, the histone malonylation level was compared under high glucose exposure in an E9 neuroepithelial cell line (NE4C). RESULTS: A total of 30 histone malonylation sites were identified in human embryonic brain tissue, including 18 novel sites. Furthermore, we found an increased histone malonylation level in brain tissues from mice with diabetes-induced NTDs. Finally, both the histone malonylation modified sites and the modified levels were proved to be increased in the NE4C treated with high glucose. CONCLUSION: Our results present a comprehensive map of histone malonylation in the human fetal brain. Furthermore, we provide experimental evidence supporting a relationship between histone malonylation and NTDs caused by high glucose-induced diabetes. These findings offer new insights into the pathological role of histone modifications in human NTDs.
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Histonas/metabolismo , Defeitos do Tubo Neural/metabolismo , Gravidez em Diabéticas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Linhagem Celular , Epigênese Genética , Feminino , Humanos , Lisina/metabolismo , Masculino , Malonatos/metabolismo , Camundongos , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/patologia , GravidezRESUMO
BACKGROUND: Neural tube defects (NTDs) are severe common birth defects that result from a failure in neural tube closure (NTC). Our previous study has shown that decreased histone methylation altered the regulation of genes linked to NTC. However, the effect of alterations in histone acetylation in human fetuses with NTDs, which are another functional posttranslation modification, remains elusive. Thus, we aimed to identify acetylation sites and changes in histone in patients with NTDs. METHODS: First, we identified histone acetylation sites between control human embryonic brain tissue and NTDs using Nano-HPLC-MS/MS. Next, we evaluated the level of histone acetylation both groups via western blotting (WB). Finally, we used LC-ESI-MS and WB to compare whether histone H4 acetylation was different in NTDs. RESULTS: A total of 43 histone acetylation sites were identified in human embryonic brain tissue, which included 16 novel sites. Furthermore, we found an increased histone acetylation and H4K5ac in tissue with NTDs. CONCLUSION: Our result present a comprehensive map of histone H4 modifications in the human fetal brain. Furthermore, we provide experimental evidence supporting a relationship between histone H4K5ac and NTDs. This offers a new insight into the pathological role of histone modifications in human NTDs.
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Histonas/genética , Histonas/metabolismo , Defeitos do Tubo Neural/genética , Acetilação , Biomarcadores , Encéfalo/embriologia , Mapeamento Encefálico/métodos , Feminino , Feto/embriologia , Feto/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Masculino , Regiões Promotoras Genéticas , Espectrometria de Massas em TandemRESUMO
The association between apoptosis and neural tube defects (NTDs) is recognized as important, however, the precise link remains to be elucidated. Epigenetic modifications in human NTDs have been detected previously. In the present study, the occurrence of epigenetic modifications in apoptosisrelated genes was investigated in a retinoic acid (RA)induced mouse NTD model. Among 84 key genes involved in programmed cell death, 13 genes, including tumor necrosis factor (Tnf), annexin A5, apoptosis inhibitor 5, Bcl2associated athanogene 3, baculoviral IAP repeatcontaining 3, caspase (Casp)12, Casp4, Casp8, lymphotoxin ß receptor, NLR family, apoptosis inhibitory protein 2, TNF receptor superfamily (Tnfrsf)1a, TNF superfamily (Tnfs)f10 and Tnfsf12, were downregulated, whereas nucleolar protein 3 was upregulated in the RAinduced NTD mice. Chromatin immunoprecipitation assays revealed that the regulatory regions of these differentially expressed TNFrelated genes showed reduced histone H3K27 acetylation in NTDs, compared with control mice without NTDs. Reverse transcriptionquantitative polymerase chain reaction revealed that H3K27acbinding to the differentially regulated genes was markedly decreased in the NTD mice, whereas binding to the unchanged genes Casp3 and Nfkb1 was unaffected. In conclusion, certain TNFrelated genes appeared to be downregulated in NTDs, possibly as a result of abnormal histone H3K27 acetylation. These results shed new light on the epigenetic dysregulation of apoptosisrelated genes in NTDs.
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Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/metabolismo , Fatores de Necrose Tumoral/metabolismo , Acetilação , Animais , Apoptose/genética , Imunoprecipitação da Cromatina , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Genômica/métodos , Masculino , Camundongos , Regiões Promotoras Genéticas , Transdução de Sinais , TranscriptomaRESUMO
BACKGROUND: Maternal diabetes related neural tube defects (NTDs) are a result of oxidative stress and apoptosis. However, the molecular mechanism behind the pathogenesis is not fully understood. Here, we report that high glucose exposure-induced epigenetic changes influence histone H4 acetylation and neuroepithelial cell proliferation. We also show that the acetyltransferase inhibitor C646 can prevent high glucose induced changes in histone H4 acetylation and neuroepithelial cell proliferation. METHODS: By using LC-MS/MS as an unbiased approach, we screened the histone acetylation profile in an E9 neuroepithelial cell line (NE-4C) under high glucose exposure. We further explored the mechanism in cells in vitro and in maternal diabetes-induced mouse embryos in vivo. RESULTS: We identified 35 core histone acetylation marks in normal E9 neuroepithelial cells, whereas high glucose exposure resulted in novel acetylation sites on H4K31 and H4K44. Acetylation levels of embryonic development associated H4K5/K8/K12/K16 increased in neuroepithelial cells exposed to high glucose in vitro and in brain tissue from maternal diabetes induced exencephalic embryos in vivo. Further, mRNA level of histone acetyltransferase CBP encoded gene Crebbp was significantly increased both in vitro and in vivo. The addition of C646, a selective inhibitor for CBP/p300, significantly rescued increase of H4K5/K8/K12/K16 acetylation levels and H3S10pi-labeled neuroepithelial cell proliferation induced by high glucose exposure. CONCLUSION: Our data provide complementary insights for potential mechanisms of maternal diabetes induced NTDs.
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Benzoatos/metabolismo , Células Neuroepiteliais/efeitos dos fármacos , Pirazóis/metabolismo , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Acetilação , Animais , Apoptose/efeitos dos fármacos , Benzoatos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida/métodos , Epigênese Genética , Feminino , Glucose/efeitos adversos , Glucose/metabolismo , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Masculino , Camundongos , Nitrobenzenos , Fragmentos de Peptídeos/antagonistas & inibidores , Gravidez , Pirazóis/farmacologia , Pirazolonas , Sialoglicoproteínas/antagonistas & inibidores , Espectrometria de Massas em Tandem/métodos , Fatores de Transcrição de p300-CBP/fisiologiaRESUMO
AIM: Neural tube defects (NTDs) are birth defects of the nervous system and are the second most frequent cause of birth defects worldwide. The etiology of NTDs is complicated and involves both genetic and environmental factors. CASP9 is an initiator caspase in the intrinsic apoptosis pathway, which in Casp9-/- mice has been shown to result in NTDs because of decreased apoptosis. The aim of this study was to evaluate the potential genetic contribution of the CASP9 gene in human NTDs. METHODS: High-throughput sequencing was performed to screen genetic variants of CASP9 genes in 355 NTD cases and 225 matched controls. Apoptosis-relevant assays were performed on transiently transfected E9 neuroepithelial cells or human embryonic kidney 293T cells, to determine the functional characteristics of NTD-specific rare variants under complete or low folic acid (FA) status. RESULTS: We found significant expression of CASP9 rare variants in NTDs and identified 4 NTD-specific missense variants. Functional assays demonstrated that a p.Y251C variant attenuates apoptosis by reducing CASP9 protein expression and decreasing activity of the intrinsic apoptosis pathway. From this, we conclude that this variant may represent a loss-of-function mutation. A 4-time recurrent p.R191G variant did not affect intrinsic apoptosis in complete medium, while it completely inhibited apoptosis induced by low FA medium. CONCLUSION: Our findings identify a genetic link for apoptosis in human NTDs and highlight the effect of gene-environment interactions in a complex disease.
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Caspase 9/genética , Caspase 9/metabolismo , Mutação , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/metabolismo , Apoptose/fisiologia , Povo Asiático/genética , Linhagem Celular , China , Estudos de Coortes , Feminino , Ácido Fólico/metabolismo , Deficiência de Ácido Fólico/metabolismo , Expressão Gênica , Interação Gene-Ambiente , Testes Genéticos , Humanos , MasculinoRESUMO
Neural tube defects (NTDs) are serious congenital malformations. Excessive maternal homocysteine (Hcy) increases the risk of NTDs, while its mechanism remains elusive. Here we report the role of histone homocysteinylation in neural tube closure (NTC). A total of 39 histone homocysteinylation sites are identified in samples from human embryonic brain tissue using mass spectrometry. Elevated levels of histone KHcy and H3K79Hcy are detected at increased cellular Hcy levels in human fetal brains. Using ChIP-seq and RNA-seq assays, we demonstrate that an increase in H3K79Hcy level down-regulates the expression of selected NTC-related genes including Cecr2, Smarca4, and Dnmt3b. In human NTDs brain tissues, decrease in expression of CECR2, SMARCA4, and DNMT3B is also detected along with high levels of Hcy and H3K79Hcy. Our results suggest that higher levels of Hcy contribute to the onset of NTDs through up-regulation of histone H3K79Hcy, leading to abnormal expressions of selected NTC-related genes.
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Histonas/metabolismo , Homocisteína/metabolismo , Defeitos do Tubo Neural/metabolismo , Adulto , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Embrião de Galinha , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Helicases/metabolismo , Feminino , Células HEK293 , Homocisteína/análogos & derivados , Humanos , Masculino , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Adulto Jovem , DNA Metiltransferase 3BRESUMO
Dishevelled (DVL/Dvl) genes play roles in canonical and noncanonical Wnt signaling, both of which are essential in neural tube closing and are involved in balancing neural progenitor growth and differentiation, or neuroepithelial cell polarity, respectively. In mouse Dvl haploinsufficiency leads to neural tube defects (NTDs), which represent the second most common birth defects. However, DVL genes' genetic contributions in human NTDs are modest. We sought to explore the molecular impact on such genes in human NTDs in a Han Chinese cohort. In 47 cases with NTDs and 61 matched controls, in brain tissues, the DVL1/2 mRNA levels were correlated with the levels of a serine/threonine protein kinase MARK2, and in 20 cases with lumbosacral spina bifida, the mRNA levels of DVL1 and MARK2 were significantly decreased; by contrast, only an intronic rare variant was found. Moreover, in an extended population, we found merely three novel rare missense variants in 1 % of individuals with NTDs. In cell-based assays, Mark2 depletion indeed reduces Dvl gene expression and interrupts neural stem cell (NSCs) growth and differentiation, which are likely to be mediated through a decrease in class IIa HDAC phosphorylation and reduced H3K4ac and H3K27ac occupancies at the Dvl1/2 promoters. Finally, the detections of folate concentration in human brain tissue and NSCs and MEF cells indicates that folate deficiency contributes to the observed decreases in Mark2 and Dvl1 expression. Our present study raises a potential common pathogenicity mechanism in human lumbosacral spina bifida about DVL genes rather than their genetic pathogenic role.
Assuntos
Proteínas Desgrenhadas/genética , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Disrafismo Espinal/genética , Transcrição Gênica , Acetilação , Proliferação de Células/fisiologia , Criança , Pré-Escolar , Proteínas Desgrenhadas/metabolismo , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Fosforilação , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Disrafismo Espinal/metabolismoRESUMO
The association between Wnt genes and neural tube defects (NTDs) is recognized, however, it remains to be fully elucidated. Our previous study demonstrated that epigenetic mechanisms are affected in human NTDs. Therefore, the present study aimed to evaluate whether Wnt2b and Wnt7b are susceptible to abnormal epigenetic modification in NTDs, using chromatin immunoprecipitation assays to evaluate histone enrichments and the MassARRAY platform to detect the methylation levels of target regions within Wnt genes. The results demonstrated that the transcriptional activities of Wnt2b and Wnt7b were abnormally upregulated in mouse fetuses with NTDs and, in the GCrich promoters of these genes, histone 3 lysine 4 (H3K4) acetylation was enriched, whereas H3K27 trimethylation was reduced. Furthermore, several CpG sites in the altered histone modification of target regions were significantly hypomethylated. The present study also detected abnormal epigenetic modifications of these Wnt genes in human NTDs. In conclusion, the present study detected abnormal upregulation in the levels of Wnt2b and Wnt7b, and hypothesized that the alterations may be due to the ectopic opening of chromatin structure. These results improve understanding of the dysregulation of epigenetic modification of Wnt genes in NTDs.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/genética , Defeitos do Tubo Neural/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Wnt/genética , Animais , Imunoprecipitação da Cromatina , Metilação de DNA/genética , Modelos Animais de Doenças , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feminino , Glicoproteínas/metabolismo , Histonas/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Disrafismo Espinal/genética , Tretinoína , Proteínas Wnt/metabolismoRESUMO
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.
Assuntos
Cílios/genética , Estudos de Associação Genética , Mutação , Defeitos do Tubo Neural/genética , Adolescente , Adulto , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Estudos de Casos e Controles , Linhagem Celular , Análise Mutacional de DNA , Feminino , Expressão Gênica , Variação Genética , Genótipo , Humanos , Masculino , Defeitos do Tubo Neural/diagnóstico , Neurogênese/genética , Fenótipo , Adulto JovemRESUMO
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.