Antisense oligonucleotides targeting the SMN2 promoter region enhance SMN2 expression in spinal muscular atrophy cell lines and mouse model.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by homozygous deletions or mutations in survival motor neuron gene 1 (SMN1). Currently, the primary therapeutic strategy for SMA is to increase the level of SMN via correcting SMN2 splicing (nusinersen and risdiplam). However, some patients with SMA do not respond to such treatments, thereby warranting a need to develop new therapeutic strategies. We have previously reported that SMN2 expression is epigenetically regulated by DNA methylation levels of the SMN2 promoter region. In the present study, we determined that methyl-CpG-binding protein 2 (MeCP2) may bind to this critical promoter region (nt-167 to 43). Antisense oligonucleotides (ASO-P1 and ASO-P2) were designed to target the key methylation sites in the SMN2 promoter region, which enhanced the overall transcription and functional protein expression levels in the SMA cell lines. These results were similar to those observed in nusinersen-treated SMA cells. Moreover, a combined treatment of ASO-P1 and ASO-NUS in SMA cell lines further increases fl-SMN2 transcript and SMN protein levels. The delivery of ASO-P1 to the central nervous system of severe SMA mice corrected the molecular, pathological, and functional phenotypes of this disease and increased survival rates. Our findings suggest that the key methylation regions in the SMN2 promoter region may be a novel therapeutic target for SMA.

A mosaic karyotype of 45,X/46,X,psu idic(Y)(q12) in a ten-year-old boy: integrating high-throughput sequencing with cytogenetic technique for precise diagnosis and genetic counselling.

BACKGROUND: Isodicentric Y chromosome (idic(Y)) is the most commonly reported aberration of the human Y chromosome, which is an important cause of abnormal sexual development. The breakpoints of isodicentric Y chromosome mostly occurred in Yq11.2 and Yp11.3, however, the breakpoints in Yq12 are relatively rare. CASE PRESENTATION: We described a 10-year-old boy presenting hypospadias, micropenis and short stature, as well as unilateral cryptorchidism without normal testicular seminiferous tubules structure by biopsy. Whole exome sequencing didn't find any pathogenic/likely pathogenic variants related to phenotypes of this patient. Copy number variation sequencing showed the duplication of whole Y chromosome. Subsequently, karyotyping and FISH analyses demonstrated his genetic diagnosis was mosaic 45,X[8]/46,X,psu idic(Y)(q12)[32], with the breakpoint in Yq12. CONCLUSIONS: Our case proved that it would be beneficial to integrate high-throughput sequencing with cytogenetic technique for precise diagnosis, treatment and genetic counselling.

The effect of folic acid deficiency on FGF pathway via Brachyury regulation in neural tube defects.

Folate deficiency in early development leads to disturbance in multiple processes, including neurogenesis during which fibroblast growth factor (FGF) pathway is one of the crucial pathways. Whether folic acid (FA) directly affects FGF pathways to influence neurodevelopment and the possible mechanism remains unclear. In this study, we presented evidence that in human FA-insufficient encephalocele, the FGF pathway was interfered. Furthermore, in Brachyury knockout mice devoid of such T-box transcription factors regulating embryonic neuromesodermal bipotency and a key component of FGF pathway, change in expression of Brachyury downstream targets, activator Fgf8 and suppressor dual specificity phosphatase 6 was detected, along with the reduction in expression of other key FGF pathway genes. By using a FA-deficient cell model, we further demonstrated that decrease in Brachyury expression was through alteration in hypermethylation at the Brachyury promoter region under FA deficiency conditions, and suppression of Brachyury promoted the inactivation of the FGF pathway. Correspondingly, FA supplementation partially reverses the effects seen in FA-deficient embryoid bodies. Lastly, in mice with maternal folate-deficient diets, aberrant FGF pathway activity was found in fetal brain dysplasia. Taken together, our findings highlight the effect of FA on FGF pathways during neurogenesis, and the mechanism may be due to the low expression of Brachyury gene via hypermethylation under FA-insufficient conditions.-Chang, S., Lu, X., Wang, S., Wang, Z., Huo, J., Huang, J., Shangguan, S., Li, S., Zou, J., Bao, Y., Guo, J., Wang, F., Niu, B., Zhang, T., Qiu, Z., Wu, J., Wang, L. The effect of folic acid deficiency on FGF pathway via Brachyury regulation in neural tube defects.

Quantitative Measurement of PARD3 Copy Number Variations in Human Neural Tube Defects.

Although more than 200 genes are known to be related to neural tube defects (NTDs), the exact molecular basis is still unclear. Evaluating the contribution of copy number variation (CNV) might be a priority because CNV involves changes in the copy number of large segments of DNA, leading to phenotypic traits and disease susceptibility. Recent studies have documented that the polarity protein partitioning defective 3 homolog (Pard3) plays an essential role in the process of neural tube closure. The aim of this study was to assess the role of PARD3 CNVs in the etiology of human NTDs. Relative quantitative PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to quantitative measurement of CNVs in 25 PARD3 exons in 202 NTD cases and 231 controls from a region of China with a high prevalence of NTDs. The results showed that microduplications ranging from 3 to 4 were evident in coding Exon 21 and Exon 25 in both case and control groups. A novel heterozygous microdeletion spanning 444 bp of Exon 14 was identified in two cases of anencephaly and is absent from all controls analyzed. Expression analyses indicated that this heterozygotic microdeletion showed no tissue specificity and led to defective expression of PARD3. Our study provides further evidence implicating PARD3 in the etiology of NTDs.

[Effect of LINE1-ORF1p overexpression on the proliferation of nephroblastoma WT_CLS1 cells].

OBJECTIVE: To prepare the LINE1-ORF1p polyclonal antibody, and to study the effect of LINE1-ORF1p on the proliferation of nephroblastoma WT_CLS1 cells. METHODS: A genetic engineering method was used to achieve prokaryotic expression of LINE1-ORF1p, and rabbits were immunized with LINE1-ORF1p to prepare polyclonal antibody. Indirect ELISA was used to evaluate antibody titer, and Western blot and immunohistochemistry were used to evaluate the specific ability of antibody to recognize LINE1-ORF1p. The eukaryotic expression vector pEGFP-N1-LINE1-ORF1 was constructed and used to transfect WT_CLS1 cells. Western blot and qRT-PCR were used to measure the protein and mRNA expression of LINE1-ORF1, respectively, and cell proliferation assay and colony-forming assay were used to evaluate the effect of LINE1-ORF1p on the proliferation of WT_CLS1 cells and the formation of tumor cell clone. RESULTS: The LINE1-ORF1p antibody prepared had a titer of >1:16 000 and could specifically recognize LINE1-ORF1p in cells and tumor tissue. WT_CLS1 cells transfected with pEGFP-N1-LINE1-ORF1 had significant increases in the mRNA and protein expression of LINE1-ORF1 and significantly enhanced cell proliferation ability and colony formation ability (P<0.05). CONCLUSIONS: LINE1-ORF1p can promote the growth of nephroblastoma cells and the formation of tumor cell clone, and may be involved in the pathogenesis of nephroblastoma.

Rare Deleterious PARD3 Variants in the aPKC-Binding Region are Implicated in the Pathogenesis of Human Cranial Neural Tube Defects Via Disrupting Apical Tight Junction Formation.

Increasing evidence that mutation of planar cell polarity (PCP) genes contributes to human cranial neural tube defect (NTD) susceptibility prompted us to hypothesize that rare variants of genes in the core apical-basal polarity (ABP) pathway are risk factors for cranial NTDs. In this study, we screened for rare genomic variation of PARD3 in 138 cranial NTD cases and 274 controls. Overall, the rare deleterious variants of PARD3 were significantly associated with increased risk for cranial NTDs (11/138 vs.7/274, P < 0.05, OR = 3.3). These NTD-specific variants were significantly enriched in the aPKC-binding region (6/138 vs. 0/274, P < 0.01). The East Asian cohort in the ExAC database and another Chinese normal cohort further supported this association. Over-expression analysis in HEK293T and MDCK cells confirmed abnormal aPKC binding or interaction for two PARD3 variants (p.P913Q and p.D783G), resulting in defective tight junction formation via disrupted aPKC binding. Functional analysis in human neural progenitor cells and chick embryos revealed that PARD3 knockdown gave rise to abnormal cell polarity and compromised the polarization process of neuroepithelial tissue. Our studies suggest that rare deleterious variants of PARD3 in the aPKC-binding region contribute to human cranial NTDs, possibly by disrupting apical tight junction formation and subsequent polarization process of the neuroepithelium.

Deregulation of the planar cell polarity genes CELSR3 and FZD3 in Hirschsprung disease.

Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of intrinsic ganglion cells in the lower intestine. Genetic factors in the pathogenesis of this disease are under active investigation. As core genes in the planar cell polarity pathway, Celsr3 and Fzd3 are believed to play vital roles in the development of the murine enteric nervous system. The potential association of CELSR3 and FZD3 with the development of HSCR in humans, however, is still unknown. We determined the genotypes of eight CELSR3 and FZD3 polymorphisms in 113 patients. Furthermore, target gene sequencing was used to search for rare mutations in the planar cell polarity genes. The mRNA and protein expression of CELSR3 and FZD3 were explored in patients with HSCR. Class III ß-tubulin in colon tissue samples was examined to elucidate enteric innervation patterns. We observed a significant association between the FZD3 rs17059206 polymorphism and HSCR susceptibility (p<0.001). In addition, five rare mutations in CELSR3 were identified in six patients with HSCR. Upregulation of CELSR3 mRNA expression was detected in 80% of aganglionic segments; a similar increase was found for FZD3 protein expression in 81.8% of aganglionic tissues, compared with the ganglionic segments. Immunohistochemical staining on tissue sections revealed obvious excess expression of both molecules in the mucosal layer. The neurite patterns were highly disorganized in the aganglionic bowel segments, with a marked reduction in the prominence of TUJ1 bundles in number, thickness, and length. Our results showed that deregulation of the planar cell polarity genes CELSR3 and FZD3 might disrupt the enteric innervation pattern and consequently contribute to the susceptibility to HSCR.

DNA methylation aberrations rather than polymorphisms of FZD3 gene increase the risk of spina bifida in a high-risk region for neural tube defects.

BACKGROUND: Animal models of neural tube defects (NTDs) have indicated roles for the Fzd3 gene and the planar cell polarity signaling pathway in convergent extension. We investigated the involvement of FZD3 in genetic and epigenetic mechanisms associated with human NTDs, especially spina bifida. We explored the effects of variants spanning the FZD3 gene in NTDs and examined the role of aberrant methylation of the FZD3 promoter on gene expression in brain tissue in spina bifida. METHODS: Six FZD3 single nucleotide polymorphisms were genotyped using a MassARRAY system in tissue from 165 NTD fetuses and 152 controls. DNA methylation aberrations in the FZD3 promoter region were detected using a MassARRAY EpiTYPER (17 CpG units from -500 to -2400 bp from the transcription start site) in brain tissue from 77 spina bifida and 74 control fetuses. RESULTS: None of the six single nucleotide polymorphisms evaluated were significantly associated with spina bifida, but the mean methylation level was significantly higher in spina bifida samples (13.70%) compared with control samples (10.91%) (p = 0.001). In terms of specific sites, DNA methylation levels were significantly higher in the spina bifida samples at 14 of the 17 CpG units, which mostly included in R2 region. FZD3 mRNA expression was negatively correlated with methylation of the FZD3 promoter region, especially the R2 region (R = 0.970; p = 0.001) in HeLa cells. CONCLUSION: The results of this study suggest that DNA methylation plays an important role in FZD3 gene expression regulation and may be associated with an increased risk of spina bifida.

Association between PTCH1 polymorphisms and risk of neural tube defects in a Chinese population.

BACKGROUND: SHH signaling pathway plays an important role in the formation of the neural plate and is involved in the regulation of the dorsoventral (DV) axis of the neural tube. Some neural tube defects (NTDs) may be caused through overactivation of the SHH signaling pathway. The PTCH1 gene, encoding a negative regulator of SHH signaling, affects neural tube closure in animal models. However, in humans, the relationship between single nucleotide polymorphisms (SNPs) of the PTCH1 gene and neural tube defects remains unclear. METHODS: MassARRAY®GENOTYPER™ was used to genotype 18 SNPs of the PTCH1 gene in 187 NTDs and 212 control samples, to determine whether PTCH1 polymorphisms are related to NTDs. MassARRAY®EpiTYPER™ was performed to assess whether methylation modifications may be associated with SNP genotypes in this Chinese population. RESULT: Increased risk for spina bifida was observed with the G allele of c.3944C>T and the T allele of c.1729™2350G>A in female patients when compared to the normal control group. High methylation levels were detected in those controls bearing the G allele of c.3944C>T. CONCLUSION: In summary, polymorphisms of the PTCH1 gene may be genetic predisposing factors for spina bifida in the population studied. In addition, methylation modifications associated with the c.3944C>T polymorphism, may provide protection.

MicroRNAs regulate the pathogenesis of CVB3-induced viral myocarditis.

AIMS: To evaluate the role of microRNAs (miRNAs) in the pathogenesis of Coxsackievirus B3 (CVB3)-induced viral myocarditis. METHODS: We detected miRNA expression profiling by microarray utilizing a mouse model on day 4 after CVB3 infection. Then we validated differentially expressed miRNAs using real-time polymerase chain reaction (PCR). We predicted target genes using miRNA target prediction databases and assessed them using mRNA microarray and qualitative reverse transcription PCR measurements. By analyzing the target function of differentially expressed miRNAs, we initially explored the regulating role of miRNAs in viral myocarditis. RESULTS: We found five differentially expressed miRNAs that are involved in regulating several important innate immune and antiviral pathways such as the Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine- cytokine receptor interaction, MAPK signaling pathway, JAK-STAT signaling pathway, and natural killer cell-mediated cytotoxicity. CONCLUSION: miRNAs regulate the pathogenesis of viral myocarditis. This study may provide a new perspective and a deeper understanding of the pathogenesis of viral myocarditis that may help with the development of novel therapies against CVB3 infection.

Establishment and validation of a predictive nomogram for the risk of premalignant lesions in children with choledochal cyst.

Background: Choledochal cyst (CDC) increases the risk (2.5%-30%) of malignancy. Metaplasia and dysplasia have been recognized as premalignant lesions among CDCs. This study aimed to evaluate the risk factors of metaplasia and dysplasia in CDC children. Methods: Two hundred and ten CDC children who underwent cyst excision and Roux-en-Y hepaticojejunostomy at our institution between July 2020 and November 2021 were included and randomly divided into the training set and validation set. Univariate and multivariate logistic regression analysis were used to identify independent risk factors of premalignant lesions in the training set and build a predictive nomogram. The performance and discriminatory abilities of the nomogram were further assessed and validated in the validation set. Results: Of the 210 CDC children, 78 (37.1%) patients developed premalignant lesions. Age (OR, 1.011, 95%CI, 1.000-1.022, P = 0.046), symptoms duration (OR, 1.021, 95%CI, 1.001-1.042, P = 0.036), cyst diameter (OR, 1.737, 95%CI, 1.328-2.273, P < 0.001), recurrent attacks of biliary pancreatitis (OR, 3.653, 95%CI, 1.205-11.076, P = 0.022), and biliary operation history (OR, 5.860, 95%CI, 1.268-27.084, P = 0.024) were identified as independent risk factors. Based on these predictors, a predictive nomogram was generated. The AUC of the nomogram was 0.873 in the training set and 0.793 in the validation set, indicating that it was robust and well calibrated. Conclusions: A novel nomogram to the individualized risk of premalignant lesions in CDC children was successfully built, on the basis of age, symptoms duration, cyst diameter, recurrent attacks of biliary pancreatitis, and biliary operation history. This nomogram, combined with the final pathological results, can help clinicians to develop more efficient follow-up strategies for the high-risk children with CDC.

Pathological findings in congenital diaphragmatic hernia on necropsy studies: A single-center case series.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) is associated with high mortality rates and significant pulmonary morbidities. The objective of this study was to delineate the histopathological features observed in necropsies of CDH patients and correlate these with their clinical manifestations. METHODS: We retrospectively reviewed the postmortem findings and corresponding clinical characteristics in eight CDH cases from 2017 to July 2022. RESULTS: The median survival time was 46 (8-624) hours. Autopsy reports showed that diffuse alveolar damage (congestion and hemorrhage) and hyaline membrane formation were the primary pathological lung changes observed. Notably, despite significant reduction in lung volume, the lung development appeared normal in 50% of the cases, while lobulated deformities were present in three (37.5%) cases. All patients displayed a large patent ductus arteriosus (PDA) and a patent foramen ovale, resulting in increased right ventricle (RV) volume, and myocardial fibers appeared slightly congested and swollen. The pulmonary vessels indicated thickening of the arterial media and adventitia. Lung hypoplasia and diffuse lung damage resulted in impaired gas exchange, while PDA and pulmonary hypertension led to RV failure, subsequent organ dysfunction and ultimately death. CONCLUSIONS: Patients with CDH typically succumb to cardiopulmonary failure, a condition driven by a complex interplay of pathophysiological factors. This complexity accounts for the unpredictable response to currently available vasodilators and ventilation therapies.

Loss-of-Function of p21-Activated Kinase 2 Links BMP Signaling to Neural Tube Patterning Defects.

Closure of the neural tube represents a highly complex and coordinated process, the failure of which constitutes common birth defects. The serine/threonine kinase p21-activated kinase 2 (PAK2) is a critical regulator of cytoskeleton dynamics; however, its role in the neurulation and pathogenesis of neural tube defects (NTDs) remains unclear. Here, the results show that Pak2-/- mouse embryos fail to develop dorsolateral hinge points (DLHPs) and exhibit craniorachischisis, a severe phenotype of NTDs. Pak2 knockout activates BMP signaling that involves in vertebrate bone formation. Single-cell transcriptomes reveal abnormal differentiation trajectories and transcriptional events in Pak2-/- mouse embryos during neural tube development. Two nonsynonymous and one recurrent splice-site mutations in the PAK2 gene are identified in five human NTD fetuses, which exhibit attenuated PAK2 expression and upregulated BMP signaling in the brain. Mechanistically, PAK2 regulates Smad9 phosphorylation to inhibit BMP signaling and ultimately induce DLHP formation. Depletion of pak2a in zebrafish induces defects in the neural tube, which are partially rescued by the overexpression of wild-type, but not mutant PAK2. The findings demonstrate the conserved role of PAK2 in neurulation in multiple vertebrate species, highlighting the molecular pathogenesis of PAK2 mutations in NTDs.

Short hairpin RNA targeting 2B gene of coxsackievirus B3 exhibits potential antiviral effects both in vitro and in vivo.

BACKGROUND: Coxsackievirus B3 is an important infectious agent of viral myocarditis, pancreatitis and aseptic meningitis, but there are no specific antiviral therapeutic reagents in clinical use. RNA interference-based technology has been developed to prevent the viral infection. METHODS: To evaluate the impact of RNA interference on viral replication, cytopathogenicity and animal survival, short hairpin RNAs targeting the viral 2B region (shRNA-2B) expressed by a recombinant vector (pGCL-2B) or a recombinant lentivirus (Lenti-2B) were tansfected in HeLa cells or transduced in mice infected with CVB3. RESULTS: ShRNA-2B exhibited a significant effect on inhibition of viral production in HeLa cells. Furthermore, shRNA-2B improved mouse survival rate, reduced the viral tissues titers and attenuated tissue damage compared with those of the shRNA-NC treated control group. Lenti-2B displayed more effective role in inhibition of viral replication than pGCL-2B in vivo. CONCLUSIONS: Coxsackievirus B3 2B is an effective target of gene silencing against coxsackievirus B3 infection, suggesting that shRNA-2B is a potential agent for further development into a treatment for enterviral diseases.

Glutamate carboxypeptidase II gene polymorphisms and neural tube defects in a high-risk Chinese population.

Glutamate carboxypeptidase II (GCPII) catalyzes the hydrolysis of N-acetylaspartylglutamate into N-acetylaspartate and glutamate in the brain. Animal experiments suggested that GCPII plays an essential role in early embryonic development. Previous studies provided conflicting results on the effect of the GCPII rs61886492 C>T (or 1561C>T) polymorphism on NTDs. In the Lvliang area of Shanxi province, where the incidence of NTDs is the highest in China, a case-control study was conducted to investigate possible association between the GCPII rs61886492 and rs202676 polymorphisms and NTD risk. Results indicated all the case and control samples displayed the rs61886492 GG genotype. Although no significant differences in rs202676 genotype or allele frequencies were found between the NTD and control groups, the combined AG+GG genotype group was significantly associated with anencephaly (p = 0.03, OR = 2.11, 95% CI, 1.11-4.01), but not with spina bifida or encephalocele. Overall, the rs202676 A>G polymorphism is a potential risk factor for anencephaly. The results of this study suggest that phenotypic heterogeneity may exist among NTDs in this Chinese population.

Increasing angiotensin-converting enzyme 1 regulated by histone 3 lysine 27 hyperacetylation in high-fat diet-induced hypertensive rat kidney.

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  = 9, fed normal chow) and a high-fat diet (HFD) group ( n  = 10, fed HFD for 16 weeks). 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.

Infliximab treatment of glycogenosis Ib with Crohn's-like enterocolitis: A case report.

BACKGROUND: Glycogen storage disease type Ib (GSD-Ib) is a glycogen metabolism disorder that leads to the manifestations of inflammatory bowel disease (IBD), especially Crohn's disease (CD)-like colitis. Although biological agents are effective for treating CD, their application in the treatment of GSD-Ib with CD-like colitis has been rarely reported. CASE SUMMARY: A 13-year-old Han male was diagnosed with GSD-Ib with CD. The patient was treated with granulocyte colony-stimulating factor. When he had symptoms of CD-like colitis, he was continuously pumped with enteral nutrition and administered oral mesalazine for 2 wk; however, the symptoms did not improve significantly. Hence, infliximab (IFX) was administered. Hitherto, the patient has been followed up for 1 year, and no clinical manifestations have been observed. After 6 mo of treatment (fifth IFX treatment), the disease activity index and all inflammatory indexes decreased, and a review of the colonoscopy data showed that the ulcers appeared smooth. CONCLUSION: In this study, the patient was successfully treated with IFX. In cases of GSD-Ib, IBD should be highly considered.

Global DNA hypomethylation is associated with NTD-affected pregnancy: A case-control study.

BACKGROUND: Neural tube defects are severe, common birth defects that result from failure of neural tube closure. They are considered to be a multifactorial disorder, and our knowledge of causal mechanisms remains limited. We hypothesized that abnormal DNA methylation occurs in NTD-affected fetuses. The correlations of global DNA methylation levels with complexity of NTDs and known risk factors of NTDs, MTHFR genotype and fever, were analyzed. METHODS: A hospital-based case-control study was performed. Epidemiologic data, pathologic diagnosis, and methylenetetrahydrofolate reductase (MTHFR) genotype analysis were completed. Array comparative genomic hybridization was used to exclude cytogenetic abnormalities. Global DNA methylation statuses were determined for both brain and skin tissue. RESULTS: Sixty-five NTD-affected fetuses and 65 normal controls matched for gestational and maternal ages were collected. In brain tissue, global DNA methylation levels were significantly decreased in cases compared with controls (4.12 vs. 4.99%; p < 0.001). DNA hypomethylation (<4.35%) resulted in a significant 5.736-fold increased risk for NTDs (95% confidence interval, 1.731-19.009; p = 0.004). Nonisolated NTDs had lower levels of global DNA methylation than did isolated NTDs (3.77 vs. 4.70%; p = 0.022). After stratifying subjects by MTHFR genotype, we observed a skewed distribution of global DNA methylation levels. For genotype C/C, global DNA methylation status was the same in the two groups (4.51 vs. 4.72%; p = 0.687). For T/T, cases had significantly lower global methylation levels than did controls (5.23 vs. 3.79%; p < 0.001). CONCLUSIONS: Global DNA hypomethylation in fetal brain tissue was associated with NTD-affected pregnancy. DNA methylation levels were correlated with NTD complexity. The MTHFR genotype contributed to global DNA hypomethylation. Birth Defects Research (Part A), 2010. (c) 2010 Wiley-Liss, Inc.

Association between rare variants in specific functional pathways and human neural tube defects multiple subphenotypes.

BACKGROUND: Neural tube defects (NTDs) are failure of neural tube closure, which includes multiple central nervous system phenotypes. More than 300 mouse mutant strains exhibits NTDs phenotypes and give us some clues to establish association between biological functions and subphenotypes. However, the knowledge about association in human remains still very poor. METHODS: High throughput targeted genome DNA sequencing were performed on 280 neural tube closure-related genes in 355 NTDs cases and 225 ethnicity matched controls, RESULTS: We explored that potential damaging rare variants in genes functioning in chromatin modification, apoptosis, retinoid metabolism and lipid metabolism are associated with human NTDs. Importantly, our data indicate that except for planar cell polarity pathway, craniorachischisis is also genetically related with chromatin modification and retinoid metabolism. Furthermore, single phenotype in cranial or spinal regions displays significant association with specific biological function, such as anencephaly is associated with potentially damaging rare variants in genes functioning in chromatin modification, encephalocele is associated with apoptosis, retinoid metabolism and one carbon metabolism, spina bifida aperta and spina bifida cystica are associated with apoptosis; lumbar sacral spina bifida aperta and spina bifida occulta are associated with lipid metabolism. By contrast, complex phenotypes in both cranial and spinal regions display association with various biological functions given the different phenotypes. CONCLUSIONS: Our study links genetic variant to subphenotypes of human NTDs and provides a preliminary but direct clue to investigate pathogenic mechanism for human NTDs.

Low folate concentration impacts mismatch repair deficiency in neural tube defects.

Aim: To know the cause of sequence variants in neural tube defect (NTD). Materials & methods: We sequenced genes implicated in neural tube closure (NTC) in a Chinese cohort and elucidated the molecular mechanism-driving mutations. Results: In NTD cases, an increase in specific variants was identified, potentially deleterious rare variants harbored in H3K36me3 occupancy regions that recruits mismatch repair (MMR) machinery. Lower folate concentrations in local brain tissues were also observed. In neuroectoderm cells, folic acid insufficiency attenuated association of Msh6 to H3K36me3, and reduced bindings to NTC genes. Rare variants in human NTDs were featured by MMR deficiency and more severe microsatellite instability. Conclusion: Our work suggests a mechanistic link between folate insufficiency and MMR deficiency that correlates with an increase of rare variants in NTC genes.