Genetic variants in UNC93B1 predispose to childhood-onset systemic lupus erythematosus.

Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.

Epigenetic Insights Into Necrotizing Enterocolitis: Unraveling Methylation-Regulated Biomarkers.

Necrotizing enterocolitis (NEC) is a multifactorial gastrointestinal disease with high morbidity and mortality among premature infants. This study aimed to identify novel methylation-regulated biomarkers in NEC intestinal tissue through multiomics analysis. We analyzed DNA methylation and transcriptome datasets from ileum and colon tissues of patients with NEC. We identify methylation-related differential genes (MrDEGs) based on the rule that the degree of methylation in the promoter region is inversely proportional to RNA transcription. These MrDEGs included ADAP1, GUCA2A, BCL2L14, FUT3, MISP, USH1C, ITGA3, UNC93A and IL22RA1. Single-cell data revealed that MrDEGs were mainly located in the intestinal epithelial part of intestinal tissue. These MrDEGs were verified through Target gene bisulfite sequencing and RT-qPCR. We successfully identified and verified the ADAP1, GUCA2A, IL22RA1 and MISP, primarily expressed in intestinal epithelial villus cells through single-cell data. Through single-gene gene set enrichment analysis, we found that these genes participate mainly in the pathological process of T-cell differentiation and the suppression of intestinal inflammation in NEC. This study enhances our understanding of the pathogenesis of NEC and may promote the development of new precision medicine methods for NEC prediction and diagnosis.

Association between CACNA1D polymorphisms and hypospadias in a southern Chinese population.

BACKGROUND: Hypospadias is a congenital genitourinary malformation, with the etiology remaining complex and poorly understood. Despite several genes have been identified to be associated with the risk of hypospadias, current understanding of the susceptibility loci for hypospadias yet remained largely improved. The CACNA1D gene encodes calcium voltage-gated channel subunit alpha 1d and may be involved in androgen signaling. However, the genetic susceptibility of CACNA1D associated with hypospadias has yet been addressed. OBJECTIVE: To evaluate the association between CACNA1D polymorphisms and the susceptibility to hypospadias. METHODS: In this study, we accessed the association between two potential regulatory SNPs (rs3774491 and rs898415) within CACNA1D and hypospadias in a cohort of southern Chinese population which comprised of 740 cases and 948 healthy individuals. Both SNP and haplotypic associations were evaluated. Bioinformatic analysis of the regulatory abilities of the CACNA1D SNPs were carried out by utilizing public ChIP-seq and DNase-seq data. The expression of Cacna1d in mouse external genitalia and testis was evaluated by qPCR. RESULTS: We found that the allele C in rs3774491 and allele G in rs898415 were significantly associated with an increased risk of hypospadias, especially for proximal hypospadias. Further model-based genotypic analyses showed that these association were prominent in additive model and recessive models. Bioinformatic analyses indicated that both SNPs were colocalized with DNase and multiple histone marker across multiple tissues, suggesting the regulatory potentials for these variants. Cacna1d is detectable in both testis and external genitalia of mouse, but the expression level was more prominent in testis than that in external genitalia, suggesting tissue-specific differences in its expression. CONCLUSION: Our findings provide evidence for CACNA1D as a novel predisposing gene for hypospadias, shedding new light on the genetic basis of malformation of urinary tract. Further investigations are warranted to elucidate the functional implication of CACNA1D underlying the development of hypospadias. LEVEL OF EVIDENCE: N/A.

Plasma anti-myosin autoantibodies in the diagnosis of necrotizing enterocolitis.

We aimed to assess whether autoantibodies can be used as biomarkers for necrotizing enterocolitis (NEC) and applied for its early diagnosis. A prospective observational study was conducted in neonates with suspected NEC abdominal distension (the developmental study), which consisted of 50 neonates finally divided into NEC (n = 24) and non-NEC (n = 26) cohorts based on follow-up results. Serum samples were collected within 48 h of illness onset and used for screening NEC-associated plasma autoantibodies by autoantigen microarray. Additionally, we validated anti-myosin autoantibodies by enzyme-linked immunosorbent assay (ELISA) in an independent validation study, for which we selected plasma samples within 48 h of onset of NEC (n = 38) and samples of gestational age- and weight-matched controls (n = 13). Autoantigen microarray revealed that both IgG and IgM anti-myosin autoantibodies in plasma from neonates with NEC were significantly higher than those in neonates with other diagnoses. ELISA showed that plasma anti-myosin autoantibodies increased in the NEC cohort, with 1.5-fold higher levels than in the non-NEC cohort. Anti-myosin autoantibodies were able to distinguish NEC from non-NEC, achieving an area under the curve (AUC) of 0.8856 (95% confidence interval (CI): 0.7918-0.9795), with sensitivity of 81.58% and specificity of 76.93%. Plasma anti-myosin autoantibodies were significantly higher in all three subtypes of NEC (P < 0.0001 for NEC I; P = 0.0018 for NEC II; P = 0.0011 for NEC III), especially in NEC stage I than that in the non-NEC controls. CONCLUSION: Anti-myosin autoantibodies may be applied as a promising diagnostic marker for NEC, especially for NEC stage I. WHAT IS KNOWN: • Intestinal damage and self-antigen exposure may lead to increased autoantibodies, and they are widely used as biomarkers for diagnosing inflammatory bowel disease. • Necrotizing enterocolitis (NEC) is a devastating disease with overwhelming inflammation and immune dysregulation. WHAT IS NEW: • Increased autoantibodies were present in patients with NEC, even before typical X-ray manifestations. • Anti-myosin autoantibodies may be applied as a promising diagnostic marker for NEC.

Establishment and identification of an animal model of Hirschsprung disease in suckling mice.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital intestinal malformation. Previous HSCR animal model needs invasive operation on adult animal. The aim of this study is to establish an early-onset animal model which is consistent with the clinical manifestation of HSCR patients. METHODS: The neonatal mice were randomly divided into the benzalkonium chloride (BAC) group, treated with BAC via enema, and the control group, treated with saline. Weight changes, excretion time of carmine, CT scan, hematoxylin-eosin staining and immunofluorescence staining were used to evaluate the effect of the model. Differentially expressed genes (DEGs) in the HSCR mice were analyzed by using DAVID 6.8 database and compared with DEGs from HSCR patients. RESULTS: The weight of mice was lower and the excretion time of carmine was longer in the BAC group. Moreover, distal colon stenosis and proximal colon enlargement appeared in the BAC group. Neurons in the distal colon decreased significantly after 4 weeks of BAC treatment and almost disappeared completely after 12 weeks. Transcriptome profiling of the mouse model and HSCR patients is similar in terms of altered gene expression. CONCLUSIONS: An economical and reliable HSCR animal model which has similar clinical characteristics to HSCR patients was successfully established. IMPACT: The animal model of Hirschsprung disease was first established in BALB/c mice. This model is an animal model of early-onset HSCR that is easy to operate and consistent with clinical manifestations. Transcriptome profiling of the mouse model and HSCR patients is similar in terms of altered gene expression.

Susceptibility of PCSK2 Polymorphism to Hirschsprung Disease in Southern Chinese Children.

Introduction: Hirschsprung's disease (HSCR) is a developmental defect of the enteric nervous system (ENS), which is caused by abnormal development of enteric neural crest cells. Its occurrence is caused by genetic factors and environmental factors. It has been reported that single nucleotide polymorphisms (SNPs) of proprotein convertase subtilisin/kexin type 2 (PCSK2) gene are associated with HSCR. However, the correlation of HSCR in southern Chinese population is still unclear. Methods: We assessed the association of rs16998727 with HSCR susceptibility in southern Chinese children using TaqMan SNP genotyping analysis of 2943 samples, including 1470 HSCR patients and 1473 controls. The association test between rs16998727 and phenotypes was performed using multivariable logistic regression analysis. Results: We got an unexpected result, PCSK2 SNP rs16998727 was not significantly different from HSCR and its HSCR subtypes: S-HSCR (OR = 1.08, 95% IC: 0.93~1.27, P_adj = 0.3208), L-HSCR (OR = 1.07, 95% IC: 0.84~1.36, P_adj = 0.5958) and TCA (OR = 0.94, 95% IC: 0.61~1.47, P_adj = 0.8001). Conclusion: In summary, we report that rs16998727 (PCSK2 and OTOR) is not associated with the risk of HSCR in southern Chinese population.

Plasma single-stranded DNA autoantibodies in the diagnosis of Hirschsprung's disease.

Background: Hirschsprung's disease (HSCR) is a neonatal enteric nervous system (ENS) disease characterized by congenital enteric ganglion cell loss. The only treatment is aganglionic bowel segment resection and innervated bowel segment reconstruction. Delayed diagnosis and treatment cause postoperative complications such as intractable constipation and enterocolitis. Existing preoperative HSCR diagnostic methods have shortcomings such as false positives, radiation and invasiveness. Methods: We used the robust linear model (RLM) for normalization and the M statistic for screening plasma human autoimmune antigen microarrays and quantitatively assessed single-stranded DNA (ssDNA) antibody levels with enzyme-linked immunosorbent assay (ELISA). Results: The autoimmune antigen microarray revealed that autoantibodies were higher in HSCR plasma than in disease control (DC) and healthy control (HC) plasma. ssDNA antibodies in HSCR plasma were significantly higher than those in DC and HC plasma. Quantitative ssDNA antibody level detection in plasma by ELISA showed that HSCR (n = 32) was 1.3- and 1.7-fold higher than DC (n = 14) and HC (n = 25), respectively. ssDNA antibodies distinguished HSCR from non-HSCR (HC and DC), achieving an area under the curve (AUC) of 0.917 (95% CI, 0.8550-0.9784), with a sensitivity of 96.99% and a specificity of 74.63%. Conclusion: ssDNA antibodies in plasma can serve as a diagnostic biomarker for HSCR in the clinic.

Association between IKBKAP polymorphisms and Hirschsprung's disease susceptibility in Chinese children.

Background: Hirschsprung's disease (HSCR) is a rare congenital disease in which enteric nervous system (ENS) in the distal intestine is absent. HSCR is a disease involving genetic factors and environmental factors. Despite a series of genes have been revealed to contribute to HSCR, many HSCR associated genes were yet not identified. Previous studies had identified that a potential susceptibility gene of HSCR was an inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein (IKBKAP). The study aimed to explore the association of genetic variants in IKBKAP and HSCR susceptibility in southern Chinese children. Methods: Single nucleotide polymorphism (SNPs) were genotyped by the Mass ARRAY iPLEX Gold system (Sequenom, San Diego, CA, USA) on all samples, which included 1,470 HSCR children (cases) and 1,473 healthy children (controls). The associations between SNPs and HSCR or clinical subtypes were assessed by comparing their allele frequencies in corresponding case and control samples. Different genetic models, including additive, recessive, and dominant models, were tested using PLINK 1.9 software. Results: Further subgroup analysis revealed rs2275630 as a total colonic aganglionosis (TCA)-specific susceptibility locus. The present study is the first to indicate that IKBKAP rs2275630 were associated with HSCR susceptibility, especially in TCA patients. Conclusions: We conclude that IKBKAP rs2275630 is a susceptibility gene of HSCR.

Susceptibility of ECE1 polymorphisms to Hirschsprung's disease in southern Chinese children.

Background: Hirschsprung's disease (HSCR) is currently considered to be a congenital gastrointestinal malformation caused mainly by genetic factors. Endothelin Converting Enzyme-1 (ECE1) has been reported to be associated with HSCR. However, the relationship between ECE1 single nucleotide polymorphism (SNP) rs169884 and HSCR in the southern Chinese population remains unknown. Methods: 1,470 HSCR patients and 1,473 controls from a southern Chinese population were recruited. The intronic SNP rs169884 in ECE1 was genotyped in all samples. We tested the association between rs169884 and HSCR under various genetic models. We also evaluated the effect of rs169884 on HSCR subtypes, including short-segment HSCR (S-HSCR), long-segment HSCR (L-HSCR) and total colonic aganglionosis (TCA). External epigenetic data were integrated to investigate the potential biological function of rs169884. Results: Chromatin states data from derived neuron cells or fetal colon tissue revealed that rs169884 might control ECE1 expression through regulating its enhancer function. We did not find a significant association between rs169884 and HSCR. For HSCR subtypes, although no significant associations were detected between rs169884 and S-HSCR (OR = 1.00, 95% CI: 0.89∼1.12, Padj = 0.77) or TCA (OR = 1.00, 95% CI: 0.72∼1.38, Padj = 0.94), we found that rs169884 could increase the risk of L-HSCR (OR = 1.23, 95% CI 1.02∼1.45, Padj = 0.024). Conclusion: These results suggested that rs169884 might play a regulatory role for ECE1 expression and increase susceptibility of L-HSCR in southern Chinese children.

Associations of CYP2B6 genetic polymorphisms with Hirschsprung's disease in a southern Chinese population.

BACKGROUND: Hirschsprung's disease (HSCR) is an enteric nervous system birth defect partially caused by a genetic disorder. Single-nucleotide polymorphisms (SNPs) of the cytochrome P450 family 2 subfamily B member 6 (CYP2B6) gene are reported to be associated with HSCR. METHODS: We evaluated the association of rs2054675, rs707265, and rs1042389 with HSCR susceptibility in southern Chinese children including 1470 HSCR patients and 1473 controls using the TaqMan SNP Genotyping Assay. RESULTS: rs2054675 C allele and the rs707265 G allele were risk SNPs for total colonic aganglionosis (OR = 1.82, 95% CI 1.29 ~ 2.55, P_adj < 0.001 and OR = 0.68, 95% CI 0.48 ~ 0.97, P_adj = 0.034). These results suggested that CYP2B6 rs2054675 and rs707265 polymorphisms were associated with increased susceptibility to the severe HSCR subtype in southern Chinese children. CONCLUSION: We suggest that CYP2B6 rs2054675 and rs707265 polymorphisms are associated with increased susceptibility to the severe HSCR subtype in southern Chinese children.

Association between ABHD1 and DOK6 polymorphisms and susceptibility to Hirschsprung disease in Southern Chinese children.

Hirschsprung disease (HSCR) is an infrequent congenital intestinal dysplasia. The known genetic variations are unable to fully explain the pathogenesis of HSCR. The α/ß-hydratase domain 1 (ABHD1) interferes with the proliferation and migration of intestinal stem cells. Docking protein 6 (DOK6) is involved in neurodevelopment through RET signalling pathway. We examined the association of ABHD1 and DOK6 genetic variants with HSCR using 1470 controls and 1473 HSCR patients from Southern Chinese children. The results clarified that DOK6 rs12968648 G allele significantly increased HSCR susceptibility, in the allelic model (p = 0.034; OR = 1.12, 95%CI = 1.01~1.24) and the dominant model (p = 0.038; OR = 1.12, 95%CI = 1.01~1.25). Clinical stratification analysis showed that rs12968648 G allele was associated with increased risk of short-segment HSCR (S-HSCR), in the allelic model (p = 0.028; OR = 1.14, 95%CI = 1.01~1.28) and the additive model (p = 0.030; OR = 1.14, 95%CI = 1.01~1.28). ABHD1 rs2304678 C allele had higher risk to develop total colonic aganglionosis (TCA) in the allelic model (p = 7.04E-03; OR = 1.67, 95%CI = 1.15~2.43) and the dominant model (p = 4.12E-03; OR = 1.93, 95%CI = 1.23~3.04). DOK6 rs12968648 and ABHD1 rs2304678 had significant intergenic synergistic effect according to logical regression (p = 0.0081; OR = 0.76, 95%CI = 0.63~0.93) and multifactor dimensionality reduction (MDR, p = 0.0045; OR = 1.25, 95%CI = 1.07~1.46). This study verified two susceptible variations of HSCR on ABHD1 and DOK6. Their roles in HSCR should be conducted in further studies.

Significant Association of rs2147555 Genetic Polymorphism in the EDNRB Gene with Hirschsprung Disease in Southern Chinese Children.

Hirschsprung disease (HSCR) is a human birth defect at the clinical setting, usually characterized by an absent enteric nervous system (ENS) from the distal bowel. The majority of HSCR cases represent a complex disorder resulting from the interaction of multiple genetic and environmental factors. Genetic events have been described to be involved in the abnormal development of the enteric nervous system. Although variants in several genes like RET and EDNRB have been suggested to contribute major risks to HSCR, very little is known about their involvement in the onset of HSCR. Here, we studied a large Chinese Han cohort consisting of 1,470 HSCR patients and 1,473 non-HSCR controls to further test whether there are more variants in EDNRB associated with HSCR. Our results provided the first evidence that rs2147555 in EDNRB confers a significant risk of HSCR in a Chinese Han population for both allelic frequencies (P = 4.16 × 10-3; OR = 1.29) and genotypic frequencies assuming either a dominant or recessive model (P = 0.011 and P = 0.027, respectively). When different subtypes of HSCR cases were analyzed, the association remained significant (OR = 1.33, P = 0.003 for short-segment HSCR; OR = 1.34, P = 0.044 for long segment HSCR).