Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Novel inherited CDX2 variant segregating in a family with diverse congenital malformations of the genitourinary system.

Anorectal malformations (ARMs) constitute a group of congenital defects of the gastrointestinal and urogenital systems. They affect males and females, with an estimated worldwide prevalence of 1 in 5000 live births. These malformations are clinically heterogeneous and can be part of a syndromic presentation (syndromic ARM) or as a nonsyndromic entity (nonsyndromic ARM). Despite the well-recognized heritability of nonsyndromic ARM, the genetic etiology in most patients is unknown. In this study, we describe three siblings with diverse congenital anomalies of the genitourinary system, anemia, delayed milestones, and skeletal anomalies. Genome sequencing identified a novel, paternally inherited heterozygous Caudal type Homeobox 2 (CDX2) variant (c.722A > G (p.Glu241Gly)), that was present in all three affected siblings. The variant identified in this family is absent from population databases and predicted to be damaging by most in silico pathogenicity tools. So far, only two other reports implicate variants in CDX2 with ARMs. Remarkably, the individuals described in these studies had similar clinical phenotypes and genetic alterations in CDX2 CDX2 encodes a transcription factor and is considered the master regulator of gastrointestinal development. This variant maps to the homeobox domain of the encoded protein, which is critical for interaction with DNA targets. Our finding provides a potential molecular diagnosis for this family's condition and supports the role of CDX2 in anorectal anomalies. It also highlights the clinical heterogeneity and variable penetrance of ARM predisposition variants, another well-documented phenomenon. Finally, it underscores the diagnostic utility of genomic profiling of ARMs to identify the genetic etiology of these defects.

Regulation of Shh/Bmp4 Signaling Pathway by DNA Methylation in Rectal Nervous System Development of Fetal Rats with Anorectal Malformation.

OBJECTIVE: To study the influence of gene methylation in the Shh/Bmp4 signaling pathway on the enteric nervous system in the rectum of rat embryos with anorectal malformations (ARMs). METHODS: Pregnant Sprague Dawley rats were divided into three groups; two groups treated with either ethylene thiourea (ETU induce ARM) or ETU+5-azacitidine (5-azaC inhibit DNA methylation) and a normal control group. The levels of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b), the methylation status of the Shh gene promoter region and the expression of the key components were detected by PCR, immunohistochemistry and western blotting. RESULTS: The expression of DNMTs in the rectal tissue of the ETU and ETU+5-azaC groups was higher than that of the control. The expression of DNMT1, DNMT3a and methylation level of the Shh gene promoter in the ETU group was higher than in the ETU+5-azaC group (P < 0.01). The methylation level of the Shh gene promoter was higher in the ETU+5-azaC group than in the control. The Shh and Bmp4 expression in the ETU and ETU+5-azaC groups were lower than in the control, and their expression in the ETU group was also lower than in the ETU+5-azaC group. CONCLUSION: The methylation status of genes in the rectum of the ARM rat model may be changed by intervention. The low methylation level of the Shh gene may promote the expression of key Shh/Bmp4 signaling pathway components.

High molecular diagnostic yields and novel phenotypic expansions involving syndromic anorectal malformations.

Evidence suggests that genetic factors contribute to the development of anorectal malformations (ARMs). However, the etiology of the majority of ARMs cases remains unclear. Exome sequencing (ES) may be underutilized in the diagnostic workup of ARMs due to uncertainty regarding its diagnostic yield. In a clinical database of ~17,000 individuals referred for ES, we identified 130 individuals with syndromic ARMs. A definitive or probable diagnosis was made in 45 of these individuals for a diagnostic yield of 34.6% (45/130). The molecular diagnostic yield of individuals who initially met criteria for VACTERL association was lower than those who did not (26.8% vs 44.1%; p = 0.0437), suggesting that non-genetic factors may play an important role in this subset of syndromic ARM cases. Within this cohort, we identified two individuals who carried de novo pathogenic frameshift variants in ADNP, two individuals who were homozygous for pathogenic variants in BBS1, and single individuals who carried pathogenic or likely pathogenic variants in CREBBP, EP300, FANCC, KDM6A, SETD2, and SMARCA4. The association of these genes with ARMs was supported by previously published cases, and their similarity to known ARM genes as demonstrated using a machine learning algorithm. These data suggest that ES should be considered for all individuals with syndromic ARMs in whom a molecular diagnosis has not been made, and that ARMs represent a low penetrance phenotype associated with Helsmoortel-van der Aa syndrome, Bardet-Biedl syndrome 1, Rubinstein-Taybi syndromes 1 and 2, Fanconi anemia group C, Kabuki syndrome 2, SETD2-related disorders, and Coffin-Siris syndrome 4.

Genome-wide identification of disease-causing copy number variations in 450 individuals with anorectal malformations.

Anorectal malformations (ARM) represent a spectrum of rare malformations originating from a perturbated development of the embryonic hindgut. Approximately 60% occur as a part of a defined genetic syndrome or within the spectrum of additional congenital anomalies. Rare copy number variations (CNVs) have been associated with both syndromic and non-syndromic forms. The present study represents the largest study to date to explore the contribution of CNVs to the expression of ARMs. SNP-array-based molecular karyotyping was applied in 450 individuals with ARM and 4392 healthy controls. CNVs were identified from raw intensity data using PennCNV. Overlapping CNVs between cases and controls were discarded. Remaining CNVs were filtered using a stringent filter algorithm of nine filter steps. Prioritized CNVs were confirmed using qPCR. Filtering prioritized and qPCR confirmed four microscopic chromosomal anomalies and nine submicroscopic CNVs comprising seven microdeletions (del2p13.2, del4p16.2, del7q31.33, del9p24.1, del16q12.1, del18q32, del22q11.21) and two microduplications (dup2p13.2, dup17q12) in 14 individuals (12 singletons and one affected sib-pair). Within these CNVs, based on their embryonic expression data and function, we suggest FOXK2, LPP, and SALL3 as putative candidate genes. Overall, our CNV analysis identified putative microscopic and submicroscopic chromosomal rearrangements in 3% of cases. Functional characterization and re-sequencing of suggested candidate genes is warranted.

miR-141-3p affects ß-catenin signaling and apoptosis by targeting Ubtd2 in rats with anorectal malformations.

Anorectal malformations (ARMs) are the most common gastrointestinal malformations. miR-141-3p was obtained from whole-transcriptome sequencing, and Ub domain-containing protein 2 (Ubtd2) was predicted as the target gene. An ARM rat model was induced using ethylenethiourea. Fluorescence in situ hybridization and immunofluorescence were used to detect the spatiotemporal expression of miR-141-3p and Ubtd2, respectively. A dual-luciferase reporter assay confirmed their targeting relationship, and cell proliferation and apoptosis were investigated after transfection in the intestinal epithelium (IEC-6). Additionally, western blotting and co-immunoprecipitation were used to examine the protein levels and the endogenous binding relationship. miR-141-3p was downregulated in the ARM group, whereas Ubtd2 increased and colocalized with TUNEL-positive cells. After miR-141-3p inhibition, protein expression of USP5 and ß-catenin was affected via Ubtd2, and USP5 could bind to both Ubtd2 and ß-catenin. Flow cytometry analysis and caspase 3/7 staining demonstrated that downregulated miR-141-3p promoted cell apoptosis through Ubtd2. In summary, targeting Ubtd2 decreased in miR-141-3p and promoted apoptosis of intestinal epithelium and regulated ß-catenin expression. This may cause aberrant apoptosis during hindgut development and mediate the imbalance of ß-catenin signaling in the cloaca, further affecting the occurrence of ARMs.

Clinical and genetic approach in the characterization of newborns with anorectal malformation.

OBJECTIVE: This study aimed to investigate clinical, surgical, and genetic data of neonates with anorectal malformation (ARM). STUDY DESIGN: A retrospective observational study was conducted on neonates with ARM as an isolated type (group 1), with ≤2 (group 2), and with ≥3 associated malformations (group 3), born between 2009 and 2020. Distribution of ARM, associated abnormalities and genetic testing were analyzed, and risk factors for adverse outcomes were identified. RESULTS: The 45 ARM cases (36% females) were divided as follows: 13 neonates belonging to group 1 (29%), 8 to group 2 (18%), and 24 to group 3 (53%). Cases were equally distributed over 11 years. Krickenbeck anatomy was: without fistula/imperforate anus (18%), perineal fistula (36%), rectourethral fistula (4%), rectovesical fistula (2%), vestibular fistula (4%), cloaca (4%), and rare ARMs (31%). Groups showed differences in anthropometric data, Krickenbeck anatomy, and intensive care burden. Additional major congenital abnormalities were prevalent specific of VATER/VACTERL spectrum (vertebral/anorectal/cardiac/tracheoesophageal/renal/limb defects), but also Hirschsprung disease was found in 3/20 biopsies (15%). The most frequent minor abnormality was a single umbilical artery. In group 3, we identified four de novo microdeletions at 8p23.2, 8q13.3, Xp22.31-p22.2, Xq28, four de novo microduplications at 1p36.32, 6p24.1-p23, 13q14.11, 15q11.2, one microdeletion at 9q33.1 inherited from the affected mother, one microdeletion at 7q35 inherited from the unaffected father, one structurally uncharacterized rearrangement involving 9p23-q34.3. Thus, we attributed the Xq28 deletion with inactivated FAM58A gene in one girl to the X-linked dominant STAR syndrome (toe syndactyly-telecanthus-anogenital/renal malformations). CONCLUSIONS: Despite the great physical and social burden on ARM patients and their parents, in the majority of cases, the etiology is largely unknown and attributed to be multifactorial. In females, STAR syndrome should be part of the differential diagnosis. Associated malformations of other organ systems interact in outcome parameters.

Transcriptome analysis reveals the spinal expression profiles of non-coding RNAs involved in anorectal malformations in rat fetuses.

BACKGROUND: Despite improvements in anorectal malformation (ARM) therapy, patients might still experience post-operative problems such as fecal incontinence, constipation, and soiling. In particular, the dysplasia of the lumbosacral spinal cord in ARM patients is a major disorder that affects fecal function post-operation. However, the pathological mechanisms involved are still unclear. METHODS: The non-coding RNAs (ncRNAs) in the lumbosacral spinal cord of fetal rats with ethylenethiourea-induced ARM were identified using RNA sequencing (RNA-seq) and examined to determine their potential function. The lumbosacral spinal cord was isolated on embryonic day 17 for subsequent RNA extraction and RNA-seq. The transcriptome data was analyzed using bioinformatics analysis, followed by validation using quantitative reverse transcription PCR. RESULTS: Compared to the control group, 26 differentially expressed microRNAs (DEMs; 22 upregulated, 4 downregulated) and 112 differentially expressed long non-coding RNAs (63 upregulated, 49 downregulated) were identified in the ARM group. Several DEMs related to development, namely miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-200a-5p, and miR-429, were selected for further analysis. Notably, compared to the control, the relative expression of miR-200 family members was highly upregulated in ARM fetal rats. Furthermore, GO and KEGG enrichment and miRNA-transcription factor-lncRNA/mRNA network analysis was explored to show molecular mechanism underlying DEMs. CONCLUSIONS: Our findings suggest the involvement of ncRNAs, especially the miR-200 family members, in the pathogenesis of lumbosacral spinal cord dysplasia in ARM fetal rats.

Re-sequencing of candidate genes FOXF1, HSPA6, HAAO, and KYNU in 522 individuals with VATER/VACTERL, VACTER/VACTERL-like association, and isolated anorectal malformation.

BACKGROUND: The acronym VATER/VACTERL association describes the combination of at least three component features (CFs): vertebral defects (V), anorectal malformations (ARM) (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb defects (L). Individuals presenting two CFs have been termed VATER/VACTERL-like. Recently, FOXF1, HSPA6, HAAO, KYNU, TRAP1, and ZIC3 have been proposed as candidate genes for VATER/VACTERL, VATER/VACTERL-like, and ARM. Re-sequencing studies identified disease-causing variants in TRAP1 and ZIC3, the contribution of other genes was not independently investigated. One affected variant carrier in FOXF1 was previously identified. Here we re-sequenced FOXF1, HSPA6, HAAO, and KYNU in 522 affected individuals. METHODS: Using molecular inversion probe (MIP) technology, re-sequencing was performed in 63 individuals with VATER/VACTERL association, 313 with VATER/VACTERL-like association, and 146 with ARM. All individuals were of European ethnicity. Variant filtering considered variants with a minor allele frequency (MAF) ≤0.01 for putative recessive disease-genes HSPA6, HAAO, and KYNU. For the putative dominant disease-gene FOXF1 we considered variants with a MAF ≤0.0001. In silico prediction tools were used for further prioritization. RESULTS: Only two variants in FOXF1 in two independently affected individuals [c.443G>T, p.(Cys148Phe); c.850T>C, p.(Tyr284His)] passed our filter criteria. One individual presented with ARM, the second presented with TE and C comprising atrial and ventricular septal defects. Sanger sequencing confirmed both variants but also their inheritance from the healthy mother. CONCLUSION: Our analysis suggests that FOXF1, HSPA6, HAAO and KYNU do not play a major role in the formation of VACTER/VACTERL phenotypes or ARM.

Wnt5a plays a critical role in anal opening in mice at an early stage of embryonic development.

PURPOSE: Anorectal malformations are associated with other organ malformations. Proximodistal elongation of the cloacal plate and anal opening at its distal end are essential for anal development. However, the anal developmental stage in which Wnt5a is directly involved remains unelucidated. Here, we attempted to identify this developmental stage; since Wnt5a is expressed in the mesoderm, and the striated muscle complex (SMC) in mice develops from the mesoderm, we also examined Wnt5a contribution to SMC development. METHODS: We established conditional knockout (CKO) mice in which Wnt5a could be knocked out using an appropriate tamoxifen dose. We evaluated the macroscopic appearance and histopathological features of Wnt5aCKO and wild-type mouse embryos. RESULTS: Wnt5aCKO mice showed phenotypes typical of Wnt5a constitutional knockout mice when Wnt5a was knocked out at E8-E11. Furthermore, the anus failed to open when Wnt5a was knocked out at E8 but opened when it was knocked out at E9 or thereafter. The caudal end of the SMC was dysplastic in Wnt5aCKO mice induced at E8, but was unaffected when mice were induced at E9 or thereafter. CONCLUSION: We suggest a critical role for Wnt5a in anal opening and SMC formation at a very early stage of embryonic development.

The Role of De Novo Variants in Formation of Human Anorectal Malformations.

Anorectal malformations (ARM) represent a rare birth defect of the hindgut that occur in approximately 1 in 3000 live births. Around 60% of ARM occur with associated anomalies including defined genetic syndromes and associations with chromosomal aberrations. The etiology of ARM is heterogeneous, with the individual environmental or genetic risk factors remaining unknown for the majority of cases. The occurrence of familial ARM and previous epidemiologic analysis suggest autosomal dominant inheritance in a substantial subset of ARM patients. The implicated mortality and reduced fecundity in patients with ARM would lead to allele loss. However, mutational de novo events among the affected individuals could compensate for the evolutionary pressure. With the implementation of exome sequencing, array-based molecular karyotyping and family-based rare variant analyses, the technologies are available to identify the respective factors. This review discusses the identification of disease-causing variants among individuals with ARM. It highlights the role of mutational de novo events.

Exome survey of individuals affected by VATER/VACTERL with renal phenotypes identifies phenocopies and novel candidate genes.

The acronym VATER/VACTERL refers to the rare nonrandom association of the following component features (CFs): vertebral defects (V), anorectal malformations (ARM) (A), cardiac anomalies (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb anomalies (L). For the clinical diagnosis, the presence of at least three CFs is required, individuals presenting with only two CFs have been categorized as VATER/VACTERL-like. The majority of VATER/VACTERL individuals displays a renal phenotype. Hitherto, variants in FGF8, FOXF1, HOXD13, LPP, TRAP1, PTEN, and ZIC3 have been associated with the VATER/VACTERL association; however, large-scale re-sequencing could only confirm TRAP1 and ZIC3 as VATER/VACTERL disease genes, both associated with a renal phenotype. In this study, we performed exome sequencing in 21 individuals and their families with a renal VATER/VACTERL or VATER/VACTERL-like phenotype to identify potentially novel genetic causes. Exome analysis identified biallelic and X-chromosomal hemizygous potentially pathogenic variants in six individuals (29%) in B9D1, FREM1, ZNF157, SP8, ACOT9, and TTLL11, respectively. The online tool GeneMatcher revealed another individual with a variant in ZNF157. Our study suggests six biallelic and X-chromosomal hemizygous VATER/VACTERL disease genes implicating all six genes in the expression of human renal malformations.

Upregulation of PPPDE1 contributes to anorectal malformations via the mitochondrial apoptosis pathway during hindgut development in rats.

Congenital anorectal malformations (ARMs) are among the most prominent deformities of the gastrointestinal tract; however, their precise aetiology remains obscure. Immunohistochemistry demonstrated that, in the ARM group, the PPPDE1-positive cells were widely distributed in the hindgut epithelial tissue from GD13 to GD16. Immunofluorescence revealed that most TUNEL-, Bax-, and Cytochrome C (Cyt C)-positive cells overlapped with PPPDE1-positive cells in the urorectal septum (URS). Western blotting and quantitative real-time RT-PCR revealed that PPPDE1 levels were significantly higher in the ARM group from GD13 to GD14 (p < 0.05). IEC-6 cells were transfected with PPPDE1 overexpression plasmid/NC (negative control) or si-PPPDE1/si-NC. Flow cytometry analysis and CCK-8 assay (used to detect apoptosis and proliferation, respectively), as well as western blotting, showed that the levels of PPPDE1 were positively correlated with the pro-apoptotic molecules Bax and Cyt C. Accordingly, aberrantly high expression of PPPDE1 caused a spatiotemporal imbalance in foetal rats with ARMs during hindgut development. Therefore, the upregulation of PPPDE1 may promote epithelial apoptosis and reduce proliferation in the hindgut via the mitochondrial apoptotic pathway. This could affect the fusion of the URS and cloacal membrane, ultimately inhibiting the hindgut development and resulting in ARMs.

Presacral neuroendocrine tumors associated with the Currarino syndrome.

Currarino syndrome (CS) is an autosomal dominant syndrome caused by mutations in MNX1 and characterized by anorectal abnormalities, partial sacral agenesis, and presacral masses. The presacral masses are typically benign; however, malignant degeneration can occur, and presacral neuroendocrine tumors (NETs) have been reported in six cases. We report three individuals from two families affected by CS in which multiple individuals developed presacral NETs. The first family, 491, had six members with features of CS, including two siblings who presented with presacral, Grade 2 NETs, one of which had metastasized to bone and lymph nodes. A germline c.874C>T (p.Arg292Trp) mutation was found in a highly conserved region of MNX1 in three affected members who underwent sequencing. A second somatic variant/deletion in MNX1 was not detected in either patient's tumor. In the second family, 342, the proband presented with an incidentally discovered presacral NET. The proband's father had previously undergone resection of a presacral NET, and so genetic testing was performed, which did not reveal an MNX1 mutation or copy number variants. The lack of a second, somatic mutation in the tumors from family 491 argues against MNX1 acting as a tumor suppressor, and the absence of a germline MNX1 mutation in family 342 suggests that other genetic and anatomic factors contribute to the development of presacral NETs. These cases highlight the variable presentation of CS, and the potential for malignancy in these patients.

Altered mRNA and lncRNA expression profiles in the striated muscle complex of anorectal malformation rats.

BACKGROUND: Striated muscle complex (SMC) dysplasia has been confirmed to contribute to postoperative defecation dysfunction of patients with anorectal malformations (ARMs). To date, the potential molecular mechanisms of SMC dysplasia underlying the development of ARMs have not been clearly explained. This study examined the expression profiles of mRNAs and lncRNAs in the malformed SMC of ARM rats using RNA sequencing (RNA-seq). METHODS: A rat model of ARMs was established by the intragastric administration of 1% ethylene thiourea (ETU) on an embryonic day 10 (E10). The rats were subjected to euthanasia and the SMC samples were collected on E19. The expression of mRNAs and lncRNAs was analyzed by RNA-seq on the Illumina HiSeq2500 platform. qRT-PCR was used to confirm the results of RNA-seq. RESULTS: Compared with the levels in control rats, 1408 mRNAs and 472 lncRNAs were differentially expressed in the SMC of E19 ARM rats. GO and KEGG pathway analyses showed that the top enriched GO terms were mainly related to muscle development and the enriched pathways were associated with muscle and synaptic development. Protein-protein interaction network analysis was also performed using the STRING database. The network map revealed the interaction between the WNT3 protein and NTRK1, NTF4, MUSK, and BMP5 proteins. Finally, the qRT-PCR results further confirmed the RNA-seq data. CONCLUSION: Our findings indicate the involvement of these dysregulated mRNAs and lncRNAs in the pathogenesis of SMC dysplasia in ARMs, providing a theoretical foundation for developing interventions to improve postoperative defecation function.

Rno_circ_0005139 regulates apoptosis by targeting Wnt5a in rat anorectal malformations.

BACKGROUND: The molecular mechanisms underlying anorectal malformations (ARM) are not fully established. Circular RNAs (circRNAs) are new born non-coding RNAs, and their role in ARM is unclear. We assumed that rno_circ_0005139 influences apoptosis and proliferation by acting as a miR-324-3p sponge, and downregulating Wnt5a in ARM. AIM: To identify the differential expression of circRNAs and mRNAs in a rat ARM model. METHODS: Sixty-six pregnant Wistar rats were randomly divided into two groups: ARM group (2-imidazolidinethione-induced) and control groups. Embryos were harvested by cesarean delivery, and anorectal tissue was taken on embryonic days 16 (E16), 17 (E17), 19 (E19), and 21 (E21). RNA sequencing and gene microarray analysis was used to identify differentially expressed circRNAs and mRNAs in the ARM in a rat model. We selected 6 circRNAs and 3 mRNAs in the Wnt signal pathway from the result of the RNA sequencing and gene microarray analysis, and quantitative reverse transcription polymerase chain reaction was performed to evaluate their tissue expression. According to bioinformatics prediction, rno_circ_0005139 acted as a miR-324-3p sponge to regulate the expression of Wnt5a. We chose rno_circ_0005139 and Wnt5a as the final candidates. We tested the function of rno_circ_0005139 and the binding sites between rno_circ_0005139 and miR-324-3p, miR-324-3p and Wnt5a by luciferase assays. Co-transfection of rno_circ_0005139 and miR-324-3p was to verify their functional consistency. RESULTS: We identified 38 upregulated and 42 downregulated circRNAs on E17 (P < 0.05), and 301 mRNAs were upregulated and 256 downregulated in the ARM on E17 (P < 0.05, fold-change > 2.0). We found that rno_circ_0006880 and rno_circ_0011386 were upregulated, whereas rno_circ_0000436, rno_circ_0005139, rno_circ_0009285, rno_circ_0014367, Wnt5a, Wnt10b, and Wnt2b were downregulated in ARM tissues. According to bioinformatics prediction, rno_circ_0005139 acted as a miR-324-3p sponge to regulate the expression of Wnt5a. We chose rno_circ_0005139 and Wnt5a as the final candidates. Because the role and molecular mechanism of rno_circ_0005139 are poorly understood, its effect on apoptosis and proliferation was investigated by in vitro plasmid transfection. A luciferase experiment showed that rno_circ_0005139 could bind with miR-324-3p, which negatively regulated Wnt5a expression. The expression of miR-324-3p was significantly higher in ARM anorectal tissues than that in control group on E17 and E19; Wnt5a expression showed the opposite trend. In addition, a miR-324-3p inhibitor attenuated the effects of rno_circ_0005139 knockdown on ARM development. CONCLUSION: Rno_circ_0005139 influences cell proliferation and apoptosis by acting as a miR-324-3p sponge, thereby downregulating Wnt5a in ARM. Accordingly, rno_circ_0005139, miR-324-3p, and Wnt5a could be targeted therapeutic factors for ARM.

Further delineation of CDC45-related Meier-Gorlin syndrome with craniosynostosis and review of literature.

Meier-Gorlin syndrome (MGS) is a rare autosomal recessive disorder characterized by the triad of short stature, microtia and absent or small patellae. We report on a patient with MGS secondary to biallelic mutations in CDC45 detected on whole exome sequencing (WES). Patients with MGS caused by mutations in CDC45 display a distinct phenotype characterized by craniosynostosis and anorectal malformation. Our patient had craniosynostosis, anorectal malformation and short stature, but did not have the microtia or patella hypoplasia. Our report also highlights the value of WES in aiding diagnosis of patients with rare genetic diseases. In conclusion, our case report and review of the literature illustrates the unique features of CDC45-related MGS as well as the benefits of WES in reducing the diagnostic odyssey for patients with rare genetic disorders.

Pathogenic copy number variants are detected in a subset of patients with gastrointestinal malformations.

BACKGROUND: Gastrointestinal atresias and urological defects are main causes of pediatric surgery in infants. As copy number variants (CNVs) have been shown to be involved in the development of congenital malformations, the aim of our study was to investigate the presence of CNVs in patients with gastrointestinal and urological malformations as well as the possibility of tissue-specific mosaicism for CNVs in the cohort. METHODS: We have collected tissue and/or blood samples from 25 patients with anorectal malformations, esophageal atresia, or hydronephrosis, and screened for pathogenic CNVs using array comparative genomic hybridization (array-CGH). RESULTS: We detected pathogenic aberrations in 2/25 patients (8%) and report a novel possible susceptibility region for esophageal atresia on 15q26.3. CNV analysis in different tissues from the same patients did not reveal evidence of tissue-specific mosaicism. CONCLUSION: Our study shows that it is important to perform clinical genetic investigations, including CNV analysis, in patients with congenital gastrointestinal malformations since this leads to improved information to families as well as an increased understanding of the pathogenesis.

Is Pouch Specific to Colon and Not Ileum?

BACKGROUND: Congenital Pouch Colon (CPC) is an anorectal anomaly with an incidence of 3.5:1 in males and females, respectively. We have earlier reported CPC to be quite prevalent in north Indian tertiary care centers. OBJECTIVE: In this article, we deliberate on the possible causes associated with CPC bringing the manifestation of the disease. In addition, we throw insights on the effective role of this congenital anomaly in Colon and provide systems genomic evaluation by comparing our recent analysis to that of Colon and Ileum based on Next-Generation Sequencing (NGS) studies. CONCLUSION: In this commentary article, we argue that a host of epigenetic factors could be the reason why the disease is manifested in colon alone. We further hypothesize on the few unmet challenges linking epigenetics to understand the genetic variants.