RESUMEN
BACKGROUND: Townes-Brocks syndrome (TBS) is a rare genetic disorder characterized by imperforate anus, dysplastic ears, thumb malformations, and other abnormalities. Previous studies have revealed that mutations in the SALL1 gene can disrupt normal development, resulting in the characteristic features of Townes-Brocks syndrome. Spalt-like transcription factors (SALLs) are highly conserved proteins that play important roles in various cellular processes, including embryonic development, cell differentiation, and cell survival. Over 400 different variants or mutations have been reported in the SALL1 gene in individuals with TBS. Most of these variants lead to the formation of premature termination codons (PTCs), also known as nonsense mutations. The majority of these PTCs occur in a specific region of the SALL1 gene called the "hotspot region", which is particularly susceptible to mutation. METHODS: In this study, we conducted whole-exome sequencing on a three-generation Chinese family with anorectal malformations. RESULTS: We identified a novel heterozygous mutation (chr16:51175376:c.757 C > T p.Gln253*) in the SALL1 gene. Molecular analysis revealed a heterozygous C to T transition at nucleotide position 757 in exon 2 of the SALL1 (NM_002968) gene. This mutation is predicted to result in the substitution of the Gln253 codon with a premature stop codon (p.Gln253*). The glutamine-rich domain forms a long alpha helix, enabling the mutant protein to interact with the wild-type SALL1 protein. This interaction may result in steric hindrance effects on the wild-type SALL1 protein. CONCLUSIONS: Our findings have expanded the mutation database of the SALL1 gene, which is significant for genetic counseling and clinical surveillance in the affected family. Furthermore, our study enhances the understanding of Townes-Brocks syndrome and has the potential to improve its diagnosis and treatment.
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Anomalías Múltiples , Ano Imperforado , Linaje , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Anomalías Múltiples/genética , Ano Imperforado/genética , Femenino , Masculino , China , Mutación , Enfermedades Raras/genética , Malformaciones Anorrectales/genética , Pueblo Asiatico/genética , Pueblos del Este de Asia , Pérdida Auditiva Sensorineural , Pulgar/anomalíasRESUMEN
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.
Asunto(s)
Malformaciones Anorrectales , Redes Reguladoras de Genes , Transducción de Señal , Transcriptoma , Animales , Malformaciones Anorrectales/genética , Malformaciones Anorrectales/metabolismo , Malformaciones Anorrectales/embriología , Transducción de Señal/genética , Transcriptoma/genética , Ratas , Femenino , Regulación del Desarrollo de la Expresión Génica , Embarazo , Embrión de Mamíferos/metabolismo , Perfilación de la Expresión Génica/métodos , Biología Computacional/métodos , Ratas Sprague-Dawley , Cloaca/embriología , Cloaca/metabolismoRESUMEN
Anorectal malformations (ARMs) represent a wide spectrum of congenital anomalies of the anus and rectum, of which more than half are syndromic. Their etiology is highly heterogeneous and still poorly understood. We report a 4-year-old girl who initially presented with an isolated ARM, and subsequently developed a global developmental delay as part of an ARID1B-related Coffin-Siris syndrome (CSS). A co-occurrence of ARMs and CSS in an individual by chance is unexpected since both diseases are very rare. A review of the literature enabled us to identify 10 other individuals with both CSS and ARMs. Among the ten individuals reported in this study, 8 had a variant in ARID1A, 2 in ARID1B, and 1 in SMARCA4. This more frequent than expected association between CSS and ARM indicates that some ARMs are most likely part of the CSS spectrum, especially for ARID1A-related CSS.
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Anomalías Múltiples , Malformaciones Anorrectales , Proteínas de Unión al ADN , Cara , Deformidades Congénitas de la Mano , Discapacidad Intelectual , Micrognatismo , Cuello , Factores de Transcripción , Humanos , Femenino , Micrognatismo/genética , Micrognatismo/patología , Preescolar , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Factores de Transcripción/genética , Cuello/anomalías , Cuello/patología , Deformidades Congénitas de la Mano/genética , Deformidades Congénitas de la Mano/patología , Anomalías Múltiples/genética , Anomalías Múltiples/patología , Proteínas de Unión al ADN/genética , Malformaciones Anorrectales/genética , Cara/anomalías , Cara/patología , ADN Helicasas/genética , Proteínas Nucleares/genética , Canal Anal/anomalías , Canal Anal/patología , FenotipoRESUMEN
Genital anomalies have been reported with VACTERL association but not considered a core feature. Acute and chronic complications stemming from unrecognized genital anomalies have been reported in adolescents and young adults with VACTERL association. We sought to determine the frequency and severity of genital anomalies in VACTERL patients and identify which core features were more frequently associated with genital anomalies. A retrospective chart review from January 2010 to October 2021 identified 211 patients with two or more core VACTERL features, 34% of whom had a genital anomaly. The majority of genital anomalies (83% of those in males and 90% in females) were classified as functionally significant (requiring surgical intervention or causing functional impairment). The frequency of genital anomalies in the VACTERL cohort was higher if anorectal malformations or renal anomalies were present in both males and females and if vertebral anomalies were present in females. Due to their functional significance, genital anomalies should be assessed in all patients with two or more core features of VACTERL association, especially in those with anorectal or renal anomalies. Most genital anomalies in males will be detected on physical examination but additional investigation is often needed to detect genital anomalies in females. The timing and type of investigation are subjects for future study.
Asunto(s)
Canal Anal , Esófago , Cardiopatías Congénitas , Riñón , Deformidades Congénitas de las Extremidades , Columna Vertebral , Tráquea , Humanos , Masculino , Femenino , Canal Anal/anomalías , Canal Anal/patología , Deformidades Congénitas de las Extremidades/patología , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/diagnóstico , Deformidades Congénitas de las Extremidades/epidemiología , Esófago/anomalías , Esófago/patología , Columna Vertebral/anomalías , Columna Vertebral/patología , Tráquea/anomalías , Tráquea/patología , Adolescente , Cardiopatías Congénitas/patología , Cardiopatías Congénitas/epidemiología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/diagnóstico , Riñón/anomalías , Riñón/patología , Adulto , Estudios Retrospectivos , Niño , Adulto Joven , Preescolar , Anomalías Urogenitales/epidemiología , Anomalías Urogenitales/genética , Anomalías Urogenitales/diagnóstico , Anomalías Urogenitales/patología , Lactante , Malformaciones Anorrectales/epidemiología , Malformaciones Anorrectales/genética , Malformaciones Anorrectales/diagnóstico , Malformaciones Anorrectales/patología , Genitales/anomalías , Genitales/patologíaRESUMEN
Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.
Asunto(s)
Malformaciones Anorrectales , Ferroptosis , Receptores de Cinasa C Activada , Transcriptoma , Animales , Receptores de Cinasa C Activada/metabolismo , Receptores de Cinasa C Activada/genética , Ferroptosis/genética , Ferroptosis/efectos de los fármacos , Ratas , Malformaciones Anorrectales/genética , Malformaciones Anorrectales/metabolismo , Malformaciones Anorrectales/patología , Femenino , Especies Reactivas de Oxígeno/metabolismo , Ratas Sprague-Dawley , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Etilenotiourea , Transducción de SeñalRESUMEN
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.
Asunto(s)
Malformaciones Anorrectales , Ano Imperforado , Deformidades Congénitas de las Extremidades , Masculino , Femenino , Humanos , Canal Anal/anomalías , Malformaciones Anorrectales/genética , Ano Imperforado/genética , Sistema Urogenital , Factor de Transcripción CDX2/genéticaRESUMEN
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.
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Malformaciones Anorrectales , Recto , Embarazo , Femenino , Ratas , Animales , Recto/anomalías , Malformaciones Anorrectales/genética , Ratas Sprague-Dawley , Canal Anal/anomalías , Metilación de ADN , Transducción de Señal , Sistema Nervioso/metabolismo , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismoRESUMEN
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.
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Malformaciones Anorrectales , Variaciones en el Número de Copia de ADN , Humanos , Malformaciones Anorrectales/genética , Aberraciones Cromosómicas , CariotipificaciónRESUMEN
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.
Asunto(s)
Malformaciones Anorrectales , Síndrome de Rubinstein-Taybi , Humanos , Malformaciones Anorrectales/diagnóstico , Malformaciones Anorrectales/genética , Patología Molecular , Fenotipo , Mutación del Sistema de Lectura , Síndrome de Rubinstein-Taybi/genética , ADN Helicasas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
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.
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Malformaciones Anorrectales , MicroARNs , Ubiquitinas , beta Catenina , Animales , Ratas , Malformaciones Anorrectales/genética , Apoptosis/genética , beta Catenina/genética , beta Catenina/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Hibridación Fluorescente in Situ , MicroARNs/genética , MicroARNs/metabolismo , Vía de Señalización Wnt , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
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.
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Malformaciones Anorrectales , Hipertelorismo , Sindactilia , Anomalías Urogenitales , Canal Anal/anomalías , Malformaciones Anorrectales/genética , Femenino , Humanos , Recién Nacido , Riñón/anomalías , Masculino , Dedos del Pie/anomalíasRESUMEN
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.
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Malformaciones Anorrectales , MicroARNs , ARN Largo no Codificante , Ratas , Animales , Malformaciones Anorrectales/genética , Perfilación de la Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/genética , Feto/metabolismoRESUMEN
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.
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3-Hidroxiantranilato 3,4-Dioxigenasa , Malformaciones Anorrectales , Factores de Transcripción Forkhead , Proteínas HSP90 de Choque Térmico , Deformidades Congénitas de las Extremidades , 3-Hidroxiantranilato 3,4-Dioxigenasa/genética , Canal Anal/anomalías , Malformaciones Anorrectales/genética , Esófago/anomalías , Factores de Transcripción Forkhead/genética , Proteínas HSP90 de Choque Térmico/genética , Cardiopatías Congénitas/genética , Humanos , Riñón/anomalías , Deformidades Congénitas de las Extremidades/genética , Columna Vertebral/anomalías , Tráquea/anomalíasRESUMEN
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.
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Canal Anal , Malformaciones Anorrectales , Desarrollo Embrionario , Proteína Wnt-5a , Canal Anal/anomalías , Animales , Malformaciones Anorrectales/genética , Cloaca , Desarrollo Embrionario/genética , Femenino , Ratones , Ratones Noqueados , Embarazo , Proteína Wnt-5a/genéticaRESUMEN
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.
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Malformaciones Anorrectales/genética , Variaciones en el Número de Copia de ADN , Malformaciones Anorrectales/diagnóstico , Malformaciones Anorrectales/epidemiología , Sitios Genéticos , Pruebas Genéticas/métodos , Humanos , MutaciónRESUMEN
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.
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Malformaciones Anorrectales/genética , Atresia Esofágica/genética , Predisposición Genética a la Enfermedad , Cardiopatías/genética , Fístula Traqueoesofágica/genética , Malformaciones Anorrectales/complicaciones , Malformaciones Anorrectales/patología , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/genética , Atresia Esofágica/complicaciones , Atresia Esofágica/patología , Femenino , Genes Ligados a X/genética , Estudios de Asociación Genética , Proteínas HSP90 de Choque Térmico/genética , Cardiopatías/complicaciones , Cardiopatías/patología , Hemicigoto , Proteínas de Homeodominio/genética , Humanos , Riñón/anomalías , Masculino , Receptores de Interleucina/genética , Fístula Traqueoesofágica/complicaciones , Fístula Traqueoesofágica/patología , Factores de Transcripción/genética , Secuenciación del ExomaRESUMEN
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.
Asunto(s)
Malformaciones Anorrectales/genética , Liasas de Carbono-Nitrógeno/genética , Tracto Gastrointestinal/metabolismo , Proteína X Asociada a bcl-2/genética , Animales , Malformaciones Anorrectales/patología , Apoptosis/genética , Proliferación Celular/genética , Citocromos c/genética , Embrión de Mamíferos , Desarrollo Fetal/genética , Tracto Gastrointestinal/crecimiento & desarrollo , Tracto Gastrointestinal/patología , Humanos , Mitocondrias/genética , Ratas , Transducción de Señal/genética , Activación Transcripcional/genéticaRESUMEN
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.
Asunto(s)
Anomalías Múltiples/genética , Canal Anal/anomalías , Anomalías del Sistema Digestivo/genética , Proteínas de Homeodominio/genética , Meningocele/genética , Tumores Neuroendocrinos/genética , Recto/anomalías , Región Sacrococcígea/anomalías , Sacro/anomalías , Siringomielia/genética , Factores de Transcripción/genética , Anomalías Múltiples/patología , Adulto , Anciano , Canal Anal/patología , Malformaciones Anorrectales/complicaciones , Malformaciones Anorrectales/genética , Malformaciones Anorrectales/patología , Anomalías del Sistema Digestivo/complicaciones , Anomalías del Sistema Digestivo/patología , Femenino , Pruebas Genéticas , Mutación de Línea Germinal/genética , Humanos , Masculino , Meningocele/complicaciones , Meningocele/patología , Persona de Mediana Edad , Tumores Neuroendocrinos/complicaciones , Tumores Neuroendocrinos/patología , Recto/patología , Región Sacrococcígea/patología , Sacro/patología , Siringomielia/complicaciones , Siringomielia/patologíaRESUMEN
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.