The broader phenotypic spectrum of congenital caudal abnormalities associated with mutations in the caudal type homeobox 2 gene.

The caudal type homeobox 2 (CDX2) gene encodes a developmental regulator involved in caudal body patterning. Only three pathogenic variants in human CDX2 have been described, in patients with persistent cloaca, sirenomelia and/or renal and anogenital malformations. We identified five patients with de novo or inherited pathogenic variants in CDX2 with clinical phenotypes that partially overlap with previous cases, that is, imperforate anus and renal, urogenital and limb abnormalities. However, additional clinical features were seen including vertebral agenesis and we describe considerable phenotypic variability, even in unrelated patients with the same recurrent p.(Arg237His) variant. We propose CDX2 variants as rare genetic cause for a multiple congenital anomaly syndrome that can include features of caudal regression syndrome and VACTERL. A causative role is further substantiated by the relationship between CDX2 and other proteins encoded by genes that were previously linked to caudal abnormalities in humans, for example, TBXT (sacral agenesis and other vertebral segmentation defects) and CDX1 (anorectal malformations). Our findings confirm the essential role of CDX2 in caudal morphogenesis and formation of cloacal derivatives in humans, which to date has only been well characterized in animals.

Maternal risk associated with the VACTERL association: A case-control study.

BACKGROUND: The VACTERL association (VACTERL) includes at least three of these congenital anomalies: vertebral, anal, cardiac, trachea-esophageal, renal, and limb anomalies. Assisted reproductive techniques (ART), pregestational diabetes mellitus, and chronic lower obstructive pulmonary disorders (CLOPD) have been associated with VACTERL. We aimed to replicate these findings and were interested in additional maternal risk factors. METHODS: A case-control study using self-administered questionnaires was performed including 142 VACTERL cases and 2,135 population-based healthy controls. Multivariable logistic regression analyses were performed to estimate confounder adjusted odds ratios (aOR) and 95% confidence intervals (95%CI). RESULTS: Parents who used invasive ART had an increased risk of VACTERL in offspring (aOR 4.4 [95%CI 2.1-8.8]), whereas the increased risk for mothers with CLOPD could not be replicated. None of the case mothers had pregestational diabetes mellitus. Primiparity (1.5 [1.1-2.1]) and maternal pregestational overweight and obesity (1.8 [1.2-2.8] and 1.8 [1.0-3.4]) were associated with VACTERL. Consistent folic acid supplement use during the advised periconceptional period may reduce the risk of VACTERL (0.5 [0.3-1.0]). Maternal smoking resulted in an almost twofold increased risk of VACTERL. CONCLUSION: We identified invasive ART, primiparity, pregestational overweight and obesity, lack of folic acid supplement use, and smoking as risk factors for VACTERL.

A Genetics-First Approach Revealed Monogenic Disorders in Patients With ARM and VACTERL Anomalies.

Background: The VATER/VACTERL association (VACTERL) is defined as the non-random occurrence of the following congenital anomalies: Vertebral, Anal, Cardiac, Tracheal-Esophageal, Renal, and Limb anomalies. As no unequivocal candidate gene has been identified yet, patients are diagnosed phenotypically. The aims of this study were to identify patients with monogenic disorders using a genetics-first approach, and to study whether variants in candidate genes are involved in the etiology of VACTERL or the individual features of VACTERL: Anorectal malformation (ARM) or esophageal atresia with or without trachea-esophageal fistula (EA/TEF). Methods: Using molecular inversion probes, a candidate gene panel of 56 genes was sequenced in three patient groups: VACTERL (n = 211), ARM (n = 204), and EA/TEF (n = 95). Loss-of-function (LoF) and additional likely pathogenic missense variants, were prioritized and validated using Sanger sequencing. Validated variants were tested for segregation and patients were clinically re-evaluated. Results: In 7 out of the 510 patients (1.4%), pathogenic or likely pathogenic variants were identified in SALL1, SALL4, and MID1, genes that are associated with Townes-Brocks, Duane-radial-ray, and Opitz-G/BBB syndrome. These syndromes always include ARM or EA/TEF, in combination with at least two other VACTERL features. We did not identify LoF variants in the remaining candidate genes. Conclusions: None of the other candidate genes were identified as novel unequivocal disease genes for VACTERL. However, a genetics-first approach allowed refinement of the clinical diagnosis in seven patients, in whom an alternative molecular-based diagnosis was found with important implications for the counseling of the families.

Maternal risk factors for the VACTERL association: A EUROCAT case-control study.

BACKGROUND: The VACTERL association (VACTERL) is the nonrandom occurrence of at least three of these congenital anomalies: vertebral, anal, cardiac, tracheoesophageal, renal, and limb anomalies. Despite suggestions for involvement of several genes and nongenetic risk factors from small studies, the etiology of VACTERL remains largely unknown. OBJECTIVE: To identify maternal risk factors for VACTERL in offspring in a large European study. METHODS: A case-control study was performed using data from 28 EUROCAT registries over the period 1997-2015 with case and control ascertainment through hospital records, birth and death certificates, questionnaires, and/or postmortem examinations. Cases were diagnosed with VACTERL, while controls had a genetic syndrome and/or chromosomal abnormality. Data collected included type of birth defect and maternal characteristics, such as age, use of assisted reproductive techniques (ART), and chronic illnesses. Multivariable logistic regression analyses were performed to estimate confounder adjusted odds ratios (aOR) with 95% confidence intervals (95% CI). RESULTS: The study population consisted of 329 VACTERL cases and 49,724 controls with recognized syndromes or chromosomal abnormality. For couples who conceived through ART, we found an increased risk of VACTERL (aOR 2.3 [95% CI 1.3, 3.9]) in offspring. Pregestational diabetes (aOR 3.1 [95% CI 1.1, 8.6]) and chronic lower obstructive pulmonary diseases (aOR 3.9 [95% CI 2.2, 6.7]) also increased the risk of having a child with VACTERL. Twin pregnancies were not associated with VACTERL (aOR 0.6 [95% CI 0.3, 1.4]). CONCLUSION: We identified several maternal risk factors for VACTERL in offspring befitting a multifactorial etiology.

Spectrum of congenital anomalies among VACTERL cases: a EUROCAT population-based study.

BACKGROUND: The VACTERL (Vertebral anomalies, Anal atresia, Cardiac malformations, Tracheo-Esophageal fistula, Renal anomalies, Limb abnormalities) association is the non-random occurrence of at least three of these congenital anomalies: vertebral, anal, cardiac, tracheo-esophageal, renal, and limb anomalies. Diagnosing VACTERL patients is difficult, as many disorders have multiple features in common with VACTERL. The aims of this study were to clearly outline component features, describe the phenotypic spectrum among the largest group of VACTERL patients thus far reported, and to identify phenotypically similar subtypes. METHODS: A case-only study was performed assessing data on 501 cases recorded with VACTERL in the JRC-EUROCAT (Joint Research Centre-European Surveillance of Congenital Anomalies) central database (birth years: 1980-2015). We differentiated between major and minor VACTERL features and anomalies outside the VACTERL spectrum to create a clear definition of VACTERL. RESULTS: In total, 397 cases (79%) fulfilled our VACTERL diagnostic criteria. The most commonly observed major VACTERL features were anorectal malformations and esophageal atresia/tracheo-esophageal fistula (both occurring in 62% of VACTERL cases), followed by cardiac (57%), renal (51%), vertebral (33%), and limb anomalies (25%), in every possible combination. Three VACTERL subtypes were defined: STRICT-VACTERL, VACTERL-LIKE, and VACTERL-PLUS, based on severity and presence of additional congenital anomalies. CONCLUSION: The clearly defined VACTERL component features and the VACTERL subtypes introduced will improve both clinical practice and etiologic research.

Exome chip association study excluded the involvement of rare coding variants with large effect sizes in the etiology of anorectal malformations.

INTRODUCTION: Anorectal malformations (ARM) are rare congenital malformations, resulting from disturbed hindgut development. A genetic etiology has been suggested, but evidence for the involvement of specific genes is scarce. We evaluated the contribution of rare and low-frequency coding variants in ARM etiology, assuming a multifactorial model. METHODS: We analyzed 568 Caucasian ARM patients and 1,860 population-based controls using the Illumina HumanExome Beadchip array, which contains >240,000 rare and low-frequency coding variants. GenomeStudio clustering and calling was followed by re-calling of 'no-calls' using zCall for patients and controls simultaneously. Single variant and gene-based analyses were performed to identify statistically significant associations, applying Bonferroni correction. Following an extra quality control step, candidate variants were selected for validation using Sanger sequencing. RESULTS: When we applied a MAF of ≥1.0%, no variants or genes showed statistically significant associations with ARM. Using a MAF cut-off at 0.4%, 13 variants initially reached statistical significance, but had to be discarded upon further inspection: ten variants represented calling errors of the software, while the minor alleles of the remaining three variants were not confirmed by Sanger sequencing. CONCLUSION: Our results show that rare and low-frequency coding variants with large effect sizes, present on the exome chip do not contribute to ARM etiology.

Uncontrolled maternal chronic respiratory diseases in pregnancy: A new potential risk factor suggested to be associated with anorectal malformations in offspring.

BACKGROUND: Chronic respiratory diseases and use of antiasthmatic medication during pregnancy may both play a role in the etiology of congenital anorectal malformations (ARM). However, it is unclear, whether the medication use or the underlying condition would be responsible. Therefore, our aim was to unravel the role of maternal chronic respiratory diseases from that of antiasthmatic medication in the etiology of ARM. METHODS: We obtained 412 ARM patients and 2,137 population-based controls from the Dutch AGORA data- and biobank. We used maternal questionnaires and follow-up telephone interviews to obtain information on chronic respiratory diseases, antiasthmatic medication use, and potential confounders. Multivariable logistic regression analyses were performed to estimate odds ratios (ORs) with 95% confidence intervals (95% CI). RESULTS: We observed higher risk estimates among women with chronic respiratory diseases with and without medication use (1.4 [0.8-2.7] and 2.0 [0.8-5.0]), both in comparison to women without a chronic respiratory disease and without medication use. Furthermore, increased ORs of ARM were found for women using rescue medication (2.4 [0.8-7.3]) or a combination of maintenance and rescue medication (2.5 [0.9-6.7]). In addition, increased risk estimates were observed for women having nonallergic triggers (2.5 [1.0-6.3]) or experiencing exacerbations during the periconceptional period (3.5 [1.4-8.6]). CONCLUSIONS: Although the 95% CIs of most associations include the null value, the risk estimates all point towards an association between uncontrolled chronic respiratory disease, instead of antiasthmatic medication use, with ARM in offspring. Further in-depth studies towards mechanisms of this newly identified risk factor are warranted.

Previous miscarriages and GLI2 are associated with anorectal malformations in offspring.

STUDY QUESTION: Are anorectal malformations (ARMs) associated with previous miscarriages or single nucleotide polymorphisms (SNPs) in the Bone Morphogenetic Protein 4 (BMP4) and GLI family zinc finger 2 (GLI2) genes? SUMMARY ANSWER: The SNP rs3738880 in GLI2 and miscarriages were associated with ARM, especially in patients with multiple congenital anomalies (MCA). WHAT IS KNOWN ALREADY: ARM are one of the most common birth defects of the gastrointestinal tract. The etiology is likely to be multifactorial, involving both environmental and genetic factors. SNPs in BMP4 and GLI2 genes were associated with ARM in non-Caucasian populations. During a patient information day, several mothers of ARM patients reported their concerns about previous miscarriages. STUDY DESIGN, SIZE, DURATION: A case-control study was performed among 427 ARM patients and 663 population-based controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: We examined the associations of ARM with SNPs in GLI2 and BMP4 using DNA samples of the children and associations with previous miscarriages using parental questionnaires. In addition, gene-gene and gene-environment interaction analyses were performed. MAIN RESULTS AND THE ROLE OF CHANCE: The SNP rs3738880 in GLI2 was associated with ARM, especially in patients with MCA (homozygous GG-genotype: odds ratio (OR): 2.1; 95% CI: 1.2, 3.7). We identified previous miscarriages as a new risk factor for ARM, especially when occurring in the pregnancy directly preceding the index pregnancy and in patients with MCA (OR: 2.1; 95% CI: 1.3, 3.5). No association with rs17563 in BMP4, nor gene-gene or gene-environment interactions were found. LIMITATIONS, REASONS FOR CAUTION: The possibility of recall errors for previous miscarriage, but we expect these errors to be limited, as a miscarriage is a major life event. In addition, potential misclassification regarding miscarriages and stillbirth, but sensitivity analyses showed that this did not influence our results. WIDER IMPLICATIONS OF THE FINDINGS: This study showed associations of ARM with rs3738880 in GLI2 and with previous miscarriages. Both associations were stronger in patients with MCA, showing the importance of stratifying the analyses by patients with isolated ARM or MCA. STUDY FUNDING/COMPETING INTERESTS: This study was funded by the Radboudumc. The authors have no conflict of interest to disclose.

Sequencing of the DKK1 gene in patients with anorectal malformations and hypospadias.

UNLABELLED: Anorectal malformations (ARM) are rare congenital malformations of the gastrointestinal tract. Approximately 60% of the patients have additional congenital malformations, such as hypospadias. A recently published article showed that deletion of one single gene, dickkopf WNT signaling pathway inhibitor-1 (Dkk1), resulted in an imperforate anus with rectourinary fistula and preputial hypospadias in mice. To determine whether DKK1 also plays a role in the etiology of ARM and hypospadias in humans, we sequenced the four exons of the DKK1 gene in 17 patients affected with both ARM and hypospadias. No new potential disease-causing variant was identified. However, we detected a known non-synonymous variant in one patient, which was predicted in silico to be damaging, and the corresponding unaffected amino acid is highly conserved. CONCLUSION: In this human study, a potential interesting non-synonymous variant was found in the DKK1 gene. Whether this variant plays a contributory role in the genesis of ARM or hypospadias would require a much larger study.

Glycosaminoglycans in the cellular uptake of drug delivery vectors - bystanders or active players?

The implementation of efficient strategies for cellular delivery is the most significant hurdle in the development of oligonucleotide and protein-based nanomedicines. Unlike small molecule drugs that enter cells by virtue of hydrophobicity or by being substrates of transporters, these macromolecules lack the capacity to cross the plasma membrane in a non-disruptive way, therefore requiring the combination with carriers that mediate entry. Remarkably, for the major part, these carriers lack distinct structural features except for a high density of positive charge. Uptake has been attributed to the ability to engage in electrostatic interactions with the lipid bilayer and negatively charged glycosaminoglycans (GAGs) of the cellular glycocalyx. However, conflicting evidence has been obtained to which degree the interaction with GAGs contributes to uptake and the molecular mechanisms involved in uptake. Also, it is not clear to which extent the same molecular mechanisms apply for the different types of cationic delivery vectors. Here, we review the available data for cationic delivery vectors, including lipoplexes, polyplexes and cell-penetrating peptides (CPPs). We show that in spite of their different molecular size and degree of positive charge, all types of vectors share major characteristics with respect to the suggested role of GAGs in uptake. Moreover, by a comparison with the role of heparan sulfates in viral uptake we propose new avenues in the search for molecular mechanisms that trigger uptake of drug delivery vehicles and discuss how these insights may translate into new design principles for nanomedicines.