Charlotte H. W. Wijers

Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association

Congenital abnormalities of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease and they are the most frequent cause of end-stage renal disease in children in the US. However, its genetic etiology remains mostly elusive. VACTERL association is a rare disorder that involves congenital abnormalities in multiple organs including the kidney and urinary tract in up to 60% of the cases. By homozygosity mapping and whole exome resequencing combined with high-throughput mutation analysis by array-based multiplex PCR and next-generation sequencing, we identified recessive mutations in the gene TNF receptor-associated protein 1 (TRAP1) in two families with isolated CAKUT and three families with VACTERL association. TRAP1 is a heat shock protein 90-related mitochondrial chaperone possibly involved in antiapoptotic and endoplasmic reticulum-stress signaling. Trap1 is expressed in renal epithelia of developing mouse kidney E13.5 and in the kidney of adult rats, most prominently in proximal tubules and in thick medullary ascending limbs of Henle’s loop. Thus, we identified mutations in TRAP1 as highly likely causing CAKUT or CAKUT in VACTERL association.

Maternal and paternal risk factors for anorectal malformations: a Dutch case-control study.

BACKGROUND Anorectal malformations (ARM) are major congenital malformations that usually require a multitude of surgical procedures at a very early age and have a large impact on the lives of patients and their parents. The causes of ARM are still largely unknown, but they are assumed to have a multifactorial etiology. A few studies focused on environmental risk factors, but evidence is still scarce. METHODS In this Dutch case-control study (1996-2008), we investigated the role of maternal and paternal risk factors in the etiology of ARM. Parents of 85 ARM cases and 650 controls filled in a questionnaire. Controls were children treated with ear ventilation tubes. RESULTS A higher occurrence of fever during the first trimester of pregnancy was found for case mothers compared to control mothers (odds ratio [OR], 5.1; 95% Confidence Interval [CI], 0.9, 28.1). Maternal occupational exposure to industrial cleaning agents and solvents increased the risk of ARM three times (OR, 2.9; 95% CI, 0.9, 9.3). Overweight (Body Mass Index [BMI] > or = 25 kg/m(2)) before pregnancy also seemed to be associated with ARM (OR, 1.8; 95% CI, 1.1, 2.8), as well as maternal multivitamin use during pregnancy (OR, 1.6; 95% CI, 1.0, 2.7), paternal smoking (OR, 1.8; 95% CI, 1.1, 2.9), and paternal occupational exposure to exhaust fumes (OR, 1.9; 95% CI, 1.0, 3.6). Reported ARM in at least one first- or second-degree family member greatly increased the risk of having a child with an ARM (OR, 40.3; 95% CI, 4.8, 342.8). CONCLUSIONS This study revealed potential risk factors for ARM, including fever during pregnancy, maternal overweight, use of multivitamins, paternal smoking, and occupational exposures, but a familial component seems important as well.

De novo microduplications at 1q41, 2q37.3, and 8q24.3 in patients with VATER/VACTERL association

The acronym VATER/VACTERL association describes the combination of at least three of the following congenital anomalies: vertebral defects (V), anorectal malformations (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb defects (L). We aimed to identify highly penetrant de novo copy number variations (CNVs) that contribute to VATER/VACTERL association. Array-based molecular karyotyping was performed in a cohort of 41 patients with VATER/VACTERL association and 6 patients with VATER/VACTERL-like phenotype including all of the patients’ parents. Three de novo CNVs were identified involving chromosomal regions 1q41, 2q37.3, and 8q24.3 comprising one (SPATA17), two (CAPN10, GPR35), and three (EPPK1, PLEC, PARP10) genes, respectively. Pre-existing data from the literature prompted us to choose GPR35 and EPPK1 for mouse expression studies. Based on these studies, we prioritized GPR35 for sequencing analysis in an extended cohort of 192 patients with VATER/VACTERL association and VATER/VACTERL-like phenotype. Although no disease-causing mutation was identified, our mouse expression studies suggest GPR35 to be involved in the development of the VATER/VACTERL phenotype. Follow-up of GPR35 and the other genes comprising the identified duplications is warranted.

First results of a European multi-center registry of patients with anorectal malformations

BACKGROUND The European consortium on anorectal malformations (ARM-NET) was established to improve the health care of patients and to identify genetic and environmental risk factors. The aim of the present study was to present the first results on clinical data of a large European cohort of ARM patients based on our registry. METHODS In 2010, the registry was established including patient characteristics and data on diagnosis, surgical therapy, and outcome regarding complications. Patients born between 2007 and 2012 were retrospectively added. A descriptive analysis of this cohort was performed. RESULTS Two hundred and three ARM patients were included. Syndromes or chromosomal abnormalities were present in 9%. Perineal fistulas were seen most in boys (42%) and girls (29%). Rare forms of ARM were found in 4% of the male and in 14% of the female patients. Forty-five percent of the patients had additional urogenital abnormalities. However, 32% of the patients were never screened for bladder abnormalities. Eight percent were never screened for renal malformations. In the majority of patients (79%), a PSARP was performed for the definitive reconstruction. CONCLUSION This collaborative effort provides a representative basis to estimate incidence of ARM types, to discuss differences and similarities in treatment, and health consequences throughout Europe.

Genetic and nongenetic etiology of nonsyndromic anorectal malformations: A systematic review

Congenital anorectal malformations (ARMs) are one of the most frequently observed birth defects of the digestive system. However, their etiology remains elusive. Therefore, we aim to summarize and critically appraise all existing literature on the genetic and nongenetic etiology of nonsyndromic ARM and to conclude with unifying hypotheses and directions for future research. A structured literature search on English language human studies was conducted in PubMed and Embase up to October 1, 2013, resulting in 112 included articles. Research on the identification of genes underlying nonsyndromic ARM is remarkably scarce. Most studies were focused on screening of candidate genes for mutations or single-nucleotide polymorphisms, which did not yield any substantial evidence. Nongenetic factors fairly consistently found to be associated with ARM are assisted reproductive techniques, multiple pregnancy, preterm delivery, low birth weight, maternal overweight or obesity, and preexisting diabetes. This review provides indications for the involvement of both genes and nongenetic risk factors in the etiology of ARM. In future studies, large cohorts of patients with ARM from national and international collaborations are needed to acquire new hypotheses and knowledge through hypothesis-generating approaches. Challenges for future studies may also lie in the investigation of gene-gene and gene-environment interactions.

Anorectal malformations and pregnancy‐related disorders: a registry‐based case–control study in 17 European regions

To identify pregnancy‐related risk factors for different manifestations of congenital anorectal malformations (ARMs).

Genome-wide association study and mouse expression data identify a highly conserved 32 kb intergenic region between WNT3 and WNT9b as possible susceptibility locus for isolated classic exstrophy of the bladder

Bladder exstrophy-epispadias complex (BEEC), the severe end of the urorectal malformation spectrum, has a profound impact on continence as well as sexual and renal functions. It is widely accepted that for the majority of cases the genetic basis appears to be multifactorial. Here, we report the first study which utilizes genome-wide association methods to analyze a cohort comprising patients presenting the most common BEEC form, classic bladder exstrophy (CBE), to identify common variation associated with risk for isolated CBE. We employed discovery and follow-up samples comprising 218 cases/865 controls and 78 trios in total, all of European descent. Our discovery sample identified a marker near SALL1, showing genome-wide significant association with CBE. However, analyses performed on follow-up samples did not add further support to these findings. We were also able to identify an association with CBE across our study samples (discovery: P = 8.88 × 10(-5); follow-up: P = 0.0025; combined: 1.09 × 10(-6)) in a highly conserved 32 kb intergenic region containing regulatory elements between WNT3 and WNT9B. Subsequent analyses in mice revealed expression for both genes in the genital region during stages relevant to the development of CBE in humans. Unfortunately, we were not able to replicate the suggestive signal for WNT3 and WNT9B in a sample that was enriched for non-CBE BEEC cases (P = 0.51). Our suggestive findings support the hypothesis that larger samples are warranted to identify association of common variation with CBE.

VATER/VACTERL association: identification of seven new twin pairs, a systematic review of the literature, and a classical twin analysis

The VATER/VACTERL association is typically defined by the presence of at least three of the following congenital malformations: vertebral anomalies, anal atresia, cardiac malformations, tracheo-esophageal fistula, renal anomalies, and limb abnormalities. The identification of 14 twin pairs with an initial diagnosis of VATER/VACTERL association at our clinical centers led to the performance of a classical twin study. This involved a thorough evaluation of these 14 twin pairs and a further 55 twin pairs identified from a systematic review of the literature. The zygosity, concordance, and malformation status of all 69 twin pairs were evaluated. Twenty-four twin pairs fulfilled the criteria for inclusion in a comparison of the concordance rates between monozygous (MZ) and dizygous (DZ) twin pairs. The pairwise concordance rates were 15% [95% confidence interval (CI) 4–42%] for MZ and 18% (95% CI 5–48%) for DZ twin pairs (P=0.53). The probandwise concordance rates were 27% (95% CI 11–52%) for MZ and 31% (95% CI 13–58%) for DZ twin pairs (P=0.40). Although based on a limited number of twin pairs, the findings of the present study are consistent with the low number of familial cases reported to date, and suggest that the role of inherited genetic factors in the majority of VATER/VACTERL cases is limited.

No major role for periconceptional folic acid use and its interaction with the MTHFR C677T polymorphism in the etiology of congenital anorectal malformations

BACKGROUND Both genetic and nongenetic factors are suggested to be involved in the etiology of congenital anorectal malformations (ARM). Maternal periconceptional use of folic acid supplements were inconsistently suggested to play a role in the prevention of ARM. Therefore, we investigated independent associations and interactions of maternal periconceptional folic acid supplement use and the infant and maternal MTHFR (methylenetetrahydrofolate reductase) C677T polymorphisms with the risk of ARM and subgroups of ARM. METHODS A case-control study was conducted among 371 nonsyndromic ARM cases and 714 population-based controls born between 1990 and 2012 using maternal questionnaires and DNA samples from mother and child. Cases were treated for ARM at departments of Pediatric Surgery of the Radboud university medical center, Sophia Childrens Hospital-Erasmus MC Rotterdam, and the University Medical Center Groningen in The Netherlands and hospitals throughout Germany. RESULTS No association with folic acid use was present (odds ratio = 1.1; 95% confidence interval: 0.8-1.4) for ARM as a group. Infant and maternal MTHFR C677T polymorphisms were weakly associated with isolated ARM in particular. Lack of folic acid supplement use in combination with infants or mothers carrying the MTHFR C677T polymorphism did not seem to increase the risk of ARM or subgroups of ARM. The relative excess risks due to interaction did not clearly indicate interaction on an additive scale either. CONCLUSION This first study investigating interactions between periconceptional folic acid supplement use and infant and maternal MTHFR C677T polymorphisms in the etiology of ARM did not provide evidence for a role of this gene-environment interaction.

Parental subfertility, fertility treatment, and the risk of congenital anorectal malformations

Background: Fertility treatment seems to play a role in the etiology of congenital anorectal malformations, but it is unclear whether the underlying parental subfertility, ovulation induction, or the treatment itself is involved. Therefore, we investigated the odds of anorectal malformations among children of subfertile parents who conceived with or without treatment compared with fertile parents. Methods: We performed a case-control study among 380 cases with anorectal malformations treated at 3 departments of pediatric surgery in The Netherlands and 1973 population-based controls born between August 1988 and August 2012. Parental questionnaires were used to obtain information on fertility-related issues and potential confounders. Results: In singletons, increased risks of anorectal malformations were observed for parents who underwent intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) treatment compared with fertile parents (odds ratio = 2.4 [95% confidence interval = 1.0–5.9] and 4.2 [1.9–8.9], respectively). For subfertile parents who conceived after IVF treatment, an elevated risk was also found when they were compared with subfertile parents who conceived without treatment (3.2 [1.4–7.2]). Among children of the latter category of parents, only the risk of anorectal malformations with other major congenital malformations was increased compared with fertile parents (2.0 [1.3–3.3]). No associations were found with intrauterine insemination or use of hormones for ovulation induction. Conclusions: We found evidence of a role of ICSI and IVF treatments in the etiology of anorectal malformations. However, subfertility without treatment increased only the risk of anorectal malformations with additional congenital malformations.