Further evidence for deletions in 7p14.1 contributing to nonsyndromic cleft lip with or without cleft palate.

BACKGROUND: Nonsyndromic cleft with or without cleft palate (nsCL/P) is a common birth defect. Although genome-wide association studies (GWAS) have identified numerous risk variants, a considerable fraction of the genetic heritability remains unknown. The aim of the present study was to replicate a previous finding that de novo deletions in a 62 kb region of chromosome 7p14 are a risk factor for nsCL/P, using an independent cohort. METHODS: Data from a published case-control GWAS cohort of 399 patients and 1318 controls were used. Copy number variant (CNV) detection in the 62 kb candidate region of 7p14 was performed using QuantiSNP. Putative CNVs in probands were verified and validated by quantitative polymerase chain reaction. Segregation analyses were performed in family members for whom DNA was available. RESULTS: Within the 62 kb candidate region, a deletion of 7.4 kb showed association with nsCL/P (13/387 cases, 20/1300 controls, plowest = 0.024, odds ratio = 2.22). In all families with a sporadic case (n = 3), the deletion occurred de novo. In multiplex families, both incomplete segregation and incomplete penetrance were observed. CONCLUSION: The present data support the hypothesis that deletions at 7p14 are a common risk factor for nsCL/P. Genome-wide CNV analyses in nsCL/P cohorts are warranted to explore the functional relevance of these deletions and their contribution to nsCL/P, and to determine exact breakpoints. Birth Defects Research (Part A) 106:767-772, 2016. © 2016 Wiley Periodicals, Inc.

CNV analysis in 169 patients with bladder exstrophy-epispadias complex.

BACKGROUND: The bladder exstrophy-epispadias complex (BEEC) represents the severe end of the congenital uro-rectal malformation spectrum. Initial studies have implicated rare copy number variations (CNVs), including recurrent duplications of chromosomal region 22q11.21, in BEEC etiology. METHODS: To detect further CNVs, array analysis was performed in 169 BEEC patients. Prior to inclusion, 22q11.21 duplications were excluded using multiplex ligation-dependent probe amplification. RESULTS: Following the application of stringent filter criteria, seven rare CNVs were identified: n = 4, not present in 1307 in-house controls; n = 3, frequency of <0.002 in controls. These CNVs ranged from 1 to 6.08 Mb in size. To identify smaller CNVs, relaxed filter criteria used in the detection of previously reported BEEC associated chromosomal regions were applied. This resulted in the identification of six additional rare CNVs: n = 4, not present in 1307 in-house controls; n = 2, frequency <0.0008 in controls. These CNVs ranged from 0.03-0.08 Mb in size. For 10 of these 13 CNVs, confirmation and segregation analyses were performed (5 of maternal origin; 5 of paternal origin). Interestingly, one female with classic bladder extrophy carried a 1.18 Mb duplication of 22q11.1, a chromosomal region that is associated with cat eye syndrome. CONCLUSIONS: A number of rare CNVs were identified in BEEC patients, and these represent candidates for further evaluation. Rare inherited CNVs may constitute modifiers of, or contributors to, multifactorial BEEC phenotypes.

WNT3 involvement in human bladder exstrophy and cloaca development in zebrafish.

Bladder exstrophy, a severe congenital urological malformation when a child is born with an open urinary bladder, is the most common form of bladder exstrophy-epispadias complex (BEEC) with an incidence of 1:30,000 children of Caucasian descent. Recent studies suggest that WNT genes may contribute to the etiology of bladder exstrophy. Here, we evaluated WNT-pathway genes in 20 bladder exstrophy patients using massively parallel sequencing. In total 13 variants were identified in WNT3, WNT6, WNT7A, WNT8B, WNT10A, WNT11, WNT16, FZD5, LRP1 and LRP10 genes and predicted as potentially disease causing, of which seven variants were novel. One variant, identified in a patient with a de novo nonsynonymous substitution in WNT3 (p.Cys91Arg), was further evaluated in zebrafish. Knock down of wnt3 in zebrafish showed cloaca malformations, including disorganization of the cloaca epithelium and expansion of the cloaca lumen. Our study suggests that the function of the WNT3 p.Cys91Arg variant was altered, since RNA overexpression of mutant Wnt3 RNA does not result in embryonic lethality as seen with wild-type WNT3 mRNA. Finally, we also mutation screened the WNT3 gene further in 410 DNA samples from BEEC cases and identified one additional mutation c.638G>A (p.Gly213Asp), which was paternally inherited. In aggregate our data support the involvement of WNT-pathway genes in BEEC and suggest that WNT3 in itself is a rare cause of BEEC.

Genome-wide association study and meta-analysis identify ISL1 as genome-wide significant susceptibility gene for bladder exstrophy.

The bladder exstrophy-epispadias complex (BEEC) represents the severe end of the uro-rectal malformation spectrum, and is thought to result from aberrant embryonic morphogenesis of the cloacal membrane and the urorectal septum. The most common form of BEEC is isolated classic bladder exstrophy (CBE). To identify susceptibility loci for CBE, we performed a genome-wide association study (GWAS) of 110 CBE patients and 1,177 controls of European origin. Here, an association was found with a region of approximately 220kb on chromosome 5q11.1. This region harbors the ISL1 (ISL LIM homeobox 1) gene. Multiple markers in this region showed evidence for association with CBE, including 84 markers with genome-wide significance. We then performed a meta-analysis using data from a previous GWAS by our group of 98 CBE patients and 526 controls of European origin. This meta-analysis also implicated the 5q11.1 locus in CBE risk. A total of 138 markers at this locus reached genome-wide significance in the meta-analysis, and the most significant marker (rs9291768) achieved a P value of 2.13 × 10-12. No other locus in the meta-analysis achieved genome-wide significance. We then performed murine expression analyses to follow up this finding. Here, Isl1 expression was detected in the genital region within the critical time frame for human CBE development. Genital regions with Isl1 expression included the peri-cloacal mesenchyme and the urorectal septum. The present study identified the first genome-wide significant locus for CBE at chromosomal region 5q11.1, and provides strong evidence for the hypothesis that ISL1 is the responsible candidate gene in this region.

Mutations in PTF1A are not a common cause for human VATER/VACTERL association or neural tube defects mirroring Danforth's short tail mouse.

Danforth's short tail (Sd) mutant mice exhibit defects of the neural tube and other abnormalities, which are similar to the human vertebral anomalies, anal atresia, cardiac defects, tracheosophageal fistula and/or esophageal atresia, renal and radial abnormalities, and limb defects (VATER/VACTERL) association, including defects of the hindgut. Sd has been shown to underlie ectopic gene expression of murine Ptf1a, which encodes pancreas-specific transcription factor 1A, due to the insertion of a retrotansposon in its 5' regulatory domain. In order to investigate the possible involvement of this gene in human VATER/VACTERL association and human neural tube defects (NTDs), a sequence analysis was performed. DNA samples from 103 patients with VATER/VACTERL and VATER/VACTERL­like association, all presenting with anorectal malformations, and 72 fetuses with NTDs, where termination of pregnancy had been performed, were included in the current study. The complete PTF1A coding region, splice sites and 1.5 kb of the 5' flanking promotor region was sequenced. However, no pathogenic alterations were detected. The results of the present study do not support the hypothesis that high penetrant mutations in these regions of PTF1A are involved in the development of human VATER/VACTERL association or NTDs, although rare mutations may be detectable in larger patient samples.

Genome-wide CNV analysis in 221 unrelated patients and targeted high-throughput sequencing reveal novel causative candidate genes for colorectal adenomatous polyposis.

To uncover novel causative genes in patients with unexplained adenomatous polyposis, a model disease for colorectal cancer, we performed a genome-wide analysis of germline copy number variants (CNV) in a large, well characterized APC and MUTYH mutation negative patient cohort followed by a targeted next generation sequencing (NGS) approach. Genomic DNA from 221 unrelated German patients was genotyped on high-resolution SNP arrays. Putative CNVs were filtered according to stringent criteria, compared with those of 531 population-based German controls, and validated by qPCR. Candidate genes were prioritized using in silico, expression, and segregation analyses, data mining and enrichment analyses of genes and pathways. In 27% of the 221 unrelated patients, a total of 77 protein coding genes displayed rare, nonrecurrent, germline CNVs. The set included 26 candidates with molecular and cellular functions related to tumorigenesis. Targeted high-throughput sequencing found truncating point mutations in 12% (10/77) of the prioritized genes. No clear evidence was found for autosomal recessive subtypes. Six patients had potentially causative mutations in more than one of the 26 genes. Combined with data from recent studies of early-onset colorectal and breast cancer, recurrent potential loss-of-function alterations were detected in CNTN6, FOCAD (KIAA1797), HSPH1, KIF26B, MCM3AP, YBEY and in three genes from the ARHGAP family. In the canonical Wnt pathway oncogene CTNNB1 (ß-catenin), two potential gain-of-function mutations were found. In conclusion, the present study identified a group of rarely affected genes which are likely to predispose to colorectal adenoma formation and confirmed previously published candidates for tumor predisposition as etiologically relevant.

Genome-wide mapping of copy number variations in patients with both anorectal malformations and central nervous system abnormalities.

BACKGROUND: Anorectal malformations (ARM) have a prevalence of around 1 in 2500 live births. In around 50% of patients, the malformation is isolated, while in the remainder it arises within the context of complex genetic abnormalities or a defined genetic syndrome. Recent studies have implicated rare copy number variations (CNVs) in both isolated and nonisolated ARM, and identified plausible candidate genes. METHODS: In the present study, array-based molecular karyotyping was performed to identify causative CNVs in 32 sporadic ARM patients with comorbid abnormalities of the central nervous system (CNS). This phenotype was selected to enrich for rare CNVs, since previous research has implicated rare CNVs in both CNS abnormalities and ARM. RESULTS: In five patients, a probable disease-causing CNV was identified (del6q14.3q16.3, del14q32.2, del17q12q21.2, and two patients with del22q11.21). In three of these patients, the CNVs were de novo. For the remaining two patients, no parental DNA was available. Deletions at 22q11.21 and 6q14.3 have been associated with both CNS abnormalities and ARM. In contrast, deletions at 14q32.2 have only been described in patients with CNS abnormalities, and the del17q12q21.2 is a novel CNV. Expression studies in mice suggest that NEUROD2 and RARA, which reside within the newly identified del17q12q21.2 region, are candidate genes for the formation of microcephaly and ARM. CONCLUSION: The present data suggest that CNVs are a frequent cause of the ARM with CNS abnormalities phenotype, and that array-analysis is indicated in such patients.

Heterozygous FGF8 mutations in patients presenting cryptorchidism and multiple VATER/VACTERL features without limb anomalies.

BACKGROUND: The acronym VATER/VACTERL association describes the combination of at least three of the following cardinal features: vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. Although fibroblast growth factor-8 (FGF8) mutations have mainly found in patients with Kallmann syndrome, mice with a hypomorphic Fgf8 allele or complete gene invalidation display, aside from gonadotropin-releasing hormone deficiency, parts or even the entire spectrum of human VATER/VACTERL association. METHODS: We performed FGF8 gene analysis in 49 patients with VATER/VACTERL association and 27 patients presenting with a VATER/VACTERL-like phenotype (two cardinal features). RESULTS: We identified two heterozygous FGF8 mutations in patients displaying either VATER/VACTERL association (p.Gly29_Arg34dup) or a VATER/VACTERL-like phenotype (p.Pro26Leu) without limb anomalies. Whereas the duplication mutation has not been reported before, p.Pro26Leu was once observed in a Kallmann syndrome patient. Both our patients had additional bilateral cryptorchidism, a key phenotypic feature in males with FGF8 associated Kallmann syndrome. Each mutation was paternally inherited. Besides delayed puberty in both and additional unilateral cryptorchidism in one of the fathers, they were otherwise healthy. Serum hormone levels downstream the gonadotropin-releasing hormone in both patients and their fathers were within normal range. CONCLUSION: Our results suggest FGF8 mutations to contribute to the formation of the VATER/VACTERL association. Further studies are needed to support this observation.

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.

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 Children's 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.

Classic bladder exstrophy: Frequent 22q11.21 duplications and definition of a 414 kb phenocritical region.

BACKGROUND: Classic bladder exstrophy (CBE) is the most common form of the bladder exstrophy and epispadias complex. Previously, we and others have identified four patients with a duplication of 22q11.21 among a total of 96 unrelated CBE patients. METHODS: Here, we investigated whether this chromosomal aberration was commonly associated with CBE/bladder exstrophy and epispadias complex in an extended case-control sample. Multiplex ligation-dependent probe amplification and microarray-based analysis were used to identify 22q11.21 duplications in 244 unrelated bladder exstrophy and epispadias complex patients (including 217 CBE patients) and 665 healthy controls. RESULTS: New duplications of variable size were identified in four CBE patients and one control. Pooling of our previous and present data (eight duplications in 313 CBE patients) yielded a combined odds ratio of 31.86 (95% confidence interval, 4.24-1407.97). Array-based sequence capture and high-throughput targeted re-sequencing established that all breakpoints resided within the low-copy repeats 22A to 22D. Comparison of the eight duplications revealed a 414 kb phenocritical region harboring 12 validated RefSeq genes. Characterization of these 12 candidate genes through whole-mount in situ hybridization of mouse embryos at embryonic day 9.5 suggested that CRKL, THAP7, and LZTR1 are CBE candidate genes. CONCLUSION: Our data suggest that duplication of 22q11.21 increases CBE risk and implicate a phenocritical region in disease formation.

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 VACTERL association with CAKUT.

De novo 13q deletions in two patients with mild anorectal malformations as part of VATER/VACTERL and VATER/VACTERL-like association and analysis of EFNB2 in patients with anorectal malformations.

Anorectal malformations (ARMs) comprise a broad spectrum of conditions ranging from mild anal anomalies to complex cloacal malformations. In 40-50% of cases, ARM occurs within the context of defined genetic syndromes or complex multiple congenital anomalies, such as VATER/VACTERL (vertebral defects [V], ARMs [A], cardiac defects [C], tracheoesophageal fistula with or without esophageal atresia [TE], renal malformations [R], and limb defects [L]) association. Here, we report the identification of deletions at chromosome 13q using single nucleotide polymorphism-based array analysis in two patients with mild ARM as part of VATER/VACTERL and VATER/VACTERL-like associations. Both deletions overlap the previously defined critical region for ARM. Heterozygous Efnb2 murine knockout models presenting with mild ARM suggest EFNB2 as an excellent candidate gene in this region. Our patients showed a mild ARM phenotype, closely resembling that of the mouse. We performed a comprehensive mutation analysis of the EFNB2 gene in 331 patients with isolated ARM, or ARM as part of VATER/VACTERL or VATER/VACTERL-like associations. However, we did not identify any disease-causing mutations. Given the convincing argument for EFNB2 as a candidate gene for ARM, analyses of larger samples and screening of functionally relevant non-coding regions of EFNB2 are warranted. In conclusion, our report underlines the association of chromosome 13q deletions with ARM, suggesting that routine molecular diagnostic workup should include the search for these deletions. Despite the negative results of our mutation screening, we still consider EFNB2 an excellent candidate gene for contributing to the development of ARM in humans.

Candidate gene association study implicates p63 in the etiology of nonsyndromic bladder-exstrophy-epispadias complex.

BACKGROUND: Bladder-exstrophy-epispadias complex (BEEC) is a severe congenital anomaly that represents a spectrum of urological abnormalities where parts or all of the distal urinary tract fail to close during development. Multiple lines of evidence strongly suggested p63 as a plausible candidate gene. We conducted a candidate gene association study to further investigate the role of p63 in human BEEC. METHODS: We conducted a family-based association study of p63 using 154 Caucasian patients with nonsyndromic BEEC and their unaffected parents. High throughput single nucleotide polymorphism (SNP) genotyping was carried out using Illumina's Golden Gate Assay for 109 selected tagging SNPs localized within p63 with a minor allele frequency > 0.01. Individual and haplotype SNP transmission disequilibrium tests were conducted using Plink and Haploview, respectively. We also examined parent-of-origin effects using paternal asymmetry tests implemented in FAMHAP (http://famhap.meb.uni-bonn.de/index.html). RESULTS: Nominally significant associations were identified between BEEC and six SNPs (rs17447782, rs1913720, rs6790167, rs9865857, rs1543969, rs4687100), and four haplotype blocks including or near these significant SNPs. Analysis of parent-of-origin effects showed significant results for seven SNPs (rs4118375, rs12696596, rs6779677, rs13091309, rs7642420, rs1913721, and rs1399774). None of these results remained significant after multiple testing correction. CONCLUSION: The altered transmission of p63 variants in nonsyndromic BEEC patients may be suggestive of its involvement in the disease etiology. Further and large multi-institutional collaborative studies are required to elucidate the role of p63 in nonsyndromic BEEC.

CNV analysis in monozygotic twin pairs discordant for urorectal malformations.

Early post-twinning mutational events can account for discordant phenotypes in monozygotic (MZ) twin pairs. Such mutational events may comprise genomic alterations of different sizes, ranging from single nucleotides to large copy-number variations (CNVs). Anorectal malformations (ARM) and the bladder exstrophy-epispadias complex (BEEC) represent the most severe end of the urorectal malformation spectrum. Recently, CNV studies in patients with sporadic ARM and the BEEC have identified de novo events that occur in specific chromosomal regions. We hypothesized that early arising, post-twinning CNVs might contribute to discordance in MZ twin pairs with ARM or the BEEC; knowledge of such CNVs might help to identify additional chromosomal regions involved in the development of these malformations. We investigated four discordant MZ twin pairs (three ARM and one BEEC) using molecular karyotyping arrays comprising 1,140,419 markers with a median marker spacing of 1.5 kb. Filtering the coding regions for possible disease-causing post-twinning de novo CNVs present only in the affected twin, but not in the unaffected twin or the parents, identified a total of 136 CNVs. These 136 CNVs were then filtered against publicly available databases and finally re-evaluated visually. No potentially causative CNV remained after applying these filter criteria. Our results suggest that post-twinning CNV events that affect coding regions of the genome did not contribute to the discordant phenotypes in MZ twin pairs that we investigated. Possible causes for the discordant phenotypes include changes in regulatory elements or smaller genetic changes within coding regions which may be detectable by whole-exome sequencing.

An incompletely penetrant novel MAFB (p.Ser56Phe) variant in autosomal dominant multicentric carpotarsal osteolysis syndrome.

Multicentric carpotarsal osteolysis syndrome (MCTO) is a rare autosomal dominant skeletal dysplasia usually presenting in early childhood with variable phenotypic features and course. Clinical manifestations comprise aggressive osteolysis of the carpal and tarsal bones in particular, an often progressive nephropathy leading to end-stage renal disease, craniofacial anomalies and mental impairment. Recently, heterozygous missense mutations in the V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (avian) (MAFB) gene have been causally related to MCTO patients in 13 unrelated families investigated. Contrary to these findings suggesting complete penetrance, in the present study, we identified a novel missense MAFB variant present not only in the patient, but also in his unaffected mother, sister and maternal grandmother. This observation demonstrates an incomplete penetrance for some MAFB mutations, thereby suggesting that modifier genes, epigenetic mechanisms or environmental factors may modulate the MCTO phenotype. This should be considered in diagnosis and genetic counseling.

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.

Isolated bladder exstrophy associated with a de novo 0.9 Mb microduplication on chromosome 19p13.12.

BACKGROUND: The exstrophy-epispadias complex (BEEC) is a urogenital birth defect of varying severity. The causes of the BEEC are likely to be heterogeneous, with individual environmental or genetic risk factors still being largely unknown. In this study, we aimed to identify de novo causative copy number variations (CNVs) that contribute to the BEEC. METHODS: Array-based molecular karyotyping was performed to screen 110 individuals with BEEC. Promising CNVs were tested for de novo occurrence by investigating parental DNAs. Genes located in regions of rearrangements were prioritized through expression analysis in mice to be sequenced in the complete cohort, to identify high-penetrance mutations involving small sequence changes. RESULTS: A de novo 0.9 Mb microduplication involving chromosomal region 19p13.12 was identified in a single patient. This region harbors 20 validated RefSeq genes, and in situ hybridization data showed specific expression of the Wiz gene in regions surrounding the cloaca and the rectum between GD 9.5 and 13.5. Sanger sequencing of the complete cohort did not reveal any pathogenic alterations affecting the coding region of WIZ. CONCLUSIONS: The present study suggests chromosomal region 19p13.12 as possibly involved in the development of CBE, but further studies are needed to prove a causal relation. The spatiotemporal expression patterns determined for the genes encompassed suggest a role for Wiz in the development of the phenotype. Our mutation screening, however, could not confirm that WIZ mutations are a frequent cause of CBE, although rare mutations might be detectable in larger patient samples. 19p13.12, microduplication, bladder exstrophy-epispadias complex, array-based molecular karyotyping, in situ hybridization analysis, copy number variations, WIZ gene.

Involvement of the WNT and FGF signaling pathways in non-isolated anorectal malformations: sequencing analysis of WNT3A, WNT5A, WNT11, DACT1, FGF10, FGFR2 and the T gene.

Anorectal malformations (ARMs) comprise a broad spectrum of anomalies, including anal atresia, congenital anal fistula and persistence of the cloaca. Research suggests that genetic factors play an important role in ARM development. However, few genetic variants have been identified. Embryogenesis is orchestrated by crosstalk of the wingless-type MMTV integration site family (WNT) and fibroblast growth factor (FGF) signaling pathways in a process that involves several intracellular cascades. Studies in mice have implicated several genes from these pathways in the etiology of ARMs. We performed sequencing analysis of seven of these previously reported genes in 78 patients with ARMs occurring within the context of at least one additional congenital anomaly. No associations were identified with variants in WNT3A, WNT5A, WNT11, DACT1, FGF10 or the T gene. In the FGFR2 gene, three novel heterozygous nucleotide substitutions were identified. Further investigations, including the study of family members, revealed that these variants were not causally related to the phenotype in the present ARM cohort. Mutations in the seven investigated genes may nonetheless be a cause of ARMs in rare cases. However, further studies should consider genes encoding other proteins in the WNT/FGF signaling pathways as possible candidates.

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.