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Background The bladder exstrophy‐epispadias complex (BEEC) is a congenital malformation of the bladder and urethra. The underlying causes of this malformation are still largely unknown; however, aside from environment, genetics is thought to play an essential role. The recurrent 22q11.2 microduplication is the most persistently detected genetic aberration found in BEEC cases. Methods We performed array comparative genomic hybridization (array‐CGH) analysis of 76 Swedish BEEC patients. Statistical analysis was performed on current dataset pooled with previously published data on the 22q11.2 microduplication in BEEC patients. We performed massive parallel sequencing (MPS) of the 22q11.2 region in 20 BEEC patients without the 22q11.2 microduplication followed by functional studies. Results We identified three additional cases with the 22q11.2 microduplication. Pooling data from this study with previously published reports showed a statistically significant enrichment of the 22q11.2 microduplication in BEEC patients (2.61% in cases vs. 0.08% in controls; OR = 32.6; p = 8.7 × 10−4). MPS of the 22q11.2 region in 20 BEEC patients without the 22q11.2 microduplication identified a novel variant in LZTR1 (p.Ser698Phe) in one patient. Functional evaluation of the LZTR1 p.Ser698Phe variant in live NIH 3T3 cells showed that the concentration and cytoplasmic mobility differ between the Lztr1wt and Lztr1mut, indicating a potential functional effect of the LZTR1mut. Conclusion Our study further emphasizes the involvement of the 22q11.2 region in BEEC development and highlights LZTR1 as a candidate gene underlying the urogenital malformation.

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. (Less)

In order to evaluate the use of the BNC2 gene in clinical practice regarding hypospadias, the presence of the impact of mutations in BNC2 gene in males who had been treated surgically for hypospadias in Sweden (N=413) and controls (N=455) were evaluated. Mutational screening was performed using Sanger sequencing and genotyping by Taqman allelic discrimination, and the findings were evaluated by disease-causing potential using Mutation Taster and PolyPhen. Nine missense mutations were identified, six of which were more common among cases than among controls, one being previously unknown. Six of these genetic variants were predicted to be possibly or probably damaging in mutational predictions and are thus potentially disease-causing. No difference was confirmed in the contribution of these findings in different severity of phenotype. Due to the complexity of the gene structure, the impact of variants was difficult to evaluate, and thus the clinical use of BNC2 in the management of 46,XY disorder of sex development (DSD) remains limited. Key words: BNC2 gene, hypospadias, mutations, polymorphism.

Item does not contain fulltext Hypospadias is a common male genital malformation and is regarded as a complex disease affected by multiple genetic as well as environmental factors. In a previous genome-wide scan for familial hypospadias, we reported suggestive linkage in nine chromosomal regions. We have extended this analysis by including new families and additional markers using non-parametric linkage. The fine mapping analysis displayed an increased LOD score on chromosome 8q24.1 and 10p15 in altogether 82 families. On chromosome 10p15, with the highest LOD score, we further studied AKR1C2, AKR1C3 and AKR1C4 involved in steroid metabolism, as well as KLF6 expressed in preputial tissue from hypospadias patients. Mutation analysis of the AKR1C3 gene showed a new mutation, c.643G>A (p.(Ala215Thr)), in a boy with penile hypospadias. This mutation is predicted to have an impact on protein function and structure and was not found in controls. Altogether, we homed in on four chromosomal regions likely to harbor genes for hypospadias. Future studies will aim for studying regulatory sequence variants in these regions.

Carnitine palmitoyl-CoA transferase-1B is a mitochondrial enzyme in the fatty acid oxidation pathway. In a previous study, CPT1B was identified as differentially expressed in the hypothalamus of two lines of chickens established by long-term selection for high (HWS) or low (LWS) body weight. Mammals have three paralogs ( CPT1a, b and c) while nonmammalian vertebrates only have two ( CPT1A, B). CPT1A is expressed in liver and CPT1B in muscle. CPT1c is expressed in hypothalamus, where it regulates feeding and energy expenditure. We identified an intronic length polymorphism, fixed for different alleles in the two populations, and mapped the hitherto missing CPT1B locus in the chicken genome assembly, to the distal tip of chromosome 1p. Based on molecular phylogeny and gene synteny we suggest that chicken CPT1B is pro-orthologous of the mammalian CPT1c. Chicken CPT1B was differentially expressed in both muscle and hypothalamus but in opposite directions: higher levels in hypothalamus but lower levels in muscle in the HWS than in the LWS line. Using an advanced intercross population of the lines, we found CPT1B expression to be influenced by a cis-acting expression quantitative trait locus in muscle. The increased expression in hypothalamus and reduced expression in muscle is consistent with an increased food intake in the HWS line and at the same time reduced fatty acid oxidation in muscle yielding a net accumulation of energy intake and storage. The altered expression of CPT1B in hypothalamus and peripheral tissue is likely to be a mechanism contributing to the remarkable difference between lines.

The Currarino syndrome (CS) consists of a sacral defect, an anorectal malformation and a pre-sacral mass. It manifests as an autosomal dominant congenital malformation in familial settings, with varying penetrance. The disease-causing gene, Motor neuron and pancreas homeobox-1 (MNX1), is known to be mutated in almost all familial cases, but due to the lack of genotype-phenotype correlation, there is a need for better clinical and molecular genetic characterization of the CS. Here, we report two novel mutations in the MNX1 gene in two cases. Each case was found to be familial upon further investigation of the other members of each family. The first affected case (a one year old boy) exhibited a missense mutation, p.Phe289Ser, in exon 3 in the highly conserved third helix of the homeodomain, which is considered to affect the DNA binding property and transcription regulation of the protein. The mutation seemed to display full penetrance of the disease in this family, but with different time of on-set. The second affected case (a 5 months old boy) displayed a 13 basepair insertion in exon 1, creating a complex frameshift mutation which results in a premature truncation of the protein that lacks the third helix homeodomain. Other members of the boy's family, who harbored the same mutation, were found to be completely asymptomatic. In conclusion, we detected two novel mutations in the MNX1 gene in cases with CS, which supports mutational analysis in the diagnosis of CS, even though the variability in the genotype and phenotype correlation maintains.

Hypospadias is a frequent congenital malformation in boys and is characterized by incomplete fusion of the urethral folds. The steroidogenic factor-1 (SF-1, NR5A1) gene plays a key role in hypothalamic-pituitary-steroidogenic organ development, and has previously been reported to be mutated in individuals with 46,XY disorder of sex development. Here, we investigated the role of SF-1 in hypospadias, a milder form of 46,XY disorder of sex development. We performed direct sequencing analysis of the SF-1 gene in 2 male Caucasian twins exhibiting very severe hypospadias, and in 95 Caucasian boys with mild and severe hypospadias. We further extended the analysis by investigating 332 mild and severe hypospadias cases and 422 male controls using TaqMan assays. Our sequencing revealed a novel heterozygous p.R313H (c.938G>A) missense mutation in each twin, and no mutations in the 95 Caucasian cases. Instead, a missense p.G146A (c.437G>C), and a silent known p.P125P (c.375C>T) polymorphism, respectively, was found in several of the latter cases. Further investigation of the 2 polymorphisms in the larger material of cases and controls showed no significant genotypic or allelic association. In conclusion, the SF-1 gene may not play a significant role in the development of hypospadias in Caucasians.

We report the identification of quantitative trait loci (QTL) affecting carcass composition, carcass length, fat deposition and lean meat content using a genome scan across 462 animals from a combined intercross and backcross between Hampshire and Landrace pigs. Data were analysed using multiple linear regression fitting additive and dominance effects. This model was compared with a model including a parent-of-origin effect to spot evidence of imprinting. Several precisely defined muscle phenotypes were measured in order to dissect body composition in more detail. Three significant QTL were detected in the study at the 1% genome-wide level, and twelve significant QTL were detected at the 5% genome-wide level. These QTL comprise loci affecting fat deposition and lean meat content on SSC1, 4, 9, 10, 13 and 16, a locus on SSC2 affecting the ratio between weight of meat and bone in back and weight of meat and bone in ham and two loci affecting carcass length on SSC12 and 17. The well-defined phenotypes in this study enabled us to detect QTL for sizes of individual muscles and to obtain information of relevance for the description of the complexity underlying other carcass traits.

Purpose We identified a girl with Hirschsprung's disease (HSCR) whose mother and grandmother had HSCR associated with multiple sclerosis (MS). The aim of this study was to outline mutations in HSCR-related genes and MS susceptibility alleles in these three individuals. Methods The phenotypes were reviewed based on medical records. The three subjects had rectosigmoid HSCR verified with histopathology. The mother and grandmother fulfilled the McDonald criteria for MS. DNA was isolated from EDTA-preserved blood according to standard procedures. Exome sequencing aiming mainly at analyzing HSCR associated genes as well as Sanger sequencing for confirmation was performed. Results All affected individuals carry a novel heterozygous nonsense mutation in the EDNRB gene (c.C397T,p.R133X,refNM_000115), changing an arginine at position 133 into a premature stop codon. None of the subjects were homozygous for the HLA risk alleles for MS. Conclusion We report a novel non-sense EDNRB gene mutation in a girl with HSCR and her mother and grandmother with HSCR and MS. We propose that this EDNRB gene mutation plays a role in the etiology of HSCR and also makes the subjects susceptible to MS.

Background/Purpose We describe a 3-day-old male infant that was operated on for annular pancreas causing duodenal stenosis. The family history revealed that the mother also underwent surgery as a neonate owing to duodenal stenosis with an annular pancreas. The aim of our study was to perform molecular investigations in this rare and familial congenital malformation. Methods We performed high-resolution (180 K) array comparative genomic hybridization using the infant's DNA and multiplex ligation-dependent probe amplification (MLPA) using the parents' and the affected mother's siblings' and parents' DNA. Results Array comparative genomic hybridization revealed previously unreported duplications on chromosome 6q24.2 and 17q22-q23.1, respectively, in the infant's DNA, and the latter duplication was also found in the healthy father using MLPA, whereas the affected mother carried the 6q24 duplication (0.7 Mb) containing parts of the utrophin gene. We further performed MLPA analysis of the 6q24 region and found that the clinically unaffected grandfather was a carrier of the microduplication, and so were 2 asymptomatic siblings of the affected mother. Conclusions The 6q24.2 mircoduplication of the utrophin gene is a potential risk factor for the development of annular pancreas, but further studies will clarify the exact role and if this is a true risk factor or a rare normal variant.

Hypospadias is a common congenital malformation in boys in which the urethral meatus opens on the underside of the penis. It is considered a complex disorder with several genes involved and the molecular etiology is just beginning to be revealed. As more than 85% of Opitz G/BBB syndrome (OS) patients with MID1 mutations are manifested with hypospadias, we have investigated the association between the MID1 gene and hypospadias. DNA from 114 hypospadias cases was analyzed with direct sequencing of the MID1 gene. Genotyping analysis was performed for the single-nucleotide polymorphism (SNP) c.1230G>A in 370 individuals with varying degrees of hypospadias and compared with 759 healthy controls. We identified one nonsense mutation c.712G>T (p.E238X), one missense mutation c.1679A>G (p.K560R) and two synonymous variants c.1230G>A (p.S410S) and c.1284T>G (p.V428V). We also detected a significant difference in the rare allele frequency of SNP c.1230G>A in hypospadias patients as compared with controls (P=0.016). Our finding suggests that hypospadias associated with hypertelorism is the mildest phenotype in OS caused by MID1 mutations.

Contains fulltext : 95737.pdf (Publisher’s version ) (Closed access) Contains fulltext : 95737a.pdf (Author’s version postprint ) (Open Access) Hypospadias is a common congenital malformation of the male external genitalia. We performed a genome-wide association study using pooled DNA from 436 individuals with hypospadias (cases) and 494 controls of European descent and selected the highest ranked SNPs for individual genotyping in the discovery sample, an additional Dutch sample of 133 cases and their parents, and a Swedish series of 266 cases and 402 controls. Individual genotyping of two SNPs (rs1934179 and rs7063116) in DGKK, encoding diacylglycerol kinase kappa, produced compelling evidence for association with hypospadias in the discovery sample (allele-specific odds ratio (OR) = 2.5, P = 2.5 x 10(1)(1) and OR = 2.3, P = 2.9 x 10, respectively) and in the Dutch (OR = 3.9, P = 2.4 x 10 and OR = 3.8, P = 3.4 x 10) and Swedish (OR = 2.5, P = 2.6 x 10 and OR = 2.2, P = 2.7 x 10) replication samples. Expression studies showed expression of DGKK in preputial tissue of cases and controls, which was lower in carriers of the risk allele of rs1934179 (P = 0.047). We propose DGKK as a major risk gene for hypospadias.

A DNA transposon integrated into -the genome of a primitive mammal some 200 million years ago and, millions of years later, it evolved an essential function in the common ancestor of all placental mammals. This protein, now named ZBED6, was recently discovered because a mutation disrupting one of its binding sites, in an intron of the IGF2 gene, makes pigs grow more muscle. These findings have revealed a new mechanism for regulating muscle growth as well as a novel transcription factor that appears to be of major importance for transcriptional regulation in placental mammals.

Hypospadias, when the urethral opening is situated on the ventral side of the penis, is a common genital malformation in boys and is partly caused by genetic factors. Mutations in the Mastermind-like domain containing 1 (MAMLD1 or CXorf6) gene have been reported in hypospadias cases. We have performed direct sequencing of the MAMLD1 gene in 99 sporadic hypospadias cases to further elucidate the role of this gene in hypospadias. Five non-synonymous mutations, one synonymous and one non-coding mutation were found. Of those, p.P286S, p.V432A, p.N589S and p.531ins3Q have previously been reported and are indicated in our study as polymorphisms. One new mutation, p.Q529K, was found in one patient with severe hypospadias and it was predicted to affect the splicing process. In our material we also found a weak association between hypospadias and the p.N589S polymorphism and in a haplotype analysis the rare alleles of p.P286S and p.N589S were more common in cases than in controls.

A single nucleotide substitution in intron 3 of IGF2 in pigs abrogates a binding site for a repressor and leads to a 3-fold upregulation of IGF2 in skeletal muscle. The mutation has major effects on muscle growth, size of the heart, and fat deposition. Here, we have identified the repressor and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (ChIP) sequencing using C2C12 cells identified about 2,500 ZBED6 binding sites in the genome, and the deduced consensus motif gave a perfect match with the established binding site in Igf2. Genes associated with ZBED6 binding sites showed a highly significant enrichment for certain Gene Ontology classifications, including development and transcriptional regulation. The phenotypic effects in mutant pigs and ZBED6-silenced C2C12 myoblasts, the extreme sequence conservation, its nucleolar localization, the broad tissue distribution, and the many target genes with essential biological functions suggest that ZBED6 is an important transcription factor in placental mammals, affecting development, cell proliferation, and growth., National Human Genome Research Institute (U.S.), Knut and Alice Wallenberg Foundation, Swedish Research Council, Forskningsrådet för miljö, areella näringar och samhällsbyggande, Fondation pour la recherche stratégique (France)

Background Meat quality traits are important in pig breeding programs, but they are difficult to include in a traditional selection program. Marker assisted selection (MAS) of meat quality traits is therefore of interest in breeding programs and a Quantitative Trait Locus (QTL) analysis is the key to identifying markers that can be used in MAS. In this study, Landrace and Hampshire intercross and backcross families were used to investigate meat quality traits. Hampshire pigs are commonly used as the sire line in commercial pig breeding. This is the first time a pedigree including Hampshire pigs has been used for a QTL analysis of meat quality traits. Results In total, we analyzed 39 meat quality traits and identified eight genome-wide significant QTL peaks in four regions: one on chromosome 3, two on chromosome 6 and one on chromosome 16. At least two of the QTLs do not appear to have been detected in previous studies. On chromosome 6 we identified QTLs for water content in M. longissimus dorsi (LD), drip loss in LD and post mortem pH decline in LD. On chromosomes 3 and 16 we identified previously undetected QTLs for protein content in LD and for freezing and cooking loss respectively. Conclusion We identified at least two new meat quality trait QTLs at the genome-wide significance level. We detected two QTLs on chromosome 6 that possibly coincide with QTLs detected in other studies. We were also able to exclude the C1843T mutation in the ryanodine receptor (RYR1) as a causative mutation for one of the chromosome 6 QTLs in this cross.