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TALPID3/KIAA0586 is an evolutionary conserved protein, which plays an essential role in protein trafficking. Its role during gastrointestinal (GI) and enteric nervous system (ENS) development has not been studied previously. Here, we analyzed chicken, mouse and human embryonic GI tissues with TALPID3 mutations. The GI tract of TALPID3 chicken embryos was shortened and malformed. Histologically, the gut smooth muscle was mispatterned and enteric neural crest cells were scattered throughout the gut wall. Analysis of the Hedgehog pathway and gut extracellular matrix provided causative reasons for these defects. Interestingly, chicken intra-species grafting experiments and a conditional knockout mouse model showed that ENS formation did not require TALPID3, but was dependent on correct environmental cues. Surprisingly, the lack of TALPID3 in enteric neural crest cells (ENCC) affected smooth muscle and epithelial development in a non-cell-autonomous manner. Analysis of human gut fetal tissues with a KIAA0586 mutation showed strikingly similar findings compared to the animal models demonstrating conservation of TALPID3 and its necessary role in human GI tract development and patterning. [ABSTRACT FROM AUTHOR] more...

Goldberg–Shprintzen syndrome (GOSHS) is caused by loss of function variants in the kinesin binding protein gene (KIFBP). However, the phenotypic range of this syndrome is wide, indicating that other factors may play a role. To date, 37 patients with GOSHS have been reported. Here, we document nine new patients with variants in KIFBP: seven with nonsense variants and two with missense variants. To our knowledge, this is the first time that missense variants have been reported in GOSHS. We functionally investigated the effect of the variants identified, in an attempt to find a genotype–phenotype correlation. We also determined whether common Hirschsprung disease (HSCR)‐associated single nucleotide polymorphisms (SNPs), could explain the presence of HSCR in GOSHS. Our results showed that the missense variants led to reduced expression of KIFBP, while the truncating variants resulted in lack of protein. However, no correlation was found between the severity of GOSHS and the location of the variants. We were also unable to find a correlation between common HSCR‐associated SNPs, and HSCR development in GOSHS. In conclusion, we show that reduced, as well as lack of KIFBP expression can lead to GOSHS, and our results suggest that a threshold expression of KIFBP may modulate phenotypic variability of the disease. [ABSTRACT FROM AUTHOR] more...

Background: Hirschsprung's disease (HSCR) is a congenital gastrointestinal disorder, characterized by enteric ganglia absence in part or entire of the colon, due to abnormal colonization and migration of enteric neural crest cells (ENCCs) during development. Currently, besides surgery which is the main therapy for HSCR, the potential of stem cell‐based transplantation was investigated as an alternative option. Although promising, it has limitations, including poor survival, differentiation, and migration of the grafted cells. We hypothesized that modulation of extracellular factors during transplantation could promote ENCCs proliferation and migration, leading to increased transplantation efficiency. Considering that the RhoA/ROCK pathway is highly involved in cytoskeletal dynamics and neurite growth, our study explored the effect of inhibition of this pathway to improve the success of ENCCs transplantation. Methods: Enteric neural crest cells were isolated from rat embryos and labeled with a GFP‐tag. Cell viability, apoptosis, differentiation, and migration assays were performed with and without RhoA/ROCK inhibition. Labeled ENCCs were transplanted into the muscle layer of an induced hypoganglionic rat model followed by intraperitoneal injections of ROCK inhibitor. The transplanted segments were collected 3 weeks after for histological analysis. Key Results: Our results showed that inhibition of ROCK increased viable cell number, differentiation, and migration of ENCCs in vitro. Moreover, transplantation of labeled ENCCs into the hypoganglionic model showed enhanced distribution of grafted ENCCs, upon treatment with ROCK inhibitor. Conclusions and Inferences: ROCK inhibitors influence ENCCs growth and migration in vitro and in vivo, and should be considered to improve the efficiency of ENCCs transplantation. [ABSTRACT FROM AUTHOR] more...

Background: Patients born with esophageal atresia (EA) have a higher incidence of infantile hypertrophic pyloric stenosis (IHPS), suggestive of a relationship. A shared etiology makes sense from a developmental perspective as both affected structures are foregut derived. A genetic component has been described for both conditions as single entities and EA and IHPS are variable components in several monogenetic syndromes. We hypothesized that defects disturbing foregut morphogenesis are responsible for this combination of malformations. Methods: We investigated the genetic variation of 15 patients with both EA and IHPS with unaffected parents using exome sequencing and SNP array‐based genotyping, and compared the results to mouse transcriptome data of the developing foregut. Results: We did not identify putatively deleterious de novo mutations or recessive variants. However, we detected rare inherited variants in EA or IHPS disease genes or in genes important in foregut morphogenesis, expressed at the proper developmental time‐points. Two pathways were significantly enriched (p < 1 × 10−5): proliferation and differentiation of smooth muscle cells and self‐renewal of satellite cells. Conclusions: None of our findings could fully explain the combination of abnormalities on its own, which makes complex inheritance the most plausible genetic explanation, most likely in combination with mechanical and/or environmental factors. As we did not find one defining monogenetic cause for the EA/IHPS phenotype, the impact of the corrective surgery could should be further investigated. [ABSTRACT FROM AUTHOR] more...

Background: Inactivating mutations in the MLH1 DNA mismatch repair (MMR) gene underlie 42% of Lynch syndrome (LS) cases. LS is a cancer predisposition causing early onset colorectal and endometrial cancer. Nonsense and frameshift alterations unambiguously cause LS. The phenotype of missense mutations that only alter a single amino acid is often unclear. These variants of uncertain significance (VUS) hinder LS diagnosis and family screening and therefore functional tests are urgently needed. We developed a functional test for MLH1 VUS termed 'oligonucleotide-directed mutation screening' (ODMS). Methods: The MLH1 variant was introduced by oligonucleotide-directed gene modification in mouse embryonic stem cells that were subsequently exposed to the guanine analogue 6-thioguanine to determine whether the variant abrogated MMR. Resuts: In a proof-of-principle analysis, we demonstrate that ODMS can distinguish pathogenic and non-pathogenic MLH1 variants with a sensitivity of >95% and a specificity of >91%. We subsequently applied the screen to 51 MLH1 VUS and identified 31 pathogenic variants. Conclusion: ODMS is a reliable tool to identify pathogenic MLH1 variants. Implementation in clinical diagnostics will improve clinical care of patients with suspected LS and their relatives. [ABSTRACT FROM AUTHOR] more...

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. [ABSTRACT FROM AUTHOR] more...

Revertant mosaicism, or “natural gene therapy”, is the phenomenon in which germline mutations are corrected by somatic events. In recent years, revertant mosaicism has been identified in all major types of epidermolysis bullosa, the group of heritable blistering disorders caused by mutations in the genes encoding epidermal adhesion proteins. Moreover, revertant mosaicism appears to be present in all patients with a specific subtype of recessive epidermolysis bullosa. We therefore hypothesized that revertant mosaicism should be expected at least in all patients with recessive forms of epidermolysis bullosa. Naturally corrected, patient-own cells are of extreme interest for their promising therapeutic potential, and their presence in all patients would open exciting, new treatment perspectives to those patients. To test our hypothesis, we determined the probability that single nucleotide reversions occur in patients’ skin using a mathematical developmental model. According to our model, reverse mutations are expected to occur frequently (estimated 216x) in each patient’s skin. Reverse mutations should, however, occur early in embryogenesis to be able to drive the emergence of recognizable revertant patches, which is expected to occur in only one per ~10,000 patients. This underestimate, compared to our clinical observations, can be explained by the “late-but-fitter revertant cell” hypothesis: reverse mutations arise at later stages of development, but provide revertant cells with a selective growth advantage in vivo that drives the development of recognizable healthy skin patches. Our results can be extrapolated to any other organ with stem cell division numbers comparable to skin, which may offer novel future therapeutic options for other genetic conditions if these revertant cells can be identified and isolated. [ABSTRACT FROM AUTHOR] more...

Lynch syndrome (LS) is a hereditary cancer predisposition caused by inactivating mutations in DNA mismatch repair (MMR) genes. Mutations in the MSH6 DNA MMR gene account for approximately 18% of LS cases. Many LS-associated sequence variants are nonsense and frameshift mutations that clearly abrogate MMR activity. However, missense mutations whose functional implications are unclear are also frequently seen in suspected-LS patients. To conclusively diagnose LS and enroll patients in appropriate surveillance programs to reduce morbidity as well as mortality, the functional consequences of these variants of uncertain clinical significance (VUS) must be defined. We present an oligonucleotide-directed mutagenesis screen for the identification of pathogenic MSH6 VUS. In the screen, the MSH6 variant of interest is introduced into mouse embryonic stem cells by site-directed mutagenesis. Subsequent selection for MMR-deficient cells using the DNA damaging agent 6-thioguanine (6TG) allows the identification of MMR abrogating VUS because solely MMR-deficient cells survive 6TG exposure. We demonstrate the efficacy of the genetic screen, investigate the phenotype of 26 MSH6 VUS and compare our screening results to clinical data from suspected-LS patients carrying these variant alleles. [ABSTRACT FROM AUTHOR] more...

Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that "oligo targeting" can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors. [ABSTRACT FROM AUTHOR] more...

TALPID3, KIAA0586, Sonic Hedgehog, enteric nervous system, neural crest cell, gastrointestinal tract, short-rib polydactyly syndrome, Joubert syndrome Keywords: TALPID3; KIAA0586; Sonic Hedgehog; enteric nervous system; neural crest cell; gastrointestinal tract; short-rib polydactyly syndrome; Joubert syndrome EN TALPID3 KIAA0586 Sonic Hedgehog enteric nervous system neural crest cell gastrointestinal tract short-rib polydactyly syndrome Joubert syndrome 1 2 2 05/04/22 20220429 NES 220429 In the original article, there was an error. [Extracted from the article] more...

The treatment for metastasised medullary thyroid cancer is still a topic of discussion. One of the main challenges remains to find effective adjuvant and palliative options for patients with metastatic disease. The diagnostic and treatment strategies for this tumour are discussed and possible new developments commented. Approaches that target rearranged during transfection (RET) are preferable to those that target RET downstream proteins as, theoretically, blocking RET downstream targets will block only one of the many pathways activated by RET. Combining several agents would seem to be more promising, in particular agents that target RET with those that independently target RET signalling pathways or the more general mechanism of tumour progression. [ABSTRACT FROM AUTHOR] more...

Despite it has been reported that several loci are involved in Hirschsprung's disease, the molecular basis of the disease remains yet essentially unknown. The study of collective properties of modules of functionally-related genes provides an efficient and sensitive statistical framework that can overcome sample size limitations in the study of rare diseases. Here, we present the extension of a previous study of a Spanish series of HSCR trios to an international cohort of 162 HSCR trios to validate the generality of the underlying functional basis of the Hirschsprung's disease mechanisms previously found. The Pathway-Based Analysis (PBA) confirms a strong association of gene ontology (GO) modules related to signal transduction and its regulation, enteric nervous system (ENS) formation and other processes related to the disease. In addition, network analysis recovers sub-networks significantly associated to the disease, which contain genes related to the same functionalities, thus providing an independent validation of these findings. The functional profiles of association obtained for patients populations from different countries were compared to each other. While gene associations were different at each series, the main functional associations were identical in all the five populations. These observations would also explain the reported low reproducibility of associations of individual disease genes across populations. [ABSTRACT FROM AUTHOR] more...

Background Childhood-onset pulmonary arterial hypertension (PAH) is rare and differs from adult-onset disease in clinical presentation, with often unexplained mental retardation and dysmorphic features (MR/DF). Mutations in the major PAH gene, BMPR2, were reported to cause PAH in only 10-16% of childhoodonset patients. We aimed to identify more genes associated with childhood-onset PAH. Methods We studied 20 consecutive cases with idiopathic or heritable PAH. In patients with accompanying MR/DF (n=6) array-comparative genomic hybridisation analysis was performed, with the aim of finding common deletion regions containing candidate genes for PAH. Three patients had overlapping deletions of 17q23.2. TBX2 and TBX4 were selected from this area as candidate genes and sequenced in all 20 children. After identifying TBX4 mutations in these children, we subsequently sequenced TBX4 in a cohort of 49 adults with PAH. Because TBX4 mutations are known to cause small patella syndrome (SPS), all patients with newly detected TBX4 mutations were screened for features of SPS. We also screened a third cohort of 23 patients with SPS for PAH. Results TBX4 mutations (n=3) or TBX4-containing deletions (n=3) were detected in 6 out of 20 children with PAH (30%). All living patients and two parents with TBX4 mutations appeared to have previously unrecognised SPS. In the adult PAH-cohort, one TBX4 mutation (2%) was detected. Screening in the cohort of (predominantly adult) SPS patients revealed no PAH. Conclusions These data indicate that TBX4 mutations are associated with childhood-onset PAH, but that the prevalence of PAH in adult TBX4 mutation carriers is low. [ABSTRACT FROM AUTHOR] more...

Hirschsprung disease (HSCR) genetics is a paradigm for the study and understanding of multigenic disorders. Association between Down syndrome and HSCR suggests that genetic factors that predispose to HSCR map to chromosome 21. To identify these additional factors, we performed a dose-dependent association study on chromosome 21 in Down syndrome patients with HSCR. Assessing 10,895 SNPs in 26 Caucasian cases and their parents led to identify two associated SNPs (rs2837770 and rs8134673) at chromosome-wide level. Those SNPs, which were located in intron 3 of the DSCAM gene within a 19 kb-linkage disequilibrium block region were in complete association and are consistent with DSCAM expression during enteric nervous system development. We replicated the association of HSCR with this region in an independent sample of 220 non-syndromic HSCR Caucasian patients and their parents. At last, we provide the functional rationale to the involvement of DSCAM by network analysis and assessment of SOX10 regulation. Our results reveal the involvement of DSCAM as a HSCR susceptibility locus, both in Down syndrome and HSCR isolated cases. This study further ascertains the chromosome-scan dose-dependent methodology used herein as a mean to map the genetic bases of other sub-phenotypes both in Down syndrome and other aneuploidies. [ABSTRACT FROM AUTHOR] more...

Loss-of-function mutations in CLMP have been found in patients with Congenital Short Bowel Syndrome (CSBS), suggesting that its encoded protein plays a major role in intestinal development. CLMP is a membrane protein that co-localizes with tight junction proteins, but its function is largely unknown. We expressed wild-type (WT)-CLMP and a mutant-CLMP (associated with CSBS) in human intestinal epithelial T84 cells that, as we show here, do not produce endogenous CLMP. We investigated the effects of WT-CLMP and mutant-CLMP proteins on key cellular processes that are important for intestinal epithelial development, including migration, proliferation, viability and transepithelial resistance. Our data showed that expression of WT-CLMP or mutant-CLMP does not affect any of these processes. Moreover, our aggregation assays in CHO cells show that CLMP does not act as a strong adhesion molecule. Thus, our data suggest that, in the in vitro model systems we used, the key processes involved in intestinal epithelial development appear to be unaffected by WT-CLMP or mutant-CLMP. Further research is needed to determine the role of CLMP in the development of the intestine. [ABSTRACT FROM AUTHOR] more...

de Vries M M, Celestino R, Castro P, Eloy C, Máximo V, van der Wal J E, Plukker J T M, Links T P, Hofstra R M W, Sobrinho-Simões M & Soares P (2012) Histopathology 61, 833-843 RET/PTC rearrangement is prevalent in follicular Hürthle cell carcinomas Aims: The molecular alterations underlying follicular Hürthle cell carcinomas (FHCCs) are largely unknown. In an attempt to clarify this issue, we analysed a series of Hürthle cell tumours for the presence of RET/PTC and PAX8/PPARG rearrangements and BRAF, HRAS and NRAS mutations. Methods and results: We investigated a series of 20 follicular Hürthle cell tumours [17 FHCCs and three follicular Hürthle cell adenomas (FHCAs)]. RET/PTC rearrangements were found in 33% of FHCAs and in 38% of FHCCs. All RET/PTC-positive FHCCs had a solid pattern of growth. PAX8/PPARG rearrangement was present in 27% of the FHCCs which displayed, in most cases, a follicular architecture. NRAS mutation was detected in one FHCC. An FHCC with a solid/microfollicular growth pattern scored positive for both RET/PTC and PAX8/PPARG rearrangement. Conclusions: Our study has shown a significant association between RET/PTC rearrangements and FHCCs with a solid growth pattern, thus raising the possibility of using tyrosine kinase inhibitors for the treatment of patients with FHCCs, which are often refractory to radioiodine treatment. [ABSTRACT FROM AUTHOR] more...

Hirschsprung disease (HSCR, aganglionic megacolon) is a complex and heterogeneous disease with an incidence of 1 in 5000 live births. Despite the multifactorial determination of HSCR in the vast majority of cases, there is a monogenic subgroup for which private rare RET coding sequence mutations with high penetrance are found (45% of HSCR familial cases). An asymmetrical parental origin is observed for RET coding sequence mutations with a higher maternal inheritance. A parent-of-origin effect is usually assumed. Here we show that a differential reproductive rate for males and females also leads to an asymmetrical parental origin, which was never considered as a possible explanation till now. In the case of HSCR, we show a positive association between penetrance of the mutation and parental transmission asymmetry: no parental transmission asymmetry is observed in sporadic RET CDS mutation carrier cases for which penetrance of the mutation is low, whereas a parental transmission asymmetry is observed in affected sib-pairs for which penetrance of the mutation is higher. This allows us to conclude that the explanation for this parental asymmetry is that more severe mutations have resulted in a differential reproductive rate between male and female carriers. [ABSTRACT FROM AUTHOR] more...

CHD7 is a member of the chromodomain helicase DNA-binding (CHD) protein family that plays a role in transcription regulation by chromatin remodeling. Loss-of-function mutations in CHD7 are known to cause CHARGE syndrome, an autosomal-dominant malformation syndrome in which several organ systems, for example, the central nervous system, eye, ear, nose, and mediastinal organs, are variably involved. In this article, we review all the currently described CHD7 variants, including 183 new pathogenic mutations found by our laboratories. In total, we compiled 528 different pathogenic CHD7 alterations from 508 previously published patients with CHARGE syndrome and 294 unpublished patients analyzed by our laboratories. The mutations are equally distributed along the coding region of CHD7 and most are nonsense or frameshift mutations. Most mutations are unique, but we identified 94 recurrent mutations, predominantly arginine to stop codon mutations. We built a locus-specific database listing all the variants that is easily accessible at . In addition, we summarize the latest data on CHD7 expression studies, animal models, and functional studies, and we discuss the latest clinical insights into CHARGE syndrome. Hum Mutat 33:1149-1160, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR] more...

Defects in the mismatch repair system lead to microsatellite instability (MSI), a feature observed in ∼ 15% of all colorectal cancers (CRCs). Microsatellite mutations that drive tumourigenesis, typically inactivation of tumour suppressors, are selected for and are frequently detected in MSI cancers. Here, we evaluated somatic mutations in microsatellite repeats of 790 genes chosen based on reduced expression in MSI CRC and existence of a coding mononucleotide repeat of 6-10 bp in length. All the repeats were initially sequenced in 30 primary MSI CRC samples and whenever frameshift mutations were identified in >20%, additional 70 samples were sequenced. To distinguish driver mutations from passengers, we similarly analyzed the occurrence of frameshift mutations in 121 intronic control repeats and utilized a statistical regression model to determine cut-off mutation frequencies for repeats of all types (A/T and C/G, 6-10 bp). Along with several know target genes, including TGFBR2, ACVR2, and MSH3, six novel candidate driver genes emerged that harbored significantly more mutations than identical control repeats. The mutation frequencies in 100 MSI CRC samples were 51% in G8 of GLYR1, 47% in T9 of ABCC5, 43% in G8 of WDTC1, 33% in A8 of ROCK1, 30% in T8 of OR51E2, and 28% in A8 of TCEB3. Immunohistochemical staining of GLYR1 revealed defective protein expression in tumors carrying biallelic mutations, supporting a loss of function hypothesis. This is a large scale, unbiased effort to identify genes that when mutated are likely to contribute to MSI CRC development. [ABSTRACT FROM AUTHOR] more...

Objective To study the influence of a possible interaction between maternal smoking and high body mass index (BMI) on the occurrence of specific congenital heart anomalies (CHA) in offspring. Design Case-control study. Setting Data from a population-based birth defects registry in the Netherlands. Patients Cases were 797 children and fetuses born between 1997 and 2008 with isolated non-syndromic CHA. They were classified into five cardiac subgroups: septal defects (n=349), right ventricular outflow tract obstructive anomalies (n=126), left ventricular outflow tract obstructive anomalies (n=139), conotruncal defects (n=115) and other CHA (n=68). Controls were 322 children and fetuses with chromosomal anomalies without cardiac anomalies. Main outcome measures Investigation of whether an interaction between maternal smoking and high BMI influences the occurrence of CHA in offspring by calculation of the synergy factors and 95% CIs. Results As opposed to smoking or high BMI alone, the risk for CHA in the offspring of women with high BMI (≥25 kg/m2) who also smoked was significantly increased. The adjusted OR was 2.65 (95% CI 1.20 to 5.87) for all CHA, 2.60 (95% CI 1.05 to 6.47) for septal defects and 3.58 (95% CI 1.46 to 8.79) for outflow tract anomalies. The interaction between maternal high BMI and smoking contributed significantly to the occurrence of all offspring-CHA combined, and to the occurrence of all cardiac subgroup anomalies except right ventricular outflow tract obstructive anomalies. Conclusions Maternal overweight and smoking may have a synergistic adverse effect on the development of the fetal heart. Overweight women who wish to become pregnant should be strongly encouraged to stop smoking and to lose weight. [ABSTRACT FROM AUTHOR] more...

Background: Dystrophic epidermolysis bullosa is a genetic blistering disorder caused by mutations in the type VII collagen gene, COL7A1. In revertant mosaicism, germline mutations are corrected by somatic events resulting in a mosaic disease distribution. This "natural gene therapy" phenomenon long has been recognized in other forms of epidermolysis bullosa but only recently in dystrophic epidermolysis bullosa. Observations: We describe a 21-year-old man with recessive dystrophic epidermolysis bullosa carrying the homozygous c.6508C>T (p.Gln2170X) nonsense mutation who reported an unaffected skin patch on his neck where blisters never had occurred. Immunofluorescent type VII collagen staining was normal in 80% of the unaffected skin biopsy; however, it was strongly reduced in the affected skin. In the unaffected skin, the somatic nucleotide substitution c.6510G>T reverted the germline nonsense codon to tyrosine (p.Gln2170Tyr), thereby restoring functional protein production. Conclusions: Revertant mosaicism is considered rare in recessive dystrophic epidermolysis bullosa. However, it might be more common than previously anticipated because our patient is the third in whom revertant mosaicism was identified in a short period of time. The correction mechanism is different than that previously reported. Systematic examination of patients with recessive dystrophic epidermolysis bullosa, therefore, will likely reveal more patients with revertant patches. This is important because the natural gene therapy phenomenon may provide opportunities for revertant cell therapy. [ABSTRACT FROM AUTHOR] more...

Objectives To determine whether offering cardiac screening to relatives of patients with left ventricular outflow tract obstructions (LVOTOs) would be justified. Background LVOTOs have been recognised as a group of congenital heart diseases with 'high heritability'. One of the LVOTOs, the bicuspid aortic valve, is often asymptomatic, but has become known to be associated with sudden, unexpected cardiac death. However, the need for cardiac screening of first-degree relatives of patients with LVOTO has not been determined owing to the lack of studies in well-defined cohorts of consecutive patients. Methods The families of a cohort of 249 consecutive paediatric patients with LVOTO were offered genetic counselling. Of 182 consenting index patients, 40 patients (22%) appeared to have associated non-cardiac congenital anomalies (LVOTO-NCA). In the other 142 patients with LVOTO, cardiac screening of 449 firstdegree relatives was performed. Results Cardiac screening disclosed a cardiac anomaly in 34 first-degree relatives (8%). In 23 (68%) of these the cardiac anomaly was a bicuspid aortic valve. Twenty-four of these anomalies were newly detected by our screening programme (71%). These 34 cardiac anomalies were found in the families of 28 index cases (20%). Conclusions This study shows that of the patients with LVOTO without NCA, 20% had (an) affected first-degree relative(s), frequently with undetected bicuspid aortic valves. These data suggest that cardiac screening of relatives of patients with LVOTO without NCA is justified. This may help prevent sudden, unexpected, cardiac death or life-threatening complications in relatives with undetected bicuspid aortic valves. [ABSTRACT FROM AUTHOR] more...

For many complex traits, genetic variants have been found associated. However, it is still mostly unclear through which downstream mechanism these variants cause these phenotypes. Knowledge of these intermediate steps is crucial to understand pathogenesis, while also providing leads for potential pharmacological intervention. Here we relied upon natural human genetic variation to identify effects of these variants on trans-gene expression (expression quantitative trait locus mapping, eQTL) in whole peripheral blood from 1,469 unrelated individuals. We looked at 1,167 published trait- or disease-associated SNPs and observed trans-eQTL effects on 113 different genes, of which we replicated 46 in monocytes of 1,490 different individuals and 18 in a smaller dataset that comprised subcutaneous adipose, visceral adipose, liver tissue, and muscle tissue. HLA single-nucleotide polymorphisms (SNPs) were 10-fold enriched for trans-eQTLs: 48% of the trans-acting SNPs map within the HLA, including ulcerative colitis susceptibility variants that affect plausible candidate genes AOAH and TRBV18 in trans. We identified 18 pairs of unlinked SNPs associated with the same phenotype and affecting expression of the same trans-gene (21 times more than expected, P<10-16). This was particularly pronounced for mean platelet volume (MPV): Two independent SNPs significantly affect the well-known blood coagulation genes GP9 and F13A1 but also C19orf33, SAMD14, VCL, and GNG11. Several of these SNPs have a substantially higher effect on the downstream trans-genes than on the eventual phenotypes, supporting the concept that the effects of these SNPs on expression seems to be much less multifactorial. Therefore, these trans-eQTLs could well represent some of the intermediate genes that connect genetic variants with their eventual complex phenotypic outcomes. [ABSTRACT FROM AUTHOR] more...

Background The inversa type of recessive dystrophic epidermolysis bullosa (RDEB-I) is a rare variant of dystrophic epidermolysis bullosa, characterised by blistering in the body flexures, trunk, and mucosa. The cause of this specific distribution is unknown. So far, 20 genotypes have been described in RDEB-I and genotype–phenotype correlations have not been studied extensively. The aim of the study was to gain more insight into the pathophysiology of this intriguing RDEB-I phenotype. Methods Twenty Dutch and British RDEB-I patients, and full genotypes in 18 of them, were identified. The literature on RDEB-I genotypes was reviewed and an extensive genotype–phenotype correlation study for RDEB-I was conducted. Results All 20 patients had generalised blistering at birth and during early infancy. In most patients, the age of transition from generalised to inversa distribution was before the age of 4 years. A spectrum of disease severity, ranging from the mildest ‘mucosal only’ phenotype to the severest phenotype with limited acral involvement, was noted. The 29 genotypes of these RDEB-I patients and those reported in the literature revealed that RDEB-I is associated with specific recessive arginine and glycine substitutions in the triple helix domain of type VII collagen. Discussion and conclusion Why these specific arginine and glycine substitutions cause the inversa distribution remains unknown. It was not possible to identify clear differences in location and nature of substituting amino acids between these mutations and missense mutations causing other RDEB phenotypes. It is hypothesised that the higher skin temperature in the affected areas plays an important role in the pathophysiology of RDEB-I. [ABSTRACT FROM PUBLISHER] more...

Hirschsprung's disease (HSCR), a congenital complex disorder of intestinal innervation, is often associated with other inherited syndromes. Identifying genes involved in syndromic HSCR cases will not only help understanding the specific underlying diseases, but it will also give an insight into the development of the most frequent isolated HSCR. The association between hydrocephalus and HSCR is not surprising as a large number of patients have been reported to show the same clinical association, most of them showing mutations in the L1CAM gene, encoding a neural adhesion molecule often involved in isolated X-linked hydrocephalus. L1 defects are believed to be necessary but not sufficient for the occurrence of the intestinal phenotype in syndromic cases. In this paper, we have carried out the molecular characterization of a patient affected with Hirschsprung's disease and X-linked hydrocephalus, with a de novo reciprocal balanced translocation t(3;17)(p12;q21). In particular, we have taken advantage of this chromosomal defect to gain access to the predisposing background possibly leading to Hirschsprung's disease. Detailed analysis of the RET and L1CAM genes, and molecular characterization of MYO18A and TIAF1, the genes involved in the balanced translocation, allowed us to identify, besides the L1 mutation c.2265delC, different additional factors related to RET-dependent and -independent pathways which may have contributed to the genesis of enteric phenotype in the present patient.European Journal of Human Genetics (2009) 17, 483–490; doi:10.1038/ejhg.2008.191; published online 29 October 2008 [ABSTRACT FROM AUTHOR] more...

Inclusions in the brain containing α-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a C. elegans model that makes it possible to monitor, in living animals, the formation of α-synuclein inclusions. In worms of old age, inclusions contain aggregated asynuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in a-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between α-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson's disease and other α-synuclein related disorders. [ABSTRACT FROM AUTHOR] more...

It has been hypothesized that the net expression of a gene is determined by the combined effects of various transcriptional system regulators (TSRs). However, characterizing the complexity of regulation of the transcriptome is a major challenge. Principal component analysis on 17,550 heterogeneous human microarray experiments revealed that 50 orthogonal factors (hereafter called TSRs) are able to capture 64% of the variability in expression in a wide range of experimental conditions and tissues. We identified gene clusters controlled in the same direction and show that gene expression can be conceptualized as a process influenced by a fairly limited set of TSRs. Furthermore, TSRs can be linked to biological functions, as we demonstrate a strong relation between TSR-related gene clusters and biological functionality as well as cellular localization, i.e. gene products of similarly regulated genes by a specific TSR are located in identical parts of a cell. Using 3,934 diverse mouse microarray experiments we found striking similarities in transcriptional system regulation between human and mouse. Our results give biological insights into regulation of the cellular transcriptome and provide a tool to characterize expression profiles with highly reliable TSRs instead of thousands of individual genes, leading to a >500-fold reduction of complexity with just 50 TSRs. This might open new avenues for those performing gene expression profiling studies. [ABSTRACT FROM AUTHOR] more...

Biallelic germline mutations of MUTYH—a gene encoding a base excision repair protein—are associated with an increased susceptibility of colorectal cancer. Whether monoallelic MUTYH mutations also increase cancer risk is not yet clear, although there is some evidence suggesting a slight increase of risk. As the MUTYH protein interacts with the mismatch repair (MMR) system, we hypothesised that the combination of a monoallelic MUTYH mutation with an MMR gene mutation increases cancer risk. We therefore investigated the prevalence of monoallelic MUTYH mutations in carriers of a germline MMR mutation: 40 carriers of a truncating mutation (group I) and 36 of a missense mutation (group II). These patients had been diagnosed with either colorectal or endometrial cancer. We compared their MUTYH mutation frequencies with those observed in a group of 134 Dutch colorectal and endometrial cancer patients without an MMR gene mutation (0.7%) and those reported for Caucasian controls (1.5%). In group I one monoallelic MUTYH mutation was found (2.5%). In group II five monoallelic germline MUTYH mutations were found (14%), four of them in MSH6 missense mutation carriers (20%). Of all patients with an MMR gene mutation, only those with a missense mutation showed a significantly higher frequency of (monoallelic) MUTYH mutations than the Dutch cancer patients without MMR gene mutations ( P=0.002) and the published controls ( P=0.001). These results warrant further study to test the hypothesis of mutations in MMR genes (in particular MSH6) and MUTYH acting together to increase cancer risk. [ABSTRACT FROM AUTHOR] more...

Methylation-associated inactivation of RASSF1A has frequently been observed in several human malignancies including sporadic colorectal and gastric cancer. However, nothing is known about the RASSF1A methylation status in the setting of MMR-deficient gastrointestinal tumours. In this study, we analysed systematically alterations in KRAS, BRAF and RASSF1A, in order to define the frequency and the pattern of these genetic/epigenetic alterations in three distinct subsets of MSI gastrointestinal tumours. Further, an association study was performed between RASSF1A methylation and the clinicopathological parameters in order to determine the profile of tumours harbouring this epigenetic event. A total of 56 MSI sporadic gastrointestinal tumours (31 colorectal and 25 gastric) and 20 MSI HNPCC analysed for KRAS/BRAF were analysed for RASSF1A promoter hypermethylation by MSP and DNA sequencing. No significant differences were found between the frequency of RASSF1A methylation in sporadic MSI colorectal and gastric carcinomas and HNPCC carcinomas (P=0.31). Methylation of RASSF1A was present in 16 of 31 (52%) sporadic MSI colorectal and 11 of 25 (44%) MSI gastric carcinomas, and in six of 20 (30%) HNPCC carcinomas. Nearly 36% of MSI sporadic colorectal carcinomas (CRCs) had RASSF1A methylation and activating mutations at KRAS and/or BRAF. In contrast, only 10 and 8% of HNPCC and sporadic gastric carcinomas, respectively, had concomitant KRAS mutations and RASSF1A methylation. The MSI sporadic gastric and CRCs with RASSF1A methylation were preferentially poorly differentiated (P=0.03, 0.05, respectively). We show that the profile of alterations RASSF1A, KRAS/BRAF is different among the three groups of MSI gastrointestinal tumours. Further, we demonstrate that MSI sporadic CRCs accumulated significantly more epigenetic/genetic alterations in RASSF1A, KRAS/BRAF than MSI sporadic gastric or HNPCC carcinomas (P=0.016). These results are likely to have therapeutic implications in the near future, due to the possibilities of using specific kinase inhibitors alone or in association with demethylating agents in MSI tumour types harbouring KRAS or BRAF mutations and RASSF1A methylation.Oncogene (2005) 24, 7630–7634. doi:10.1038/sj.onc.1208906 published online 11 July 2005 [ABSTRACT FROM AUTHOR] more...

Recently, it was shown that the oncogenic activation of BRAF, a member of the RAS/RAF family of kinases, by the V600E mutation is characteristic for sporadic colon tumors with microsatellite instability. Further, it was shown to associate with the silencing of the mismatch repair (MMR) gene MLH1 by hypermethylation. Moreover, BRAF mutations proved to be absent in tumors from hereditary nonpolyposis colorectal cancer syndrome (HNPCC) families with germline mutations in the MMR genes MLH1 and MSH2. These data suggest that the oncogenic activation of BRAF is involved only in sporadic colorectal tumorigenesis. In order to further support this hypothesis, we have extended the analysis of the BRAF gene to a different subset of HNPCC families without germline mutations in MLH1 and MSH2. BRAF-V600E mutations were analysed by automatic sequencing in 38 tumors from HNPCC families with germline mutations in the MSH6 gene and also in HNPCC (suspected) families that do not have mutations in the MMR genes MLH1, MSH2 and MSH6. All patients belong to different families. No mutations were detected in 14 tumors from HNPCC patients with germline mutations in MSH6. Further, no mutations of BRAF were found in tumors from 23 MMR-negative families, from which 13 fulfilled the Amsterdam criteria (HNPCC) and 10 were suspected for HNPCC as they were positive for the Bethesda criteria. Overall, our data reinforce the concept that BRAF is not involved in the colorectal tumorigenesis of HNPCC. The detection of a positive BRAF-V600E mutation in a colorectal cancer suggests a sporadic origin of the disease and the absence of germline alterations of MLH1, MSH2 and also of MSH6. These findings have a potential impact in the genetic testing for HNPCC diagnostics and suggest a potential use of BRAF as exclusion criteria for HNPCC or as a molecular marker of sporadic cancer.Oncogene (2005) 24, 3995–3998. doi:10.1038/sj.onc.1208569 Published online 21 March 2005 [ABSTRACT FROM AUTHOR] more...

PURPOSE: Recently, we found a certain haplotype in the human leukocyte antigen Class III subregion to be associated with breast cancer. Epidemiologic studies have shown that breast cancer and colorectal cancer have several risk factors in common. In view of these studies and because polymorphisms located in the human leukocyte antigen III region have been found to be associated with colorectal cancer, we wondered whether the same region also is involved in colorectal cancer susceptibility. METHODS: The human leukocyte antigen region was genotyped with 14 microsateffite markers in germline DNA from 643 colorectal cancer patients and 841 family-based controls. Association analyses and the Haplotype Sharing Statistic were used to search for differences between patients and controls. Sub- group analyses were performed for gender, age at diagnosis, and localization of the tumor. RESULTS: The Haplotype Sharing Statistic analysis revealed neither a difference in mean haplotype sharing between all patients and controls, nor in any of the subgroups. The single allele, genotype, and two-locus association analyses for all patients and for the different subgroups did not show an association with cob- rectal cancer for the 14 microsatellite markers. Also, no association was observed between the tumor necrosis factor-beta polymorphism and colorectal cancer. CONCLUSIONS: No association was observed between commonly occurring haplotypes and alleles in the human leukocyte antigen region and colorectal cancer risk. [ABSTRACT FROM AUTHOR] more...

Patients with Hirschsprung disease (HSCR) do not always receive a genetic diagnosis after routine screening in clinical practice. One of the reasons for this could be that the causal mutation is not present in the cell types that are usually tested—whole blood, dermal fibroblasts or saliva—but is only in the affected tissue. Such mutations are called somatic, and can occur in a given cell at any stage of development after conception. They will then be present in all subsequent daughter cells. Here, we investigated the presence of somatic mutations in HSCR patients. For this, whole-exome sequencing and copy number analysis were performed in DNA isolated from purified enteric neural crest cells (ENCCs) and blood or fibroblasts of the same patient. Variants identified were subsequently validated by Sanger sequencing. Several somatic variants were identified in all patients, but causative mutations for HSCR were not specifically identified in the ENCCs of these patients. Larger copy number variants were also not found to be specific to ENCCs. Therefore, we believe that somatic mutations are unlikely to be identified, if causative for HSCR. Here, we postulate various modes of development following the occurrence of a somatic mutation, to describe the challenges in detecting such mutations, and hypothesize how somatic mutations may contribute to 'missing heritability' in developmental defects. [ABSTRACT FROM AUTHOR] more...

Analyzes the functional consequences of two distinct RET photo-oncogene mutations causing Hirschsprung's disease. Coding of a transmembrane receptor by the gene; Residues essential for the recognition of RET; Effects of the mutations on the ability of RET to transduce downstream signals. more...