Bi-allelic Variations of SMO in Humans Cause a Broad Spectrum of Developmental Anomalies Due to Abnormal Hedgehog Signaling.

The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report bi-allelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans.

L1 syndrome diagnosis complemented with functional analysis of L1CAM variants located to the two N-terminal Ig-like domains.

L1CAM gene mutations cause neurodevelopmental disorders collectively termed L1 syndrome. Insufficient information about L1CAM variants complicates clinical prognosis, genetic diagnosis and genetic counseling. We combined clinical data, in silico effect predictions and functional analysis of four L1CAM variants, p.I37N, p.T38M, p.M172I and p.D202Y, located to the two N-terminal Ig-like domains present in five families with symptoms of L1 syndrome. Software tools predicted destabilizing effects of p.I37N and p.D202Y but results for p.T38M and p.M172I were inconsistent. Cell surface expression of mutant proteins L1-T38M, L1-M172I and L1-D202Y was normal. Conversely, L1-I37N accumulated in the endoplasmic reticulum (ER) and showed temperature-sensitive protein maturation suggesting that p.I37N induces protein misfolding. L1CAM-mediated cell-cell aggregation was severely impaired by L1CAM variants p.I37N, p.M172I and p.D202Y but was preserved by the variant p.T38M. Our experimental data indicate that protein misfolding and accumulation in the ER affect function of the L1CAM variant p.I37N whereas the variants p.M172I and p.D202Y impair homophilic interaction at the cell surface.

ATP5PO levels regulate enteric nervous system development in zebrafish, linking Hirschsprung disease to Down Syndrome.

Hirschsprung disease (HSCR) is a complex genetic disorder characterized by the absence of enteric nervous system (ENS) in the distal region of the intestine. Down Syndrome (DS) patients have a >50-fold higher risk of developing HSCR than the general population, suggesting that overexpression of human chromosome 21 (Hsa21) genes contribute to HSCR etiology. However, identification of responsible genes remains challenging. Here, we describe a genetic screening of potential candidate genes located on Hsa21, using the zebrafish. Candidate genes were located in the DS-HSCR susceptibility region, expressed in the human intestine, were known potential biomarkers for DS prenatal diagnosis, and were present in the zebrafish genome. With this approach, four genes were selected: RCAN1, ITSN1, ATP5PO and SUMO3. However, only overexpression of ATP5PO, coding for a component of the mitochondrial ATPase, led to significant reduction of ENS cells. Paradoxically, in vitro studies showed that overexpression of ATP5PO led to a reduction of ATP5PO protein levels. Impaired neuronal differentiation and reduced mitochondrial ATP production, were also detected in vitro, after overexpression of ATP5PO in a neuroblastoma cell line. Finally, epistasis was observed between ATP5PO and ret, the most important HSCR gene. Taken together, our results identify ATP5PO as the gene responsible for the increased risk of HSCR in DS patients in particular if RET variants are also present, and show that a balanced expression of ATP5PO is required for normal ENS development.

Building a brain in the gut: development of the enteric nervous system.

The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, is an essential component of the gut neuromusculature and controls many aspects of gut function, including coordinated muscular peristalsis. The ENS is entirely derived from neural crest cells (NCC) which undergo a number of key processes, including extensive migration into and along the gut, proliferation, and differentiation into enteric neurons and glia, during embryogenesis and fetal life. These mechanisms are under the molecular control of numerous signaling pathways, transcription factors, neurotrophic factors and extracellular matrix components. Failure in these processes and consequent abnormal ENS development can result in so-called enteric neuropathies, arguably the best characterized of which is the congenital disorder Hirschsprung disease (HSCR), or aganglionic megacolon. This review focuses on the molecular and genetic factors regulating ENS development from NCC, the clinical genetics of HSCR and its associated syndromes, and recent advances aimed at improving our understanding and treatment of enteric neuropathies.

CHD7 mutations and CHARGE syndrome: the clinical implications of an expanding phenotype.

BACKGROUND: CHARGE syndrome is a highly variable, multiple congenital anomaly syndrome, of which the complete phenotypic spectrum was only revealed after identification of the causative gene in 2004. CHARGE is an acronym for ocular coloboma, congenital heart defects, choanal atresia, retardation of growth and development, genital hypoplasia, and ear anomalies associated with deafness. This typical combination of clinical features is caused by autosomal dominant mutations in the CHD7 gene. OBJECTIVE: To explore the emerging phenotypic spectrum of CHD7 mutations, with a special focus on the mild end of the spectrum. METHODS: We evaluated the clinical characteristics in our own cohort of 280 CHD7 positive patients and in previously reported patients with CHD7 mutations and compared these with previously reported patients with CHARGE syndrome but an unknown CHD7 status. We then further explored the mild end of the phenotypic spectrum of CHD7 mutations. RESULTS: We discuss that CHARGE syndrome is primarily a clinical diagnosis. In addition, we propose guidelines for CHD7 analysis and indicate when evaluation of the semicircular canals is helpful in the diagnostic process. Finally, we give updated recommendations for clinical surveillance of patients with a CHD7 mutation, based on our exploration of the phenotypic spectrum and on our experience in a multidisciplinary outpatient clinic for CHARGE syndrome. CONCLUSION: CHARGE syndrome is an extremely variable clinical syndrome. CHD7 analysis can be helpful in the diagnostic process, but the phenotype cannot be predicted from the genotype.

Haplotype sharing test maps genes for familial cardiomyopathies.

Identifying a mutation in a heterogeneous disease such as inherited cardiomyopathy is a challenge because classical methods, like linkage analysis, can often not be applied as there are too few meioses between affected individuals. However, if affected individuals share the same causal mutation, they will also share a genomic region surrounding it. High-density genotyping arrays are able to identify such regions shared among affected individuals. We hypothesize that the longest shared haplotype is most likely to contain the disease-causing mutation. We applied this method to two pedigrees: one with arrhythmogenic right ventricular cardiomyopathy (ARVC) and one with dilated cardiomyopathy (DCM), using high-density genome-wide SNP arrays. In the ARVC pedigree, the largest haplotype was on chromosome 12 and contained a causative PKP2 mutation. In the DCM pedigree, a causative MYH7 mutation was present on a large shared haplotype on chromosome 14. We calculated that a pedigree containing at least seven meioses has a high chance of correctly detecting the mutation-containing haplotype as the largest. Our data show that haplotype sharing analysis can assist in identifying causative genes in families with low penetrance Mendelian diseases, in which standard tools cannot be used due to lack of sufficient pedigree information.

Tumours with loss of MSH6 expression are MSI-H when screened with a pentaplex of five mononucleotide repeats.

BACKGROUND: microsatellite instability (MSI) is commonly screened using a panel of two mononucleotide and three dinucleotide repeats as recommended by a consensus meeting on MSI tumours held at the National Cancer Institute (Bethesda, MD, USA). According to these recommendations, tumours are classified as MSI-H when at least two of the five microsatellite markers show instability, MSI-L when only one marker shows instability and MSS when none of the markers show instability. Almost all MSI-H tumours are characterised by alterations in one of the four major proteins of the mismatch repair (MMR) system (MLH1, MSH2, MSH6 or PMS2) that renders them MMR deficient, whereas MSI-L and MSS tumours are generally MMR proficient. However, tumours from patients with a pathogenic germline mutation in MSH6 can sometimes present an MSI-L phenotype with the NCI panel. The MSH6 protein is not involved in the repair of mismatches of two nucleotides in length and consequently the three dinucleotide repeats of the NCI panel often show stability in MSH6-deficient tumours. METHODS: a pentaplex panel comprising five mononucleotide repeats has been recommended as an alternative to the NCI panel to determine tumour MSI status. Several studies have confirmed the sensitivity, specificity and ease of use of the pentaplex panel; however, its sensitivity for the detection of MSH6-deficient tumours is so far unknown. Here, we used the pentaplex panel to evaluate MSI status in 29 tumours known to harbour an MSH6 defect. RESULTS: MSI-H status was confirmed in 15 out of 15 (100%) cases where matching normal DNA was available and in 28 out of 29 (97%) cases where matching DNA was not available or was not analysed. CONCLUSION: these results show that the pentaplex assay efficiently discriminates the MSI status of tumours with an MSH6 defect.

COGENT (COlorectal cancer GENeTics): an international consortium to study the role of polymorphic variation on the risk of colorectal cancer.

It is now recognised that a part of the inherited risk of colorectal cancer (CRC) can be explained by the co-inheritance of low-penetrance genetic variants. The accumulated experience to date in identifying these variants has served to highlight difficulties in conducting statistically and methodologically rigorous studies and follow-up analyses. The COGENT (COlorectal cancer GENeTics) consortium includes 20 research groups in Europe, Australia, the Americas, China and Japan. The overarching goal of COGENT is to identify and characterise low-penetrance susceptibility variants for CRC through association-based analyses. In this study, we review the rationale for identifying low-penetrance variants for CRC and our proposed strategy for establishing COGENT.

Fine mapping of the 9q31 Hirschsprung's disease locus.

Hirschsprung's disease (HSCR) is a congenital disorder characterised by the absence of ganglia along variable lengths of the intestine. The RET gene is the major HSCR gene. Reduced penetrance of RET mutations and phenotypic variability suggest the involvement of additional modifying genes in the disease. A RET-dependent modifier locus was mapped to 9q31 in families bearing no coding sequence (CDS) RET mutations. Yet, the 9q31 causative locus is to be identified. To fine-map the 9q31 region, we genotyped 301 tag-SNPs spanning 7 Mb on 137 HSCR Dutch trios. This revealed two HSCR-associated regions that were further investigated in 173 Chinese HSCR patients and 436 controls using the genotype data obtained from a genome-wide association study recently conducted. Within one of the two identified regions SVEP1 SNPs were found associated with Dutch HSCR patients in the absence of RET mutations. This ratifies the reported linkage to the 9q31 region in HSCR families with no RET CDS mutations. However, this finding could not be replicated. In Chinese, HSCR was found associated with IKBKAP. In contrast, this association was stronger in patients carrying RET CDS mutations with p = 5.10 x 10(-6) [OR = 3.32 (1.99, 5.59)] after replication. The HSCR-association found for IKBKAP in Chinese suggests population specificity and implies that RET mutation carriers may have an additional risk. Our finding is supported by the role of IKBKAP in the development of the nervous system.

Medullary thyroid carcinoma and biomarkers: past, present and future.

The clinical management of patients with persistent or recurrent medullary thyroid carcinoma (MTC) is still under debate, because these patients either have a long-term survival, due to an indolent course of the disease, or develop rapidly progressing disease leading to death from distant metastases. At this moment, it cannot be predicted what will happen within most individual cases. Biomarkers, indicators which can be measured objectively, can be helpful in MTC diagnosis, molecular imaging and treatment, and/or identification of MTC progression. Several MTC biomarkers are already implemented in the daily management of MTC patients. More research is being aimed at the improvement of molecular imaging techniques and the development of molecular systemic therapies. Recent discoveries, like the prognostic value of plasma calcitonin and carcino-embryonic antigen doubling-time and the presence of somatic RET mutations in MTC tissue, may be useful tools in clinical decision making in the future. In this review, we provide an overview of different MTC biomarkers and their applications in the clinical management of MTC patients.

Hirschsprung disease, associated syndromes and genetics: a review.

Hirschsprung disease (HSCR, aganglionic megacolon) represents the main genetic cause of functional intestinal obstruction with an incidence of 1/5000 live births. This developmental disorder is a neurocristopathy and is characterised by the absence of the enteric ganglia along a variable length of the intestine. In the last decades, the development of surgical approaches has importantly decreased mortality and morbidity which allowed the emergence of familial cases. Isolated HSCR appears to be a non-Mendelian malformation with low, sex-dependent penetrance, and variable expression according to the length of the aganglionic segment. While all Mendelian modes of inheritance have been described in syndromic HSCR, isolated HSCR stands as a model for genetic disorders with complex patterns of inheritance. The tyrosine kinase receptor RET is the major gene with both rare coding sequence mutations and/or a frequent variant located in an enhancer element predisposing to the disease. Hitherto, 10 genes and five loci have been found to be involved in HSCR development.

Evaluation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify PMS2 mutation carriers.

Until recently, no prediction models for Lynch syndrome (LS) had been validated for PMS2 mutation carriers. We aimed to evaluate MMRpredict and PREMM5 in a clinical cohort and for PMS2 mutation carriers specifically. In a retrospective, clinic-based cohort we calculated predictions for LS according to MMRpredict and PREMM5. The area under the operator receiving characteristic curve (AUC) was compared between MMRpredict and PREMM5 for LS patients in general and for different LS genes specifically. Of 734 index patients, 83 (11%) were diagnosed with LS; 23 MLH1, 17 MSH2, 31 MSH6 and 12 PMS2 mutation carriers. Both prediction models performed well for MLH1 and MSH2 (AUC 0.80 and 0.83 for PREMM5 and 0.79 for MMRpredict) and fair for MSH6 mutation carriers (0.69 for PREMM5 and 0.66 for MMRpredict). MMRpredict performed fair for PMS2 mutation carriers (AUC 0.72), while PREMM5 failed to discriminate PMS2 mutation carriers from non-mutation carriers (AUC 0.51). The only statistically significant difference between PMS2 mutation carriers and non-mutation carriers was proximal location of colorectal cancer (77 vs. 28%, p < 0.001). Adding location of colorectal cancer to PREMM5 considerably improved the models performance for PMS2 mutation carriers (AUC 0.77) and overall (AUC 0.81 vs. 0.72). We validated these results in an external cohort of 376 colorectal cancer patients, including 158 LS patients. MMRpredict and PREMM5 cannot adequately identify PMS2 mutation carriers. Adding location of colorectal cancer to PREMM5 may improve the performance of this model, which should be validated in larger cohorts.

MEIS and PBX homeobox proteins in ovarian cancer.

Three amino-acid loop extension (TALE) homeobox proteins MEIS and PBX are cofactors for HOX-class homeobox proteins, which control growth and differentiation during embryogenesis and homeostasis. We showed that MEIS and PBX expression are related to cisplatin resistance in ovarian cancer cell lines. Therefore, MEIS1, MEIS2 and PBX expression were investigated immunohistochemically in a tissue microarray (N=232) of ovarian cancers and ovarian surface epithelium (N=15). Results were related to clinicopathologic characteristics and survival. All cancers expressed MEIS1, MEIS2 and PBX in nucleus and cytoplasm. MEIS1 and 2 only stained nuclear in surface epithelium. Nuclear MEIS2 was negatively related to stage, grade and overall survival in univariate analyses. Additionally, MEIS and PBX RNA expression in ovarian surface epithelium and other normal tissues and ovarian cancer versus other tumour types using public array data sets were studied. In ovarian cancer, MEIS1 is highly expressed compared to other cancer types. In conclusion, MEIS and PBX are extensively expressed in ovarian carcinomas and may play a role in ovarian carcinogenesis.

A novel susceptibility locus for Hirschsprung's disease maps to 4q31.3-q32.3.

We report on a multigenerational family with isolated Hirschsprung's disease (HSCR). Five patients were affected by either short segment or long segment HSCR. The family consists of two main branches: one with four patients (three siblings and one maternal uncle) and one with one patient. Analysis of the RET gene, the major gene involved in HSCR susceptibility, revealed neither linkage nor mutations. A genome wide linkage analysis was performed, revealing suggestive linkage to a region on 4q31-q32 with a maximum parametric multipoint LOD score of 2.7. Furthermore, non-parametric linkage (NPL) analysis of the genome wide scan data revealed a NPL score of 2.54 (p = 0.003) for the same region on chromosome 4q (D4S413-D4S3351). The minimum linkage interval spans a region of 11.7 cM (12.2 Mb). No genes within this chromosomal interval have previously been implicated in HSCR. Considering the low penetrance of disease in this family, the 4q locus may be necessary but not sufficient to cause HSCR in the absence of modifying loci elsewhere in the genome. Our results suggest the existence of a new susceptibility locus for HSCR at 4q31.3-q32.3.

NDRG4, an early detection marker for colorectal cancer, is specifically expressed in enteric neurons.

BACKGROUND: Promoter methylation of N-myc Downstream-Regulated Gene 4 (NDRG4) in fecal DNA is an established early detection marker for colorectal cancer (CRC). Despite its connection to CRC, NDRG4 is predominantly studied in brain and heart, with little to no knowledge about its expression or role in other organs. In this study, we aimed to determine the whole-body expression of NDRG4, with a focus on the intestinal tract. METHODS: We investigated NDRG4 expression throughout the body by immunohistochemistry, Western Blotting and in situ mRNA hybridization using tissues from NDRG4 wild-type, heterozygous and knockout mice and humans. In addition, we explored cell-specific expression of NDRG4 in murine whole-mount gut preparations using immunofluorescence and confocal microscopy. KEY RESULTS: NDRG4 is specifically expressed within nervous system structures throughout the body. In the intestinal tract of both mouse and man, NDRG4 immunoreactivity was restricted to the enteric nervous system (ENS), where it labeled cell bodies of the myenteric and submucosal plexuses and interconnecting nerve fibers. More precisely, NDRG4 expression was limited to neurons, as NDRG4 always co-localized with HuC/D (pan-neuronal marker) but never with GFAP (an enteric glial cell marker). Furthermore, NDRG4 was expressed in various neuropeptide Y positive neurons, but was only found in a minority (~10%) of neurons expressing neuronal nitric oxide synthase. CONCLUSIONS AND INFERENCES: NDRG4 is exclusively expressed by central, peripheral and enteric neurons/nerves, suggesting a neuronal-specific role of this protein. Our findings raise the question whether NDRG4, via the ENS, an understudied component of the tumor microenvironment, supports CRC development and/or progression.

Cellular effects of imatinib on medullary thyroid cancer cells harboring multiple endocrine neoplasia Type 2A and 2B associated RET mutations.

BACKGROUND: Activating mutations in the RET gene, which encodes a tyrosine kinase receptor, often cause medullary thyroid carcinoma (MTC). Surgical resection is the only curative treatment; no effective systemic treatment is available. We evaluated imatinib, a tyrosine kinase inhibitor currently used to treat chronic myelogenous leukemia and gastrointestinal stromal tumors, as a potential drug for systemic treatment of MTC, in 2 MTC-derived cell lines expressing multiple endocrine neoplasia-associated mutant RET receptors. METHODS: We determined RET expression and Y1062 phosphorylation using Western blot analysis and quantitative polymerase chain reaction. We determined the effects on cell proliferation by a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and we used fluorescence-activated cell sorter analysis with annexin V/propidium iodide staining to study imatinib-induced cell-cycle arrest, apoptosis, and cell death. RESULTS: Imatinib inhibited RET Y1062 phosphorylation in a dose-dependent manner after 1.5 hours of exposure. After 16 hours both RET Y1062 phosphorylation and protein expression levels were affected. Dose-dependent decreases in cell proliferation of both cell lines after exposure to imatinib with inhibitory concentration of 50% levels of 23 +/- 2 micromol/L and 25 +/- 4 micromol/L were seen. These values are high, compared with those for chronic myelogenous leukemia and gastrointestinal stromal tumors. We further could show that imatinib induced cell-cycle arrest, and apoptotic and nonapoptotic cell death. CONCLUSIONS: Imatinib inhibits RET-mediated MTC cell growth affecting RET protein levels in vitro in a dose-dependent manner. The concentration of imatinib necessary to inhibit RET in vitro, however, makes it impossible to conclude that imatinib monotherapy will be a good option for systemic therapy of MTC.

[Prophylactic thyroidectomy in children who are carriers of a multiple endocrine neoplasia type 2 mutation: description of 20 cases and recommendations based on the literature]. / Profylactische thyreoïdectomie bij kinderen die drager zijn van een mutatie van multipele endocriene neoplasie type 2: beschrijving van 20 casussen en aanbevelingen op grond van de literatuur.

OBJECTIVE: Evaluation of treatment of children who are proven carriers of a multiple endocrine neoplasia type 2 (MEN 2)-associated rearranged during transfection (RET) gene mutation. DESIGN: Retrospective case study and review of the literature. METHOD: Between 1976 and 2005, 6 boys and 14 girls with a proven RET mutation or biochemical indication of MEN 2 had thyroid surgery at the University Medical Center, Groningen, The Netherlands. The median age was 10 years (range: 0-08). Preoperative assessment, surgical procedure, pathological findings, postoperative complications and treatment results were studied and compared with data from the literature. RESULTS: All 20 children underwent total thyroidectomy. In 17 children with preoperatively abnormal basal or stimulated calcitonin levels, total thyroidectomy was combined with tracheo-oesophageal exploration (n = 6) or central compartment dissection (n = 11). C-cell hyperplasia was found in 19 cases (95%) and medullary thyroid carcinoma in 14 (70%; aged 3-18 years). Lymph-node metastases were found in 3 children (15%), all over the age of 10. They underwent additional selective lateral neck dissection, unilateral in 2 cases and bilateral in 1. Two children developed hypoparathyroidism postoperatively, no recurrent laryngeal-nerve palsy was observed. All patients are clinically free of disease after a median follow-up of 9 years (range: 0.6-27). The patients with node metastases still have biochemical evidence of disease. The literature indicates that the progression of the malignant transformation to medullary thyroid carcinoma is connected to the type of RET-mutation. The treatment plan depends on the type of mutation. CONCLUSION: Medullary thyroid cancer occurs at a very young age in carriers ofgermline RET mutations. In patients with high-risk mutations prophylactic thyroidectomy is likely to be recommended before the child reaches the age of 2. Elective central lymph-node dissection can be omitted in this instance. After this age, however, the risk of lymph-node metastases increases and, for cases with increased basal or stimulated calcitonin levels, total thyroidectomy with central compartment dissection is indicated.

BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing.

BACKGROUND: According to the international criteria for hereditary non-polyposis colorectal cancer (HNPCC) diagnostics, cancer patients with a family history or early onset of colorectal tumours showing high microsatellite instability (MSI-H) should receive genetic counselling and be offered testing for germline mutations in DNA repair genes, mainly MLH1 and MSH2. Recently, an oncogenic V600E hotspot mutation within BRAF, a kinase encoding gene from the RAS/RAF/MAPK pathway, has been found to be associated with sporadic MSI-H colon cancer, but its association with HNPCC remains to be further clarified. METHODS: BRAF-V600E mutations were analysed by automatic sequencing in colorectal cancers from 206 sporadic cases with MSI-H and 111 HNPCC cases with known germline mutations in MLH1 and MSH2. In addition, 45 HNPCC cases showing abnormal immunostaining for MSH2 were also analysed. RESULTS: The BRAF-V600E hotspot mutation was found in 40% (82/206) of the sporadic MSI-H tumours analysed but in none of the 111 tested HNPCC tumours or in the 45 cases showing abnormal MSH2 immunostaining. CONCLUSIONS: Detection of the V600E mutation in a colorectal MSI-H tumour argues against the presence of a germline mutation in either the MLH1 or MSH2 gene. Therefore, screening of these mismatch repair (MMR) genes can be avoided in cases positive for V600E if no other significant evidence, such as fulfilment of the strict Amsterdam criteria, suggests MMR associated HNPCC. In this context, mutation analysis of the BRAF hotspot is a reliable, fast, and low cost strategy which simplifies genetic testing for HNPCC.

Two cases of the caudal duplication anomaly including a discordant monozygotic twin.

We present two unrelated patients with various duplications in the caudal region. One patient presented with a duplication of the distal spine from L4, left double ureter, duplication of the vagina and cervix, and duplication of the distal colon. The second patient was diagnosed with a duplication of the colon, bladder, vagina and uterus. The first patient had an unaffected monozygotic twin sister. Dominguez et al. [1993: Am J Dis Child 147:1048-1052] presented six similar cases, and introduced the name "caudal duplication syndrome." The pathogenesis of the caudal duplication anomaly is unclear. The possibility of a polytopic primary developmental field defect or a disruptive sequence are discussed. On the other hand, somatic or germline mutations in certain developmental genes could be involved, as illustrated by the mouse mutations disorganisation and fused. DNA-analysis of the AXIN1 gene, the human homologue of the gene responsible for fused, performed in our first patient, did not show any apparent pathogenic mutation.