A comparison between the abdominal and femoral adipose tissue proteome of overweight and obese women.

Body fat distribution is an important determinant of cardiometabolic health. Lower-body adipose tissue (AT) has protective characteristics as compared to upper-body fat, but the underlying depot-differences remain to be elucidated. Here, we compared the proteome and morphology of abdominal and femoral AT. Paired biopsies from abdominal and femoral subcutaneous AT were taken from eight overweight/obese (BMI ≥ 28 kg/m2) women with impaired glucose metabolism after an overnight fast. Proteins were isolated and quantified using liquid chromatography-mass spectrometry, and protein expression in abdominal and femoral subcutaneous AT was compared. Moreover, correlations between fat cell size and the proteome of both AT depots were determined. In total, 651 proteins were identified, of which 22 proteins tended to be differentially expressed between abdominal and femoral AT after removal of blood protein signals (p < 0.05). Proteins involved in cell structure organization and energy metabolism were differently expressed between AT depots. Fat cell size, which was higher in femoral AT, was significantly correlated with ADH1B, POSTN and LCP1. These findings suggest that there are only slight differences in protein expression between abdominal and femoral subcutaneous AT. It remains to be determined whether these differences, as well as differences in protein activity, contribute to functional and/or morphological differences between these fat depots.

Diet-induced weight loss decreases adipose tissue oxygen tension with parallel changes in adipose tissue phenotype and insulin sensitivity in overweight humans.

BACKGROUND/OBJECTIVES: Although adipose tissue (AT) hypoxia is present in rodent models of obesity, evidence for this in humans is limited. Here, we investigated the effects of diet-induced weight loss (WL) on abdominal subcutaneous AT oxygen tension (pO2), AT blood flow (ATBF), AT capillary density, AT morphology and transcriptome, systemic inflammatory markers and insulin sensitivity in humans. SUBJECTS/METHODS: Fifteen overweight and obese individuals underwent a dietary intervention (DI), consisting of a 5-week very-low-calorie diet (VLCD, 500 kcal day-1; WL), and a subsequent 4-week weight stable diet (WS). Body composition, AT pO2 (optochemical monitoring), ATBF (133Xe washout), and whole-body insulin sensitivity were determined, and AT biopsies were collected at baseline, end of WL (week 5) and end of WS (week 9). RESULTS: Body weight, body fat percentage and adipocyte size decreased significantly during the DI period. The DI markedly decreased AT pO2 and improved insulin sensitivity, but did not alter ATBF. Finally, the DI increased AT gene expression of pathways related to mitochondrial biogenesis and non-mitochondrial oxygen consumption. CONCLUSIONS: VLCD-induced WL markedly decreases abdominal subcutaneous AT pO2, which is paralleled by a reduction in adipocyte size, increased AT gene expression of mitochondrial biogenesis markers and non-mitochondrial oxygen consumption pathways, and improved whole-body insulin sensitivity in humans.

Weight loss-induced cellular stress in subcutaneous adipose tissue and the risk for weight regain in overweight and obese adults.

BACKGROUND/OBJECTIVE: Weight loss is often followed by weight regain after the dietary intervention (DI). Cellular stress is increased in adipose tissue of obese individuals. However, the relation between cellular stress and weight regain is unclear. Previously, we observed increased adipose tissue cellular stress of participants regaining weight compared with participants maintaining weight loss. In the current study, we further investigated the relation between weight regain and changes in the expression of stress-related genes and stress protein levels to determine possible predictors of weight regain. PARTICIPANTS/METHODS: In this randomized controlled trial, sixty-one healthy overweight/obese participants followed a DI of either a 5-week very-low-calorie diet (500 kcal per day) or a 12-week low-calorie diet (1250 kcal per day; WL period) with a subsequent 4-week weight stable diet (WS period), and a 9-month follow-up. The WL and WS period taken together was named the DI. Abdominal subcutaneous adipose tissue biopsies were collected in 53 participants for microarray and liquid chromatography-mass spectrometry analysis. RNA and protein levels for a broad set of stress-related genes were correlated to the weight regain percentage. RESULTS: Different gene sets correlated to weight regain percentage during WS and DI. Bioinformatics clustering suggests that during the WS phase-defined genes for actin filament dynamics, glucose handling and nutrient sensing are related to weight regain. HIF-1 (hypoxia-inducible factor-1) is indicated as an important regulator. With regard to DI, clustering of correlated genes indicate that LGALS1, ENO1 and ATF2 are important nodes for conferring risk for weight regain. CONCLUSIONS: Our present findings indicate that the risk for weight regain is related to expression changes of distinct sets of stress-related genes during the first 4 weeks after returning to energy balance, and during the DI. Further research is required to investigate the mechanistic significance of these findings and find targets for preventing weight regain.

Adipose tissue gene expression is differentially regulated with different rates of weight loss in overweight and obese humans.

BACKGROUND/OBJECTIVES: Moderate weight loss (WL) can ameliorate adverse health effects associated with obesity, reflected by an improved adipose tissue (AT) gene expression profile. However, the effect of rate of WL on the AT transcriptome is unknown. We investigated the global AT gene expression profile before and after two different rates of WL that resulted in similar total WL, and after a subsequent weight stabilization period. SUBJECTS/METHODS: In this randomized controlled trial, 25 male and 28 female individuals (body mass index (BMI): 28-35 kg m-2) followed either a low-calorie diet (LCD; 1250 kcal day-1) for 12 weeks or a very-low-calorie diet (VLCD; 500 kcal day-1) for 5 weeks (WL period) and a subsequent weight stable (WS) period of 4 weeks. The WL period and WS period together is termed dietary intervention (DI) period. Abdominal subcutaneous AT biopsies were collected for microarray analysis and gene expression changes were calculated for all three periods in the LCD group, VLCD group and between diets (ΔVLCD-ΔLCD). RESULTS: WL was similar between groups during the WL period (LCD: -8.1±0.5 kg, VLCD: -8.9±0.4 kg, difference P=0.25). Overall, more genes were significantly regulated and changes in gene expression appeared more pronounced in the VLCD group compared with the LCD group. Gene sets related to mitochondrial function, adipogenesis and immunity/inflammation were more strongly upregulated on a VLCD compared with a LCD during the DI period (positive ΔVLCD-ΔLCD). Neuronal and olfactory-related gene sets were decreased during the WL period and DI period in the VLCD group. CONCLUSIONS: The rate of WL (LCD vs VLCD), with similar total WL, strongly regulates AT gene expression. Increased mitochondrial function, angiogenesis and adipogenesis on a VLCD compared with a LCD reflect potential beneficial diet-induced changes in AT, whereas differential neuronal and olfactory regulation suggest functions of these genes beyond the current paradigm.

Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism.

There is growing interest in the potential health benefits of diets that involve regular periods of fasting. While animal studies have provided compelling evidence that feeding patterns such as alternate-day fasting can increase longevity and reduce incidence of many chronic diseases, the evidence from human studies is much more limited and equivocal. Additionally, although several candidate processes have been proposed to contribute to the health benefits observed in animals, the precise molecular mechanisms responsible remain to be elucidated. The study described here examined the effects of an extended fast on gene transcript profiles in peripheral blood mononuclear cells from ten apparently healthy subjects, comparing transcript profiles after an overnight fast, sampled on four occasions at weekly intervals, with those observed on a single occasion after a further 24 h of fasting. Analysis of the overnight fasted data revealed marked inter-individual differences, some of which were associated with parameters such as gender and subject body mass. For example, a striking positive association between body mass index and the expression of genes regulated by type 1 interferon was observed. Relatively subtle changes were observed following the extended fast. Nonetheless, the pattern of changes was consistent with stimulation of fatty acid oxidation, alterations in cell cycling and apoptosis and decreased expression of key pro-inflammatory genes. Stimulation of fatty acid oxidation is an expected response, most likely in all tissues, to fasting. The other processes highlighted provide indications of potential mechanisms that could contribute to the putative beneficial effects of intermittent fasting in humans.

Heritability and genetic etiology of habitual physical activity: a twin study with objective measures.

Twin studies with objective measurements suggest habitual physical activity (HPA) are modestly to highly heritable, depending on age. We aimed to confirm or refute this finding and identify relevant genetic variants using a candidate gene approach. HPA was measured for 14 days with a validated triaxial accelerometer (Tracmor) in two populations: (1) 28 monozygotic and 24 dizygotic same-sex twin pairs (aged 22 ± 5 years, BMI 21.8 ± 3.4 kg/m(2), 21 male, 31 female pairs); (2) 52 and 65 unrelated men and women (aged 21 ± 2 years, BMI 22.0 ± 2.5 kg/m(2)). Single nucleotide polymorphisms (SNPs) in PPARD, PPARGC1A, NRF1 and MTOR were considered candidates. Association analyses were performed for both groups separately followed by meta-analysis. Structural equation modeling shows significant familiality for HPA, consistent with a role for additive genetic factors (heritability 57 %, 95 % CI 32-74 %, AE model) or common environmental factors (47 %, 95 % CI 23-65 %, CE model). A moderate heritability was observed for the time spent on low- and high-intensity physical activity (P ≤ 0.05), but could not be confirmed for the time spent on moderate-intensity physical activity. For PPARD, each additional effect allele was inversely associated with HPA (P ≤ 0.01; rs2076168 allele C) or tended to be associated with HPA (P ≤ 0.05; rs2267668 allele G). Linkage disequilibrium existed between those two SNPs (alleles A/G and A/C, respectively) and meta-analysis showed that carriers of the AA GC haplotype were less physically active than carriers of the AA AA and AA AC haplotypes combined (P = 0.017). For PPARGC1A, carriers of AA in rs8192678 spent more time on high-intensity physical activity than GG carriers (P = 0.001). No associations were observed with SNPs in NRF1 and MTOR. In conclusion, HPA may be modestly heritable, which is confirmed by an association with variants in PPARD.

The effects of 30 days resveratrol supplementation on adipose tissue morphology and gene expression patterns in obese men.

Polyphenolic compounds, such as resveratrol, have recently received widespread interest because of their ability to mimic effects of calorie restriction. The objective of the present study was to gain more insight into the effects of 30 days resveratrol supplementation on adipose tissue morphology and underlying processes. Eleven healthy obese men were supplemented with placebo and resveratrol for 30 days (150 mg per day), separated by a 4-week washout period in a double-blind randomized crossover design. A postprandial abdominal subcutaneous adipose tissue biopsy was collected to assess adipose tissue morphology and gene expression using microarray analysis. Resveratrol significantly decreased adipocyte size, with a shift toward a reduction in the proportion of large and very-large adipocytes and an increase in small adipocytes. Microarray analysis revealed downregulation of Wnt and Notch signaling pathways and upregulation of pathways involved in cell cycle regulation after resveratrol supplementation, suggesting enhanced adipogenesis. Furthermore, lysosomal/phagosomal pathway and transcription factor EB were upregulated reflecting an alternative pathway of lipid breakdown by autophagy. Further research is necessary to investigate whether resveratrol improves adipose tissue function.

Circulating ACE is a predictor of weight loss maintenance not only in overweight and obese women, but also in men.

BACKGROUND: Circulating angiotensin-converting enzyme (ACE) was identified as a predictor of weight loss maintenance in overweight/obese women of the Diogenes project. OBJECTIVE: To investigate whether ACE acted also as a predictor in men of the Diogenes study and to compare it with that in women. DESIGN: Subjects, who lost ≥ 8% of body weight induced by low-caloric diet in an 8-week weight loss period, were assigned to weight loss maintenance with dietary intervention for 6 months. SUBJECTS: 125 overweight/obese healthy men from eight European countries who completed whole intervention. MEASUREMENTS: Concentrations and activity of serum ACE at baseline and after the 8-week weight loss, in addition to anthropometric and physiological parameters. RESULTS: Serum ACE concentration decreased by 11.3 ± 10.6% during the weight loss period in men. A greater reduction is associated with less body weight regain during the maintenance period (r=0.227, P=0.012). ACE change was able to predict a weight regain ≤ 20% after 6 months, with an odds ratio of 1.59 (95% confidence interval (CI): 1.09-2.33, P=0.016) for every 10% reduction, which was independent of body mass index and weight loss. The prediction power was weaker in men than in women, but without a significant sex difference (P=0.137). In pooled subjects (N=218), the odds ratio was 1.96 (95% CI: 1.46-2.64, P<0.001). CONCLUSIONS: A greater reduction of ACE during weight loss is favorable for weight maintenance in both men and women. This can offer useful information for personalized advice to improve weight loss maintenance. It also confirms the role of ACE in the metabolic pathways of weight regulation.

Expression of genes involved in lipid metabolism in men with impaired glucose tolerance: impact of insulin stimulation and weight loss.

BACKGROUND: The impaired glucose tolerance (IGT) state is characterized by insulin resistance. Disturbances in fatty acid (FA) metabolism may underlie this reduced insulin sensitivity. The aim of this study was to investigate whether the prediabetic state is accompanied by changes in the expression of genes involved in FA handling during fasting and in insulin-mediated conditions and to study the impact of weight loss. METHODS: Seven IGT men and 5 men with normal glucose tolerance (NGT), comparable in terms of age and BMI, participated in the study. The 5 IGT men followed a 12-week weight loss program. Muscle biopsies were taken and the expression of 6 genes was investigated. RESULTS: Subjects had a reduction of 15.5 ± 4.3 kg in body weight. Baseline gene expression was not different between NGT and IGT men. After a hyperinsulinemic clamp, there was an overall upregulation of PGC1α, SREBP-1c, SREBP-2, and ACC-2. The upregulation of SREBP-2 was more pronounced in IGT men (p = 0.049). Weight loss significantly increased insulin sensitivity by 71%, which was not reflected in altered gene expression profiles. CONCLUSIONS: SREBP-2 shows altered insulin responsiveness in IGT men compared with NGT men, while there were no differences in basal gene expression.

SNP analyses of postprandial responses in (an)orexigenic hormones and feelings of hunger reveal long-term physiological adaptations to facilitate homeostasis.

BACKGROUND: The postprandial responses in (an)orexigenic hormones and feelings of hunger are characterized by large inter-individual differences. Food intake regulation was shown earlier to be partly under genetic control. OBJECTIVE: This study aimed to determine whether the postprandial responses in (an)orexigenic hormones and parameters of food intake regulation are associated with single nucleotide polymorphisms (SNPs) in genes encoding for satiety hormones and their receptors. DESIGN: Peptide YY (PYY), glucagon-like peptide 1 and ghrelin levels, as well as feelings of hunger and satiety, were determined pre- and postprandially in 62 women and 41 men (age 31+/-14 years; body mass index 25.0+/-3.1 kg/m(2)). Dietary restraint, disinhibition and perceived hunger were determined using the three-factor eating questionnaire. SNPs were determined in the GHRL, GHSR, LEP, LEPR, PYY, NPY, NPY2R and CART genes. RESULTS: The postprandial response in plasma ghrelin levels was associated with SNPs in PYY (215G>C, P<0.01) and LEPR (326A>G and 688A>G, P<0.01), and in plasma PYY levels with SNPs in GHRL (-501A>C, P<0.05) and GHSR (477G>A, P<0.05). The postprandial response in feelings of hunger was characterized by an SNP-SNP interaction involving SNPs in LEPR and NPY2R (668A>G and 585T>C, P<0.05). Dietary restraint and disinhibition were associated with an SNP in GHSR (477G>A, P<0.05), and perceived hunger with SNPs in GHSR and NPY (477G>A and 204T>C, P<0.05). CONCLUSIONS: Part of the inter-individual variability in postprandial responses in (an)orexigenic hormones can be explained by genetic variation. These postprandial responses represent either long-term physiological adaptations to facilitate homeostasis or reinforce direct genetic effects.

Epigenetic manifestations in diet-related disorders.

Epigenetic phenomena are changes in phenotype that are due to resetting of gene expression under the influence of the environment or genetic factors without changing the DNA sequence. Usually this resetting occurs at a certain stage in life and remains fixed thereafter. In humans, evidence for epigenetic involvement in diet-related complex traits and disorders is accumulating. The fetal origins theory indicates that nutrition can influence the later life risk for certain common disorders like the metabolic syndrome. In parent-of-origin effects, the risk for a common disorder like type I diabetes depends on the sex of the parent who transmits genetic risk factors. Interestingly, both dietary and genetic factors can exert their epigenetic influence over several generations. Imprinting, i.e. silencing of one copy of an autosomal pair of genes, can be part of the mechanism pointing to the importance of DNA methylation. In addition, chromatin modifications have been shown to be involved in epigenetic manifestations. The intriguing possibility that diet may influence the direction and extent of epigenetic changes opens new ways for prevention or treatment of common disorders. At the same time, maternal nutrition might be used to actively direct fetal development with consequences for later life performance such as cognitive abilities. More knowledge on those novel applications is needed. This will in part come from novel strategies to map the epigenomic regions, allowing the identification of more genes involved in epigenetics and allowing the study of their response to nutrition.

Spina bifida and genetic factors related to myo-inositol, glucose, and zinc.

BACKGROUND: Myo-inositol, glucose and zinc and related genetic factors are suggested to be implicated in the etiology of spina bifida. We investigated the biochemical concentrations of these nutrients and polymorphisms in the myo-inositol transporter SLC5A11, myo-inositol synthase ISYNA1, and zinc transporter SLC39A4 in association with spina bifida risk. METHODS: Seventy-six spina bifida triads only were ascertained. In mothers, fathers, and spina bifida children polymorphisms determined were SLC5A11 (544C > T), ISYNA1 (1029A > G), and SLC39A4 (1069C > T). Serum myo-inositol and glucose, and red blood cell zinc concentrations were determined in mothers and spina bifida children. Transmission disequilibrium tests (TDT) were applied to determine associations between the polymorphisms and spina bifida. Associations between biochemical values and genotypes were studied by one-way analysis of variance (ANOVA). Interactions between alleles, biochemical values, and environmental factors were analyzed by conditional logistic regression. RESULTS: No association between SLC5A11, ISYNA1, and SLC39A4 and spina bifida was shown, chi2SLC5A11=0.016, P=0.90; chi2SYNA1=1.52, P=0.22; chi2SLC39A4=0.016, P=0.90; and degrees of freedom (df)=1. Maternal glucose concentrations were comparable for the SLC5A11 genotypes. Significantly lower myo-inositol concentrations were observed in mothers with SLC5A11 CC-genotype, mean (SD) 14.2 (2.6)micromol/L compared to SLC5A11 TT-genotype, 17.0 (3.4)micromol/L, P <0.05 . No significant associations were observed between ISYNA1 and myo-inositol and glucose, and between SLC39A4 and zinc. A significant interaction was demonstrated between a maternal glucose < 4.5 mmol/L and ISYNA1 1029A > G polymorphism on spina bifida risk. CONCLUSION: The combination of maternal glucose < 4.5 mmol/L and ISYNA1 1029A > G polymorphism protects against spina bifida offspring. Moreover, maternal SLC5A11 544C > T polymorphism contributes to the serum myo-inositol concentration. Larger studies should confirm these findings.

No evidence for paternal mtDNA transmission to offspring or extra-embryonic tissues after ICSI.

There is a risk that ICSI may increase the transmission of mtDNA diseases to children born after this technique. Knowledge of the fate and transmission of paternal mitochondrial DNA is important since mutations in mitochondrial DNA have been described in oligozoospermic males. We have used an adaptation of solid phase mini-sequencing to exclude the presence of levels of paternal mtDNA >0.001% in ICSI families. This method is more sensitive than those used in previous studies and is sufficient to detect the likely paternal contribution (approximately 0.1-0.5% from simple calculations of expected dilution during fertilization). Using this method, we were able to detect concentrations as low as 0.001% paternal mtDNA in a maternal mtDNA background. No paternal mtDNA was detected in the embryonic (blood or buccal swabs) tissue of children born after ICSI nor in extra-embryonic tissue (placenta or umbilical cord). In conclusion, we did not detect paternal mtDNA in blood, buccal swabs, placenta or umbilical cord of children born after ICSI. We have found no evidence that ICSI increases the risk of paternal transmission of mtDNA and hence of mtDNA disorders.

Hereditary neuropathy with liability to pressure palsies with a small deletion interrupting the PMP22 gene.

Hereditary neuropathy with liability to pressure palsies is associated with a deficiency in the Peripheral Myelin Protein 22 (PMP22). Most hereditary neuropathy with liability to pressure palsies cases are caused by a deletion of a 1.5 Mb region on chromosome 17p11.2-12 encompassing the PMP22 gene. We describe a hereditary neuropathy with liability to pressure palsies family that lacks the common deletion, but carries a small deletion spanning the 3' region of the PMP22 gene, causing only a partial deletion of one copy of the gene.

Promoter haplotype combinations of the platelet-derived growth factor alpha-receptor gene predispose to human neural tube defects.

Neural tube defects (NTDs), including anencephaly and spina bifida, are multifactorial diseases that occur with an incidence of 1 in 300 births in the United Kingdom. Mouse models have indicated that deregulated expression of the gene encoding the platelet-derived growth factor alpha-receptor (Pdgfra) causes congenital NTDs (refs. 2-4), whereas mutant forms of Pax-1 that have been associated with NTDs cause deregulated activation of the human PDGFRA promoter. There is an increasing awareness that genetic polymorphisms may have an important role in the susceptibility for NTDs (ref. 6). Here we identify five different haplotypes in the human PDGFRA promoter, of which the two most abundant ones, designated H1 and H2 alpha, differ in at least six polymorphic sites. In a transient transfection assay in human bone cells, the five haplotypes differ strongly in their ability to enhance reporter gene activity. In a group of patients with sporadic spina bifida, haplotypes with low transcriptional activity, including H1, were under-represented, whereas those with high transcriptional activity, including H2 alpha, were over-represented. When testing for haplotype combinations, H1 homozygotes were fully absent from the group of sporadic patients, whereas H1/H2 alpha heterozygotes were over-represented in the groups of both sporadic and familial spina bifida patients, but strongly under-represented in unrelated controls. Our data indicate that specific combinations of naturally occurring PDGFRA promoter haplotypes strongly affect NTD genesis.

Identification and characterization of an Xq26-q27 duplication in a family with spina bifida and panhypopituitarism suggests the involvement of two distinct genes.

We investigated a family with a duplication, dup(X)q26-q27, that was present in two brothers, their mother, and their maternal grandmother. The brothers carrying the duplication displayed spina bifida and panhypopituitarism, whereas a third healthy brother inherited the normal X chromosome. Preferential inactivation of the X chromosome containing the duplication was evident in healthy carrier females. We determined the boundaries of the Xq26-q27 duplication. Via interphase FISH analysis we narrowed down each of the two breakpoint regions to approximately 300-kb intervals. The proximal breakpoint is located in Xq26.1 between DXS1114 and HPRT and is contained in YAC yWXD599, while the distal breakpoint is located in Xq27.3 between DXS369 and DXS1200 and contained in YAC yWXD758. The duplication comprises about 13 Mb. Evidence from the literature points to a predisposing gene for spina bifida in Xq27. We hypothesize that the spina bifida in the two brothers may be due to interruption of a critical gene in the Xq27 breakpoint region. Several candidate genes were mapped to the Xq27 critical region but none was shown to be disrupted by the duplication event. Recently, M. Lagerström-Fermér et al. (1997, Am. J. Hum. Genet. 60, 910-916) reported on a family with X-linked recessive panhypopituitarism associated with a duplication in Xq26; however, no details were reported on the extent of the duplication. Our study corroborates their hypothesis that X-linked recessive panhypopituitarism is likely to be caused by a gene encoding a dosage-sensitive protein involved in pituitary development. We place the putative gene between DXS1114 and DXS1200, corresponding to the interval defined by the duplication in the present family.

A deletion encompassing Zic3 in bent tail, a mouse model for X-linked neural tube defects.

Bent tail is a mouse model for human neural tube defects. Bent tail mice are characterized by a shortened, kinked tail. We have observed numerous aberrations in Bent tail embryos including exencephaly, rotation defects and occasionally omphalocele, orofacial schisis and situs abnormalities. Exencephaly was seen in >10% of all embryos and resulted from a closure defect of the hindbrain. Bent tail maps to the proximal part of the X chromosome. By haplotype analysis we have appointed the Bent tail locus to a 1.1 cM interval between markers DXMit159 and DXMit143. Subsequent analysis has revealed the presence of a deletion in all affected animals. The deletion is approximately 1 Mb in size and encompasses the gene for ZIC:3, a zinc finger transcription factor expressed in murine neuroectoderm and dorsal axial mesoderm during neurulation. ZIC:3 is a homolog of the Drosophila segmentation gene odd-paired. Although the Bent tail phenotype probably is the result of the deletion of several genes, combining data on ZIC:3 expression and function of ZIC: genes in the mouse shows that deletion of Zic3 alone is compatible with a major role of this gene in the congenital malformations of the Bent tail mouse. In man, mutations in ZIC3 are associated with situs abnormalities. These patients occasionally also show spina bifida, indicating that genetic variation in human ZIC3 may contribute to other congenital malformations, including neural tube defects.