Doreen Zegers

Wnt Signaling and the Control of Human Stem Cell Fate

Wnt signaling determines major developmental processes in the embryonic state and regulates maintenance, self-renewal and differentiation of adult mammalian tissue stem cells. Both β-catenin dependent and independent Wnt pathways exist, and both affect stem cell fate in developing and adult tissues. In this review, we debate the response to Wnt signal activation in embryonic stem cells and human, adult stem cells of mesenchymal, hematopoetic, intestinal, gastric, epidermal, mammary and neural lineages, and discuss the need for Wnt signaling in these cell types. Due to the vital actions of Wnt signaling in developmental and maintenance processes, deregulation of the pathway can culminate into a broad spectrum of developmental and genetic diseases, including cancer. The way in which Wnt signals can feed tumors and maintain cancer stem stells is discussed as well. Manipulation of Wnt signals both in vivo and in vitro thus carries potential for therapeutic approaches such as tissue engineering for regenerative medicine and anti-cancer treatment. Although many questions remain regarding the complete Wnt signal cell-type specific response and interplay of Wnt signaling with pathways such as BMP, Hedgehog and Notch, we hereby provide an overview of current knowledge on Wnt signaling and its control over human stem cell fate.

Association of the BDNF Val66Met variation with obesity in women

Brain-derived neurotrophic factor (BDNF) has been implied in the regulation of food intake. In the present study, we genotyped the Val66Met polymorphism in a Belgian cohort of 532 obese women and 197 healthy female controls and were, for the first time, able to show an association of the 66Met allele with obesity, at least in our female cohort.

A gene variant of PNPLA3, but not of APOC3, is associated with histological parameters of NAFLD in an obese population

Mechanisms explaining the relationship in non‐alcoholic fatty liver disease (NAFLD), obesity, and insulin resistance are poorly understood. A genetic basis has been suggested. We studied the association between the genes patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) and apolipoprotein C3 (APOC3) and metabolic and histological parameters of NAFLD in obese patients.

Association between polymorphisms of the Nesfatin gene, NUCB2, and obesity in men.

Nesfatin-1, which originates from its precursor protein nucleobindin-2 (NUCB2), is a novel appetite-regulating molecule that might be associated with the melanocortin signalling pathway in the hypothalamus. The secreted protein appears to play an important role in metabolic control through its anorexigenic and anti-hyperglycemic effects. Therefore, we hypothesized that polymorphisms in the NUCB2 gene might influence the susceptibility for the development of obesity. In this study, we investigated the association of NUCB2 polymorphisms with the development of obesity in an extensive Caucasian population comprising 1049 obese subjects and 315 normal weight control individuals. We selected 8 tagSNPs, which after additional analysis of 6 multi-marker tests, cover most information on common genetic variation in the selected region. We found association with obesity for 3 SNPs (rs1330, rs214101 and rs757081) and 3 multi-marker tests, only when analyzing the male population separately. We subsequently performed linear regression analysis, again in the male population only, and found that several SNPs were associated with BMI, weight and fat free mass. These data indicate that polymorphisms in the NUCB2 gene could play an important role in the protection against the development of obesity in male subjects and might have an influence on energy homeostasis. Nevertheless, further research including replication of our results and elucidation of the molecular mechanism remains necessary.

Identification of three novel genetic variants in the melanocortin-3 receptor of obese children.

The melanocortin‐3 receptor (MC3R), a G‐protein‐coupled receptor expressed in the hypothalamus, is a key component of the leptin‐melanocortin pathway that regulates energy homeostasis. It is suggested that an MC3R defect leads to an increased feed efficiency, by which nutrients are partitioned preferentially into fat. In this study, we hypothesized that early‐onset obesity could be induced by mutations in MC3R. To investigate this hypothesis, we screened the entire coding region of the MC3R gene for mutations in obese subjects. A total of 404 overweight and obese children and adolescents, 86 severely obese adults (BMI ≥40 kg/m2), and 150 normal‐weight control adults were included. Besides three synonymous coding variations in the MC3R gene (S69S, L95L, I226I), we were able to identify three novel heterozygous, nonsynonymous, coding mutations (N128S, V211I, L299V) in three unrelated obese children. None of these mutations were found in any of the control subjects. Functional studies assessing localization and signaling properties of the mutant receptors provided proof for impaired function of the L299V mutated receptor, whereas no conclusive evidence for functional impairment of the N128S and V211I mutated receptors could be established. First, these results provide supporting evidence for a role of the MC3R gene in the pathogenesis of obesity in a small subset of patients. Second, they show that caution is called for the interpretation of newly discovered mutations in MC3R.

Association study of MC4R with complex obesity and replication of the rs17782313 association signal.

Recently, genome-wide association studies have discovered several single nucleotide polymorphisms (SNPs) involved in the etiology of complex obesity. A variant downstream from the melanocortin-4 receptor gene (MC4R), a gene known to be involved in monogenic obesity, was reported to be highly associated with BMI. In the present study, we performed a replication study with the previously reported SNP rs17782313. We also included 3 tagSNPs (rs8087522, rs11872992, and rs1943226) for the MC4R gene region in our study to understand the role of this gene in complex obesity. We genotyped all 4 SNPs in a population of 1049 obese cases (mean BMI=38.2±6.2) and 312 healthy lean individuals (mean BMI 22.0±1.7). We could confirm that rs17782313 is highly associated with complex obesity in our population (odds ratio=1.42, 95% CI 1.14-1.77, P=0.002). Furthermore, we found this SNP to be associated with BMI (B=0.92, 95% CI 0.19-1.65, P=0.01) and body weight (B=2.44, 95% CI 0.28-4.60, P=0.03). In addition, we could also detect an association between rs11872992 and complex obesity (odds ratio=0.74, 95% CI 0.57-0.98, P=0.03). Through conditional analysis, we demonstrate that this effect is independent from the rs17782313 association signal. No associations with obesity could be found for rs8087522 and rs1943226. In conclusion, we could replicate the previously reported association between rs17782313 and complex obesity. Furthermore, our data do not support the hypothesis that a SNP in MC4R causes the rs17782313 association signal.

Identification and Functional Characterization of Novel Mutations in the Melanocortin-4 Receptor

Objective: Melanocortin-4 receptor (MC4R) deficiency is the most common cause of monogenic obesity. In the present study, we screened the MC4R gene for mutations in a population of overweight and obese children and adolescents. Method: Cross-sectional mutation analysis of 112 overweight/obese children and adolescents and 121 lean individuals. Results: We identified 11 sequence variations, 5 of which were present in our control population or had been previously reported as polymorphisms. The remaining 6 variations are disease-causing mutations including 2 novel ones: a I186V mutation and a F280L mutation. The 4 previously described mutations (D90N, M200V, P260Q, Q307X) were identified in single probands. Using confocal imaging, we demonstrated that F280L and P260Q cause intracellular retention of the mutant receptor. No difference in cell surface expression could be detected for the I186V mutation. Using a cAMP responsive luciferase vector, we demonstrated that the receptor with I186V is unable to activate its intracellular signaling pathway while the P260Q mutation causes reduced activation of the receptor. Conclusion: We detected MC4R deficiency in 6 patients from our cohort, amounting to a prevalence of 5.3%. Two novel mutations were identified. We also confirmed that intracellular retention is a common pathogenic effect of MC4R mutations.

Mutation screen of the SIM1 gene in pediatric patients with early-onset obesity

Background:The transcription factor SIM1 (Single-minded 1) is involved in the control of food intake and in the pathogenesis of obesity. In mice, Sim1 is involved in the development of the paraventricular nucleus, and Sim1 deficiency leads to severe obesity and hyperphagia. In humans, chromosomal abnormalities in the SIM1 gene region have been reported in obese individuals. Furthermore, recent data also suggest that loss-of-function point mutations in SIM1 are responsible for SIM1 haplo-insufficiency that is involved in causing human obesity. In this study, we therefore wanted to expand the evidence regarding the involvement of SIM1 mutations in the pathogenesis of severe early-onset obesity.Methods:We screened 561 severely overweight and obese children and adolescents and 453 lean adults for mutations in the coding region of the SIM1 gene. Mutation screening in all patients and lean individuals was performed by high-resolution melting curve analysis combined with direct sequencing. To evaluate the effect of the mutations on SIM1 transcriptional activity, luciferase reporter assays were performed.Results:Mutation analysis identified four novel nonsynonymous coding variants in SIM1 in four unrelated obese individuals: p.L242V, p.T481K, p.A517V and p.D590E. Five synonymous variants, p.P57P, p.F93F, p.I183I, p.V208V and p.T653T, were also identified. Screening of the lean control population revealed the occurrence of four other rare SIM1 variants: p.G408R, p.R471P, p.S492P and p.S622F. For variants p.T481K and p.A517V, which were found in obese individuals, a decrease in SIM1 transcriptional activity was observed, whereas the transcriptional activity of all variants found in lean individuals resembled wild type.Conclusions:In this study, we have demonstrated the presence of rare SIM1 variants in both an obese pediatric population and a population of lean adult controls. Further, we have shown that functional in vitro analysis of SIM1 variants may help in distinguishing benign variants of no pathogenic significance from variants which contribute to the obesity phenotype.

Identification of mutations in the NUCB2/nesfatin gene in children with severe obesity.

Nesfatin-1 is the N-terminal fragment of nucleobindin-2 (NUCB2) that was identified as a novel satiety molecule in rodents. The protein is reported to exert anorexigenic effects and appears to play an important role in hypothalamic pathways regulating energy homeostasis and food intake. In this study, we hypothesized that mutations in the nesfatin encoding gene NUCB2 might cause obesity in humans. Therefore, we screened the entire coding region of the NUCB2 gene for mutations in a population of 471 obese children and adolescents. Mutation analysis of NUCB2 identified a total of seven sequence variants of which four were previously reported as polymorphisms. The remaining three variants included ex9+6G>C, L125H and K178X and were found in 3 unrelated individuals in the obese population only (0.6%). Biochemical experiments including ELISA and western blot were performed on plasma samples of the obese patient carrying the nonsense mutation K178X. However, neither NUCB2/nesfatin-1 immunoreactive plasma levels of the patient, nor expression of full length NUCB2 differed significantly from matched obese control individuals. In conclusion, we have identified the first genetic variants in the NUCB2 gene in obese individuals, although further functional characterization will be essential to verify disease causality of the mutations.

CNV analysis and mutation screening indicate an important role for the NPY4R gene in human obesity.

Genome‐wide copy number variation (CNV) analyses have associated the 10q11.22 CNV with obesity. As the NPY4R gene is the most interesting candidate gene in this region, it was hypothesized that both genetic and structural variation in NPY4R might be implicated in the pathogenesis of obesity.