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Institute of Biology, Centre National de la Recherche Scientifique 8090 and University Hospital, Pasteur Institute of Lille (J.D., F.V., E.D., A.A., C.D., K.C., J.W., P.B., P.F.), F-59019 Lille, France; Department of Internal Medicine, University Hospital (A.A., G.W.), 1010 Lausanne, Switzerland; Nutrition Department, Hôtel Dieu (B.G.-G., K.C.), 75181 Paris, France; Pediatric Endocrinology Department, Jeanne de Flandre Hospital (J.W.), 59037 Lille, France; Barts and The London Genome Center, Queen Mary’s College, University of London (P.F.), London EC1 M6BQ, United Kingdom
Address all correspondence and requests for reprints to: Prof. P. Froguel, Centre National de la Recherche Scientifique 8090, Institut Pasteur de Lille, 1 rue Calmette, F-59019 Lille Cedex, France. E-mail: froguel{at}mail-good.pasteur-lille.fr
Abstract
A linkage between obesity-related phenotypes and the 2p21–23 locus has been reported previously. The urocortin (UCN) gene resides at this interval, and its protein decreases appetite behavior, suggesting that UCN may be a candidate gene for susceptibility to obesity. We localized the UCN gene by radiation hybrid mapping, and the surrounding markers were genotyped in a collection of French families. Evidence for linkage was shown between the marker D2S165 and leptin levels (LOD score, 1.34; P = 0.006) and between D2S2247 and the z-score of body mass index (LOD score, 1.829; P = 0.0019). The gene was screened for SNPs in 96 obese patients. Four new variants were established. Two single nucleotide polymorphisms were located in the promoter (-535 A
G, -286 GA), one in intron 1 (+31 CG), and one in the 3'-untranslated region (+34 CT). Association studies in cohorts of 722 unrelated obese and 381 control subjects and transmission disequilibrium tests, performed for the two frequent promoter polymorphisms, in 120 families (894 individuals) showed that no association was present between these variants and obesity, obesity-related phenotypes, and diabetes. Thus, our analyses of the genetic variations of the UCN gene suggest that, at least in French Caucasians, they do not represent a major cause of obesity.
OBESITY IS A common multifactorial disorder and a major risk factor for type 2 diabetes, hypertension, dyslipidemia, and coronary heart disease (1). Obesity may be the result of interaction between environmental and genetic factors (2, 3). Epidemiological studies have shown that 30–70% of the variation in body weight may be attributed to genetic factors. In a collection of French obese families, a genome-wide scan in affected sibling pairs shows evidence for linkage of serum leptin levels with markers on chromosome 2p (LOD score, 2.68) (4). Moreover, Comuzzie et al. (5, 6) presented evidence of linkage with leptin level at the same locus in an African American population. This region of chromosome 2p contains, among others, the urocortin gene (UCN) encoding a neuropeptide related to the CRF and expressed in heart, brain, thymus, and spleen (7). UCN is the second endogenous mammalian ligand identified for the CRF receptors (8, 9, 10). It stimulates the production and/or release of ACTH through cAMP-dependent mechanisms (11, 12). Intracerebroventricular injections of UCN reduced food intake in rats (13, 14), and ip injections reduced body weight in ob/ob mice (15). To examine the possible contribution of the UCN gene to obesity, we physically mapped the UCN gene and performed linkage analyses using polymorphic markers around the gene. In addition, we screened this gene for mutations in 96 French obese patients and performed association studies with identified genetic variants of the UCN gene.
Subjects and Methods
Subjects and phenotypes
DNA samples of obese probands and their families are part of a collection realized with French subjects recruited from the Department of Nutrition at the Hôtel Dieu Hospital (Paris, France; 46%) and by a multimedia campaign at the Institut Pasteur de Lille (Lille, France; 54%). Obesity was assessed by body mass index (BMI) and the z-score of BMI (i.e. the SD of the BMI corrected for age and sex). The 158 nuclear families that have at least 1 morbidly obese proband (BMI, >40 kg/m2) and 1 or several subjects with a BMI greater than 27 kg/m2 in the sibship (264 sibling pairs) have been previously described (4). Among those, 120 families, consisting of 894 individuals, were included in the transmission disequilibrium tests analyses. In addition, 722 unrelated obese subjects (mean age, 49.1 ± 14.7 yr; BMI, 34.5 ± 3.9 kg/m2; 316 men and 406 women) and 381 nonobese nondiabetic subjects (mean age, 56.8 ± 13.5 yr; BMI, 22.9 ± 2.4kg/m2; 151 men and 230 women) have been included in association studies. Each individual gave informed consent, and the ethics committee of Paris (Hôtel Dieu and Cochin) approved the protocols.
Physical mapping
The human UCN gene was mapped with the Genebridge 4 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL) and two designed primer sets (set 1, 5'-TACAGACGCTCGCCGACAAC-3' and 5'-GTCCCGCCTGCCTCATGGTC-3'; set 2, 5'-GCGGGGAGGGGAGCGGCG-3' and 5'-GGTCTTCAGTACTTTTAT-3') amplified a 302-bp intronic fragment and a 579-bp fragment of exon 2, respectively. Data were submitted to the web site http://www-genome.wi.mit.edu/cgibin/contig/rhmapper.pl, at the Whitehead Institute (Cambridge, MA) for computational and statistical analysis.
Genotyping
Genotyping was performed for four microsatellite markers around the UCN locus (D2S2170, D2S2247, D2S165, and D2S2255) as previously described (4). The identified single nucleotide polymorphisms were genotyped using Light Cycler technology (16) (Roche Molecular Biochemicals, http:/biochem.roche.com).
Mutation screening
We screened for mutations the two exons of the UCN gene, its intron-exon boundaries, the proximal promoter from -1285 nucleotides vs. ATG, and the 3'-untranslated region (3'UTR) up to 80 nucleotides. For the five PCR fragments designed, the primers and annealing temperatures are described in Table 1
. From families contributing to the previously described linkage to leptin levels at D2S165 (4), 96 obese subjects were selected (mean age, 41.4 ± 14.6 yr; BMI, 35.1 ± 10.5 kg/m2; 29 men and 67 women). Mutation screening was performed by direct sequencing as previously described (17).
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We used the regressive approach proposed by Haseman and Elston and implemented in the MapMaker Sibs package (18). This method regresses a function of the sibling’s phenotypes with the proportion of identical alleles shared by descent between the two paired siblings. Association studies were performed in 722 unrelated obese subjects and 381 nonobese nondiabetic subjects. Genotype frequencies were compared using the 
2 test. Continuous clinical and biological variables were analyzed using either one-way ANOVA or Wilcoxon and Kruskal-Wallis tests depending on the shapes of the distribution curves. Hardy-Weinberg’s equilibrium was tested by the 2 test. Transmission of obesity-related phenotypes (serum leptin levels adjusted for age and sex, z-score of BMI) with the risk allele from parents to affected siblings was determined by the transmission disequilibrium test (19).
Results
RH mapping allowed to assign the urocortin gene to chromosome 2, 5.98 cR3000 downstream from marker D2S2247 and 1.61 cR3000 upstream from marker WI-4431 (primer set 1 and set 2; LOD score, >13). As expected from previous studies (4), analyses with the dichotomous obese status showed no evidence of linkage with the D2S2170, D2S2247, D2S165, and D2S2255 markers (Table 2). In contrast, multipoint quantitative trait analyses for these markers showed some evidence of linkage to serum leptin level and z-score of BMI (Table 2), which were supported by single point results. The strongest evidence for linkage showed D2S165 (LOD score, 1.34) for leptin level and D2S2247 (LOD score, 1.82) for z-score of BMI. Informativity at the markers in this region calculated by GeneHunter was satisfactorily high (0.81).
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G -535, GA -286), a CG +31 variant in intron 1, and a CT +34 variant in the 3'-untranslated region. In the initial screening of 96 subjects, the AG (-535) variant in the promoter was detected in a total of six subjects, the GA (-286) in three subjects, and the 3'UTR CT +34 variant and the CG +31 variant in intron 1, respectively, in two and one subjects.
Frequencies of the four genetic variants are presented in Table 3. None of the polymorphisms exhibited a significant deviation from the Hardy Weinberg equilibrium, and no significant linkage disequilibrium between the five polymorphisms was present. None of the variants appeared to be associated with obesity, obesity-related phenotypes, or diabetes (data not shown). To perform segregation analyses in families with an identified polymorphism, all available members were genotyped. For the polymorphisms AG (-535) this is 76 families (514 subjects), for GA (-286) this is 26 families (212 subjects), for 3'UTR CT (+34) this is 15 families (133 subjects), and for intron 1 CG (+31) this is 3 families (35 subjects). Between 4 and 43 subjects were genotyped per family. None of the variants presented evidence of a cosegregation with obesity or diabetes (data not shown). Moreover, transmission disequilibrium tests were performed for the relatively frequent AG -535 and GA -286 polymorphisms, and no significant association was found between obesity-related phenotypes, normalized leptin, and these variants (data not shown).
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We, and others previously reported a linkage between serum leptin level and markers on chromosome 2p (4, 5, 6). At least two interesting candidate genes for obesity lie at this locus: the POMC and UCN genes. Mutation screening and association studies previously performed allowed us to exclude a major role for the POMC gene in obesity (20). By RH mapping, we located the UCN gene between the D2S2247 (46.9 cM) and the D2S165 (47.43 cM) markers. The POMC gene, mapped close to the D2S171 marker (20), may lie at least 1.5 cM telomeric to the UCN gene. The described results of our analyses show a linkage between the microsatellite markers surrounding the UCN gene locus and variations in the leptin level, the z-score of the BMI. Different markers achieved different levels of significance according to their differences in heterozygosity. By a screening for SNPs of 96 subjects from families contributing to the linkage, 4 new polymorphisms were detected. In the performed association studies, none of the variants was associated with either obesity or obesity-related phenotypes (BMI, z-score of BMI, leptin level, and waist to hip ratio) or diabetes. Notwithstanding the evidence of linkage at the 2p21–23 locus with obesity-related phenotypes that remain indirect evaluations of fat mass, our study suggests that the UCN gene has no major role in susceptibility to obesity, at least in French Caucasians. Nevertheless, variations in functional sequences, which may modify UCN gene expression and/or a nearby gene, may be responsible for the reported linkage. Moreover, 2 other corticotropin-releasing factor-related peptides have been identified: UCN II and UCN III peptides. The UCN II gene that is located at 3p21.3 is expressed centrally. The UCN II peptide binds selectively to the CRF-R2 receptor (21). Functional studies have shown that it is involved in central autonomic and appetite control. Likewise, UCN III peptide binds selectively to CRF-R2, however, with a lower affinity than UCN II. UCN III expression profiles are consistent with a potential role in behavioral function (22). Therefore, we cannot exclude that genetic variations resulting in a UCN I deficiency may be counterbalanced by expression of the UCN II and III peptides. Moreover, these latter are potential candidate genes for obesity as well. Further investigations will be required to clarify the roles of these components in the complex regulatory network of food intake.
Acknowledgments
We thank our patients for participating in this study.
Footnotes
This work was supported by a grant from Eli Lilly \|[amp ]\| Co.
Abbreviations: BMI, Body mass index; RH, radiation hybrid; UCN, urocortin; UTR, untranslated region.
Received August 10, 2001.
Accepted November 5, 2001.
References
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  M. Ciullo, T. Nutile, C. Dalmasso, R. Sorice, C. Bellenguez, V. Colonna, M. G. Persico, and C. Bourgain Identification and Replication of a Novel Obesity Locus on Chromosome 1q24 in Isolated Populations of Cilento Diabetes, March 1, 2008; 57(3): 783 - 790. [Abstract] [Full Text] [PDF]
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