Frequency of and interaction between polymorphisms in the ... - Nature

International Journal of Obesity (2000) 24, 1239±1245 ß 2000 Macmillan Publishers Ltd All rights reserved 0307±0565/00 $15.00 www.nature.com/ijo

Frequency of and interaction between polymorphisms in the b 3-adrenergic receptor and in uncoupling proteins 1 and 2 and obesity in Germans D Evans1*, S Minouchehr1, G Hagemann1, WA Mann1, D Wendt1, A Wolf2 and U Beisiegel1 1

Medizinische Klinik, UniversitaÈtskrankenhaus Eppendorf, Hamburg, Germany; and 2Klinik fuÈr Allgemeinchirurgie, Evangelisches und Johanniter Klinikum, Dinslaken, Germany

OBJECTIVE: To determine the role of polymorphisms in the genes for b 3-adrenergic receptor (b b3-AR) and in uncoupling proteins 1 and 2 (UCP-1, UCP-2) in obesity. DESIGN: Association study with three polymorphisms and obesity. SUBJECTS: Two hundred and thirty-six morbidly obese patients who underwent gastric banding surgery, 381 patients from the medical clinic and 198 healthy blood donors. 0 MEASUREMENTS: The frequencies of the W64R in b 3-AR, the ÿ3826A?G in UCP-1 and the 45 bp insertion in the 3 untranslated region of exon 8 in UCP-2 polymorphisms were determined. RESULTS: There were no signi®cant differences in the frequencies of the b 3-AR and UCP-1 polymorphisms between obese (body mass index, BMI > 30 kg=m2) and lean subjects. Lean, but not obese, carriers of the R allele of b 3-AR had a signi®cantly higher BMI. The mean age of obese subjects (excluding diabetics) who were carriers of the G allele of the UCP-1 polymorphism, 36 y, was signi®cantly younger than wild-type, 40 y (P ˆ 0.007). This effect was not seen in lean subjects. The effect of the G allele on the mean age of obese subjects was more apparent in subjects who were also carriers of the R allele of the b 3-AR polymorphism. The frequency of the ins allele of UCP-2 was signi®cantly higher in obese subjects, 0.31, than in lean, 0.24 (P ˆ 0.002) and carriers of the ins allele had a signi®cantly higher BMI, 38 vs 35 (P ˆ 0.005). There was no association between any of the polymorphisms and type II diabetes. CONCLUSION: In a German population, there was no association between the W64R in b 3-AR or the ÿ3826A?G in UCP-1 polymorphisms 0and obesity. However, they act synergistically to accelerate the development of obesity. The 45 bp insertion in the 3 untranslated region of exon 8 in UCP-2 polymorphism is associated with obesity. International Journal of Obesity (2000) 24, 1239±1245 Keywords: polymorphisms; obesity; b3-adrenergic receptor; uncoupling proteins 1 and 2

Introduction The risk of becoming obese has a strong genetic component.1 Since single gene defects such as those in the leptin gene are very rare,2 it is likely that a combination of polymorphisms in one or more candidate genes may contribute to the development of most cases of obesity. Reduced basal metabolic rate (BMR) is one of the factors associated with weight gain in humans3 and, since it is in part genetically determined,4 variation in genes involved in determining BMR may contribute to the genetic basis of obesity. One such gene is that for the b3-adrenergic receptor. A variant, W64R, in the human b3-adrenergic receptor gene has been associated with obesity. How-

*Correspondence: D Evans, Medizinische Klinik, UniversitaÈtskrankenhaus, Eppendorf, Martinistrasse 52, 20251 Hamburg, Germany. E-mail: [email protected] Received 19 October 1999; revised 9 March 2000; accepted 7 April 2000

ever this association is a matter of some controversy. In a recent review article,5 Strosberg identi®ed some 15 studies in which an association was found and eight studies in which no association was found. In a recent large population-based study of 1259 randomly chosen Germans, no association was found between the W64R polymorphism and obesity.6 In a metaanalysis of 31 studies, Fujisawa et al 7 concluded that carriers of the R allele may have an average increase of BMI of 0.3 kg=m2 compared with normal homozygous subjects. Moreover, the polymorphism has been associated with low BMR and a greater tendency to gain weight in a number of studies and thus may contribute to an increase in body mass index (BMI) in subjects pre-disposed to obesity through lifestyle or other genetic factors.8 ± 10 Uncoupling proteins (UCP) are a family of proteins whose function is to uncouple oxidative phosphorylation of ADP to ATP leading to the generation of heat.11 UCP-1 is expressed in brown adipose tissue,12 UCP-2 in most tissues including white adipose tissue and UCP-3 in skeletal muscle.13,14 The ÿ3826A?G

b 3-AR and UCP polymorphisms and obesity D Evans et al

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polymorphism in UCP-1 has been associated in some, but not all studies, with a tendency to weight gain and increased BMI.15 ± 19 Recent studies have suggested an additive effect between the W64R polymorphism and the ÿ3826A?G polymorphism in UCP-1.15 ± 19 Linkage has been reported between markers close to the UCP-2 gene and obesity,13 with resting metabolic rate in French Canadians20 and with a prediabetic phenotype in Pima Indians.21 Two polymorphisms have been reported in the human UCP-2 gene, an ala?val substitution in exon 4 and a 45 bp insertion 0 in the 3 untranslated region of exon 8 (ins allele), which in some populations have been associated with resting metabolic rate and obesity.22 ± 27 In this study we report on the incidence of these three polymorphisms and their interaction in ethnic Germans, with a special emphasis on those with morbid obesity.

Krankenhaus, Eppendorf, Hamburg, who served as normal controls. Patients gave informed consent and the study was approved by the Ethics Commission of È rztekammer Hamburg. The clinical details of the the A subjects are given in Table 1. For ethical reasons the blood donors had to remain anonymous and we were therefore unable to obtain their BMI; instead serum leptin levels were determined as an indicator of the degree of obesity. As expected they were signi®cantly lower than those that were measured in obese subjects.

Materials and methods

Assay of plasma leptin

Study subjects

Subjects, ethnic Germans, were recruited from three sources: (1) 236 morbid obese patients undergoing gastric banding surgery at the Evangelisches Krankenhaus, Dinslaken, 35 of whom had type II diabetes; (2) 381 patients from the Medical Clinic of the UniversitaÈts-Krankenhaus, Eppendorf, Hamburg; these patients form a heterogeneous group including outpatients attending the lipid and diabetes outpatient clinics and inpatients; and (3) 198 blood donors from the Blood Transfusion Service of the UniversitaÈts-

DNA Analysis

DNA was extracted from the buffy coat obtained from 10 ml EDTA blood using standard methods. The W64R genotype was determined as described by Gagnon et al,28 the UCP-1 polymorphism as described by Valve et al 16 and the 45 bp insertion polymorphism of UCP-2 as described by Walder et al.23

Plasma leptin levels were determined using the Quantikine TM Human leptin immunoassay manufactured by R&D Systems Inc. of Minneapolis, USA. Statistical analysis

Values are given as means with standard deviations. Statistical analysis was performed using Student's unpaired t-test, ANOVA and w2 test on Statistica software. A P-value of 0.05 or less was considered signi®cant. Relative risk and odds ratios were calculated as described by Large et al.29

Table 1 Clinical characteristics and allele frequencies for each patient group Morbid obese

Clinical data Age (y) BMI (kg=m2) Leptin Type 2 diabetes bA3 W64R WW WR RR R frequency UCP-1ÿ3826A?G AA AG GG G frequency UCP-2 45bp insertion del=del del=ins ins=ins ins frequency

Clinic

Blood donors

Male (n ˆ 44)

Female (n ˆ 191)

Male (n ˆ 243)

Female (n ˆ 137)

Male (n ˆ 137)

Female (n ˆ 61)

39  7.2 52  9.8 32  13.7a 7

38  9.3 51  7.8 61  23.2b 28

55  14.5 27  5.3 10  10.8c 78

57  17.9 25  4.9 18  13.3d 52

51  6.1 NA 7  5.0 0

50  5.8 NA 18  12.8 0

38 5 0 0.06

160 24 2 0.08

202 30 1 0.07

111 22 1 0.09

109 21 0 0.08

48 6 0 0.06

23 17 4 0.28

97 80 14 0.28

130 99 14 0.26

70 55 12 0.29

76 49 12 0.27

36 21 4 0.24

17 19 8 0.40*

95 83 14 0.29

135 95 13 0.25

78 48 12 0.26

75 57 5 0.24

32 27 2 0.24

Values are means with standard deviation. NA ˆ not available; a n ˆ 19; bn ˆ 69; cn ˆ 51; dn ˆ 37; en ˆ 137; fn ˆ 61. *P ˆ 0.005 and 0.004 for difference in allele frequency to blood donors and clinic respectively. International Journal of Obesity

b 3-AR and UCP polymorphisms and obesity D Evans et al

Results The frequencies of the W64R in b3-adrenergic receptor (b3-AR), the ÿ3826A?G in UCP-1 and the 45 bp 0 insertion in the 3 untranslated region of exon 8 in UCP-2 polymorphisms in each subject group according to sex are given in Table 1. There were no signi®cant differences in the allele frequencies of the b3-AR and UCP-1 polymorphisms between the groups. Morbid obese patients of both sexes showed an increase in the frequency of the ins allele of the UCP-2 polymorphism over blood donors, which in the case of males was statistically signi®cant, P ˆ 0.002. Combining the sexes, when the morbid obese patients were divided into those over and those under 40 y of age, then the frequency of the ins allele was signi®cantly higher amongst the older patients, 0.38 vs 0.27, P ˆ 0.01 (Table 2). Morbid obese patients homozygous for the ins allele were signi®cantly older (41 8.0, n ˆ 22) than del=del homozygotes (37 8.31, n ˆ 112), P ˆ 0.026 (Figure 1) with heterozygotes being intermediate (39 9.76, n ˆ 101). As we were unable to obtain the BMI of the blood donors, to further investigate the association between these polymorphisms and obesity we recruited a heterogeneous group of patients from the Medical Clinic, University Hospital, Hamburg. Since the W64R in b3-AR has been associated with type 2 diabetes and since there have been reports of genetic

linkage between markers in the vicinity of the UCP-2 gene and a prediabetic phenotype,13,21 a number of patients with type 2 diabetes were included. The subjects were divided into those with and without type 2 diabetes, irrespective of their degree of obesity. For each of the polymorphisms there was an increased frequency in the rarer allele in subjects with type 2 diabetes but in no case was this statistically signi®cant (Table 3). The subject groups were combined and then divided into obese, with a BMI of 30 or more, and lean, with a BMI of less than 30. The blood donors were included amongst the lean controls. The frequencies of the polymorphisms in the obese and lean groups were compared (Table 4). The allele frequencies for each of the polymorphisms were in Hardy ± Weinberg equilibrium. There was no statistically signi®cant differences in the allele frequencies of W64R in b3-AR or the ÿ3826A?G in UCP-1 between obese and lean subjects. In the case of the UCP-2 polymorphism there was a statistically signi®cant association with obesity, the frequency of the ins allele being higher in obese subjects than lean, 0.31 vs 0.22, P ˆ 0.002. The relative risk for obesity for ins=ins homozygotes was 2.1 with an odds ratio of 2.2 and for carriers of the ins allele the relative risk was 1.2 and the odds ratio 1.4. Excluding the blood donors from the lean group did not affect this ®nding; for the obese subjects the ins allele frequency was 0.31 and

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Table 2 UCP-2 polymorphism and age in patients with morbid obesity (BMI > 40 kg=m2)

Over 40 y, n ˆ 83 40 y and younger, n ˆ 152

del=del

del=ins

ins=ins

ins frequency

32 (39%) 80 (53%)

39 (47%) 62 (41%)

12 (14%) 10 (7%)

0.38* 0.27

*For difference in allele frequency P ˆ 0.01.

Figure 1 Scatterplot showing the range of ages for each genotype of the 45 bp insertion polymorphism of UCP-2. The mean value for each genotype is indicated. The difference in age between del=del and ins=ins homozygotes is signi®cant, P ˆ 0.026. International Journal of Obesity

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Table 3 Polymorphism frequencies and type II diabetes W64R in b3-adrenergic receptor (b3-AR) Type 2 diabetes, n ˆ 154 No diabetes, n ˆ 626

WW 132 537

WR 20 89

RR 2 2

R frequency 0.08 0.07

ÿ3826A?G in uncoupling protein 1 (UCP-1) Type 2 diabetes, n ˆ 165 No diabetes, n ˆ 648

AA 84 348

AG 68 253

GG 13 47

G frequency 0.28 0.27

del=del 88 343

del=ins 61 267

ins=ins 16 38

ins frequency 0.28 0.26

45 bp insertion in uncoupling protein 2 (UCP-2) Type 2 diabetes, n ˆ 165 No diabetes, n ˆ 648

Table 4 Polymorphism frequencies in obese (BMI > 30 kg=m2) and lean subjects W64R in b3-adrenergic receptor (b3-AR) BMI > 30, n ˆ 294 BMI < 30, n ˆ 488 ÿ3826A?G in uncoupling protein 1 (UCP-1) BMI > 30, n ˆ 305 BMI < 30, n ˆ 508 45 bp insertion in uncoupling protein 2 (UCP-2) BMI > 30, n ˆ 305 BMI < 30, n ˆ 508

WW 253 416 AA 166 266

WR 39 70 AG 116 205

RR 2 2 GG 23 37

R frequency 0.07 0.08 G frequency 0.27 0.27

del=del 145 286

del=ins 130 198

ins=ins 30 24

ins frequency 0.31 0.24*

*Difference in allele frequency P ˆ 0.002.

for the lean it was 0.24, P ˆ 0.004. When the obese and lean groups (excluding blood donors) were further divided into those aged 40 and over and those less than 40 and the allele frequencies determined, a similar result was obtained to that found for the morbid obese patients. The frequency of the ins allele (0.36) was signi®cantly higher amongst the older obese subjects than in younger ones (0.27), P ˆ 0.02. Although the older lean subjects had a higher frequency of the ins allele, 0.25 vs 0.18, this was not statistically signi®cant, P ˆ 0.08. The mean BMI for each genotype is presented in Table 5. Since the subjects formed a heterogeneous group, including patients with dyslipidemia and type 2 diabetes, which are associated with an increase in BMI, we performed an ANOVA with the polymorphism as independent variable, BMI as dependent variable and with sex, age, type 2 diabetes, serum total cholesterol and triglycerides as co-variants. Only the UCP-2 polymorphism had a signi®cant affect, P ˆ 0.005. Young ( < 40 y of age) lean patients who were carriers of the R allele of the b3-AR polymorphism had a signi®cantly higher BMI than older lean

patients, 26( 2.2) vs 23(  2.4), P ˆ 0.02. This association was not seen in the obese patients. Obese subjects (excluding diabetics) who were carriers of the G allele of the ÿ3826A?G UCP-1 were signi®cantly younger (AA 40, AG=GG 36, P ˆ 0.005, Figure 2), an effect not seen in lean subjects (AA 50, AG=GG 51, P ˆ 0.68). This effect was stronger in obese men (AA 46  11.4, AG 37  5.5, GG 37  15.1, P ˆ 0.003) than women (AA 38  10.2, 36  7.9, GG 36  10.8, P ˆ 0.23). It has been reported that the W64R b3-AR and the ÿ3826A?G UCP-1 polymorphisms may act synergistically,15 ± 19 therefore obese patients were divided into carriers and non-carriers of the R allele of the b3AR W64R polymorphism. The effect of the UCP-1 polymorphism on the age of obese patients was much greater in the presence of the R allele. In the absence of the R allele, the mean age of AA homozygotes was 39, n ˆ 102 and G carriers, 37, n ˆ 87 (P ˆ 0.12), whereas in the presence of the R allele, the mean age of AA homozygotes was 45, n ˆ 17 and G carriers, 33, n ˆ 13, P ˆ 0.006 (Figure 2).

Table 5 Polymorphisms and BMI

Discussion

Gene

Genotype

n

Mean BMI  s.d.

bA-3

WW WR RR AA AG GG del=del del=ins ins=ins

501 80 3 314 245 44 316 240 47

36  14.0 36  13.2 38  14.7 36  13.9 36  13.5 37  14.9 35  13.2 37  14.4 38  13.6

UCP 1 UCP 2

International Journal of Obesity

The overall frequency of the W64R polymorphism in the b3-AR amongst the 780 subjects typed here, 0.07 (WW 85.6%, WR 13.8%, RR 0.5%) was the same as that obtained from a population-based survey of 1259 Germans (WW 88.3%, WR 10.8%, RR 0.8%).6 Consistent with this study, we also found no association with either obesity or type 2 diabetes. In the group of non-obese subjects who were younger than 40 there

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Figure 2 Scatterplot showing the range of ages according to genotypes of the W64R polymorphism in the b3-adrenergic receptor gene and the ÿ3826A?G polymorphism in the UCP-1 gene. Upper panel, UCP-1 polymorphism alone; middle panel, UCP-1 polymorphism and wild-type W64R polymorphism; and lower panel, UCP-1 polymorphism and carriers of the W64R polymorphism.

was a small, but statistically signi®cant, association between carriers of the R allele and higher BMI. Given that any affect of the polymorphism on BMI

is probably very slight, in the order of 0.3 kg=m2,7 it is in this sub-group of patients that it would be most apparent, as such a modest effect on BMI would International Journal of Obesity

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probably not be visible within a morbidly obese population. We observed no association between the ÿ3826A?G UCP-1 polymorphism and obesity or BMI. This is consistent with the ®ndings reported by other groups.15,30 Obese subjects who were carriers of the G allele were signi®cantly younger. Oppert et al 30 have reported that carriers of the G allele show an increase tendency to gain weight. That obese carriers of the G allele are younger is not inconsistent with these ®ndings since an increased tendency for weight gain would result in an accelerated development of obesity. Obese subjects who were carriers of the G allele who were also carriers of the R allele of the b3AR polymorphism were signi®cantly younger than those who were not. A synergistic effect of the two polymorphisms has been reported by a number of groups.15 ± 19 In this study we report on a signi®cant association 0 of the 45 bp insertion in the 3 untranslated region of exon 8 polymorphism in UCP-2 with obesity, particularly amongst older patients. This polymorphism has been the subject of four recent reports with con¯icting ®ndings.23,25 ± 27 The frequency of the ins allele in 483 French morbid obese with a mean age of 48, 0.25, was not signi®cantly increased over 113 normal weight control subjects, 0.23.27 The ins allele frequency of 0.29 did not differ between 68 obese and 104 normal weight German children and there was no association with BMI.26 In contrast in a cross-sectional survey of 453 South Indian subjects with a mean age of 45 the frequency of the ins allele was 0.19 and there was a signi®cant association between the ins allele and increased BMI in women but not in men.25 This association was more signi®cant, P ˆ 0.0001 vs 0.015 in an additional group of 143 South Indians, both men and women, who were parents of a group of NIDDM probands with mean ages of 68 and 58, respectively. The frequency of the ins allele in 780 full-blooded Pima Indians was 0.41 and there was no association with obesity.23 However, when the 139 subjects over 45 y of age were analyzed separately there was a signi®cant association between the ins allele and increased BMI. Taken together these data suggest that the association of the polymorphism on BMI and obesity is probably a modest one which requires a number of years to manifest itself. This is consistent with the suggestion, made by Cassel et al,25 that the polymorphism plays a role in the susceptibility to weight gain and for a role in affecting the metabolic rate suggested by Walder et al.23 In the French study, however, there was no association between the polymorphism and metabolic data and tendency to weight gain in morbidly obese patients who were heterozygous for the polymorphism, no ins=ins homozygotes were present in the French population.27 Although linkage has been reported between markers close to the UCP-2 gene and diabetes13 and to a pre-diabetic phenotype in Pima Indians,21 we found

International Journal of Obesity

no association between the polymorphism and diabetes. This lack of association with type 2 diabetes is consistent with the reports of Cassell et al,25 Walder et al 23 and Otabe et al,27 none of whom found an association between this polymorphism and diabetes. In addition, no association could be found between a second polymorphism in UCP-2, A55V, and diabetes.22 It would seem improbable that variation in the UCP-2 gene plays an important role in the development of type 2 diabetes. In conclusion, in this study we show that, although there was no direct association between the W64R in b3-AR and the ÿ3826A?G in UCP-1 polymorphisms and obesity, carriers of the polymorphism tended to develop obesity at a younger age, and the two polymorphisms act synergistically. We ®nd an association 0 between the 45 bp insertion in the 3 untranslated region of exon 8 polymorphism in UCP-2 with obesity and BMI, especially in older obese subjects. Acknowledgements

We thank Dr Wittkopf of the Blood Transfusion Centre, UniversitaÈtskrankenhaus Eppendorf, Hamburg for provision of the control blood samples. This work was supported by grants from the Deutsche Forschungsgemeinschaft to DE (Ev 29=2-1) and AM (Ma 1101=4-1) and KliFo 258=10=2. References

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9 Widen E, Lehto M, Kanninen T, Walston J, Shuldiner AR, Groop LC. Association of a polymorphism in the b3-adrenergic receptor gene with features of the insulin resistence syndrome in Finns. New Engl J Med 1995; 333: 348 ± 351. 10 Clement K, Vaisse C, Manning B, Basdevant A, Guy-Grand B, Ruiz J, Silver KD, Shuldiner AR, Froguel P, Strosberg AD. Genetic variation in the b3-adrenergic receptor and an increased capacity to gain weight in patients with morbid obesity. New Engl J Med 1995; 333: 352 ± 354. 11 Flier JS, Lowell BB. Obesity research springs a proton leak. Nature Genet 1997; 15: 223 ± 224. 12 Klaus S, Casteilla L, Bouillaud F, Ricquier D. The uncoupling protein UCP: a membraneous mitochondrial ion carrier exclusively expressed in brown adipose tissue. Int J Biochem 1991; 23: 791 ± 801. 13 Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levy-Meyrueis, C, Bouillaud F, Seldin MF, Surwit RS, Riquier D, Warden CH. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nature Genet 15: 269 ± 272. 14 Vidal-Puig A, Solanes G, Grujic D, Flier JS, Lowell BB. UCP3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue. Biochem Biophys Res Commun 1997; 235: 79 ± 82. 15 Clement K, Ruiz J, Cassard-Doulcier AM, Bouilland F, Ricquier D, Basdevant A, Guy-Grand B, Froguel P. Additive effectÿ3826A?G variant of uncoupling protein gene and the trp64arg mutation of the b3-adrenergic receptor gene on weight gain in morbid obesity. Int J Obes Relat Metab Disord 1996; 20: 1062 ± 1066. 16 Valve R, Heikkinen S, Rissanen A, Laakso M, Uusitupa M. Synergistic effect of polymorphisms in uncoupling protein 1 and b3-adrenergic receptor genes on basal metabolic rate in obese Finns. Diabetologia 1998; 41: 357 ± 361. 17 Esterbauer H, Oberko¯er H, Liu Y-M, Breban D, Hell E, Krempler F, Patsch W. Uncoupling protein-1 mRNA expression in obese human subjects: the role of sequence variations at the uncoupling protein-1 gene locus. J Lipid Res 1998; 39: 834 ± 844. 18 Kogure A, Yoshida T, Sakane N, Umekawa T, Takakura Y, Kondo M. Synergistic effect of polymorphisms in uncoupling protein and b3-adrenergic receptor genes on weight loss in obese Japanese. Diabetologia 1998; 41: 1399. 19 Fumeron F, Durack-Brown I, Betoulle D, Cassard-Doulcier AM, Tuzet S, Bouillaud F, Melchoir JC, Ricquier D, Apfelbaum M. Polymorphisms of uncoupling protein (UCP) and b3adrenergic receptor genes in obese people submitted to a low calorie diet. Int J Obes Relat Metab Disord 1996; 20: 1051 ± 1054. 20 Bouchard C, Perusse L, Chagnon YC, Warden C, Ricquier D. Linkage between markers in the vicinity of the uncoupling protein-2 gene and resting metabolic rate in humans. Hum Mol Genet 1997; 6: 1887 ± 1889.

21 Pratley RE, Thompson DB, Bogardus C. An autosomal gene scan for loci linked to prediabetic phenotypes in Pima Indians. J Clin Invest 1998; 101: 1757 ± 1764. 22 Urhammer SA, Dalgaard LT, Sorensen TIA, Moller AM, Andersen T, Tybjaerg-Hansen A, Hansen T, Clausen JO, Vestergaard H, Pedersen O. Mutational analysis of the coding region of the uncoupling protein 2 gene in obese NIDDM patients: impact of a common amino acid polymorphism on juvenile and maturity onset forms of obesity and insulin resistance. Diabetologia 1997; 40: 1227 ± 1230. 23 Walder K, Norman RA, Hanson RL, Schrauwen P, Neverova M, Jenkinson CP, Easlick J, Warden CH, Pecquer C, Raimbault S, Ricquier D, Harper M, Silver K, Schuldner AR, Solanes G, Lowell BR, Chung WK, Leibel RL, Pratley R, Ravussin E. Association between uncoupling protein polymorphisms (UCP2-UCP3) and energy metabolism=obesity in Pima Indians. Hum Mol Genet 1998; 7: 1431 ± 1435. 24 Klannemark M, Orho M, Groop LC. No relationship between identi®ed variants in the uncoupling protein 2 gene and energy expenditure. Eur J Endocrinol 1998; 139: 217 ± 233. 25 Cassell PG, Neverova M, Janmohamed S, Uwakwe N, Qureshi A, McCarthya MI, Saker PJ, Albon L, Kopelman P, Noonan K, Easlick J, Ramachandran A, Snehalatha C, Pecquer C, Ricquier D, Warden CH, Hitman GA. An uncoupling protein 2 gene variant is associated with a raised body mass index but not type II diabetes. Diabetologia 1999; 42: 688 ± 692. 26 Tu N, Chen H, Winnikes U, Reinert I, Marmann G, Pirke KM, Lentes K-U. Structural organization and mutational analysis of the human uncoupling protein-2 (hucp2) gene. Pharmac Lett 1999; 64: 41 ± 50. 27 Otabe S, Clement K, Rich N, Warden C, Pecqueur C, Neverova M, Raimbault S, Guy-Grand B, Basdevant A, Ricquier D, Froguel P, Vasseur F. Mutation screening of the human UCP-2 gene in normoglycemic and NIDDM morbidly obese patients. Diabetes 1998; 47: 840 ± 842. 28 Gagnon J, Mauriege P, Roy S, SjoÈstroÈm D, Chagnon YC, Dionne FT, Oppert J-M, Perusse L, SjoÈstroÈm L, Bouchard C. The trp64arg polymorphism of b3-adrenergic receptor gene has no effect on obesity phenotypes in the Quebec family study and Swedish obese subjects cohorts. J Clin Invest 1996; 98: 2086 ± 2093. 29 Large V, HellstroÈm L, Reynisdottir S, LoÈnnqvist F, Eriksson P, Lannfelt L, Arner P. Human beta-2 adrenoceptor gene polymorphisms are highly frequent in obesity and associate with altered adipocyte beta-2 adrenoceptor function. J Clin Invest 1997; 100: 3005 ± 3013. 30 Oppert JM, Vohl MC, Chagnon M, Dionne FT, CassardDoulcier AM, Ricquier D, Perusse L, Bouchard C. DNA polymorphisms in the uncoupling protein (UCP) gene and human body fat. Int J Obes Relat Metab Disord 1994; 18: 526 ± 531.

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