Mutation Screening and Association Studies of the Human UCP 3 Gene in Normoglycemic and NIDDM Morbidly Obese Patients

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1 Mutation Screening and Association Studies of the Human UCP 3 Gene in Normoglycemic and NIDDM Morbidly Obese Patients Shuichi OTABE, Karine CLEMENT, Séverine DUBOIS, Frederic LEPRETRE, Veronique PELLOUX, Rudolph LEIBEL, Wendy CHUNG, Philippe BOUTIN, Bernard GUY-GRAND, Philippe FROGUEL and Francis VASSEUR short running title: Polymorphisms of the UCP 3 gene From Institute of Biology of Lille-CNRS EP10 France (SO, KC, SD, FL, FV), Department of Nutrition Hotel-Dieu Paris France (KC, VP, PF, BGG), Medical Faculty of Lille and CHRU of Lille France (PF, VF), Columbia University Department of Pediatrics New York USA (RL, WC). Address correspondence and reprint requests to Dr. Philippe FROGUEL, Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP.. 245, Lille cedex, France Phone: (+33) Fax.: (+33) e mail: froguel@xenope.pasteur-lille.fr Key words: Uncoupling protein (UCP), mutation, polymorphism, obesity, NIDDM Abbreviations: UCP uncoupling protein, SSCP single-strand conformation polymorphism, NIDDM non insulin diabetes mellitus, QTL quantitative trait loci, RFLP restriction fragment length of polymorphism. Abstract

2 To examine whether a genetic variation of the uncoupling protein 3 (UCP3) gene is involved in human obesity and NIDDM, we have screened by single-strand conformation polymorphism (SSCP) and direct sequencing, the 7 exons and the intron exon junction including the non-translated exon 1 of this gene. Moreover, we performed association studies between some of the detected variants and control subjects and morbidly obese patients. The study included 382 unrelated morbidly obese patients without or with NIDDM and 118 control subjects. We detected a total of six genetic polymorphisms including two aminoacid changes: a missense mutation (Val-9-Met change, GTG-ATG) in exon 2, two silent mutations (Ala-83-Ala, GCC-GCT and Tyr-99-Tyr, TAT-TAC) in exon 3, a mutation in intron 4 (C->T 36 bp 5' of splice site), a silent mutation (Tyr-210-Tyr, TAC-TAT) in exon 5, and a missense mutation (Arg-308-Trp change, CGG-TGG) in exon 7. The two types of missense mutations in exon 2 and exon 7 were absent from 118 control subjects and 96 non obese NIDDM patients, and were each detected in a single patient within the 226 morbidly obese subjects screened by SSCP. Moreover, association studies of the Tyr-99-Tyr in exon 3, the C->T 36 bp 5' of splice site in intron 4 and the Tyr-210-Tyr in exon 5 were performed by PCR-restriction fragment length of polymorphism (RFLP) in morbidly obese and control subjects. In the morbidly obese group, the patients homozygous (CC genotype) for Tyr-99-Tyr presented a significant higher BMI, a higher maximal reached BMI and minimal reached BMI (after dietetic intervention) than heterozygotes and wild type subjects (p=0.02, p=0.04 and p=0.03, respectively). Moreover, both the CC genotype of the intron 4 variant and the CC genotype of the exon 5 variant carriers in the morbidly obese group under a recessive model were significantly associated with NIDDM in comparison with other genotype (p=0.01 and p=0.04, respectively). Therefore the human UCP3 gene might play a role in morbid obesity and NIDDM associated with morbid obesity, at least in Caucasians.

3 UCPs are inner mitochondrial membrane transporters which dissipate the proton gradient, releasing stored energy as heat[nicholls, 1984 #9; Klingenberg, 1990 #5], and are therefore considered as potentially important determinants of defence against obesity. Three distinct UCP were identified so far: UCP 1[Klingenberg, 1990 #5; Himms-Hagen, 1997 #27] is mostly expressed in brown adipose tissue which is an important site of energy expenditure. UCP 1 seems unlikely to be a major gene involved in weight regulation of most large-sized adult animals and humans living in thermoneutral environment, where little brown adipose tissue exists[garruti, 1992 #43]. Recently two other human uncoupling proteins UCP 2 and UCP 3 were consecutively identified from a human muscle cdna library, and acting as mediator of adaptive thermogenesis in humans[fleury, 1997 #3; Vidal-Puig, 1997 #19; Boss, 1997 #2]. In comparison to UCP 1, the UCP 2 and 3 are widely expressed in adult human tissues. Concerning UCP 3, the gene is expressed abundantly in human skeletal muscle which is believed to be an important site for regulated energy expenditure and minimally in human heart and other critical organs. Moreover, recent studies presented a linkage between markers (D11S911,D11S916 and D11S11321) on chromosome 11 (11q13) in the vicinity of the UCP 2 and 3 gene and resting metabolic rate (RMR), percentage body fat (%FAT) and fat mass (FM)[Bouchard, 1997 #28; Solanes, 1997 #30]. In previous works, we demonstrated that UCP 2 gene is unlikely to play a major role in morbid obesity, as none of the polymorphisms detected in the coding sequences of the UCP 2 gene were associated with morbid obesity [Otabe, #45]. Therefore, in this study we performed the screening of the human UCP 3 gene and association studies to investigate whether a genetic variation of this gene is involved in human obesity and NIDDM.

4 RESEARCH DESIGN AND METHODS Subjects. A total of 382 unrelated morbidly obese patients (Mean age: X±Y y, mean BMI: X±Y kg/m2, sex ratio F/M=X/Y) without (X) or with NIDDM (Y) were randomly selected from a well described collection of independent French patients recruited from the Department of Nutrition at the Hotel-Dieu Hospital in Paris. We considered as NIDDM patients those receiving an antidiabetic oral treatment, or an insulin treatment started at least two years after the diagnosis of hyperglycemia and those with fasting plasma glucose over 7.8 mmol/l. For the remaining subjects, a standard 75 g oral glucose tolerance test was performed and the diagnosis of diabetes was made according to the WHO criteria. A control group of 118 unrelated non-obese (BMI<27 kg/m2) non diabetic subjects, selected from French pedigrees has been included (Mean age: 57.1±11.4y, mean BMI: 22.5±4.4 kg/m2, sex ratio F/M=64/54). For detection of new variants, SSCP screening was performed on 226 morbidly obese patients (mean age: 46.9±12.1, mean BMI: 46.8±6.7, sex ratio F/M=182/44) and the 118 control subjects. Direct sequencing was performed on 73 morbidly obese patients (Mean age: 48.2±11.9y, mean BMI: 47.9±7.8 kg/m2, sex ratio F/M=52/21) and 38 control subjects.(mean age: 57.1±11.9y, mean BMI: 23.3±2.6 kg/m2, sex ratio F/M=21/17). For an association study with 2 identified variants, a group of 211 morbidly obese subjects (BMI 40 kg/m2) was chosen from previous selected 382 obese patients and genotyped for 2 variants located in exon 5 and intron 4. Clinical characteristics of these subjects are sex ratio f/m 171/41, age 47.0±12 years, BMI:47±7.0 kg/m of these morbidly obese subjects also had chronic hyperglycemia: 76 subjects presented overt NIDDM, 38 subjects had impaired glucose tolerance (IGT), and 97 subjects presented normal glucose tolerance.

5 Methods. The 7 exons and intron exon junctions of the UCP 3 gene were screened for mutations by the automated fluorescent SSCP technique and direct sequencing [Boutin, 1997 #34]. For the detection of three variants (Tyr-99-Tyr in exon 3, the C->T 36 bp 5' of splice site in intron 4 and Tyr-210-Tyr in exon 5), PCR amplification were carried out in a final reaction volume of 25 µl, containing 200 ng human genomic DNA, 10 mmol/l Tris-HCl (ph 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl2, 200 Mmol/l of each dntp, 150 nmoles of each primer (Table 1) and 1.25 units of Taq polymerase (Applied Biosystems, Forster City, CA). PCR cycling conditions were: an initial denaturation at 94 C for 5 minutes, followed by 35 cycles at 94 C for 40 seconds, 30 seconds at anennealing tempertature, 30 seconds at 72 C and a final extension of 15 minutes at 72 C. The PCR products were digested at 37 C for 12 h by Mae II for the Tyr-99-Tyr, MboI for the C->T 36 bp 5' of splice site in intron 4 and Rsa I (Boehringer Mannheim GmbH Germany) for the Tyr-210-Tyr variant, since the nucleotide change resulted in the loss or gain of a site for a restriction endonuclease. Fragments were resolved on 4 % agarose gel and visualised by ethidium bromide staining. Statistics analysis Categorical variables were compared between groups using the chi square test. Fisher's exact test was used in calculations involving small samples. The wilcoxon/kriskal wallis test was used as a non-parametric method. A multivariate logistic regression analysis was performed to test for possible relationships between genetic variants and clinical parameters. RESULTS Screening of UCP3 gene. A total of six genetic variants were detected in our study. A misense mutation (Val-9-Met change, GTG-ATG) in exon 2, two silent mutations (Ala-83-Ala, GCC-GCT and Tyr-99-Tyr, TAT-TAC) in exon 3, a

6 mutation of intron 4 (C->T 36 bp 5' of splice site), a silent mutation (Tyr-210- Tyr, TAC-TAT) in exon 5, and a missense mutation (Arg-308-Trp change, CGG- TGG) in exon 7 were found by SSCP and direct sequencing. The two types of missense mutations in exon 2 and exon 7 were absent from 118 control subjects and detected each in a single morbidly obese patient within the 226 that were screened. Unfortunately, other members of these probands' families with these missense mutation were not available to carry out the segregation analysis in our study. The exon 3 G304A variant (R. Leibel, personal communication), the G->A variant (R. Leibel, personal communication) in the 3' first bp of intron 6 which would destroy the splice site necessary to make the "long" form of UCP3[Solanes, 1997 #30], and the exon 7 C844T variant (R. Leibel, personal communication) were not detected in our population of subjects and controls, although these variants offered from Dr Leibel R.L. could be detected clearly by our SSCP protocol. Association study with morbidly obese subjects associated with and without NIDDM. For all 3 markers, the variant were similarly distributed among male and female subjects (data not shown). a. UCP3-exon 3 variant (Tyr-99-Tyr) 382 morbidly obese subjects were genotyped : 254 (0.67) were TT (wild), 115 (0.30) were TC (heterozygotes) and 13(0.03) were CC (homozygotes). 103 control subjects were genotyped : 81 (0.79) were TT, 20 (0.19) were TC and 2 (0.02) were CC. The frequency of homozygous CC bearers was higher in the obese group (3%) than in the control group (1%) (chi2=9.9, p=0.05 and p=0.06 with pearson correction). A recessive model (homozygous vs. heterozygous + wild) was used showing that CC bearers have a higher BMI (52.7±8.5 kg/m2) than heterozygotes + wild subjects (47.5±7.5 kg/m2, p=0.02). They had also a higher maximal reached BMI during like (54±8.5 vs. 50.2±8.8 kg/m2, p=0.04). These morbidly obese subjects with the CC genotype had no significant trend of

7 higher weight at age 20 (88.0±25 vs. 78.3±31, p=0.1) and after the age of 20 their minimal reached weight under dietetic intervention was also higher (77.0 ± 21 vs ±, p= 0.03). In contrast, the presence of the C allele was not associated with an higher weight gain during adult life (Their weight gain were defined by the difference between the measured weight and the reported weight at the age of 20 years (dw-w20). b. the mutation of intron 4 (C->T 36 bp 5' of splice site) 211 morbidly obese subjects were genotyped : 125 (0.59) were TT (wild), 75 (0.36) were CT (heterozygotes) and 11(0.05) were CC (homozygotes). 100 control subjects were genotyped : 62 (0.62) were TT, 31 (0.31) were TC and 7 (0.07) were CC. No association was found between the mutation of intron 4 and obesity (chi2=1 p=0.6). Codominant and recessive models were tested. The weight related phenotypes (BMI, maximal reached BMI, weight at 20 years and minimal reached BMI after dietetic intervention) were similar in the 3 genotyped groups even if data were adjusted on age (data not shown). In contrast, a significant association with the diabetic status was observed under a codominant model (Chi2=8.2, p=0.016) (Table 2 a) and a recessive model (Chi2=7;4, p=0.006) (Table 2 b). c. UCP3-exon 5 variant (Tyr-210-Tyr) 211 morbidly obese subjects were genotyped : 43 (0.20) were TT (wild), 115 (0.55) were CT (heterozygotes) and 43 (0.25) were CC (homozygotes). 105 control subjects were genotyped : 27 (0.26) were TT, 54 (0.51) were TC and 24 (0.23) were CC. No association was found between this UCP3 exon 5 variant and obesity (chi2=1.9, p=0.38). Codominant and recessive models were tested. Weight related phenotypes, years and minimal reached BMI (after dietetic intervention) were similar in the 3 genotyped groups under recessive and codominant models (data not shown). In contrast, a trend toward an association with the diabetic

8 status was observed under a recessive model (Chi2=4, p=0.04) (Table 2 a) and a codominant model (Chi2=4.0, p=0.12) (Table 2 b). DISCUSSION The mouse region syntenic to human 11q13 is on chromosome 7 and has been linked using quantitative trait locus (QTL) analysis to obesity and non insulin dependent diabetes mellitus in a number of models[warden, 1995 #35; Surwit, 1995 #36; Taylor, 1996 #37]. Mapping of UCP 2 to this region raised the possibility that UCP 2 was the relevant gene[fleury, 1997 #3]. Bouchard and coworkers[bouchard, 1997 #28] presented a close and high linkage between markers (D11S911, D11S916 and D11S1321) in the vicinity of the UCP 2 gene and resting metabolic rate (RMR). Genes for UCP 2 and UCP 3 are highly homologous and are close on the chromosome 11[Solanes, 1997 #30]. With two tightly linked candidate genes, it was important to clarify the existence of pathological mutations as causes of obesity and NIDDM. However, UCP 2 is expressed at high levels in many sites not thought to mediate adaptive thermogenesis, especially immune systems such as spleen, lymph node, thymus and gastrointestinal tract whose role in mediating regulated energy expenditure is unclear[fleury, 1997 #3; Gimeno, 1997 #42; Boss, 1997 #2; Vidal-Puig, 1997 #19]. On the other hand, UCP 3, which is selectively and abundantly expressed in skeletal muscle [Boss, 1997 #2; Vidal-Puig, 1997 #19; Gong, 1997 #31] is believed to be an important site for regulated energy expenditure and may be more relevant than UCP 2 as a mediator of adaptive thermogenesis in humans. Recently, the screening of the coding region of the UCP 2 gene in 35 obese NIDDM patients was reported by SSCP analysis of muscle UCP 2 cdna, and one missense mutation was identified. However, it was demonstrated that the polymorphism was not implicated in the pathogenesis of juvenile or maturity onset obesity or insulin resistance in Caucasians[Urhammer, 1997 #29]. Then, the mutation screening of all exons including a non-translated exon and the

9 intron/exon junctions of the human UCP 2 gene by direct sequencing in 72 morbidly obese patients without or with NIDDM allowed us to describe 3 new nucleotide changes and a 45bp insertion/deletion polymorphism. As none of these polymorphisms of the UCP 2 gene was associated with morbid obesity and NIDDM, it is unlikely that UCP 2 plays a major role in morbid obesity, at least in French Caucasian [Otabe, #45]. As for another more important candidate gene, the present study demonstrated a total of six genetic variants including three silent mutations: Ala-83-Ala, Tyr-99-Tyr and Tyr-210-Tyr, an intron 4 mutation (C->T 36 bp 5' of splice site), and two missense mutations: Val-9-Met and Arg- 308-Trp, although the Tyr-99-Tyr variant was recently reported [Urhammer, 1998 #44]. The patients that presented these two misense mutations had both hyperinsulinemia, unfortunately as other members of the families were unavailable, we could not investigate the segregation of these mutations. The role of these mutations in morbid obesity and NIDDM would deserve further investigations. Interestingly, the association studies of three variants suggested that the homozygote subjects for Tyr-99-Tyr variant for the C allele in exon 3 have a significant higher BMI at any age, and showed that the existence of the c allele in the 36 bp 5' of splice site (intron 4) and the homozygocity of Tyr-210- Tyr for the C allele was significantly associated with the diabetic status. Indeed, we could demonstrate the correlation between these all three variant and morbidly obese patients in our studies. Each of these variants is a silent mutation or a variant of intron with no obvious biological effect. However, it might be in linkage disequilibrium with a nearby functionally relevant mutation, either in UCP3 gene including the promoter region or elsewhere. In conclusion, although missense mutations in exon 2 and 7 and linkage analysis of the these three polymorphysms in large families would deserve further investigations, variations in the UCP3 gene could contribute to weight gain and a risk factor for NIDDM in morbidly obese patients due to other possible additive interaction between genetic and environmental factors.

10 ACKNOWLEDGEMENT We thank Amar Abderrhamani and Suren Budhan for helpful suggestions. TABLE 1 The list of primers for sequence and RFLP and annealing temperature Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Primer sequence 5'-TGT-AAA-ACG-ACG-GCC-AGT-GCC-ATC-CAA-TCC-CTG-CTG-3' 5'-CAG-GAA-ACA-GCT-ATG-ACC-TGA-AAG-CCT-CCA-ATG-AAA-3' 5'-TGT-AAA-ACG-ACG-GCC-AGT-GCC-AGA-CAT-CAC-TCC-ATC-3' 5'-CAG-GAA-ACA-GCT-ATG-ACC-ACT-AGC-CCC-TCC-TTC-CAT-3' 5'-TGT-AAA-ACG-ACG-GCC-AGT-CAC-CCC-CTG-CTA-TTG-TCC-3' 5'-CAG-GAA-ACA-GCT-ATG-ACC-GTC-TCT-TGA-CCC-ACA-CAC-TTT-3' 5'-TGT-AAA-ACG-ACG-GCC-AGT-GCA-GCC-CCG-CAG-AGA-ACA-3' 5'-CAG-GAA-ACA-GCT-ATG-ACC-TCC-CTG-CCC-CAG-CCT-GAG-3' 5'-TGT-AAA-ACG-ACG-GCC-AGT-TTG-CAG-CCA-GGG-CAT-CCA-TTT-C-3' 5'-CAG-GAA-ACA-GCT-ATG-ACC-AGC-GAG-TTC-TGG-GTT-CCC-TC-3' 5'-TGT-AAA-ACG-ACG-GCC-AGT-TAT-GGA-CAG-AAC-ACA-AAT-GC-3' 5'-CAG-GAA-ACA-GCT-ATG-ACC-TCT-CTG-GGA-GGG-AGT-GCT-3' 5'-TGT-AAA-ACG-ACG-GCC-AGT-GTT-GGT-TGT-TTT-TCT-TAT-CA-3' 5'-CAG-GAA-ACA-GCT-ATG-ACC-GTC-TGT-GTC-CAT-GTG-TGC-GT-3' Annealing tempertature ( C) Tyr-99-Tyr 5'-CCC-CTT-CTT-GCC-TTC-CCA-TCT-G-3' 55 5'-GTA-CAC-CTG-CTT-GAC-GGA-GAC-3' Variant of intron 4 Tyr-210-Tyr 5'-TTC-CCA-TTC-CTC-CCT-CCC-GA-3' 5'-AGC-GAG-TTC-TGG-GTT-CCC-TC-3' 5'- TCA-AGG-AGA-AGC-TGC-TGG-AGT-3' 5'-AGC-GAG-TTC-TGG-GTT-CCC-TC-3' Table1.

11 Table 2 Association with the diabetic status in morbidly obese a. under a codominant model NIDDM others intron 4 tt ct cc 68 (0.59) 36 (0.32) 10 (0.09) 57 (0.59) 39 (0.40) 1 (0.01) exon 5 tt ct cc 22 (0.19) 57 (0.50) 35 (0.31) 21 (0.22) 58 (0.60) 18 (0.19) 2 2 Chi = 8.2, p=0.016 NIDDM = diabetic (pooling true NIDDM and glucose intolerant) Chi = 4.0, p=0.12 b. under a recessive model NIDDM tt + ct intron 4 cc 104 (0.91) 10 (0.09) tt+ ct exon 5 cc 79 (0.70) 35 (0.30) others 96 (0.99) 1 (0.01) 79 (0.81) 18 (0.19) Chi = 7.4, p= under a recessive model (with fishers exact test used for small sample p = 0.01) 2 2 Chi = 4, p= 0.04 under a recessive model (with fishers exact test used for small sample p = 0.03) Table 2.

12 TABLE 1 The list of variants in the human UCP 3 gene Exon Location Codon Nucleotide change (amino acid) 2 9 GTG (Val) ATG (Met) 3 83 CCC (Ala) GCC (Ala) 3 99 TAT (Tyr) TAC (Tyr) Intron 4 36 bp 5' of splice site ACC ATC TAT (Tyr) TAT (Tyr) CGG (Alg) TGG (Trp) Table 3.

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir-484 30 20 10 0 **** **** NCP mir- 484P NCP mir- 484P b Relative