OBJECTIVE

Single nucleotide polymorphisms (SNPs) in intron 1 of fat mass– and obesity-associated gene (FTO) are strongly associated with human adiposity, whereas Fto−/− mice are lean and Fto+/− mice are resistant to diet-induced obesity. We aimed to determine whether FTO mutations are disproportionately represented in lean or obese humans and to use these mutations to understand structure-function relationships within FTO.

RESEARCH DESIGN AND METHODS

We sequenced all coding exons of FTO in 1,433 severely obese and 1,433 lean individuals. We studied the enzymatic activity of selected nonsynonymous variants.

RESULTS

We identified 33 heterozygous nonsynonymous variants in lean (2.3%) and 35 in obese (2.4%) individuals, with 8 mutations unique to the obese and 11 unique to the lean. Two novel mutations replace absolutely conserved residues: R322Q in the catalytic domain and R96H in the predicted substrate recognition lid. R322Q was unable to catalyze the conversion of 2-oxoglutarate to succinate in the presence or absence of 3-methylthymidine. R96H retained some basal activity, which was not enhanced by 3-methylthymidine. However, both were found in lean and obese individuals.

CONCLUSIONS

Heterozygous, loss-of-function mutations in FTO exist but are found in both lean and obese subjects. Although intron 1 SNPs are unequivocally associated with obesity in multiple populations and murine studies strongly suggest that FTO has a role in energy balance, it appears that loss of one functional copy of FTO in humans is compatible with being either lean or obese. Functional analyses of FTO mutations have given novel insights into structure-function relationships in this enzyme.

Genome-wide association studies have revealed that single nucleotide polymorphisms (SNPs) within the first intron of fat mass– and obesity-associated gene (FTO) are strongly associated with adiposity (1,,4). These associations have been largely consistent across multiple different ethnicities including Europeans (5,,8), Asians (9,11), Hispanics (12,13), and Africans (13,14). At present, it remains unclear whether these SNPs influence the expression or splicing of the FTO gene and/or whether the unequivocal association with obesity is causally related to alterations in expression or function of FTO. If the effect of the intron 1 SNPs is through FTO, it also remains unclear whether the obesity risk SNPs result in a loss or a gain of FTO function. It remains possible that the SNPs influence the expression and/or function of neighboring genes and that such an effect might underlie the association with adiposity. There are several examples in metabolic disease where common variants close to a particular gene are associated with alterations in risk of common phenotypes such as fat mass or risk of obesity, whereas rare loss- or gain-of-function mutations in the same gene are associated with a more severe version of the same metabolic phenotype (e.g., MCR4 [5,15,,,19], POMC [20,21], BDNF [22,24], and PCSK1 [25,26]). When information is available from both types of variants, clarity of mechanistic understanding is greatly enhanced. To establish whether nonsynonymous variants of FTO might be enriched in either lean or obese subjects, we sequenced the FTO coding region and intron-exon boundaries in a large group of subjects with severe obesity and a similarly sized group of individuals with lifelong leanness. We have recently established that FTO encodes a 2-oxoglutarate (2-OG) Fe2+-dependent dioxygenase (27). In this study, we have examined the functional enzymatic properties of naturally occurring variants to gain further insights into structure-function relationships in the human enzyme.

FTO was sequenced in 1,433 severely obese subjects. All adults had BMI >40 kg/m2, and all obese children had a BMI >97th percentile for sex and age (Table 1). Three hundred forty-five French morbidly obese adults were recruited by the CNRS UMR8090 at Lille and the Department of Nutrition of Paris Hotel Dieu Hospital. Thirty-three French morbidly obese adults were patients of the CHRU Lille hospital. We also sequenced 287 Swiss morbidly obese adults, all of them recruited after gastric surgery in Zurich, Switzerland. Eighty-eight morbidly obese adult patients were recruited at the Antwerp University Hospital. Three hundred ninety-nine French obese children were recruited in CNRS UMR8090, and 281 obese children were recruited as part of the Genetics of Obesity Study (GOOS) cohort (28). A total of 1,221 lean adult subjects were part of the French DESIR (Data from an Epidemiological Study on the Insulin Resistance syndrome) general prospective study (29). Selection criteria were BMI <23 kg/m2 at inclusion. We included 212 lean French children selected from the Stanislas study (30). Inclusion criteria was BMI z score (zBMI) <90th percentile for sex and age according to the Eastern Cooperative Oncology Group (ECOG) (31). All patients were of European ancestry. The study protocol was approved by all local ethics committees, and informed consent was obtained from each subject before participating in the study.

TABLE 1

Clinical characteristics for obese and lean subjects

n (male/female)Age (years)BMI (kg/m2)zBMI
Obese (adults: BMI >40 kg/m2; children: BMI >97th percentile)     
    French adults 378 (79/299) 43.15 ± 11.52 51.63 ± 9.17 — 
    Swiss adults 287 (79/208) 41.56 ± 11.21 52.32 ± 5.80 — 
    Belgian adults 88 (36/52) 45.45 ± 11.21 54.79 ± 5.20 — 
    French children 399 (188/211) 10.56 ± 3.23 — 5.01 ± 1.04 
    English children 281 (135/146) 11.5 ± 2.40 — 4.6 ± 1.80 
Lean (adults: BMI <23 kg/m2; children: BMI <90th percentile)     
    French adults 1,221 (378/843) 43.26 ± 9.37 20.42 ± 1.28 — 
    French children 212 (96/116) 11.77 ± 2.42 — 0.10 ± 0.98 
n (male/female)Age (years)BMI (kg/m2)zBMI
Obese (adults: BMI >40 kg/m2; children: BMI >97th percentile)     
    French adults 378 (79/299) 43.15 ± 11.52 51.63 ± 9.17 — 
    Swiss adults 287 (79/208) 41.56 ± 11.21 52.32 ± 5.80 — 
    Belgian adults 88 (36/52) 45.45 ± 11.21 54.79 ± 5.20 — 
    French children 399 (188/211) 10.56 ± 3.23 — 5.01 ± 1.04 
    English children 281 (135/146) 11.5 ± 2.40 — 4.6 ± 1.80 
Lean (adults: BMI <23 kg/m2; children: BMI <90th percentile)     
    French adults 1,221 (378/843) 43.26 ± 9.37 20.42 ± 1.28 — 
    French children 212 (96/116) 11.77 ± 2.42 — 0.10 ± 0.98 

Data are means ± SD unless otherwise indicated.

Genotyping of the FTO rs9939609 SNP.

Genotyping of the FTO rs9939609 SNP was performed using a Taqman SNP Genotyping Assay (Applied Biosystems) on an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions.

Sequencing.

We screened the nine exons of the FTO by direct nucleotide sequencing. We amplified overlapping PCR fragments according to primers and PCR-optimized conditions (available from the authors upon request). PCR amplifications were inspected for single bands of expected sizes on agarose gels before purification with Agencourt AMPure on Biomek NX (Beckman Coulter). Sequencing was achieved using the automated ABI Prism 3730xl DNA Sequencer in combination with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit 3.1 (Applied Biosystems), and purification of sequencing reaction was performed with Agencourt CleanSEQ on Biomek NX (Beckman Coulter). Sequences were assembled and analyzed with Sequencher software (GeneCodes).

Statistical analysis.

The comparison of prevalence has been tested with Fisher exact test, and the reported P values are two-sided. P values of <0.05 were considered to indicate statistical significance.

Cloning of human FTO.

Wild-type human FTO cDNA was amplified (Expand DNA polymerase; Roche) using XhoI-tagged primers with the first strand of the reverse-transcribed cDNA (Superscript II; Invitrogen) from human brain total RNA as a template. Using the XhoI sites, the resulting PCR product was cloned into an NH2-terminal 6× His-tagged bacterial expression vector (pET302/NT-His; Invitrogen). The FTO mutants were generated from cloned wild-type FTO using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to manufacturer's protocols. The mutations were confirmed by DNA sequencing using Big Dye terminator chemistry (Applied Biosystems) and electrophoresis on an ABI 3730 automated DNA sequencer. Sequences were assembled and examined using Sequencher software (GeneCodes). All primer sequences are available upon request.

Human wild-type and mutant FTO protein production.

Human wild-type and mutant FTO protein production was carried out as previously described (32). Briefly, expression plasmids were transformed into Escherichia coli BL21-Gold (DE3; Stratagene) and cultured in 2 l Luria-Bertani broth and 50 μg/ml carbenicillin to A600 1.0 at 37°C. Expression of the cloned gene was induced by addition of 0.5 mmol/l isopropyl-β-d-1-thiogalactopyranoside (IPTG) at 15°C for 4 h. The cells were harvested and washed in PBS, and pellets were stored at −80°C. Cells were resuspended in 40 ml of lysis buffer (50 mmol/l HEPES-KOH, pH 8, 2 mmol/l β-mercaptoethanol, 5% glycerol, and 300 mmol/l NaCl), prior to addition of lysozyme (2 mg/ml). After 30 min of incubation on ice, cells were sonicated and the lysate was clarified by centrifugation at 15000g for 20 min. The lysate was supplemented with imidazole (10 mmol/l) and loaded onto a 1.5-ml Ni-NTA (nickel–nitrilotriacetic acid)–agarose column (Qiagen) previously equilibrated in lysis buffer containing 10 mmol/l imidazole. The column was washed with 30 ml of lysis buffer containing 10 mmol/l imidazole, followed by 7.5 ml of lysis buffer containing 40 mmol/l imidazole. The FTO protein was eluted in lysis buffer containing 250 mmol/l imidazole. The eluate was then loaded onto a 30-kDa concentrator (Sartorius Stedium Biotech) and centrifuged at 2500g for 30 min. Then 2 ml of buffer (20 mmol/l HEPES, pH 8, 5% glycerol, 50 mmol/l NaCl) was added to the concentrated fraction, which was concentrated again down to 100 μl by centrifugation at 2500g. FTO preparations were aliquoted and then stored at −80°C. FTO proteins were visualized by SDS–10% PAGE and stained with Coomassie blue.

Assays of FTO activity.

Conversion of 14C-2-oxoglutarate to 14C-succinate by 2-OG Fe2+-dependent DNA dioxygenases has been assayed previously (33). This method was modified and optimized to assay FTO as previously described (32). To measure the uncoupled reaction (no prime substrate present), 1.5 μmol/l FTO was assayed in 10 μl reaction mixture containing 50 mmol/l HEPES-KOH, pH 7, 50 μg/ml BSA, 4 mmol/l ascorbate, 75 μmol/l Fe(NH4)2(SO4)2, and 20 μmol/l [5-14C]-2-oxoglutarate (30 mCi/mmol from Moravek Biochemicals) and incubated at 37°C for various times. To measure stimulation of this activity by 3-methylthymidine, 1 mmol/l 3-methylthymidine (Moravek Biochemicals) was included in the assay mix. The reaction was stopped by adding 5 μl stop solution containing 20 mmol/l succinate, 20 mmol/l 2-OG, followed by 5 μl 160 mmol/l dinitrophenylhydrazine, which precipitates 2-OG. This mix was incubated at room temperature for 30 min. An additional 10 μl 1 M 2-OG was added and incubated for a further 30 min. The precipitate was removed by centrifugation. Clear supernatant (10 μl) was scintillation counted to monitor the 14C-succinate generated.

Prevalence of nonsynonymous mutations in lean and obese subjects.

To identify potential loss-of-function mutations, we screened the nine coding exons of FTO by direct nucleotide sequencing in 1,433 extremely obese subjects (753 adults with a BMI >40 kg/m2 and 680 children with a BMI >97th percentile) and in 1,433 lean subjects (1,221 adults with a BMI <23 kg/m2 and 212 children recruited with a zBMI <90th percentile) according to the ECOG (31), with an average zBMI close to the 50th percentile (zBMI = 0.1 ± 0.98) (Table 1). Additionally, we also genotyped the FTO rs9939609 SNP (2) in all of the patients. Genotype call rate was >99% in both lean and obese subjects, and genotypic distribution obeyed the Hardy-Weinberg equilibrium in the lean control subgroup (P = 0.58). We genotyped 363 duplicated DNA samples and observed a concordance rate of 100%. In the lean subgroup, genotype counts were 532 (TT), 668 (TA), and 224 (AA). In the obese subgroup, genotype counts were 367 (TT), 665 (TA), and 390 (AA). As expected, the obesity risk A allele frequency was 11.6% higher in obese versus lean subjects (obese: 50.8%; lean: 39.2%; OR 1.60 [1.44–1.78]; P = 1.2 × 10−18).

A total of 26 different rare (frequency <1% in the present cohort) nonsynonymous variants were identified in FTO (Table 2). The prevalence of nonsynonymous variants was similar between the two groups, with 33 lean (2.3%) and 35 obese (2.4%) individuals carrying mutations, and no significant differences in prevalence were observed between children and adults (Table 2). We found that a subset of those was unique to each group. Eight (A134T, G187A, M223V, A241T, H419R, E471G, I492V, and V493F) nonsynonymous mutations were identified uniquely in the obese cohort, whereas 11 (P5L, E24K, R80P, P93R, V94I, N143S, I148R, D189N, E234D, R316Q, and P399H) were identified uniquely in the lean cohort (Table 3). G187A (n = 2), A241T (n = 2), and V493F (n = 4) were identified in multiple obese subjects (Table 3). Seven variants (R96H, L146M, A163T, V201I, S256N, R322Q, and A405V) were present in both cohorts. The prevalence of synonymous mutations was not significantly different in obese compared with lean subjects (0.8% vs. 0.4%, P > 0.05; Table 4). All mutations were identified in heterozygous form, and no nonsense variants were reported in the studied populations. Thus, there is no obvious enrichment of nonsynonymous mutations in lean or obese individuals.

TABLE 2

Summary of all nonsynonymous mutations found in obese and lean subjects

Nonsynonymous mutationsObese childrenObese adultsAll obese subjectsLean childrenLean adultsAll lean subjects
P5L     1 
E24K     1 
R80P     1 
P93R     1 
V94I     1 
R96H  1  1 
A134T  1    
N143S     1 
L146M 2 3 
I148R     1 
A163T 7 5 
G187A  2    
D189N     1 
V201I  2 2 
M223V  1    
E234D     1 
A241T  2    
S256N 8 9 
R316Q     1 
R322Q  1  1 
P399H     1 
A405V  1  1 
H419R  1    
E471G  1    
I492V  1    
V493F 4    
Prevalence of mutation carriers (%) 1.32 3.45 2.44 3.77 2.05 2.31 
Nonsynonymous mutationsObese childrenObese adultsAll obese subjectsLean childrenLean adultsAll lean subjects
P5L     1 
E24K     1 
R80P     1 
P93R     1 
V94I     1 
R96H  1  1 
A134T  1    
N143S     1 
L146M 2 3 
I148R     1 
A163T 7 5 
G187A  2    
D189N     1 
V201I  2 2 
M223V  1    
E234D     1 
A241T  2    
S256N 8 9 
R316Q     1 
R322Q  1  1 
P399H     1 
A405V  1  1 
H419R  1    
E471G  1    
I492V  1    
V493F 4    
Prevalence of mutation carriers (%) 1.32 3.45 2.44 3.77 2.05 2.31 

Data in boldface indicate combined adult and children numbers.

TABLE 3

Summary of nonsynonymous mutations unique to the lean or the obese group

Nonsynonymous mutationsn
Obese subjects  
    A134T 
    G187A 
    M223V 
    A241T 
    H419R 
    E471G 
    I492V 
    V493F 
    Prevalence of mutations (%) 0.91 
Lean subjects  
    P5L 
    E24K 
    R80P 
    P93R 
    V94I 
    N143S 
    I148R 
    D189N 
    E234D 
    R316Q 
    P399H 
    Prevalence of mutations (%) 0.77 
Nonsynonymous mutationsn
Obese subjects  
    A134T 
    G187A 
    M223V 
    A241T 
    H419R 
    E471G 
    I492V 
    V493F 
    Prevalence of mutations (%) 0.91 
Lean subjects  
    P5L 
    E24K 
    R80P 
    P93R 
    V94I 
    N143S 
    I148R 
    D189N 
    E234D 
    R316Q 
    P399H 
    Prevalence of mutations (%) 0.77 
TABLE 4

Summary of all synonymous mutations found in lean and obese subjects

Synonymous mutationsObese subjects (n)Lean subjects (n)
T6T 
P33P — 
L44L — 
P93P — 
A174A — 
E217E — 
I334I — 
Q336Q — 
V374V — 
C456C — 
L464L — 
I492I — 
Prevalence of mutations (%) 0.84 0.42 
Synonymous mutationsObese subjects (n)Lean subjects (n)
T6T 
P33P — 
L44L — 
P93P — 
A174A — 
E217E — 
I334I — 
Q336Q — 
V374V — 
C456C — 
L464L — 
I492I — 
Prevalence of mutations (%) 0.84 0.42 

Functional properties of nonsynonymous mutations.

FTO is a member of the AlkB homologue (ABH) protein family, which numbers nine homologues in humans (27). Most 2-OG and Fe2+-dependent dioxygenases slowly catalyze the conversion of 2-OG to succinate even in the absence of their prime substrate (33), and this so-called uncoupled reaction may be stimulated by substrates or their analogues. We have previously reported that murine Fto is capable of demethylating the base 3-methylthymine in the context of single-stranded DNA (27), but that reaction proceeds rather slowly. Human FTO can also accomplish this reaction, but again at a very slow turnover rate (34). We have previously exploited the ability of wild-type human FTO to efficiently catalyze the conversion of 2-OG to succinate in a manner that is stimulated by the presence of the free nucleoside 3-methylthymidine (32), and have used this assay throughout this study.

Analysis based on a three-dimensional (3D) homology model (27) comparing FTO with the known 3D structure of three members of the family (ABH2, ABH3, and AlkB) (35) suggests that FTO can be divided in several structural and functional regions (Fig. 1). The more conserved NH2-terminal segment contains 1) a putative nuclear localization signal present only in FTO (36), 2) a double-stranded β-helix in a “jellyroll fold” containing all the catalytic apparatus that is well conserved in all ABHs, 3) a substrate recognition lid that is very different in each ABH member but phylogenetically well conserved among species within each paralog, and 4) two long insertions, absent in other ABH members, that are of variable lengths in different species and are substantially less conserved than the rest of the protein. The COOH-terminal domain, whose structure and function remain unknown, is absent in all other ABH members but contains several residues that are absolutely conserved among highly diverse species.

FIG. 1.

Predicted structural and functional regions of FTO based on a 3D homology model of FTO (27).

FIG. 1.

Predicted structural and functional regions of FTO based on a 3D homology model of FTO (27).

Two of the naturally occurring mutations that we detected, namely R316Q (found in one lean subject) and R322Q (found in one lean and one obese), replace absolutely conserved residues in the catalytic domain (Fig. 2). The model predicts R316 and R322 to be involved in 2-OG coordination, by forming stabilizing salt bridges with the carboxylates of this cosubstrate. We have previously studied the enzymatic property of R316Q (32), and in this study we have examined R322Q compared with wild-type FTO (Fig. 3 A). As predicted, R322Q, like R316Q (32), was completely unable to convert 2-OG to succinate in either the absence or presence of 3-methylthymidine.

FIG. 2.

Distribution of nonsynonymous mutations along FTO exons and functional domains. Highlighted mutants are those that have been assayed in the uncoupled reaction in the absence and presence of 3-methylthymidine. Mutants that were inactive in the uncoupled reaction assay with 3-methylthymidine are shown in red, whereas those that did not affect this reaction are shown in blue.

FIG. 2.

Distribution of nonsynonymous mutations along FTO exons and functional domains. Highlighted mutants are those that have been assayed in the uncoupled reaction in the absence and presence of 3-methylthymidine. Mutants that were inactive in the uncoupled reaction assay with 3-methylthymidine are shown in red, whereas those that did not affect this reaction are shown in blue.

FIG. 3.

Activity of wild-type (WT) and mutant FTO proteins. Decarboxylation of 14C-2-oxoglutarate to 14C-succinate by 1.5 μmol/l FTO in the absence or presence of 3-methylthymidine was measured at various time intervals. A: WT1 and R322Q FTO proteins. B: WT1 and R96H FTO proteins. C: WT2, P5L, V94I, and A241T FTO proteins. D: WT3 and V493F FTO proteins. WT1, 2, and 3 are three different wild-type FTO preparations made at the same time as the mutant proteins with which they are assayed in A, B, C, or D. Open symbols indicate without 3-methylthymidine; closed symbols indicate with 1 mmol/l 3-methylthymidine (also indicated by − and +). E: SDS–10% PAGE of FTO protein preparations stained with Coomassie blue. Per lane, 3 μg total protein was loaded. FTO and size markers are indicated.

FIG. 3.

Activity of wild-type (WT) and mutant FTO proteins. Decarboxylation of 14C-2-oxoglutarate to 14C-succinate by 1.5 μmol/l FTO in the absence or presence of 3-methylthymidine was measured at various time intervals. A: WT1 and R322Q FTO proteins. B: WT1 and R96H FTO proteins. C: WT2, P5L, V94I, and A241T FTO proteins. D: WT3 and V493F FTO proteins. WT1, 2, and 3 are three different wild-type FTO preparations made at the same time as the mutant proteins with which they are assayed in A, B, C, or D. Open symbols indicate without 3-methylthymidine; closed symbols indicate with 1 mmol/l 3-methylthymidine (also indicated by − and +). E: SDS–10% PAGE of FTO protein preparations stained with Coomassie blue. Per lane, 3 μg total protein was loaded. FTO and size markers are indicated.

One mutation, R96H (detected in one lean and one obese subject) occurs at an absolutely conserved residue within the putative “substrate recognition lid” of the protein (Fig. 1). In other members of the ABH family, this region of the molecule is known to be required for binding of the primary substrate but is not involved in interactions with the cosubstrate 2-OG. Notably, in contrast to R322Q, R96H retained some ability to convert 2-OG to succinate, but this activity was not enhanced by the presence of 3-methylthymidine (Fig. 3 B), a finding consistent with the hypothesis that R96 functions as part of the primary substrate recognition lid.

We also tested the enzymatic activity of several other variants representing the different regions of FTO (P5L, V94I, I148R, M223V, E234D, A241T, A405V, I492V, and V493F) (Fig. 2). None of these variants, all of which are located in less conserved positions, had any significant impact on enzymatic activity (Fig. 3 C and D and data not shown).

We have previously demonstrated that murine Fto localizes to the nucleus (27). Bioinformatic analysis suggests that a sequence of 18 amino acid residues in the NH2-terminal of FTO encodes a putative nuclear localization signal (Fig. 1). P5L affects a residue within this putative nuclear localization domain. COS-7 cells were transfected with a construct expressing WT FTO-GFP (wild-type FTO green fluorescent protein), P5L FTO-GFP mutant protein, or GFP alone (mock-GFP). WT FTO-GFP and P5L FTO-GFP colocalized with DAP1, whereas GFP alone was seen in both the cytoplasm and the nucleus (supplementary Fig. 1, available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0703/DC1). Thus, P5L does not appear to impair the nuclear localization of the protein.

Recent genome-wide association efforts have been highly successful in identifying a large number of common genetic variants reliably associated with important human diseases and quantitative traits (2,32,37). Understanding the precise biological mechanisms underlying such associations will provide a major scientific challenge that will require a multiplicity of approaches in humans and in model organisms. FTO exemplifies many of these challenges. Although SNPs in intron 1 of FTO are unequivocally associated with adiposity in multiple populations, there is still no evidence that those SNPs influence the expression or splicing of FTO itself. FTO is located adjacent to other genes, the expression or function of which could conceivably influence energy balance (10,36,38). Recent findings in mice rendered null for Fto support the notion that Fto itself has an important influence on energy balance. Fto-null mice are small, are lean, have an increased metabolic rate, and are hyperphagic, whereas Fto+/− mice are resistant to diet-induced obesity (39). Given that information, it is reasonable to speculate that loss-of-function mutations in FTO might be more common in lean rather than obese humans. To test this hypothesis, we sequenced FTO in a large number of lean and obese subjects and found that 1) nonsynonymous mutations were equally common in both the obese and lean cohorts and 2) heterozygous mutations that severely impaired enzymatic activity of FTO were found in both lean and obese individuals with no other obvious major clinical phenotypes. Our findings illustrate the importance, when sequencing “candidate” genes whose candidacy derives largely from genome-wide association studies in extreme phenotypes, of studying both ends of the spectrum of a quantitative trait. (If we had considered FTO to be an “obesity gene” and sequenced only obese subjects, the finding of loss-of-function mutations would have been misleading.) From our human genetic data, we can conclude that heterozygosity for a severely dysfunctional FTO allele is compatible with being either lean or obese in humans. Understanding the relationship between SNPs in intron 1 of FTO with human adiposity will require other approaches. In collaboration with Dr. Laurence Colleaux from Hopital Necker Paris and others, we have recently found a consanguineous Israeli-Arab family in which nine siblings were homozygous for the R316Q mutation in FTO (32). All homozygous carriers were severely growth retarded, had multiple congenital malformations, and died in infancy. Although heterozygous parents of these children had no obvious metabolic phenotype, ongoing efforts are directed at establishing measures of adiposity in heterozygous versus wild-type relatives (32).

Our studies have brought new insights into aspects of the structure-function relationship of the human FTO enzyme. Human FTO can catalyze the uncoupled reaction (2-OG to succinate) and is stimulated by 3-methylthymidine (32), and we have used this as a robust test of FTO's catalytic activity. As predicted, we found that the arginine at position 322 is essential for the catalytic activity of human FTO, and we have made similar observations for R316Q (32). Mutation of the arginine corresponding to human 316 in mouse Fto (R313A) also generated an inactive enzyme (27). Perhaps more interestingly, we have demonstrated that mutation of R96 to histidine in human FTO produces an enzyme that is capable of some basal conversion of 2-OG to succinate but is insensitive to the effects of 3-methylthymidine. These findings are consistent with R96 being part of the so-called substrate recognition lid that is responsible for substrate fixation and selectivity. Thus, the lid is well conserved among species orthologues, but has diverged substantially in the various paralogs of ABH to accommodate different substrates. For instance, R131 at this position in the lid of ABH3 is positioned to interact with 1-meA N3, and R131A in ABH3 abolishes the activity toward 1-me-A containing single-stranded DNA (27).

Although all of the other nonsynonymous variants we tested appeared to have normal enzymatic activity, we should express caution about categorically stating that these are fully wild type in function. First, we do not know what the true natural primary substrate(s) of FTO is (are), and it is entirely possible that mutations that affect FTO's actions on this elusive substrate will not necessarily impair the uncoupled reaction. Second, although we have demonstrated that FTO, in vitro, possesses dioxygenase activity, whether it has any other biological role, enzymatic or otherwise, is yet to be determined. This could potentially be of relevance to V493F, a COOH-terminal mutation that was found only in the obese population but was found in four different individuals (Fig. 2), and yet did not impact on the ability of FTO to convert 2-OG to succinate or its stimulation by 3-methylthymidine (Fig. 3 D). The COOH-terminal region of FTO, although highly conserved across species, is not shared with other members of the ABH family, and its structure and function are unknown.

We noted two potentially interesting observations regarding the COOH-terminal region of the protein. First, on a “mutations per nucleotide” basis, nonsynonymous mutations are found ∼3 times less frequently in the COOH-terminus than in the rest of the molecule. Second, although nonsynonymous variants elsewhere in the molecule are found equally in obese and lean subjects, eight such variants found in the COOH-terminus were detected in obese subjects and only two in lean (Fig. 2). These preliminary observations are of potential interest and might lead to a better understanding of FTO function and its role in energy homeostasis localization.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This study was supported by le Conseil Régional Nord Pas de Calais/FEDER, the Agence Nationale de la Recherche, Cancer Research U.K., EC FP6 Marie Curie, the U.K. Medical Research Council Centre for Obesity and Related metabolic Disorders (MRC-CORD), the Wellcome Trust, and the National Institute for Health Research Cambridge Biomedical Research Centre.

No potential conflicts of interest relevant to this article were reported.

We acknowledge Peter Robins for assistance with protein purification and enzyme assays.

1
Dina
C
,
Meyre
D
,
Gallina
S
,
Durand
E
,
Körner
A
,
Jacobson
P
,
Carlsson
LM
,
Kiess
W
,
Vatin
V
,
Lecoeur
C
,
Delplanque
J
,
Vaillant
E
,
Pattou
F
,
Ruiz
J
,
Weill
J
,
Levy-Marchal
C
,
Horber
F
,
Potoczna
N
,
Hercberg
S
,
Le Stunff
C
,
Bougnères
P
,
Kovacs
P
,
Marre
M
,
Balkau
B
,
Cauchi
S
,
Chèvre
JC
,
Froguel
P
:
Variation in FTO contributes to childhood obesity and severe adult obesity
.
Nat Genet
2007
;
39
:
724
726
2
Frayling
TM
,
Timpson
NJ
,
Weedon
MN
,
Zeggini
E
,
Freathy
RM
,
Lindgren
CM
,
Perry
JR
,
Elliott
KS
,
Lango
H
,
Rayner
NW
,
Shields
B
,
Harries
LW
,
Barrett
JC
,
Ellard
S
,
Groves
CJ
,
Knight
B
,
Patch
AM
,
Ness
AR
,
Ebrahim
S
,
Lawlor
DA
,
Ring
SM
,
Ben-Shlomo
Y
,
Jarvelin
MR
,
Sovio
U
,
Bennett
AJ
,
Melzer
D
,
Ferrucci
L
,
Loos
RJ
,
Barroso
I
,
Wareham
NJ
,
Karpe
F
,
Owen
KR
,
Cardon
LR
,
Walker
M
,
Hitman
GA
,
Palmer
CN
,
Doney
AS
,
Morris
AD
,
Smith
GD
,
Hattersley
AT
,
McCarthy
MI
:
A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity
.
Science
2007
;
316
:
889
894
3
Hinney
A
,
Nguyen
TT
,
Scherag
A
,
Friedel
S
,
Bronner
G
,
Muller
TD
,
Grallert
H
,
Illig
T
,
Wichmann
HE
,
Rief
W
,
Schafer
H
,
Hebebrand
J
:
Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants
.
PLoS ONE
2007
;
2
:
e1361
4
Scuteri
A
,
Sanna
S
,
Chen
WM
,
Uda
M
,
Albai
G
,
Strait
J
,
Najjar
S
,
Nagaraja
R
,
Orru
M
,
Usala
G
,
Dei
M
,
Lai
S
,
Maschio
A
,
Busonero
F
,
Mulas
A
,
Ehret
GB
,
Fink
AA
,
Weder
AB
,
Cooper
RS
,
Galan
P
,
Chakravarti
A
,
Schlessinger
D
,
Cao
A
,
Lakatta
E
,
Abecasis
GR
:
Genome-wide association scan shows genetic variants in the FTO gene are associated with obesity-related traits
.
PLoS Genet
2007
;
3
:
e115
5
Loos
RJ
,
Lindgren
CM
,
Li
S
,
Wheeler
E
,
Zhao
JH
,
Prokopenko
I
,
Inouye
M
,
Freathy
RM
,
Attwood
AP
,
Beckmann
JS
,
Berndt
SI
Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial
Jacobs
KB
,
Chanock
SJ
,
Hayes
RB
,
Bergmann
S
,
Bennett
AJ
,
Bingham
SA
,
Bochud
M
,
Brown
M
,
Cauchi
S
,
Connell
JM
,
Cooper
C
,
Smith
GD
,
Day
I
,
Dina
C
,
De
S
,
Dermitzakis
ET
,
Doney
AS
,
Elliott
KS
,
Elliott
P
,
Evans
DM
,
Sadaf
Farooqi I
,
Froguel
P
,
Ghori
J
,
Groves
CJ
,
Gwilliam
R
,
Hadley
D
,
Hall
AS
,
Hattersley
AT
,
Hebebrand
J
,
Heid
IM
,
KORA
,
Lamina
C
,
Gieger
C
,
Illig
T
,
Meitinger
T
,
Wichmann
HE
,
Herrera
B
,
Hinney
A
,
Hunt
SE
,
Jarvelin
MR
,
Johnson
T
,
Jolley
JD
,
Karpe
F
,
Keniry
A
,
Khaw
KT
,
Luben
RN
,
Mangino
M
,
Marchini
J
,
McArdle
WL
,
McGinnis
R
,
Meyre
D
,
Munroe
PB
,
Morris
AD
,
Ness
AR
,
Neville
MJ
,
Nica
AC
,
Ong
KK
,
O'Rahilly
S
,
Owen
KR
,
Palmer
CN
,
Papadakis
K
,
Potter
S
,
Pouta
A
,
Qi
L
Nurses' Health Study
Randall
JC
,
Rayner
NW
,
Ring
SM
,
Sandhu
MS
,
Scherag
A
,
Sims
MA
,
Song
K
,
Soranzo
N
,
Speliotes
EK
Diabetes Genetics Initiative
Syddall
HE
,
Teichmann
SA
,
Timpson
NJ
,
Tobias
JH
,
Uda
M
SardiNIA Study
Vogel
CI
,
Wallace
C
,
Waterworth
DM
,
Weedon
MN
Wellcome Trust Case Control Consortium
Willer
CJ
FUSION, Wraight
Yuan
X
,
Zeggini
E
,
Hirschhorn
JN
,
Strachan
DP
,
Ouwehand
WH
,
Caulfield
MJ
,
Samani
NJ
,
Frayling
TM
,
Vollenweider
P
,
Waeber
G
,
Mooser
V
,
Deloukas
P
,
McCarthy
MI
,
Wareham
NJ
,
Barroso
I
,
Jacobs
KB
,
Chanock
SJ
,
Hayes
RB
,
Lamina
C
,
Gieger
C
,
Illig
T
,
Meitinger
T
,
Wichmann
HE
,
Kraft
P
,
Hankinson
SE
,
Hunter
DJ
,
Hu
FB
,
Lyon
HN
,
Voight
BF
,
Ridderstrale
M
,
Groop
L
,
Scheet
P
,
Sanna
S
,
Abecasis
GR
,
Albai
G
,
Nagaraja
R
,
Schlessinger
D
,
Jackson
AU
,
Tuomilehto
J
,
Collins
FS
,
Boehnke
M
,
Mohlke
KL
:
Common variants near MC4R are associated with fat mass, weight and risk of obesity
.
Nat Genet
2008
;
40
:
768
775
6
Hunt
SC
,
Stone
S
,
Xin
Y
,
Scherer
CA
,
Magness
CL
,
Iadonato
SP
,
Hopkins
PN
,
Adams
TD
:
Association of the FTO gene with BMI
.
Obesity (Silver Spring)
2008
;
16
:
902
904
7
Price
RA
,
Li
WD
,
Zhao
H
:
FTO gene SNPs associated with extreme obesity in cases, controls and extremely discordant sister pairs
.
BMC Med Genet
2008
;
9
:
4
8
Peeters
A
,
Beckers
S
,
Verrijken
A
,
Roevens
P
,
Peeters
P
,
Van Gaal
L
,
Van Hul
W
:
Variants in the FTO gene are associated with common obesity in the Belgian population
.
Mol Genet Metab
2008
;
93
:
481
484
9
Hotta
K
,
Nakata
Y
,
Matsuo
T
,
Kamohara
S
,
Kotani
K
,
Komatsu
R
,
Itoh
N
,
Mineo
I
,
Wada
J
,
Masuzaki
H
,
Yoneda
M
,
Nakajima
A
,
Miyazaki
S
,
Tokunaga
K
,
Kawamoto
M
,
Funahashi
T
,
Hamaguchi
K
,
Yamada
K
,
Hanafusa
T
,
Oikawa
S
,
Yoshimatsu
H
,
Nakao
K
,
Sakata
T
,
Matsuzawa
Y
,
Tanaka
K
,
Kamatani
N
,
Nakamura
Y
:
Variations in the FTO gene are associated with severe obesity in the Japanese
.
J Hum Genet
2008
;
53
:
546
553
10
Stratigopoulos
G
,
Padilla
SL
,
LeDuc
CA
,
Watson
E
,
Hattersley
AT
,
McCarthy
MI
,
Zeltser
LM
,
Chung
WK
,
Leibel
RL
:
Regulation of Fto/Ftm gene expression in mice and humans
.
Am J Physiol Regul Integr Comp Physiol
2008
;
294
:
R1185
R1196
11
Chang
YC
,
Liu
PH
,
Lee
WJ
,
Chang
TJ
,
Jiang
YD
,
Li
HY
,
Kuo
SS
,
Lee
KC
,
Chuang
LM
:
Common variation in the fat mass and obesity-associated (FTO) gene confers risk of obesity and modulates BMI in the Chinese population
.
Diabetes
2008
;
57
:
2245
2252
12
Villalobos-Comparan
M
,
Teresa Flores-Dorantes
M
,
Teresa Villarreal-Molina
M
,
Rodriguez-Cruz
M
,
Garcia-Ulloa
AC
,
Robles
L
,
Huertas-Vazquez
A
,
Saucedo-Villarreal
N
,
Lopez-Alarcon
M
,
Sanchez-Munoz
F
,
Dominguez-Lopez
A
,
Gutierrez-Aguilar
R
,
Menjivar
M
,
Coral-Vazquez
R
,
Hernandez-Stengele
G
,
Vital-Reyes
VS
,
Acuna-Alonzo
V
,
Romero-Hidalgo
S
,
Ruiz-Gomez
DG
,
Riano-Barros
D
,
Herrera
MF
,
Gomez-Perez
FJ
,
Froguel
P
,
Garcia-Garcia
E
,
Teresa Tusie-Luna
M
,
Aguilar-Salinas
CA
,
Canizales-Quinteros
S
:
The FTO gene is associated with adulthood obesity in the Mexican population
.
Obesity (Silver Spring)
2008
;
16
:
2296
2301
13
Wing
MR
,
Ziegler
J
,
Langefeld
CD
,
Ng
MC
,
Haffner
SM
,
Norris
JM
,
Goodarzi
MO
,
Bowden
DW
:
Analysis of FTO gene variants with measures of obesity and glucose homeostasis in the IRAS Family Study
.
Hum Genet
2009
;
125
:
615
626
14
Grant
SF
,
Li
M
,
Bradfield
JP
,
Kim
CE
,
Annaiah
K
,
Santa
E
,
Glessner
JT
,
Casalunovo
T
,
Frackelton
EC
,
Otieno
FG
,
Shaner
JL
,
Smith
RM
,
Imielinski
M
,
Eckert
AW
,
Chiavacci
RM
,
Berkowitz
RI
,
Hakonarson
H
:
Association analysis of the FTO gene with obesity in children of Caucasian and African ancestry reveals a common tagging SNP
.
PLoS ONE
2008
;
3
:
e1746
15
Chambers
JC
,
Elliott
P
,
Zabaneh
D
,
Zhang
W
,
Li
Y
,
Froguel
P
,
Balding
D
,
Scott
J
,
Kooner
JS
:
Common genetic variation near MC4R is associated with waist circumference and insulin resistance
.
Nat Genet
2008
;
40
:
716
718
16
Geller
F
,
Reichwald
K
,
Dempfle
A
,
Illig
T
,
Vollmert
C
,
Herpertz
S
,
Siffert
W
,
Platzer
M
,
Hess
C
,
Gudermann
T
,
Biebermann
H
,
Wichmann
HE
,
Schäfer
H
,
Hinney
A
,
Hebebrand
J
:
Melanocortin-4 receptor gene variant I103 is negatively associated with obesity
.
Am J Hum Genet
2004
;
74
:
572
581
17
Stutzmann
F
,
Vatin
V
,
Cauchi
S
,
Morandi
A
,
Jouret
B
,
Landt
O
,
Tounian
P
,
Levy-Marchal
C
,
Buzzetti
R
,
Pinelli
L
,
Balkau
B
,
Horber
F
,
Bougnères
P
,
Froguel
P
,
Meyre
D
:
Non-synonymous polymorphisms in melanocortin-4 receptor protect against obesity: the two facets of a Janus obesity gene
.
Hum Mol Genet
2007
;
16
:
1837
1844
18
Vaisse
C
,
Clement
K
,
Guy-Grand
B
,
Froguel
P
:
A frameshift mutation in human MC4R is associated with a dominant form of obesity
.
Nat Genet
1998
;
20
:
113
114
19
Yeo
GS
,
Farooqi
IS
,
Aminian
S
,
Halsall
DJ
,
Stanhope
RG
,
O'Rahilly
S
:
A frameshift mutation in MC4R associated with dominantly inherited human obesity
.
Nat Genet
1998
;
20
:
111
112
20
Challis
BG
,
Pritchard
LE
,
Creemers
JW
,
Delplanque
J
,
Keogh
JM
,
Luan
J
,
Wareham
NJ
,
Yeo
GS
,
Bhattacharyya
S
,
Froguel
P
,
White
A
,
Farooqi
IS
,
O'Rahilly
S
:
A missense mutation disrupting a dibasic prohormone processing site in pro-opiomelanocortin (POMC) increases susceptibility to early-onset obesity through a novel molecular mechanism
.
Hum Mol Genet
2002
;
11
:
1997
2004
21
Krude
H
,
Biebermann
H
,
Luck
W
,
Horn
R
,
Brabant
G
,
Grüters
A
:
Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans
.
Nat Genet
1998
;
19
:
155
157
22
Gray
J
,
Yeo
GS
,
Cox
JJ
,
Morton
J
,
Adlam
AL
,
Keogh
JM
,
Yanovski
JA
,
El Gharbawy
A
,
Han
JC
,
Tung
YC
,
Hodges
JR
,
Raymond
FL
,
O'rahilly
S
,
Farooqi
IS
:
Hyperphagia, severe obesity, impaired cognitive function, and hyperactivity associated with functional loss of one copy of the brain-derived neurotrophic factor (BDNF) gene
.
Diabetes
2006
;
55
:
3366
3371
23
Han
JC
,
Liu
QR
,
Jones
M
,
Levinn
RL
,
Menzie
CM
,
Jefferson-George
KS
,
Adler-Wailes
DC
,
Sanford
EL
,
Lacbawan
FL
,
Uhl
GR
,
Rennert
OM
,
Yanovski
JA
:
Brain-derived neurotrophic factor and obesity in the WAGR syndrome
.
N Engl J Med
2008
;
359
:
918
927
24
Thorleifsson
G
,
Walters
GB
,
Gudbjartsson
DF
,
Steinthorsdottir
V
,
Sulem
P
,
Helgadottir
A
,
Styrkarsdottir
U
,
Gretarsdottir
S
,
Thorlacius
S
,
Jonsdottir
I
,
Jonsdottir
T
,
Olafsdottir
EJ
,
Olafsdottir
GH
,
Jonsson
T
,
Jonsson
F
,
Borch-Johnsen
K
,
Hansen
T
,
Andersen
G
,
Jorgensen
T
,
Lauritzen
T
,
Aben
KK
,
Verbeek
AL
,
Roeleveld
N
,
Kampman
E
,
Yanek
LR
,
Becker
LC
,
Tryggvadottir
L
,
Rafnar
T
,
Becker
DM
,
Gulcher
J
,
Kiemeney
LA
,
Pedersen
O
,
Kong
A
,
Thorsteinsdottir
U
,
Stefansson
K
:
Genome-wide association yields new sequence variants at seven loci that associate with measures of obesity
.
Nat Genet
2009
;
41
:
18
24
25
Benzinou
M
,
Creemers
JW
,
Choquet
H
,
Lobbens
S
,
Dina
C
,
Durand
E
,
Guerardel
A
,
Boutin
P
,
Jouret
B
,
Heude
B
,
Balkau
B
,
Tichet
J
,
Marre
M
,
Potoczna
N
,
Horber
F
,
Le Stunff
C
,
Czernichow
S
,
Sandbaek
A
,
Lauritzen
T
,
Borch-Johnsen
K
,
Andersen
G
,
Kiess
W
,
Körner
A
,
Kovacs
P
,
Jacobson
P
,
Carlsson
LM
,
Walley
AJ
,
Jørgensen
T
,
Hansen
T
,
Pedersen
O
,
Meyre
D
,
Froguel
P
:
Common nonsynonymous variants in PCSK1 confer risk of obesity
.
Nat Genet
2008
;
40
:
943
945
26
Jackson
RS
,
Creemers
JW
,
Ohagi
S
,
Raffin-Sanson
ML
,
Sanders
L
,
Montague
CT
,
Hutton
JC
,
O'Rahilly
S
:
Obesity and impaired prohormone processing associated with mutations in the human prohormone convertase 1 gene
.
Nat Genet
1997
;
16
:
303
306
27
Gerken
T
,
Girard
CA
,
Tung
YC
,
Webby
CJ
,
Saudek
V
,
Hewitson
KS
,
Yeo
GS
,
McDonough
MA
,
Cunliffe
S
,
McNeill
LA
,
Galvanovskis
J
,
Rorsman
P
,
Robins
P
,
Prieur
X
,
Coll
AP
,
Ma
M
,
Jovanovic
Z
,
Farooqi
IS
,
Sedgwick
B
,
Barroso
I
,
Lindahl
T
,
Ponting
CP
,
Ashcroft
FM
,
O'Rahilly
S
,
Schofield
CJ
:
The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase
.
Science
2007
;
318
:
1469
1472
28
O'Rahilly
S
,
Farooqi
IS
,
Yeo
GS
,
Challis
BG
:
Minireview: human obesity-lessons from monogenic disorders
.
Endocrinology
2003
;
144
:
3757
3764
29
Balkau
B
:
[An epidemiologic survey from a network of French Health Examination Centres, (D.E.S.I.R.): epidemiologic data on the insulin resistance syndrome]
.
Rev Epidemiol Sante Publique
1996
;
44
:
373
375
[in French]
30
Visvikis-Siest
S
,
Siest
G
:
The STANISLAS Cohort: a 10-year follow-up of supposed healthy families: gene-environment interactions, reference values and evaluation of biomarkers in prevention of cardiovascular diseases
.
Clin Chem Lab Med
2008
;
46
:
733
747
31
Poskitt
EM
:
Defining childhood obesity: the relative body mass index (BMI): European Childhood Obesity group
.
Acta Paediatr
1995
;
84
:
961
963
32
Boissel
S
,
Reish
O
,
Proulx
K
,
Kawagoe-Takaki
H
,
Sedgwick
B
,
Yeo
GS
,
Meyre
D
,
Golzio
C
,
Molinari
F
,
Kadhom
N
,
Etchevers
HC
,
Saudek
V
,
Farooqi
IS
,
Froguel
P
,
Lindahl
T
,
O'Rahilly
S
,
Munnich
A
,
Colleaux
L
:
Loss-of-function mutation in the dioxygenase-encoding FTO gene causes severe growth retardation and multiple malformations
.
Am J Hum Genet
2009
;
85
:
106
111
33
Kaule
G
,
Günzler
V
:
Assay for 2-oxoglutarate decarboxylating enzymes based on the determination of [1–14C]succinate: application to prolyl 4-hydroxylase
.
Anal Biochem
1990
;
184
:
291
297
34
Jia
G
,
Yang
CG
,
Yang
S
,
Jian
X
,
Yi
C
,
Zhou
Z
,
He
C
:
Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO
.
FEBS Lett
2008
;
582
:
3313
3319
35
Sundheim
O
,
Vågbø
CB
,
Bjørås
M
,
Sousa
MM
,
Talstad
V
,
Aas
PA
,
Drabløs
F
,
Krokan
HE
,
Tainer
JA
,
Slupphaug
G
:
Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage
.
EMBO J
2006
;
25
:
3389
3397
36
Peters
T
,
Ausmeier
K
,
Rüther
U
:
Cloning of Fatso (Fto), a novel gene deleted by the Fused toes (Ft) mouse mutation
.
Mamm Genome
1999
;
10
:
983
986
37
Wellcome Trust Case Control Consortium
.
Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls [see comment]
.
Nature
2007
;
447
:
661
678
38
Peters
T
,
Ausmeier
K
,
Dildrop
R
,
Rüther
U
:
The mouse Fused toes (Ft) mutation is the result of a 1.6-Mb deletion including the entire Iroquois B gene cluster
.
Mamm Genome
2002
;
13
:
186
188
39
Fischer
J
,
Koch
L
,
Emmerling
C
,
Vierkotten
J
,
Peters
T
,
Brüning
JC
,
Rüther
U
:
Inactivation of the Fto gene protects from obesity
.
Nature
2009
;
458
:
894
898

Supplementary data