all adults had bmi > 40 kg / m , 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 .
we also sequenced 287 swiss morbidly obese adults , all of them recruited after gastric surgery in zurich , switzerland .
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 ) .
inclusion criteria was bmi z score ( zbmi ) < 90th percentile for sex and age according to the eastern cooperative oncology group ( ecog ) ( 31 ) .
the study protocol was approved by all local ethics committees , and informed consent was obtained from each subject before participating in the study .
clinical characteristics for obese and lean subjects data are means  sd unless otherwise indicated .
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 .
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 ) .
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 . 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 .
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 g / ml carbenicillin to a600 1.0 at 37c .
expression of the cloned gene was induced by addition of 0.5 mmol / l isopropyl--d-1-thiogalactopyranoside ( iptg ) at 15c for 4 h. the cells were harvested and washed in pbs , and pellets were stored at 80c .
cells were resuspended in 40 ml of lysis buffer ( 50 mmol / l hepes - koh , ph 8 , 2 mmol / l -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 15000 g 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 eluate was then loaded onto a 30-kda concentrator ( sartorius stedium biotech ) and centrifuged at 2500 g 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 l by centrifugation at 2500 g .
conversion of c-2-oxoglutarate to c - succinate by 2-og fe - 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 mol / l fto was assayed in 10 l reaction mixture containing 50 mmol / l hepes - koh , ph 7 , 50 g / ml bsa , 4 mmol / l ascorbate , 75 mol / l fe(nh4)2(so4)2 , and 20 mol / l [ 5-c]-2-oxoglutarate ( 30 mci / mmol from moravek biochemicals ) and incubated at 37c for various times . to measure stimulation of this activity by 3-methylthymidine , 1
the reaction was stopped by adding 5 l stop solution containing 20 mmol / l succinate , 20 mmol / l 2-og , followed by 5 l 160 mmol / l dinitrophenylhydrazine , which precipitates 2-og .
an additional 10 l 1 m 2-og was added and incubated for a further 30 min .
clear supernatant ( 10 l ) was scintillation counted to monitor the c - succinate generated .
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 .
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 ) .
the comparison of prevalence has been tested with fisher exact test , and the reported p values are two - sided .
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 .
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 g / ml carbenicillin to a600 1.0 at 37c .
expression of the cloned gene was induced by addition of 0.5 mmol / l isopropyl--d-1-thiogalactopyranoside ( iptg ) at 15c for 4 h. the cells were harvested and washed in pbs , and pellets were stored at 80c .
cells were resuspended in 40 ml of lysis buffer ( 50 mmol / l hepes - koh , ph 8 , 2 mmol / l -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 15000 g 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 eluate was then loaded onto a 30-kda concentrator ( sartorius stedium biotech ) and centrifuged at 2500 g 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 l by centrifugation at 2500 g .
conversion of c-2-oxoglutarate to c - succinate by 2-og fe - 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 mol / l fto was assayed in 10 l reaction mixture containing 50 mmol / l hepes - koh , ph 7 , 50 g / ml bsa , 4 mmol / l ascorbate , 75 mol / l fe(nh4)2(so4)2 , and 20 mol / l [ 5-c]-2-oxoglutarate ( 30 mci / mmol from moravek biochemicals ) and incubated at 37c for various times . to measure stimulation of this activity by 3-methylthymidine , 1
the reaction was stopped by adding 5 l stop solution containing 20 mmol / l succinate , 20 mmol / l 2-og , followed by 5 l 160 mmol / l dinitrophenylhydrazine , which precipitates 2-og .
an additional 10 l 1 m 2-og was added and incubated for a further 30 min .
clear supernatant ( 10 l ) was scintillation counted to monitor the c - succinate generated .
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 / m and 680 children with a bmi > 97th percentile ) and in 1,433 lean subjects ( 1,221 adults with a bmi < 23 kg / m 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 [ 1.441.78 ] ; p = 1.2  10 ) .
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 ) .
eight ( a134 t , g187a , m223v , a241 t , h419r , e471 g , 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 ) , a241 t ( n = 2 ) , and v493f ( n = 4 ) were identified in multiple obese subjects ( table 3 ) .
seven variants ( r96h , l146 m , a163 t , 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 .
summary of all nonsynonymous mutations found in obese and lean subjects data in boldface indicate combined adult and children numbers .
summary of nonsynonymous mutations unique to the lean or the obese group summary of all synonymous mutations found in lean and obese subjects fto is a member of the alkb homologue ( abh ) protein family , which numbers nine homologues in humans ( 27 ) .
most 2-og and fe - 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 .
the more conserved nh2-terminal segment contains 1 ) a putative nuclear localization signal present only in fto ( 36 ) , 2 ) a double - stranded -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 . 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 .
as predicted , r322q , like r316q ( 32 ) , was completely unable to convert 2-og to succinate in either the absence or presence of 3-methylthymidine .
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 .
decarboxylation of c-2-oxoglutarate to c - succinate by 1.5 mol / l fto in the absence or presence of 3-methylthymidine was measured at various time intervals . a : wt1 and r322q 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 : sds10% page of fto protein preparations stained with coomassie blue . per lane , 3 g total protein was loaded .
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 .
3b ) , 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 , a241 t , 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 .
bioinformatic analysis suggests that a sequence of 18 amino acid residues in the nh2-terminal of fto encodes a putative nuclear localization signal ( fig .
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 .
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 / m and 680 children with a bmi > 97th percentile ) and in 1,433 lean subjects ( 1,221 adults with a bmi < 23 kg / m 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 [ 1.441.78 ] ; p = 1.2  10 ) .
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 ) .
eight ( a134 t , g187a , m223v , a241 t , h419r , e471 g , 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 ) , a241 t ( n = 2 ) , and v493f ( n = 4 ) were identified in multiple obese subjects ( table 3 ) .
seven variants ( r96h , l146 m , a163 t , 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 .
summary of all nonsynonymous mutations found in obese and lean subjects data in boldface indicate combined adult and children numbers .
summary of nonsynonymous mutations unique to the lean or the obese group summary of all synonymous mutations found in lean and obese subjects
fto is a member of the alkb homologue ( abh ) protein family , which numbers nine homologues in humans ( 27 ) .
most 2-og and fe - 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 .
the more conserved nh2-terminal segment contains 1 ) a putative nuclear localization signal present only in fto ( 36 ) , 2 ) a double - stranded -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 . 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 .
as predicted , r322q , like r316q ( 32 ) , was completely unable to convert 2-og to succinate in either the absence or presence of 3-methylthymidine .
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 .
decarboxylation of c-2-oxoglutarate to c - succinate by 1.5 mol / l fto in the absence or presence of 3-methylthymidine was measured at various time intervals .
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 : sds10% page of fto protein preparations stained with coomassie blue . per lane , 3 g total protein was loaded .
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 .
3b ) , 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 , a241 t , 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 .
bioinformatic analysis suggests that a sequence of 18 amino acid residues in the nh2-terminal of fto encodes a putative nuclear localization signal ( fig .
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 .
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 .
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 .
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 .
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 .
these preliminary observations are of potential interest and might lead to a better understanding of fto function and its role in energy homeostasis localization .
 objectivesingle 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 methodswe 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.resultswe 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.conclusionsheterozygous , 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 . 
RESEARCH DESIGN AND METHODS
Genotyping of the FTO rs9939609 SNP.
Sequencing.
Statistical analysis.
Cloning of human FTO.
Human wild-type and mutant FTO protein production.
Assays of FTO activity.
RESULTS
Prevalence of nonsynonymous mutations in lean and obese subjects.
Functional properties of nonsynonymous mutations.
DISCUSSION
Supplementary Material