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Am J Hum Genet. Jul 10, 2009; 85(1): 106–111.
PMCID: PMC2706958

Loss-of-Function Mutation in the Dioxygenase-Encoding FTO Gene Causes Severe Growth Retardation and Multiple Malformations

Abstract

FTO is a nuclear protein belonging to the AlkB-related non-haem iron- and 2-oxoglutarate-dependent dioxygenase family. Although polymorphisms within the first intron of the FTO gene have been associated with obesity, the physiological role of FTO remains unknown. Here we show that a R316Q mutation, inactivating FTO enzymatic activity, is responsible for an autosomal-recessive lethal syndrome. Cultured skin fibroblasts from affected subjects showed impaired proliferation and accelerated senescence. These findings indicate that FTO is essential for normal development of the central nervous and cardiovascular systems in human and establish that a mutation in a human member of the AlkB-related dioxygenase family results in a severe polymalformation syndrome.

Main Text

Fto was originally identified in mice as one of the genes encoded by the 1.6 Mb deletion causing the fused toes (Ft) mutant, an autosomal-dominant mouse mutation generated by a transgene integration into region D of mouse Chr 8.1 In addition to Fto, the deleted segment also contains three members of the Iroquois gene family (Irx3, Irx5, and Irx6, also known as the IrxB complex) as well as the Fts/Aktip and Ftm/Rpgrip1l genes. Mice heterozygous for the Ft mutation are characterized by partial syndactyly of forelimbs and thymic hyperplasia.1 Ft/Ft embryos die in midgestation and present many abnormalities including limb polydactyly and distal truncations, major brain and heart defects, delay or absence of neural tube closure, and facial structure hypoplasia.2,3

Interestingly, a human genome-wide search for type 2 diabetes susceptibility genes identified the strong association of a series of single nucleotide polymorphisms (SNPs), in tight linkage disequilibrium with rs9939609 and located in the first intron of the FTO gene (MIM 610966), with higher body mass index (BMI) and obesity risk in European cohorts.4,5 This association was replicated in various European cohorts and several studies have been performed trying to identify association between the obesity risk allele and energy intake/expenditure or eating behavior trait.6–8 However, how FTO variants modulate components of the energy balance remains elusive so far. FTO has been recognized as a member of the AlkB-related family of non-haem iron- and 2-oxoglutarate-dependent dioxygenases.9 These non-heme iron enzymes, which require Fe2+ as a cofactor and 2-oxoglutarate and dioxygen as cosubstrates, reverse alkylated DNA and RNA damages by oxidative demethylation.10–14 Fto-deficient mice have been recently described.15 Homozygous mutant mice are viable but postnatal death occurs frequently and loss of Fto leads to postnatal growth retardation, significant reduction in adipose tissue, and lean body mass.

We ascertained a large Palestinian Arab consanguineous multiplex family in which nine affected individuals presented with a previously unreported polymalformation syndrome (Figure 1A). The study was approved by the supreme Israeli Helsinki Review Board (33/07-08) and informed consent was obtained from all family members. All affected individuals had postnatal growth retardation, microcephaly, severe psychomotor delay, functional brain deficits, and characteristic facial dysmorphism. In some patients, structural brain malformations, cardiac defects, genital anomalies, and cleft palate were also observed (Table 1). Early lethality resulting from intercurrent infection or unidentified cause occurred at 1–30 months of age. Extensive biochemical, metabolic, and genetic analyses failed to diagnose any previously known inherited disorders. Because the pedigree suggested an autosomal-recessive mode of inheritance, we performed a genome-wide autozygosity screen with the Perkin Elmer Biosystems linkage mapping set (version 1) and identified a unique region of shared homozygosity on chromosome 16q12 (Figure S1 available online). Further genotype and haplotype analyses reduced the critical region to a 6.5 Mb interval between loci D16S411 and D16S3140 (maximum LOD score Zmax = 4.16 at θ = 0 at the D16S3136 locus). No other genomic region showed consistent linkage. This genetic interval encompasses 28 genes that were all systematically analyzed at both DNA and/or RNA levels (Table S1). We identified a homozygous single-nucleotide variation at cDNA position 947 (c.947G→A) within the FTO gene (Figure 1B). The c.947G→A transition predicts a p.R316Q substitution. Notably, this amino acid residue is absolutely conserved across all known FTO paralogs and AlkB orthologs (Figure 1C) and is involved in 2-oxoglutarate coordination by forming stabilizing salt bridges with the carboxylates of this cosubstrate.9 This variant cosegregated with the disease and was not found in 730 control chromosomes, including 378 chromosomes from individuals of Palestinian Arab origin. We subsequently undertook to estimate the frequency of FTO sequence variants within the general population. FTO coding exons and intron-exon boundaries sequence was determined in 1492 controls of European descent without overt disease or syndromic features. No nonsense variant was identified and the prevalence of missense variants was 0.87%. In addition, neither homozygous missense mutation nor composite heterozygous mutations were detected in this large series of control subjects (data not shown), further supporting the hypothesis that the R316Q mutation is the disease-causing defect. To estimate the prevalence of FTO mutations, 27 unrelated children with partially overlapping clinical features were tested but no mutation in the FTO gene was identified, strengthening the idea that the new syndrome described here is a very rare condition.

Figure 1
Genetic Analysis of a Family with Ante- and Postnatal Growth Retardation and a Severe Polymalformative Syndrome
Table 1
Major Clinical Features in Affected Individuals with c.947G→A Mutation in FTO

FTO belongs to the AlkB-related protein family and was shown to localize in the nucleus.9 Immunofluorescence experiments performed on patient and control fibroblasts demonstrated that the mutation did not affect its nuclear localization (data not shown). Murine and human Fto have been shown to demethylate 3-methylthymine and 3-methyluracil residues in single-stranded DNA and RNA in vitro, albeit with a relatively low efficiency. Fto was less active on the more common forms of methylated DNA base damage, 1-methyladenine and 3-methylcytosine.9–14,16 Moreover, the recombinant murine Fto protein mutated in the residue corresponding to human R316 amino acid displays a much reduced or absent DNA demethylation activity. To investigate the functional consequences of the R316Q mutation, we tested the catalytic activity of the wild-type and mutant R316Q FTO proteins in vitro. Two different assays were used: the first followed the conversion of the cosubstrate 2-oxoglutarate to succinate and the second monitored the ability of FTO to demethylate 3-methylthymine in DNA. Most 2-oxoglutarate- and Fe2+-dependent dioxygenases slowly catalyze the conversion of 2-oxoglutarate to succinate even in the absence of their prime substrate,16 and this uncoupled reaction may be stimulated by substrates or their analogs. We optimized conditions to assay the ability of human recombinant FTO protein to convert 14C-2-oxoglutarate to 14C-succinate and found that this activity was stimulated 6- to 8- fold by free 3-methylthymidine (Figure 2A). Other nucleosides such as 1-methyldeoxyadenosine and thymidine or the base 3-methylthymine did not stimulate the activity (data not shown). Interestingly, the R316Q FTO mutant protein had no detectable ability to catalyze the uncoupled reaction and also was not stimulated by 3-methylthymidine (Figure 2A). The ability of FTO to demethylate 14C-labeled 3-methylthymine in single-stranded poly(dT) substrate was assayed by measuring the release of 14C-formaldehyde. The optimum pH for the demethylation of 3-methylthymine in DNA by AlkB, ABH2 (MIM 610602), and ABH3 (MIM 610603) is 6 to 6.5 and this is also the case for human FTO. Wild-type FTO11,16,17 actively but slowly demethylated this substrate whereas the mutant R316Q protein was inactive in the assay (Figure 2B). The defective activity of FTO, as observed in both assays, is most likely due to the inability of the mutant protein to interact with the cosubstrate 2-oxoglutarate.

Figure 2
Biochemical Analyses of the Wild-Type and Mutant FTO Proteins

To gain insight into the function of the FTO gene product and the pathophysiology of the disease, we investigated the expression of FTO in human embryos and adult tissues. RT-PCR showed an ubiquitous expression in all fetal and adult tissues tested, as has been observed in mice (data not shown).18 Human embryo sections were hybridized in situ at stages Carnegie (C)15, 33–36 days postfertilization (dpf) and C18 (44–46 dpf) with antisense probes against FTO and sense probes as negative controls. We observed a nearly ubiquitous expression of FTO, with higher expression in the central nervous system and the liver. In the heart, the mitral and semilunar valves and the wall of the pulmonary trunk stained as did the ventricular myocardium, and a strong and uniform expression was observed in the developing pituitary and the frontonasal and mandibular mesenchyme (Figure S2). It is worth noting that this wide spatiotemporal pattern of expression is consistent with the broad spectrum of clinical manifestations of the disease (Table 1).

Interestingly, the cultured skin fibroblasts of patient III.15 displayed an altered morphology (including hypertrophy), an increased number of vacuoles and cellular debris (Figure 3A), a reduced life span, and decreased proliferative abilities when compared to controls (Figure 3B). Cellular expression of the senescence-associated β-galactosidase (SA-β-Gal) is thought to be a reliable indicator of the switch mechanism that occurs when cells enter the senescent phenotype.19 Notably, a significantly elevated number of SA-β-Gal-positive cells, increasing with passage numbers, was found in patient cultured cells compared to passage-matched control fibroblasts (Figure 3C). Although further analyses on skin fibroblasts of additional patients are necessary to validate these findings, taken together these data could yet suggest that the defective FTO activity may cause premature senescence-like phenotype.

Figure 3
Cell Morphology and Proliferative Abilities of Cultured Skin Fibroblasts Harboring the R316Q FTO Mutation

Although repair of alkylated nucleic acid by oxidative demethylation is crucial to maintain genome integrity, no AlkB-related protein mutations have been hitherto identified in human. The results reported here provide therefore the first example of a human disorder related to the defect of an AlkB-related protein. The Escherichia coli AlkB protein catalyzes the oxidative demethylation of 1-methyladenine, 3-methylcytosine, and 3-methylthymine in DNA and RNA. Eight mammalian orthologs of AlkB have been previously identified (ABH1 to 8),20 and FTO is the ninth member of this family. Of this family, only ABH2 and ABH3 have been demonstrated to be highly active in repairing methylation damage to DNA.13,14 In comparison, ABH1 (MIM 605345) has a modest activity on 3-methylcytosine in single-stranded DNA or RNA21 whereas FTO has a similar low activity on 3-methylthymine in these substrates.9,16 Mice lacking either Abh2 or Abh3 have no overt phenotype but the Abh2−/− mice accumulate significant levels of 1-methyladenine in their genomes.22 Notably, Abh1−/− mice are viable but have intrauterine growth retardation and defects in placental differentiation.23

Surprisingly, although homozygous Fto−/− mice have postnatal growth retardation, significant reduction in adipose tissue, and lean body mass, they are not, however, reported to have any developmental abnormalities in the central nervous or cardiovascular systems.15 Thus, humans homozygous for a catalytically inactive FTO and Fto null mice share the growth retardation phenotype but both mutations differ in the impact on development of central nervous and cardiovascular systems. There are several possible reasons for the different findings in our patients versus the Fto−/− mouse. First, major phenotypic differences between rodent models and humans harboring identical mutations are well described. For example, maturity-onset diabetes of the young subtype 3 (MODY3 [MIM 600496]) is caused by heterozygous mutations in the transcription factor hepatocyte nuclear factor (HNF)-1α (MIM 142410).24 By contrast, mice with heterozygous mutations in Hnf-1α gene are phenotypically normal.25 Second, it is possible that the presence, in the nucleus, of a mutant catalytically inactive FTO might have biochemical consequences over and above that seen with complete deficiency of Fto. These could include toxic “gain-of-function” effects or dominant-negative interference with other related family members. Finally, it is formally possible that the phenotype seen in the human subjects is due to a combination of the mutant FTO and a second, as yet undetected, mutation in the 6 Mb critical region. The fact that no mutations were detected on sequencing of the coding regions and splice junctions of all 28 known and putative genes in that linked region makes the chances of such a second mutation very small.

A cluster of variants located within the first intron of FTO has recently been strongly and reproducibly associated with human adiposity.4,5 Yet, whether these variants influence the risk of obesity directly, via the altered expression of FTO or through other mechanisms, remains questionable. In this regard, it is worth noting that homozygosity for the R316Q FTO mutation caused severe growth retardation. Detailed anthropometric measurements are not available on unaffected family members but, noticeably, none of the parents were clinically obese. Given the results from the Fto-deficient mice, it might be hypothesized that humans heterozygous for a loss-of-function mutation in FTO might actually be relatively resistant to becoming obese. Future studies of the body composition of carrier and noncarrier relatives will be required to clarify the relationship between FTO variant heterozygosity and adiposity.

The molecular mechanisms whereby the mutant FTO leads to the severe phenotype observed in our patients remain unknown. The fact that (1) FTO is a member of the Alk-B-related family of dioxygenases, (2) at least two of the mammalian members of this family have established roles in DNA repair, and (3) FTO can demethylate 3-methylthymine in the context of DNA might suggest that FTO plays a role in the maintenance of genome integrity. If that were the case, one could easily understand how FTO defect relates to developmental malformations and the accelerated senescence observed in cultured fibroblasts derived from the patients. However, it should also be pointed out that (1) several members of the mammalian Alk-B-related family have no established role in DNA repair, (2) FTO demethylates 3-methylthymine slowly and inefficiently, suggesting that this is not its true physiological substrate, (3) 3-methylthymine is a very rare lesion in vivo, and (4) defective repair of rare methylated bases in DNA can hardly be related to the striking increase in energy expenditure observed in Fto-deficient mice. Taken together, this suggests that FTO roles in the nucleus are presumably not restricted to DNA repair.

Because of its association with obesity, FTO is currently the subject of intense interest. The finding of obesity resistance in Fto-deficient mice has led to the suggestion that interference with Fto enzymatic function might be a novel and interesting antiobesity therapeutic strategy. The discovery that humans homozygous for an enzymatically inactive mutant form of FTO have multiple congenital abnormalities suggests that any program of research exploring the therapeutic utility of FTO inhibitors should incorporate a particularly careful assessment of teratogenicity and other toxic effects.

Acknowledgments

We are grateful to the patients for their participation in the study. We acknowledge Jean Philippe Jais for LOD score calculation, Carron Sher for participation to clinical evaluation, Uli Rüther for kindly providing FTO antibodies, Céline Cluzeau for help with western blotting, Peter Robins for purification of FTO protein, and Marcella Ma and Debbie Lyon for additional technical assistance. This study was supported by the Centre National de la Recherche Scientifique, the Agence Nationale de la Recherche, the Région Ile-de-France, EU FP6 EUGENE2 (LSHM-CT-2004-512013), EC FP6 Marie Curie, the Medical Research Council UK, Cancer Research UK, the Wellcome Trust, and NIHR Cambridge Biomedical Research Centre.

Supplemental Data

Document S1. Two Figures and One Table:

Web Resources

The URLs for data presented herein are as follows:

References

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