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.

Figure 1

Genetic Analysis of a Family with Ante- and Postnatal Growth Retardation and a Severe Polymalformative Syndrome (A) Pedigree of the family. Filled symbols and slashes indicate affected and deceased infants. (B) Electrophoregrams showing the variation of FTO gene sequence in an affected infant and a healthy control. (C) Part of the multiple sequence alignment of FTO representative orthologs (H.s, Homo sapiens; Ma.m, Macaca mulatta; R.n, Rattus norvegicus; Mu.m, Mus musculus; C.f, Canis familiaris; E.c, Equus caballus; Or.a, Ornithorhynchus anatinus; Ov.a, Ovis aries; B.t, Bos taurus; M.d, Monodelphis domestica; G.g, Gallus gallus; D.r, Danio rerio; X.t, Xenopus tropicalis; ABH2 H.s, human ABH2; ABH3 H.s, Human ABH3; AlkB E.c, E. coli AlkB). The conserved residues are highlighted and the amino acid highlighted in red is the absolutely conserved arginine involved in the R316Q mutation in patients (Ai, affected infant). Blue strands labeled with roman numerals identify three of the eight β strands that form the conserved double-stranded β-helix of the 2OG-oxygenases.

Figure 2

Biochemical Analyses of the Wild-Type and Mutant FTO Proteins (A) Purified FTO was added to 10 μl reaction mixture containing 50 mM HEPES.KOH (pH 7), 50 μg/ml BSA, 4 mM ascorbate, 75 μM Fe(NH₄)₂(SO₄)₂, and 20 μM [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 mM 3-methylthymidine (Moravek Biochemicals) was included in the assay mix. The reaction was stopped by adding 5 μl stop solution containing 20 mM succinate, 20 mM 2-oxoglutarate followed by 5 μl 160 mM dinitrophenylhydrazine, which precipitates 2-oxoglutarate. This mix was incubated at room temperature for 30 min. An additional 10 μl 1M 2-oxoglutarate 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. Time course of activity of 1.5 μM FTO: open and closed squares, wild-type FTO protein; open and closed triangles, mutant R316Q FTO protein. Open symbols and dotted line: without 3-methylthymidine; closed symbols and solid line: with 3-methylthymidine (1 mM). (B) A DNA substrate containing ¹⁴C-3-methylthymine was prepared by treating poly(dT) with [¹⁴C]-methyl iodide (54 Ci/mmole, Amersham Biosciences) as previously described and had a specific activity of 1580 cpm/μg poly(dT). FTO was added to the ¹⁴C-methylated poly(dT) substrate (1000 cpm) in a 100 μl reaction mix containing 50 mM MES-HCl (pH 6), 75 μM Fe(NH₄)₂(SO₄)₂, 100 μM 2-oxoglutarate, 2 mM ascorbate, 10 μg/ml bovine serum albumin and incubated at 20°C for various times. All assays were performed in triplicate. To stop the reaction, EDTA was added to a final concentration of 10 mM. The polynucleotide substrate was then ethanol precipitated in the presence of carrier calf thymus DNA. Two-thirds of the ethanol-soluble radioactive material was monitored by scintillation counting. Equal volumes of the protein preparations were assayed. Standard error of the mean is shown for each time point. Closed square, 2 μM wild-type FTO protein; closed triangle, 2 μM mutant R316Q FTO protein; open circle, “mock prep” prepared in the absence of recombinant FTO expression. Equal volumes of the protein preparations were also examined by SDS-10% PAGE.

Figure 3

Cell Morphology and Proliferative Abilities of Cultured Skin Fibroblasts Harboring the R316Q FTO Mutation (A) Fibroblast morphology. The arrow shows altered cell morphology and enlarged cell size of a patient fibroblast. (B) Fibroblast growth curves. Fibroblasts were seeded at a density of 5000 cells/well in 12-well dishes. The number of cells per dish was determined with a CASY cell counter on days 2, 5, 7, 9, and 11 after seeding. Filled and open symbols correspond to patient and control fibroblasts, respectively. Data correspond to the mean of three replicates. Standard error of the mean is shown for each point. (C) Senescence-associated β-galactosidase assay. Fibroblasts were seeded on 6-well dishes at a density of 130,000 cells/well. Senescent fibroblasts were stained with the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology). Percentages of β-galactosidase-positive cells for passages 7, 9, and 13 were calculated on the basis of approximately 100 cells. Data correspond to the mean of three replicates. Standard error of the mean is shown for each point.