Rare recessive loss-of-function methionyl-tRNA synthetase mutations presenting as a multi-organ phenotype

Eline van Meel, Daniel J Wegner, Paul Cliften, Marcia C Willing, Frances V White, Stuart Kornfeld, F Sessions Cole, Eline van Meel, Daniel J Wegner, Paul Cliften, Marcia C Willing, Frances V White, Stuart Kornfeld, F Sessions Cole

Abstract

Background: Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to its cognate transfer RNA and therefore plays an essential role in protein biosynthesis.

Methods: We used exome sequencing, aminoacylation assays, homology modeling, and immuno-isolation of transfected MARS to identify and characterize mutations in the methionyl-tRNA synthetase gene (MARS) in an infant with an unexplained multi-organ phenotype.

Results: We identified compound heterozygous mutations (F370L and I523T) in highly conserved regions of MARS. The parents were each heterozygous for one of the mutations. Aminoacylation assays documented that the F370L and I523T MARS mutants had 18 ± 6% and 16 ± 6%, respectively, of wild-type activity. Homology modeling of the human MARS sequence with the structure of E. coli MARS showed that the F370L and I523T mutations are in close proximity to each other, with residue I523 located in the methionine binding pocket. We found that the F370L and I523T mutations did not affect the association of MARS with the multisynthetase complex.

Conclusion: This infant expands the catalogue of inherited human diseases caused by mutations in aminoacyl-tRNA synthetase genes.

Figures

Figure 1
Figure 1
Liver and bone marrow pathology. A: The patient’s bone marrow (left photo) contains megakaryocytes (arrow) and numerous myeloid cells (chevron), while erythroid cells are difficult to identify. In contrast, erythroid cells (curved arrows) are readily apparent in control bone marrow (right photo). B: The patient’s liver (left photo) shows lobular disarray with hepatocyte ballooning (arrowhead), canalicular cholestasis (arrow) and zone 1 steatosis (chevron). Iron deposition (star) is present in macrophages and hepatocytes. Control liver (right photo) for comparison. C: Electron micrograph of hepatocyte shows mild pleomorphism of mitochondria (arrows), which contain predominantly flattened, straight and curved cristae. These are non-specific findings that can be seen in normal liver.
Figure 2
Figure 2
Exome sequencing reveals mutations in highly conserved regions of the catalytic domain of MARS. A: MARS mutations in the patient and her parents. The patient is compound heterozygous for the mutations c.1108 T > C, F370L (paternally inherited) and c.1568 T > C, I523T (maternally inherited), see arrows. B: Protein sequence alignment of MARS orthologs. The mutated residues are indicated by arrows and marked in red when conserved. C: Schematic representation of the different domains of MARS. The locations of the mutations, which are in the catalytic domain (orange), are indicated by arrows. The GST-like domain is shown in blue and the tRNA binding domain in green.
Figure 3
Figure 3
MARS mutations F370L and I523T significantly impair MARS activity. A: F370L and I523T MARS are expressed in HEK293 cells. Western blots show that wild-type and the mutant forms of MARS were expressed at similar levels (upper panel) with equal amounts of protein loaded, as reflected by the equal levels of actin (lower panel). Endogenous MARS in mock treated cells was only detected after prolonged exposure (see Additional file 2: Figure S1). B: Measurement of MARS activity, using the aminoacylation assay, shows greatly reduced activity of both MARS mutants. The activities of F370L and I523T MARS are represented as the percentages of wild-type MARS activity (set to 100%). The values are the averages of 3 independent experiments, ± the standard deviations.
Figure 4
Figure 4
Location of the mutated residues I523 and F370 in human MARS. A: Homology modeling of human MARS (purple) with E. coli MARS (pink, PDB 1PFU) in complex with methionine phosphinate (grey, with elements colored in red (oxygen), orange (phosphorus), yellow (sulfur) and blue (nitrogen)). B: Close-up of the methionine binding pocket. Residues that interact with methionine are represented by sticks (pink). Residue I523 of human MARS (purple sticks) is located close to the methionine binding pocket, but does not directly interact with methionine. C: Residue F370 (purple sticks) is located in an alpha-helix close to residue I523.
Figure 5
Figure 5
F370L and I523T MARS associate with the multisynthetase complex. A: Coomassie staining of wild-type, F370L and I523T MARS-FLAG purified fractions. MARS-FLAG is indicated (arrow). The asterisk shows a degradation product of MARS (~75 kD), which is more pronounced after purification of wild-type MARS-FLAG. However, the ratio of the 100 kD full length MARS and the partially degraded form was not established in the wild-type and MARS mutants. MARS was not immunoprecipitated from cell lysates that expressed I523T MARS-FLAG with a nonsense mutation in the FLAG tag that abolished binding to the anti-FLAG antibody (see Additional file 3: Figure S2). B: Components of the multisynthetase complex are co-purified upon immuno-isolation of wild-type, F370L or I523T MARS. Western blotting shows the presence of similar levels of MARS, KARS and RARS (upper, middle and lower panel, respectively) after immuno-isolation of MARS-FLAG from HEK293 cells.

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Source: PubMed

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