tRNA methyltransferase homolog gene TRMT10A mutation in young onset diabetes and primary microcephaly in humans

Abstract

We describe a new syndrome of young onset diabetes, short stature and microcephaly with intellectual disability in a large consanguineous family with three affected children. Linkage analysis and whole exome sequencing were used to identify the causal nonsense mutation, which changed an arginine codon into a stop at position 127 of the tRNA methyltransferase homolog gene TRMT10A (also called RG9MTD2). TRMT10A mRNA and protein were absent in lymphoblasts from the affected siblings. TRMT10A is ubiquitously expressed but enriched in brain and pancreatic islets, consistent with the tissues affected in this syndrome. In situ hybridization studies showed that TRMT10A is expressed in human embryonic and fetal brain. TRMT10A is the mammalian ortholog of S. cerevisiae TRM10, previously shown to catalyze the methylation of guanine 9 (m(1)G9) in several tRNAs. Consistent with this putative function, in silico topology prediction indicated that TRMT10A has predominant nuclear localization, which we experimentally confirmed by immunofluorescence and confocal microscopy. TRMT10A localizes to the nucleolus of β- and non-β-cells, where tRNA modifications occur. TRMT10A silencing induces rat and human β-cell apoptosis. Taken together, we propose that TRMT10A deficiency negatively affects β-cell mass and the pool of neurons in the developing brain. This is the first study describing the impact of TRMT10A deficiency in mammals, highlighting a role in the pathogenesis of microcephaly and early onset diabetes. In light of the recent report that the type 2 diabetes candidate gene CDKAL1 is a tRNA methylthiotransferase, the findings in this family suggest broader relevance of tRNA methyltransferases in the pathogenesis of type 2 diabetes.

Figure 1. Diabetes and microcephaly in a large consanguineous family.

(A) Three siblings presented with young onset diabetes and microcephaly with intellectual disability (black symbols); arrow shows the proband. Hatched symbols represent relatives with adult onset diabetes and the blue hatched symbol gestational diabetes. The double line indicates consanguinity. M/M denotes two mutant alleles, N/M one normal and one mutant allele. (B) A head MRI in the proband at age 26 years showed a small brain with normal architecture and normal gyration. (C) The radiograph of the hands showed normally shaped bones and absence of epiphyseal dysplasia.

Figure 2. Patients are homozygous for a nonsense mutation in TRMT10A and lose TRMT10A expression.

(A) Sanger sequencing at the level of the mutation identified by whole exome sequencing in proband. The mutation c.379 G>A; p.Arg127Stop changes a CGA codon (Arginine) into a TGA codon (Stop), and is found homozygous in the proband (P), heterozygous in unaffected mother (M) and absent in an unrelated control subject (C). (B) TRMT10A protein and (C) mRNA expression was examined by Western blot and real-time PCR in lymphoblast from three controls (CT1-3), two patients homozygous for the nonsense mutation (P1 and P2) and one heterozygous carrier (HET). α-Tubulin was used as loading control and TRMT10A mRNA expression was normalized to the geometric mean of the reference genes GAPDH, actin and OAZ1 expression.

Figure 3. TRMT10A mRNA and protein expression is enriched in brain and pancreatic islets.

TRMT10A mRNA (A) and protein (B–C) expression in rat tissues and islets was examined by real-time PCR and Western blot. TRMT10A mRNA expression was normalized to the geometric mean of the reference genes GAPDH, actin, and OAZ1, and protein was normalized to β-actin. Results are means ± SE of n = 3. ***p<0.001 for the comparison islets vs all other tissues, #p<0.05 for islets and brain vs liver, kidney, spleen lung and fat by one-way ANOVA followed by paired t test with Bonferroni correction for multiple comparisons.

Figure 4. TRMT10A expression profile in fetal telencephalon at 11 GW.

(A) TRMT10A antisense (AS) probe and (B) TRMT10A sense (S) probe as a negative control. Scale bar: 1 mm. (C) TRMT10A is expressed throughout the thickness of the dorsal telencephalon at 11 GW, with higher expression in the ventricular zone (VZ, red arrow) and marginal zone (MZ, black arrow). Scale bar: 100 µm. (D) Cresyl Violet staining on adjacent section. (E) Higher resolution of MZ (black arrow) and VZ (red arrow).

Figure 5. TRMT10A has predominant nuclear localization.

INS-1E cells were transfected with a vector encoding hrGFP alone or fused to TRMT10A (TRMT10A-hrGFP). 48 h after transfection RG9MTD2 subcellular localization was examined by confocal microscopy (A). Nuclei were stained with Hoechst 33342. Pictures were taken at 40× magnification, zoom 3×. The presence of the recombinant fusion protein was confirmed by Western blot using an antibody against hTRMT10A (B). NT denotes non-transfected. The figure is representative of three independent experiments.

Figure 6. TRMT10A protein expression is enriched in the nucleolus of β- and non-β-cells.

Endogenous TRMT10A was detected by immunofluorescence using anti-hTRMT10A antibody in dispersed rat (A) and human islet cells (B–C). Anti-fibrillarin antibody was used to immunolabel the nucleolus. Nuclei were stained with Hoechst 33342. Pictures were taken at 40× magnification and are representative of two independent experiments.

Figure 7. TRMT10A knockdown enhances total protein biosynthesis in rat β-cells.

Total protein synthesis was measured in INS-1E cells transfected with control siRNA (siCT) or two siRNAs targeting rat TRMT10A (siTRMT10A #1 and #2). Protein biosynthesis was corrected by total protein content and expressed as % of siCT. INS-1E cells treated for 2 h with the inhibitor of translation cycloheximide (CY, 10 µM) were used as positive control (n = 4). *CY vs CT, § siTRMT10A vs siCT p<0.05 by paired t test.

Figure 8. TRMT10A knockdown sensitizes β-cells to FFA-, high glucose- and ER stress-induced apoptosis.

INS-1E cells (A–E), primary rat β-cells (F) and dispersed human islets (G) were transfected with control siRNA (siCT) or siRNAs targeting rat (siTRMT10A #1 and #2) or human TRMT10A (siTRMT10A #3 and #4). 48 h after transfection cells were exposed or not (CT) to oleate (OL), palmitate (PAL) and 28 mM glucose (G28), or to the chemical ER stressors cyclopiazonic acid (CPA), tunicamycin (TU) and brefeldin (BR), for 24 (A–B) or 16 h (C–E). Apoptosis was examined by propidium iodide and Hoechst 33342 staining (A–D, F–G) or Western blot for cleaved caspase-3 (E). Results are means ± SE (n = 3–5). The blots are representative of 4 independent experiments. * Treated vs CT; § siTRMT10A vs siCT. One symbol p<0.05, two p<0.01, three p<0.001 by paired t test with Bonferroni correction for multiple comparisons.