THUMPD1 bi-allelic variants cause loss of tRNA acetylation and a syndromic neurodevelopmental disorder

Abstract

Covalent tRNA modifications play multi-faceted roles in tRNA stability, folding, and recognition, as well as the rate and fidelity of translation, and other cellular processes such as growth, development, and stress responses. Mutations in genes that are known to regulate tRNA modifications lead to a wide array of phenotypes and diseases including numerous cognitive and neurodevelopmental disorders, highlighting the critical role of tRNA modification in human disease. One such gene, THUMPD1, is involved in regulating tRNA N4-acetylcytidine modification (ac4C), and recently was proposed as a candidate gene for autosomal-recessive intellectual disability. Here, we present 13 individuals from 8 families who harbor rare loss-of-function variants in THUMPD1. Common phenotypic findings included global developmental delay, speech delay, moderate to severe intellectual deficiency, behavioral abnormalities such as angry outbursts, facial dysmorphism, and ophthalmological abnormalities. We demonstrate that the bi-allelic variants identified cause loss of function of THUMPD1 and that this defect results in a loss of ac4C modification in small RNAs, and of individually purified tRNA-Ser-CGA. We further corroborate this effect by showing a loss of tRNA acetylation in two CRISPR-Cas9-generated THUMPD1 KO cell lines. In addition, we also show the resultant amino acid substitution that occurs in a missense THUMPD1 allele identified in an individual with compound heterozygous variants results in a marked decrease in THUMPD1 stability and RNA-binding capacity. Taken together, these results suggest that the lack of tRNA acetylation due to THUMPD1 loss of function results in a syndromic form of intellectual disability associated with developmental delay, behavioral abnormalities, hearing loss, and facial dysmorphism.

Keywords: N4-acetylcytidine; NAT10; RNA acetylation; THUMPD1; ac4C; developmental disorder; intellectual disability; tRNA biology; tRNA modifications.

Figure 1

Clinical features of individuals with bi-allelic THUMPD1 variants Photographs of five individuals from our cohort, showing facial dysmorphisms. Each individual is referenced with the corresponding identifier used throughout the manuscript: Individuals F1:II:1 (A, B) and F1:II:2 (C) are brothers and show down-slanting palpebral fissures, broad and flat philtrum, hypoplastic columella, and low-set ears. Individual F6:II:1 (F) has a wide mouth, epicanthus, ptosis, and narrow palpebral fissures. Individual F8:II.1 (G, H) shows scaphocephaly, frontal bossing, deep-set ears, hypotelorism, and intermittent bilateral esotropia.

Figure 2

Family pedigrees of individuals with bi-allelic THUMPD1 variants and a schematic of THUMPD1 with positions of the corresponding mutated amino acid residues indicated (A) Pedigrees of eight unrelated families with affected members indicated as filled circles (females) and squares (males) with denoted THUMPD1 variants and the corresponding protein alterations. Double horizontal lines indicate consanguinity. Individual F2:II.6 has a different disease (Rahman syndrome) but is able to attend school with borderline performance, good behavior, and normal development for his age. (B) Schematic representation of the human THUMPD1 protein sequence with THUMP-domain highlighted in green. The nature and positions of the variants are shown above the primary sequence diagram (colored blue for missense mutations, orange for nonsense mutations, and red for frameshift variants).

Figure 3

Bi-allelic c.706C>T THUMPD1 (p.Gln236^∗) causes a lack of ac4C modification in tRNA (A) Western blot analysis of the protein cell extracts prepared from two control individuals and individual F2:II.1 (15DG1395) lymphoblasts probed with anti-THUMPD1 antibody and anti-tubulin antibody (as loading control). (B) THUMPD1 mRNA expression in two control individuals and individual F2:II.1 (15DG1395) lymphoblasts as determined by RT-qPCR of the corresponding RNA samples (relative to control #1). Samples were analyzed in triplicate, error bars represent ± SD. ^∗p(adjusted) < 0.05, ^∗∗∗p(adjusted) < 0.001 as determined by a Brown-Forsythe and Welch one-way ANOVA with Benjamini-Hochberg correction (FDR < 5%). (C) LC-MS nucleoside modification analysis of small RNA samples from individual F2:II.1 (15DG1395) lymphoblasts compared (normalized) to control #1 small RNA samples. Alterations (fold change) for each nucleoside modification was calculated as a ratio of the corresponding value in variant-containing RNA to control sample RNA normalized based on the abundance of all four standard non-modified nucleosides (A, C, G, and U). Samples were run in duplicate, error bars represent ± SD. (D) LC-MS nucleoside modification analysis of the purified tRNA-Ser-CGA1-1 samples prepared from control #1 (two top panels) and individual F2:II.1 (15DG1395) (two bottom panels) lymphoblasts. The ac4C peaks are shown in green and unmodified cytidine peaks are in blue (as controls). LC elution times are shown above each peak.

Figure 4

THUMPD1 KO in HEK293T cells causes loss of ac4C modification in small RNA samples (A) Schematic representation of the THUMPD1 KO in HEK293T cell line. sgRNA was designed to disrupt THUMPD1 nucleotide sequence in exon 2 to produce an aberrant PTC-containing transcript (see Table S1 for gRNA sequence). (B) Sanger DNA sequencing data of the THUMPD1 exon 2 genomic region obtained from selected THUMPD1 KO clone (top) and WT HEK293T cells (bottom). Underlined DNA sequence in WT HEK293T sample is complementary to the designed sgRNA. Dotted vertical line in both sequences indicates a Cas9 target cleavage site. (C) ICE analysis (Synthego) results of the THUMPD1 exon 2 genomic DNA region from a selected THUMPD1 KO clone. Target nucleotide sequence complementary to sgRNA is in red font and dotted vertical line indicates a Cas9 target cleavage site. The indel type and percentage of detected sequence are shown on the left. (D) Western blot analysis of the protein cell extracts prepared from HEK293T control and two independent THUMPD1 KO cell lines probed with anti-THUMPD1 antibody and anti-GAPDH antibody (as loading control). (E) THUMPD1 mRNA expression in HEK293T control and THUMPD1 KO cells determined by RT-qPCR of the corresponding RNA samples. Samples were analyzed in triplicate, error bars represent ± SD. ^∗∗∗∗p < 0.0001, as determined by a Welch’s t test. (F) Northern immunoblot analysis of the large and small RNA samples prepared from HEK293T control (WT) and THUMPD1 KO cells. Positions of 18S rRNA and tRNA on the gel are shown by the arrows on the left. (G) LC-MS nucleoside modification analysis of the small RNA samples prepared from HEK293T control and THUMPD1 KO cells. Alterations (fold change) for each nucleoside modification was calculated as a ratio of the corresponding value in THUMPD1 KO RNA to HEK293T control RNA normalized based on the abundance of all standard non-modified nucleosides (A, C, G, and U). Samples were run in duplicate, error bars represent ± SD.

Figure 5

THUMPD1 p.Pro164Ser affects its stability and binding to RNA (A) Proline at position 164 (outlined with a black box) within THUMP domain (positions 147–254 [in red] is highly conserved in primary sequences of THUMPD1 orthologs), including model organisms Homo sapiens, Mus musculus, Rattus norvegicus, Danio rerio, Drosophila melanogaster, and Saccharomyces cerevisiae. (B) Coomassie blue-stained gel showing purified WT and p.Pro164Ser (P164S) mutant proteins. Protein molecular marker sizes are shown on the left side of the gel. (C) Thermal denaturation profile from a protein thermal shift assay of purified THUMPD1 WT (black line) and P164S mutant (red line) proteins. (D) A plot first derivative of the fluorescence emission as a function of temperature (−dF/dT) (from the data in C). The protein melting temperature (T_m) of the P164S mutant protein (red line) is ∼5°C lower than the WT protein (black line). (E) EMSA gel image testing the binding of purified THUMPD1 WT protein (left) and P164S mutant protein (right) to tRNA-Ser-CGA. The ascending triangles in black show an increasing concentration of THUMPD1 in the EMSA assay samples from 0 nM to 1,000 nM. (F) Quantitation of the EMSA of THUMPD1 binding to tRNA-Ser-CGA from (E) showed that purified THUMPD1 WT protein (black line) had substantially higher affinity/binding to tRNA than P164S mutant (red line). Samples were run in duplicate, error bars represent ± SD.