Alternative titles; symbols
HGNC Approved Gene Symbol: TRNT1
Cytogenetic location: 3p26.2 Genomic coordinates (GRCh38): 3:3,126,940-3,153,435 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 3p26.2 | Retinitis pigmentosa and erythrocytic microcytosis | 616959 | Autosomal recessive | 3 |
| Sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay | 616084 | Autosomal recessive | 3 |
The CCA-adding enzyme TRNT1 (EC 2.7.7.72) is an essential enzyme that catalyzes the addition of the CCA terminus to the 3-prime end of tRNA precursors. This reaction is a fundamental prerequisite for mature tRNAs to become aminoacylated and to participate in protein biosynthesis (Lizano et al., 2007).
By searching an EST database for sequences similar to the bovine CCA-adding enzyme, followed by PCR and 5-prime RACE, Nagaike et al. (2001) cloned human TRNT1. The deduced 434-amino acid protein has an N-terminal mitochondria-targeting signal sequence and conserved DXD and RRD motifs involved in nucleotide incorporation activity. The mature protein begins with leu27 and has a calculated molecular mass of 47 kD.
Lizano et al. (2007) cloned a splice variant of TRNT1 that uses an alternative 5-prime donor splice site in exon 6, resulting in a deletion of 60 bases. The deduced protein lacks the 20 amino acids that form helices 11 and 12 and their connecting and flanking sequences. Database analysis indicated that this splice variant is conserved in eutherian mammals and fish.
DeLuca et al. (2016) observed expression of trnt1 in the adult retina of zebrafish. Using antisense probes for whole-mount in situ hybridization, they found that zebrafish trnt1 is expressed ubiquitously during early embryogenesis. By 2 days postfertilization (dpf), expression is enriched in the brain, cardiovascular region, and pectoral fin buds, and in proliferative cells of the retina. At 3 dpf, trnt1 transcript expression is maintained in the brain, retina, and cardiac regions. By 5 dpf, expression is observed in the head and trunk neuromasts, zebrafish sensory structures analogous to the auditory system of mammals.
Nagaike et al. (2001) showed that purified recombinant human TRNT1 could incorporate both ATP and CTP into a test substrate, 3-prime CA-truncated yeast tRNA(phe). The T-arm of the substrate tRNA was required for efficient CCA addition by TRNT1. Unlike the E. coli ortholog, human TRNT1 could use both cytoplasmic and mitochondrial tRNA substrates, since it recognized the general T-arm structure rather than a specific sequence.
Lizano et al. (2007) found that the TRNT1 isoform lacking helices 11 and 12 could incorporate C residues into yeast tRNA(phe), but it could not add the terminal A residue to complete the CCA motif. Structure/function analysis showed that this isoform had reduced affinity for ATP and that helices 11 and 12 were required for a structural rearrangement that permitted binding of ATP to the single nucleotide-binding pocket.
Nagaike et al. (2001) determined that the TRNT1 gene has 7 exons and spans about 20 kb.
By genomic sequence analysis, Nagaike et al. (2001) mapped the TRNT1 gene to chromosome 3p25.1. They identified TRNT1 pseudogenes on chromosomes 1 and 22.
Sideroblastic Anemia with B-Cell Immunodeficiency, Periodic Fevers, and Developmental Delay
In 15 patients from 13 families with sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD; 616084), Chakraborty et al. (2014) identified homozygous or compound heterozygous mutations in the TRNT1 gene (see, e.g., 612907.0001-612907.0006). The first mutations were found in consanguineous families by linkage analysis and candidate gene sequencing; subsequent mutations were found by whole-exome analysis or direct sequencing of the TRNT1 gene. There were 3 frameshift alleles, 3 splicing variants, and 7 missense mutations. Knockdown of TRNT1 by siRNA in wildtype human fibroblasts caused cytotoxicity and apoptosis, suggesting that complete loss of TRNT1 function is lethal. In vitro functional expression studies showed that the missense mutants could only partially rescue the growth defect in a yeast strain with a defective ortholog of TRNT1 (cca1), consistent with a partial loss of function. However, there was some variability in residual function of the mutants, suggesting that the phenotypic severity may depend upon the degree of CCA-adding enzyme loss of function.
Retinitis Pigmentosa and Erythrocytic Microcytosis
In 2 brothers and an unrelated man with retinitis pigmentosa and erythrocytic microcytosis (RPEM; 616959), DeLuca et al. (2016) identified compound heterozygosity for mutations in the TRNT1 gene (612907.0007-612907.0010). Western blot analysis of protein extracted from patient fibroblasts showed significantly decreased levels of TRNT1 protein compared to controls, although the authors noted that the protein level was not as diminished as in published cases of SIFD, consistent with the milder phenotype in their patients. Suppression of trnt1 expression in zebrafish recapitulated several features of human SIFD; however, when levels of trnt1 were titrated, visual dysfunction was observed in the absence of other phenotypes. DeLuca et al. (2016) concluded that hypomorphic TRNT1 mutations can cause a recessive disease that is almost entirely limited to the retina.
DeLuca et al. (2016) demonstrated that zebrafish trnt1 expression is found in all of the organs affected in human SIFD (616084) and RPEM (616959). Using morpholino oligonucleotides to reduce endogenous Trnt1 activity, the authors observed cardiovascular defects and reduced eye size, as well as reduced staining of hemoglobin-containing red blood cells. Zebrafish neuromasts, which form the counterpart to the human auditory system, showed significantly reduced touch responses. To evaluate vision, DeLuca et al. (2016) tested trnt1 morphants with normal gross morphology and retention of touch response for their response to light, and observed a lower response rate compared to controls. The authors noted that, like their human patients with RPEM, these fish exhibited reduced visual function in the absence of detectable syndromic features. The visual defects were only partially rescued by the addition of exogenous human TRNT1 RNA, suggesting that the visual response is more sensitive to Trnt1 levels than other defects.
In 2 sibs, born of consanguineous Pakistani parents, with sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD; 616084), Chakraborty et al. (2014) identified a homozygous c.569G-T transversion in exon 5 of the TRNT1 gene, resulting in an arg190-to-ile (R190I) substitution at a highly conserved residue in the active site. The mutation, which was found by linkage analysis and candidate gene sequencing, was not found in the Exome Sequencing Project database. In vitro functional expression studies showed that the missense R190I mutant could only partially rescue the growth defect in a yeast strain with a defective ortholog of TRNT1 (cca1), consistent with a partial loss of function. A homozygous R190I mutation was also found in another Pakistani patient with the disorder.
In a Caucasian Hispanic patient, born of consanguineous parents, with sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD; 616084), Chakraborty et al. (2014) identified a homozygous c.668T-C transition in exon 6 of the TRNT1 gene, resulting in an ile223-to-thr (I223T) substitution at a highly conserved residue in the active site. The mutation, which was found by linkage analysis and candidate gene sequencing, was found at a very low frequency (0.0077%) in the Exome Sequencing Project database. In vitro functional expression studies showed that the missense I223T mutant could only partially rescue the growth defect in a yeast strain with a defective ortholog of TRNT1 (cca1), consistent with a partial loss of function. Direct sequencing of subsequent patients with the disorder found that 5 unrelated patients of Caucasian or Afro-Caribbean origin were compound heterozygous for I223T and another pathogenic mutation (see, e.g., 612907.0003 and 612907.0006).
In 2 unrelated Caucasian patients with sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD; 616084), Chakraborty et al. (2014) identified compound heterozygous mutations in the TRNT1 gene: a C-to-G transversion in intron 6 (c.1057-7C-G), predicted to result in a splicing defect, frameshift, and premature termination, and I223T (612907.0002).
In a Caucasian patient with sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD; 616084), Chakraborty et al. (2014) identified compound heterozygous mutations in the TRNT1 gene: a c.497T-C transition in exon 5, resulting in a leu166-to-ser (L166S) substitution, and a c.461C-T transition in exon 4, resulting in a thr154-to-ile (T154I; 612907.0005) substitution. Both mutations occurred at highly conserved residues in the active site. In vitro functional expression studies showed that both missense mutants could only partially rescue the growth defect in a yeast strain with a defective ortholog of TRNT1 (cca1), consistent with a partial loss of function; however, the degree of residual enzyme activity varied somewhat between the 2 mutants. Neither mutation was present in the Exome Sequencing Project database.
For discussion of the thr154-to-ile (T154I) mutation in the TRNT1 gene that was found in compound heterozygous state in a patient with sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD; 616084) by Chakraborty et al. (2014), see 612907.0004.
In a patient of Afro-Caribbean origin with sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD; 616084), Chakraborty et al. (2014) identified compound heterozygous mutations in the TRNT1 gene: a 4-bp insertion (c.1142_1143insATGT) in exon 8, resulting in a frameshift and premature termination (Trp381fs), and I223T (612907.0002).
In a 21-year-old man with retinitis pigmentosa and erythrocytic microcytosis (RPEM; 616959), DeLuca et al. (2016) identified compound heterozygosity for mutations in the TRNT1 gene. The first mutation was a 1-bp deletion (c.1246delA; chr3.3,189,786Adel, GRCh37) in exon 8, causing a frameshift predicted to result in a protein with 10 incorrect amino acids that is truncated by 7 amino acids. The second mutation was a 3-bp in-frame deletion (c.126_128delAGA) in exon 2, resulting in loss of the poorly conserved glu43 residue (612907.0008), located on the surface of the TRNT1 structure within the head domain, in a helix-turn-helix secondary structure motif. Structural analysis was consistent with this variant having a relatively mild effect on TRNT1 function, thus explaining the milder phenotype in this patient. In addition, Western blot analysis of protein extracted from patient fibroblasts showed significantly decreased levels of TRNT1 protein compared to controls, although the authors noted that the protein level was not as diminished as in published cases of SIFD (616084). The proband's unaffected parents were each heterozygous for 1 of the mutations; the 3-bp deletion was also found in the ExAC database at a frequency of 0.008%. Past medical history in this patient included juvenile rheumatoid arthritis and recurrent fevers in early childhood, which resolved after treatment with methotrexate.
For discussion of the 3-bp in-frame deletion (c.126_128delAGA; chr3.3,170,850AGAdel, GRCh37) in exon 2 of the TRNT1 gene, resulting in deletion of glutamic acid at codon 43 (E43del), that was found in compound heterozygous state in a patient with retinitis pigmentosa and erythrocytic microcytosis (RPEM; 616959) by DeLuca et al. (2016), see 612907.0007.
In 2 brothers with retinitis pigmentosa and erythrocytic microcytosis (RPEM; 616959), DeLuca et al. (2016) identified compound heterozygosity for mutations in the TRNT1 gene. The first mutation was a 1-bp insertion (c.1246insA; chr3.3,189,785_3,189,786AG-AAG, GRCh37) in exon 8, causing a frameshift predicted to result in a protein with 6 incorrect amino acids that is truncated by 9 amino acids. The second mutation was a splice site mutation (c.609-26T-C) in intron 5; analysis of PCR products from 1 of the brothers revealed an additional 14-bp product that was absent from controls. Bidirectional sequencing confirmed the addition of 14 nucleotides to the spliced RNA, predicted to result in the addition of 4 incorrect amino acids followed by a premature termination codon. The 1-bp insertion was found at a frequency of 0.07% in Europeans in the ExAC database, whereas the splice site mutation was not found in 257 controls from the same geographic area or in the 1000 Genomes Project or ExAC databases. Western blot analysis of protein extracted from patient fibroblasts showed significantly decreased levels of TRNT1 protein compared to controls, although the authors noted that the protein level was not as diminished as in published cases of SIFD (616084), consistent with the milder phenotype observed in the brothers.
For discussion of the splice site mutation (c.609-26T-C; chr3.3,188,088T-C, GRCh37) in intron 5 of the TRNT1 gene that was found in compound heterozygous state in 2 brothers with retinitis pigmentosa and erythrocytic microcytosis (RPEM; 616959) by DeLuca et al. (2016), see 612907.0009.
Chakraborty, P. K., Schmitz-Abe, K., Kennedy, E. K., Mamady, H., Naas, T., Durie, D., Campagna, D. R., Lau, A., Sendamarai, A. K., Wiseman, D. H., May, A., Jolles, S., and 23 others. Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD). Blood 124: 2867-2871, 2014. [PubMed: 25193871] [Full Text: https://doi.org/10.1182/blood-2014-08-591370]
DeLuca, A. P., Whitmore, S. S., Barnes, J., Sharma, T. P., Westfall, T. A., Scott, C. A., Weed, M. C., Wiley, J. S., Wiley, L. A., Johnston, R. M., Schnieders, M. J., Lentz, S. R., Tucker, B. A., Mullins, R. F., Scheetz, T. E., Stone, E. M., Slusarski, D. C. Hypomorphic mutations in TRNT1 cause retinitis pigmentosa with erythrocytic microcytosis. Hum. Molec. Genet. 25: 44-56, 2016. [PubMed: 26494905] [Full Text: https://doi.org/10.1093/hmg/ddv446]
Lizano, E., Schuster, J., Muller, M., Kelso, J., Morl, M. A splice variant of the human CCA-adding enzyme with modified activity. J. Molec. Biol. 366: 1258-1265, 2007. [PubMed: 17204286] [Full Text: https://doi.org/10.1016/j.jmb.2006.12.016]
Nagaike, T., Suzuki, T., Tomari, Y., Takemoto-Hori, C., Negayama, F., Watanabe, K., Ueda, T. Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases. J. Biol. Chem. 276: 40041-40049, 2001. [PubMed: 11504732] [Full Text: https://doi.org/10.1074/jbc.M106202200]