Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD)

Pranesh K. Chakraborty, Klaus Schmitz-Abe, Erin K. Kennedy, Hapsatou Mamady, Turaya Naas, Danielle Durie, Dean R. Campagna, Ashley Lau, Anoop K. Sendamarai, Daniel H. Wiseman, Alison May, Stephen Jolles, Philip Connor, Colin Powell, Matthew M. Heeney, Patricia-Jane Giardina, Robert J. Klaassen, Caroline Kannengiesser, Isabelle Thuret, Alexis A. Thompson, Laura Marques, Stephen Hughes, Denise K. Bonney, Sylvia S. Bottomley, Robert F. Wynn, Ronald M. Laxer, Caterina P. Minniti, John Moppett, Victoria Bordon, Michael Geraghty, Paul B. M. Joyce, Kyriacos Markianos, Adam D. Rudner, Martin Holcik and Mark D. Fleming

Key Points

  • SIFD is a syndromic form of congenital sideroblastic anemia associated with immunodeficiency, periodic fevers, and developmental delay.

  • SIFD is due to partial loss-of-function mutations in the CCA-adding enzyme TRNT1.


Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy.


The congenital sideroblastic anemias (CSAs) are a heterogeneous group of syndromic and nonsyndromic inherited diseases characterized by pathologic iron deposition in the mitochondria of red blood cell precursors in the bone marrow. All identified causative CSA genes encode structural RNAs or proteins that are involved in 1 of 3 mitochondrial pathways: heme synthesis, mitochondrial iron-sulfur cluster biogenesis, and mitochondrial protein synthesis.1 We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers and developmental delay (SIFD). Variably severe sensorineural hearing loss, cardiomyopathy, and central nervous system abnormalities also occurred in some patients.2 SIFD pedigrees indicated an autosomal recessive mode of inheritance. Here, we extend the cohort of patients described with SIFD, and, using whole exome sequencing and a novel method of identity by descent mapping, identify the causative gene as TRNT1, a template-independent RNA polymerase required for the maturation of cytosolic and mitochondrial transfer RNAs (tRNAs).

Materials and methods

Ethics approval

The work was completed with the approval of the institutional review boards at Boston Children's Hospital and the Children's Hospital of Eastern Ontario.

Genomic analyses

We automated the discovery process using a custom-built, rule-based “Variant Explorer” pipeline using copy number variation, family linkage as well as population level homozygosity to aid interpretation of the results (K.S.-A. and K.M., unpublished). For the analysis, we analyzed 180 (113 affected) samples from multiplex families or singletons with CSA.

siRNA knockdowns

Small interfering RNA (siRNA) transfections of fibroblasts were performed using lipofectamine RNAiMAX according to the protocol provided by the manufacturer (Invitrogen). The cells were transfected TRNT1 siRNA (Hs_TRNT1, Qiagen SI00751464 [#4], SI04142691 [#6], SI04235056 [#7], SI04301857 [#8]), or a nonsilencing control siRNA (Dharmacon, 5′-UUCUCCGAACGUGUCACGUdTdT-3). Cells were collected for analysis at 24, 48, or 72 hours posttransfection.


See supplemental Methods on the Blood Web site.

Kinetic measurement of cytotoxicity and Caspase-3/7 activation

Normal human skin fibroblast cells were transfected with increasing concentrations of siRNA for 24 hours in a 96-well plate. Cells were then incubated with either 100 nM YOYO-1 dye (Life Technologies) or 1 μM of Cell Player reagent (Essen BioScience) and the was incubation monitored for 48 hours using the INCUCYTE ZOOM Live-Cell Imaging System as described by the manufacturer (Essen BioScience, MI). The fraction of YOYO-1 and Caspase 3/7–positive cells was measured after treatment with 0.0625% Triton X-100X for the YOYO-1 assay or 1 μM Vybrant Green DNA (Life Technologies, Invitrogen) for the Caspase 3/7 assay.

Overexpression and purification of native and variant TRNT1 proteins

HEK293T cells were transfected with either wild-type TRNT1-FLAG plasmid (pTrueORF, Origene, USA) or mutated plasmid DNA in antibiotic-free Opti-MEM with lipofectamine 2000 (Invitrogen). After 48 hours, lysates were immunoprecipitated with Flag-beads (Sigma-Aldrich) as previously described.3 Before assay, protein was quantified using BCA protein assay (Pierce), checked for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

CCA-adding enzyme assays

In vitro transcription of Bacilla subtilis tRNAAsp lacking 3 nucleotides (CCA) at the 3′-terminus was performed using the plasmid G73 (a gift of Dr. Alan M. Weiner, Department of Biochemistry, University of Washington, Seattle, WA)4 and CCA-adding enzyme activity assayed using either in-gel or glass fiber assays measuring the incorporation of [α32P]-adenosine triphosphate as described in detail in the supplemental Methods.

Yeast strain construction and plate-based assays

See the supplemental Methods and supplemental tables.

Statistical analyses

All results are expressed as mean ± standard error of the mean with a minimum of 3 biological replicates, unless otherwise noted. The Student t test was used to determine statistical significance (Graph Pad Prism 5).

Results and discussion

We performed genome-wide Affymetrix 6.0 SNP analysis on 6 SIFD probands (Table 1; patients 2, 3, 6, 7, 8, and 10) and 10 of their parents. We mapped the disease locus by analyzing genotypes for overlapping regions of identity by descent. We identified a region on chromosome 3p26.1 in which both consanguineous probands (patients 6 and 8) were homozygous for distinct haplotypes (supplemental Figure 1). Subsequent linkage analysis in another consanguineous Pakistani kindred (patients 1A and 1B, parents, and 2 unaffected siblings) was also consistent with linkage to 3p26.1, resulting in a cumulative logarithm of odds score of 3.38. Affected members of this pedigree shared a partially overlapping, identical homozygous haplotype with patient 8, also Pakistani. On this basis, we were able to narrow the disease interval to a 1.04-Mb, 5-cM region containing 4 genes, including TRNT1. Given its role in mitochondrial tRNA metabolism, TRNT1 was considered the primary candidate gene. Independently, whole exome sequencing was performed on patient 7. Rare variants in mitochondrial proteins annotated in the MitoCarta5 database were examined, and revealed biallelic, missense variants in TRNT1. By Sanger sequencing amplified exons, we found biallelic TRNT1 mutations in all but 1 of the other SIFD patients previously described (Table 1) as well as 5 additional affected individuals from 4 families (Table 1, patients 11, 12A, 12B, 13, and 14). In patient 4, we identified only 1 uncommon variant. We did not identify a small deletion or copy number variation in this patient using a custom Nimblegen 720k array spanning the TRNT1 locus. In aggregate, we identified 3 frameshift alleles, 3 splicing variants, and 7 unique missense TRNT1 alleles: p.T154I, p.M158V, p.L166S, p.R190I, p.I223T, p.I326T, and p.K416E. Except for p.K416E, each of the missense variants occurs in a highly conserved residue and received a PolyPhen2 score >0.95, indicating a likely damaging mutation.6 Nonetheless, K416 is highly conserved in mammals, suggesting its potential pathogenicity. Five of the missense mutations (p.T154I, p.M158V, p.L166S, p.R190I, and p.I223T) cluster in the active site, and 2 (p.I326T and p.K416E) are in the less well-conserved C-terminal region (supplemental Figure 2).7 Only 1 missense variant, p.I223T, occurs in the National Heart, Lung, and Blood Institute Exome Sequencing Variant Project (, and then only at an allele frequency of 0.0077%. We did not identify any uncommon variants in TRNT1 in 58 other unrelated probands with nonsyndromic and phenotypically distinct syndromic CSA.8

Table 1

SIFD patients and TRNT1 mutations

TRNT1 encodes the human CCA-adding enzyme, an RNA polymerase required for the posttranscriptional, template-independent addition of 2 cytosines and 1 adenosine to the 3′ end of all tRNA molecules, which is necessary for tRNA aminoacylation.9 CCA-adding enzymes are also implicated in tRNA quality control and the stress response.10-13 TRNT1 encodes the only human CCA-adding activity and is responsible for the maturation of both cytosolic and mitochondrial tRNAs.11

siRNA knockdown of TRNT1 in wild-type human fibroblasts caused cytotoxicity and apoptosis (supplemental Figure 3), suggesting that eliminating TRNT1 function altogether is lethal, whereas mutations only impairing function could be disease-associated. To determine the functional significance of the disease-associated variants, we examined the effect of the variants in patient-derived skin fibroblasts and in yeast. Only full-length TRNT1 transcript was detected from control human skin fibroblasts (supplemental Figure 4a). The c.608+1 G>T and del1054_1056+10 variants resulted in aberrant splicing (supplemental Figure 4a-c). The c.1057-7C>G mutation found in 3 individuals (patients 2, 3, and 9) is strongly predicted by the Human Splicing Finder14 to result in a new splice acceptor site that, if employed, inserts 6 nucleotides in the complementary DNA upstream of exon 8, encoding the insertion of sequential threonine and TAG stop codons (p.D352_S353insTX) and premature termination of the protein (supplemental Figure 4d). Unfortunately, no primary material was available from any of these patients to validate this prediction.

We examined the ability of TRNT1 mutants to complement budding yeast cells harboring a temperature-sensitive mutation in CCA1 (cca1-1, originally named ts352),7,15 and in a CCA1 deletion strain. Although expression of the wild-type human TRNT1 in the cca1-1 strain fully restored growth at a nonpermissive temperature (Figure 1A), expression of the mutant TRNT1 alleles provided only partial rescue (Figure 1B). Furthermore, as demonstrated by tetrad dissections, only the wild-type yeast protein and the human p.I326T mutant were able to rescue the lethal deletion of CCA1 through meiosis in haploid yeast (supplemental Figure 5a). Consequently, we rescued the lethal deletion of CCA1 with wild-type and mutant TRNT1 plasmids using a plasmid shuffle assay (Figure 1C; supplemental Figure 5b,c). We found that TRNT1 with any of the 5 missense mutations that cluster near the active site of the enzyme all complemented growth to varying degrees as compared with wild-type human TRNT1. The p.R190I and p.I223T variants grew particularly poorly on nonfermentable glycerol medium, indicating a defect in mitochondrial function. Two mutants, p.I326T and p.K416E, not near the active site, complemented the cca1∆ strain as efficiently as yeast CCA1 itself, suggesting that they may be milder alleles than the other missense mutations.

Figure 1

Mutations in TRNT1 in SIFD patients are loss-of-function alleles. (A) Yeast cca1-1 cells were transformed with centromeric plasmids encoding yeast CCA1, human TRNT1, or an empty vector control (EV) and grown at permissive (22°C) or nonpermissive (37°C) temperatures overnight in selective media. The optical density of the sample measured at a wavelength of 600 nm (OD600) value for the empty vector was subtracted from each value and the ratio of OD600 at 37°C/22°C was calculated and normalized so that the relative growth rate of cells harboring a CCA1 plasmid is 1 (mean ± standard error of the mean, with minimum n = 5). The ability of human TRNT1 to rescue growth at the nonpermissive temperature was not statistically different from CCA1 as determined by Student paired t test. (B) Patient TRNT1 mutants partially rescue growth impairment of cca1-1 cells. cca1-1 cells were transformed with EV, wild-type TRNT1, or TRNT1 mutants (p.T154I, p.L166S, p.R190I, p.M158V, p.I223T, and p.I326T) and grown as in (a). The OD600 value for the EV control was subtracted from each value and the ratio of OD600 at 37°C/22°C was calculated and normalized so that the relative growth rate of cells harboring a TRNT1 plasmid is 1 (mean ± standard error of the mean, with minimum n = 3; Student paired t test *P ≤ .05). (C) Complemented cca1∆ strains that contain only the TRNT-CEN-TRP1 plasmid were isolated from SC-trp + 5-FOA plates (supplemental Figure 15B). Ten-fold serial dilutions of 106 cells of the indicated genotypes were spotted on YEP + 2% dextrose or 3% glycerol plates at the indicated temperatures. Wild-type and most TRNT1 mutants have growth defects and temperature sensitivity, with the exception of TRNT1-p.K416E and TRNT-p.I326T, which are indistinguishable from CCA1 cells. (D) Two different concentrations of purified TRNT1-FLAG protein with patient-specific mutations were incubated with [α32P]-adenosine triphosphate and tRNAAsp lacking the terminal CCA sequence at 37°C for 5 minutes as described in the supplemental Methods and the reactions were terminated by addition of loading dye and resolved on a denaturing 5% polyacrylamide/8M urea gel, dried, and exposed to a phosphoimager screen. Other than the T154I variant, all of the mutants have negligible activity in this assay.

Although TRNT1 mRNA levels were comparable between wild type and mutants, protein levels were variable in wild-type and cca1-1 yeast (supplemental Figure 6a,b). This was equally true of TRNT1 expression in patient fibroblast cells (supplemental Figure 6c-e), suggesting that mutations in TRNT1 may also affect TRNT1 proteostasis. In support of this hypothesis, we also found that rescued cca1∆ strains selected for high expression of TRNT1 (supplemental Figure 1a,b).

All but 1 of the missense mutations impaired the ability of TRNT1 to catalyze the formation of the CCA trinucleotide, in vitro (Figure 1D; supplemental Figure 8a-c). Most mutant proteins had no detectable activity, whereas the activity of the p.T154I and p.K416E alleles were ∼60% and equal to the wild-type, respectively. Because of the limited numbers of patients, we were unable to perform a systematic genotype-phenotype correlation; however, we note that the siblings with the p.K416E allele have a mild phenotype, with predominantly neurological abnormalities, periodic fevers, and infections, but minimal anemia and longer survival. Similarly, patient 7, carrying the p.T154I variant, is neither transfusion- nor intravenous immunoglobulin–dependent. Patient 10, carrying the p.I326T allele, also has a variant phenotype having few neurological issues and not requiring intravenous immunoglobulins. Altogether, these genetic and functional assays indicate that all SIFD patients have TRNT1 mutations and an individual patient may present with a range of clinical severity of the constituent phenotypes depending upon the degree of CCA-adding enzyme loss of function.


Contribution: P.K.C., K.M., A.D.R., M.H., and M.D.F. designed and supervised the study; R.F.W., D.H.W., A.M., S.J., P.C., C.P., M.M.H., P.J.G., R.J.K., C.K., I.T., A.A.T., L.M., S.H., D.K.B., S.S.B., R.M.L., C.P.T., J.M., C.M., V.B., M.T.G., P.K.C., and M.D.F. characterized SIFD syndrome and collected clinical data and patient samples; K.S.-A. was supervised by K.M. and developed the informatics pipeline and identified the causative genetic defect by way of linkage studies; D.R.C. and A.L. performed mutation analysis in CSA patients; P.K.C. and M.T.G. identified the causative genetic defect post exomic sequencing; E.K.K., supervised by A.D.R., M.H., and P.K.C., designed and performed the yeast CCA knockout and rescue experiments; T.N., supervised by M.H. and P.K.C., designed and performed the siRNA knockdown experiments; H.M., supervised by A.D.R., M.H., and P.K.C., designed and performed the adenylation assays; D.D., supervised by A.D.R., M.H., and P.K.C., performed the temperature-sensitive yeast rescue experiments with advice from A.D.R. and P.B.M.J.; A.K.S. created the figure depicting the locations of the missense mutations on the TRNT1 X-ray crystal structure; E.K.K., H.M., T.N., and D.D. prepared the draft methods; and P.K.C., A.D.R., M.H., and M.D.F. prepared the final manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Pranesh K. Chakraborty, Newborn Screening Ontario, Department of Pediatrics, Children’s Hospital of Eastern Ontario, 401 Smyth Rd K1H 8L1, Ottawa, ON, Canada; e-mail: pchakraborty{at}; and Mark D. Fleming, Department of Pathology, Boston Children's Hospital, 300 Longwood Ave, Bader 124.1, Boston, MA 02115; e-mail: mark.fleming{at}


The authors acknowledge the patients with SIFD and their families, Dr Kym Boycott and the infrastructure of the Finding of Rare Disease Genes Canada Consortium, particularly Jeremy Schwartzentruber for analysis of whole-exome sequence analysis for patient 7. The authors thank Dr Dennis Bulman, Dr Amanda Smith, Dr Matthew Lines, Dr Nehal Thakor, Urszula Liwak, Mame Daro Faye, and Lynn Courteau (University of Ottawa, Ottawa, ON, Canada) for critical review of data and scientific advice and Drs. Tony Rupar and Jack Rip (Western University, London, ON, Canada) for invaluable advice in the design of the adenylation assay. The G7324 plasmid used to prepare the tRNA substrate was a generous gift of Dr Alan M. Weiner (Department of Biochemistry, University of Washington, Seattle, WA); the temperature-sensitive yeast strain ts35218 was a gift from Dr Anita Hopper (Ohio State University, OH).

This work was supported by the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) (A.D.R.); grants from CIHR, NSERC, and the Cancer Research Society (M.H.); CIHR, the Academic Health Sciences Centre Innovation fund, and the Ontario Ministry of Health and Long Term Care Fund (P.K.C.); the National Institutes of Health (grant R01 DK087992) (K.M.); a National Institute for Social Care and Health Research Fellowship (S.J.); and the Llandough Haematology Development Fund (A.M.).


  • P.K.C., K.S.-A., E.K.K., H.M., T.N., and D.D. contributed equally to the design and execution of this work; D.R.C. and A.L. contributed equally to the execution of this work; and P.K.C., A.M.R., M.H., and M.D.F. jointly directed this work.

  • The online version of this article contains a data supplement.

  • There is an Inside Blood Commentary on this article in this issue.

  • The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

  • Submitted August 8, 2014.
  • Accepted August 22, 2014.


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