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

Abstract

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.

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 tRNA^Asp 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.