Hypomorphic mutations in TRNT1 cause retinitis pigmentosa with erythrocytic microcytosis

Abstract

Retinitis pigmentosa (RP) is a highly heterogeneous group of disorders characterized by degeneration of the retinal photoreceptor cells and progressive loss of vision. While hundreds of mutations in more than 100 genes have been reported to cause RP, discovering the causative mutations in many patients remains a significant challenge. Exome sequencing in an individual affected with non-syndromic RP revealed two plausibly disease-causing variants in TRNT1, a gene encoding a nucleotidyltransferase critical for tRNA processing. A total of 727 additional unrelated individuals with molecularly uncharacterized RP were completely screened for TRNT1 coding sequence variants, and a second family was identified with two members who exhibited a phenotype that was remarkably similar to the index patient. Inactivating mutations in TRNT1 have been previously shown to cause a severe congenital syndrome of sideroblastic anemia, B-cell immunodeficiency, recurrent fevers and developmental delay (SIFD). Complete blood counts of all three of our patients revealed red blood cell microcytosis and anisocytosis with only mild anemia. Characterization of TRNT1 in patient-derived cell lines revealed reduced but detectable TRNT1 protein, consistent with partial function. Suppression of trnt1 expression in zebrafish recapitulated several features of the human SIFD syndrome, including anemia and sensory organ defects. When levels of trnt1 were titrated, visual dysfunction was found in the absence of other phenotypes. The visual defects in the trnt1-knockdown zebrafish were ameliorated by the addition of exogenous human TRNT1 RNA. Our findings indicate that hypomorphic TRNT1 mutations can cause a recessive disease that is almost entirely limited to the retina.

Figure 1.

Experimental structure of TRNT1 (gray) with overlay (green) showing a computational estimate of Glu43 deletion. Inset: area around Glu43 deletion with side chain of Glu43 shown in the experimental structure. The minor change in the modeled structure is consistent with a small functional impact of the allele.

Figure 2.

Color fundus photographs (A, B, D, E, G, H) and OCT (C, F, I) from individuals P1 (A–C), P2 (D–F) and P3 (G–I) affected with TRNT1-associated autosomal-recessive RP. The color photographs reveal vascular narrowing in all three individuals, subtle bone spicule-like pigmentation in P1 and cystoid macular edema in P2 and P3. OCT reveals thinning of the photoreceptor layers in all three individuals and marked cystoid macular edema in P2 and P3.

Figure 3.

Photomicrograph of a Wright's stained peripheral blood smear from Patient 1 (A) demonstrating microcytosis, hypochromia, anisocytosis and numerous eliptocytes. A normal blood smear (B) is provided for comparison (100×).

Figure 4.

Expression of WT and mutant TRNT1 proteins in control and patient fibroblasts. (A) Western blot analysis depicting TRNT1 expression in dermal fibroblasts obtained from a normal control, an RP control (i.e. patient with non-TRNT1-associated RP) and three compound heterozygous patients (gel was loaded with 20 µg of total protein). GAPDH served as loading control. (B) Quantification by densitometry of lysates between groups for TRNT1 (n = 4) normalized to GAPDH. Data presented as mean ± SEM. Statistical significance between control and patient fibroblasts determined by one-way ANOVA–Tukey, post hoc test, (*) P-value < 0.05, n = 4. An overall diminution of TRNT1 protein and a second 47 kDa band present only in cells from patients with TRNT1 mutations was detected (see the Supplementary Material, Fig. S2D).

Figure 5.

trnt1 expression and knockdown in zebrafish embryos. (A) Zebrafish trnt1 gene structure noting the location of MOs used for knockdown. (B) Zebrafish trnt1 transcript is broadly expressed throughout the embryo up to 1 dpf. (C) By 2 dpf, trnt1 becomes enriched in the brain, pectoral fin, blood and heart (inset). (D) Section through the central retina of 3 dpf larva showing trnt1 expression. (E) trnt1 expression in blood forming regions and the neuromast. (F) RT-PCR of RNA isolated from control and morphant embryos showing altered splicing in MO-injected embryos through 5 dpf. Uninjected WT embryos are used as control. Phenotypic range of morphological defects observed in trnt1 morphants at 5 dpf. (G) WT-like, (H) reduced eye size and blood flow and (I) eye and cardiovascular defects. Blue bar represents the penetrance of morphological defects with MO dose: 6.5 ng generating 80% defective, 4.5 ng generating 60% defective and 1.5 ng inducing defects in 15% of injected embryos.

Figure 6.

trnt1 knockdown affects tRNA maturation and visual behaviors. (A) RNA-Seq analysis of control and morphant embryos evaluating nucleotide triplet CCA addition of all annotated tRNAs. (B) Vision Startle Reflex Response. (C) Violin plot showing the distribution of the number of responses for control, knockdown and hTRNT1 RNA-injected larvae. ***P-value < 1e-6 MO versus WT; *P-value = 0.001032 MO versus MO+RNA; **P-value = 1.492e-6 MO+RNA versus WT by the Mann–Whitney test.