SLC7A14 linked to autosomal recessive retinitis pigmentosa

Abstract

Retinitis pigmentosa (RP) is characterized by degeneration of the retinal photoreceptors and is the leading cause of inherited blindness worldwide. Although few genes are known to cause autosomal recessive RP (arRP), a large proportion of disease-causing genes remain to be revealed. Here we report the identification of SLC7A14, a potential cationic transporter, as a novel gene linked to arRP. Using exome sequencing and direct screening of 248 unrelated patients with arRP, we find that mutations in the SLC7A14 gene account for 2% of cases of arRP. We further demonstrate that SLC7A14 is specifically expressed in the photoreceptor layer of the mammalian retina and its expression increases during postnatal retinal development. In zebrafish, downregulation of slc7a14 expression leads to an abnormal eye phenotype and defective light-induced locomotor response. Furthermore, targeted knockout of Slc7a14 in mice results in retinal degeneration with abnormal ERG response. This suggests that SLC7A14 has an important role in retinal development and visual function.

Figure 1. Pedigree and clinical characteristics of a patient with arRP.

(a) A simplified pedigree of the W60 family. The arrow pointing to the filled symbol indicates the index proband patient; the oblique lines indicate deceased subjects; the number in the box or circle indicates the number of unaffected siblings. M, mutation; W, wild-type. (b) Fundoscopy of the proband patient revealed extensive degeneration in both eyes. A representative image is shown. (c) OCT of the proband patient determined that the retinal thickness was significantly decreased. (d) The proband patient exhibited an extinguished response to an ERG.

Figure 2. Identification of mutations in SLC7A14 in patients with arRP.

(a) Sequence chromatograms illustrate the causal lesions that were identified in five unrelated patients with arRP. Each mutation leads to alteration of a highly conserved amino acid residue. (b) The identified mutations in the nontransmembrane (line) and transmembrane (column) domains of SLC7A14 predicted by SMART algorithms.

Figure 3. Expression analysis and functional assay of the SLC7A14 mutation.

(a) High expression of SLC7A14 was observed in the retina, brain and spinal cord. (b) SLC7A14 is expressed as early as stage E18.5 and expression increases during postnatal development of the retina. (c) Immunohistochemical analysis of the retina showed that SLC7A14 (green) is expressed in the photoreceptor layer. Rhodopsin (red) is expressed in the outer segment of rod photoreceptors. DAPI (blue) indicates DNA in the cells’ nuclei. Scale bar, 50 μm. (d) In vitro assay elucidated the mutation effect on Slc7a14 subcellular localization. Wild-type Slc7a14 is colocalized with LysoTracker, whereas the p.G330R mutant does not and exhibits a remarkable change with a diffuse distribution of the signal in the cytoplasm, demonstrating that the mutation affects protein subcellular localization. Scale bar, 50 μm.

Figure 4. Knockdown of SLC7A14 in zebrafish.

(a) Injection of 1 nl of a 0.25 mM solution of slc7a14-MO dramatically reduces eye size in zebrafish. (b) At 2dpf, 4dpf and 5dpf, the eye-to-body ratio of fish injected with the slc7a14-MO was significantly smaller than that of fish injected with the control MO. N=6, Dunnett test, ***P<0.001. (c) Light-induced locomotor response with sudden light-to-dark transitions demonstrated significant visual impairment in zebrafish injected with the slc7a14-MO. N=6 in each group. (d) Dunnett tests of the light-induced locomotor responses indicate that significant differences exist between the responses of fish with small eyes and those with normal eyes during the light-ON phase. N=10, Dunnett test, **P<0.01. Bars in the graph represent standard errors of the mean (s.e.m.).

Figure 5. Generation and characterization of Slc7a14-knockout mice.

(a) Schematic of the Slc7a14 gene knockout strategy in mice. (b) ERG testing demonstrated a lower b-amplitude and a longer a-wave potential time in 2-month-old Slc7a14^−/− mice when compared with age-matched controls. N=3. Bar in the graph represents s.e.m. Student’s t-test, *P<0.05; ***P<0.001. (c) The 6-month-old KO mice displayed severely decline of ERG responses.