A novel homozygous missense mutation p.P388S in TULP1 causes protein instability and retinitis pigmentosa

Abstract

Purpose: Retinitis pigmentosa (RP) is an inherited retinal disorder that results in the degeneration of photoreceptor cells, ultimately leading to severe visual impairment. We characterized a consanguineous family from Southern India wherein a 25 year old individual presented with night blindness since childhood. The purpose of this study was to identify the causative mutation for RP in this individual as well as characterize how the mutation may ultimately affect protein function.

Methods: We performed a complete ophthalmologic examination of the proband followed by exome sequencing. The likely causative mutation was identified and modeled in cultured cells, evaluating its expression, solubility (both with western blotting), subcellular distribution, (confocal microscopy), and testing whether this variant induced endoplasmic reticulum (ER) stress (quantitative PCR [qPCR] and western blotting).

Results: The proband presented with generalized and parafoveal retinal pigmented epithelium (RPE) atrophy with bone spicule-like pigmentation in the midperiphery and arteriolar attenuation. Optical coherence tomography scans through the macula of both eyes showed atrophy of the outer retinal layers with loss of the ellipsoid zone, whereas the systemic examination of this individual was normal. The proband's parents and sibling were asymptomatic and had normal funduscopic examinations. We discovered a novel homozygous p.Pro388Ser mutation in the tubby-like protein 1 (TULP1) gene in the individual with RP. In cultured cells, the P388S mutation does not alter the subcellular distribution of TULP1 or induce ER stress when compared to wild-type TULP1, but instead significantly lowers protein stability as indicated with steady-state and cycloheximide-chase experiments.

Conclusions: These results add to the list of known mutations in TULP1 identified in individuals with RP and suggest a possible unique pathogenic mechanism in TULP1-induced RP, which may be shared among select mutations in TULP1.

Figure 1

Clinical characterization of the patient. A, B: Fundus photographs of the patient’s right and left eyes showing parafoveal RPE atrophy, bone spicule-like pigmentation, and arteriolar attenuation. C, D:Fundus autofluorescence images showing parafoveal hypoautofluorescence corresponding to the area of RPE atrophy and a patchy decrease in autofluorescence throughout the retina in both eyes. E, F: Optical coherence tomography (OCT) scans through the macula showing outer retinal atrophy with loss of the ellipsoid zone. G–L: Fundus photographs, autofluorescence, and OCT images of an age-matched control subject.

Figure 2

Pedigree and in silico analysis of the pathogenic mutation. A: Pedigree of the consanguineous family with variant segregation based on Sanger sequencing. B: Multiple sequence alignment of TULP1 amino acid residues across species. Arrow indicates highlighted TULP1 residue. Alignments were performed using Clustal Omega multiple sequence alignment software. C: In silico prediction findings related to the P388S mutation.

Figure 3

Sub-cellular localization of WT TULP1 and P388S TULP1. Representative confocal microscopy images of human embryonic kidney (HEK-293A) cells transfected with (A) green fluorescent protein (peGFP-C1), B: wild-type (WT) TULP1 enhanced GFP (eGFP), or (C) P388S TULP1 eGFP constructs (green) and stained with AlexaFluor 633 phalloidin (red). Scale bar = 50 μm. TULP1 eGFP images are representative n≥5 biologic, independent replicates. Phalloidin images were representative of n≥3 separate independent wells of a single transfection experiment.

Figure 4

Characterization of the P388S TULP1 variant. A: Western blot of wild-type (WT) and P388S TULP1 enhanced green fluorescent protein (eGFP) levels in soluble and insoluble fractions. B: Quantification of WT and P388S TULP1 eGFP expression in soluble and insoluble fractions of western blot in (A), n≥5, mean ± standard deviation (SD; **p<0.01, one-sample t test versus hypothetical value of 1 [i.e., unchanged]). C: Quantitative PCR (qPCR) of TULP1 mRNA expression from WT TULP1 eGFP- and P388S TULP1 eGFP-transfected HEK-293A cells. Representative data of n≥3 independent experiments, mean ± SD; n.s., not significant.

Figure 5

Cycloheximide chase of WT and P388S TULP1. A, B: Western blots of wild-type (WT) and P388S TULP1 enhanced green fluorescent protein (eGFP) stability in human embryonic kidney (HEK)-293A cells treated with 25 μM cycloheximide (CHX) and harvested at the indicated time points. C: Quantification of western blot from (A) and (B) showing percentage of TULP1 remaining over time when treated with CHX. (●) indicates WT TULP1 eGFP, and (■) indicates P388S TULP1 eGFP. n = 3 independent experiments, mean ± standard deviation (SD); *p<0.05, **p<0.01, two-tailed t test compared to each WT value, n.s., not significant.

Figure 6

P388S TULP1 does not activate the ER stress response. A: Quantitative PCR (qPCR) of hHSPA5, hDNAJB9, and hASNS transcript levels with TaqMan probes in wild-type (WT) TULP1 enhanced green fluorescent protein- (eGFP-) or P388S TULP1 eGFP-expressing cells. B: Western blot showing GRP78 expression in eGFP-, WT TULP1 eGFP-, or P388S TULP1 eGFP-transfected cells. One microgram per milliliter Tm was used as a positive control to analyze GRP78 induction. C: Quantification of western blot in (B). n = 3 biologic independent experiments, mean ± standard deviation (SD); *p<0.05, **p<0.01, one-sample t test versus hypothetical value of 1 (i.e., unchanged), n.s., not significant.