Introduction: TULP1 exemplifies the remarkable clinical and genetic heterogeneity observed in inherited retinal dystrophies. Our research describes the clinical and molecular characteristics of a patient manifesting an atypical retinal dystrophy pattern, marked by the identification of both a previously unreported and a rarely encountered TULP1 variant. Methods: Whole-exome sequencing was performed to identify potential causative variants. The pathogenicity of the identified TULP1 variants was evaluated through in silico predictors and a minigene splice assay, specifically designed to assess the effect of the unreported TULP1 variant. Results: We identified two TULP1 gene variants in a patient exhibiting unusual and symmetrical alterations in both retinas, characterized by an increase in autofluorescence along the distribution of retinal vessels. These variants included a known rare missense variant, c.1376T>C, and a novel splice site variant, c.822G>T. For the latter variant (c.822G>T), we conducted a minigene splice assay that demonstrated the incorporation of a premature stop codon. This finding suggests a likely activation of the nonsense-mediated mRNA decay mechanism, ultimately resulting in the absence of protein production from this allele. Segregation analysis confirmed that these variants were in trans. Discussion: Our data support that individuals with biallelic TULP1 variants may present with a unique pattern of macular degeneration and periarteriolar vascular pigmentation. This study highlights the importance of further clinical and molecular characterization of TULP1 variants to elucidate genotype-phenotype correlations in the context of inherited retinal dystrophies.

Cancer-associated retinopathy (CAR) is a potentially blinding disease triggered by autoimmunity to cancer antigens at distant sites. It may masquerade as immune-related adverse events from the use of immune checkpoint inhibitors (ICIs). We present a patient with an underlying tubby-related protein 1 (TULP1) cancer-associated retinopathy who lost vision following initiation of atezolizumab for small-cell lung cancer. This 75-year-old man presented with no light perception, paramacular and peripheral retinal pigmentary changes, attenuated outer retina, and extinguished rod and cone responses. The visual loss followed the induction of atezolizumab therapy. Possible atezolizumab-associated acute macular neuroretinopathy was considered, and atezolizumab was discontinued. Vision improved on oral corticosteroid and deteriorated when corticosteroid was tapered quickly. Retinal autoantibody serology testing was negative for both anti-recoverin and anti-enolase and was positive for anti-TULP1 autoantibodies. Re-induction of atezolizumab concomitant with high-dose oral and intravitreal corticosteroids resulted in visual recovery at the three-month follow-up. These findings suggest that ICI therapy for cancer can exacerbate the retinal dysfunction in a patient with underlying autoimmunity from cancer. Patients with a high risk of CAR may need to be evaluated for retinal autoantibodies before initiation of ICI.

Photoreceptors are highly compartmentalized cells with large amounts of proteins synthesized in the inner segment (IS) and transported to the outer segment (OS) and synaptic terminal. Tulp1 is a photoreceptor-specific protein localized to the IS and synapse. In the absence of Tulp1, several OS-specific proteins are mislocalized and synaptic vesicle recycling is impaired. To better understand the involvement of Tulp1 in protein trafficking, our approach in the current study was to physically isolate Tulp1-containing photoreceptor compartments by serial tangential sectioning of retinas and to identify compartment-specific Tulp1 binding partners by immunoprecipitation followed by liquid chromatography tandem mass spectrometry. Our results indicate that Tulp1 has two distinct interactomes. We report the identification of: (1) an IS-specific interaction between Tulp1 and the motor protein Kinesin family member 3a (Kif3a), (2) a synaptic-specific interaction between Tulp1 and the scaffold protein Ribeye, and (3) an interaction between Tulp1 and the cytoskeletal protein microtubule-associated protein 1B (MAP1B) in both compartments. Immunolocalization studies in the wild-type retina indicate that Tulp1 and its binding partners co-localize to their respective compartments. Our observations are compatible with Tulp1 functioning in protein trafficking in multiple photoreceptor compartments, likely as an adapter molecule linking vesicles to molecular motors and the cytoskeletal scaffold.

Photoreceptor disc component (PRCD) is a small protein which is exclusively localized to photoreceptor outer segments, and is involved in the formation of photoreceptor outer segment discs. Mutations in PRCD are associated with retinal degeneration in humans, mice, and dogs. The purpose of this work was to identify PRCD-binding proteins in the retina. PRCD protein-protein interactions were identified when implementing the Ras recruitment system (RRS), a cytoplasmic-based yeast two-hybrid system, on a bovine retina cDNA library. An interaction between PRCD and tubby-like protein 1 (TULP1) was identified. Co-immunoprecipitation in transfected mammalian cells confirmed that PRCD interacts with TULP1, as well as with its homolog, TUB. These interactions were mediated by TULP1 and TUB highly conserved C-terminal tubby domain. PRCD localization was altered in the retinas of TULP1- and TUB-deficient mice. These results show that TULP1 and TUB, which are involved in the vesicular trafficking of several photoreceptor proteins from the inner segment to the outer segment, are also required for PRCD exclusive localization to photoreceptor outer segment discs.

Tubby-like proteins (TULPs) are characterized by a conserved C-terminal domain that binds phosphoinositides. Collectively, mammalian TULP1-4 proteins play essential roles in intracellular transport, cell differentiation, signaling, and motility. Yet, little is known about how the function of these proteins is regulated in cells. Here, we present the protein-protein interaction network of TULP3, a protein that is responsible for the trafficking of G-protein-coupled receptors to cilia and whose aberrant expression is associated with severe developmental disorders and polycystic kidney disease. We identify several protein interaction nodes linked to TULP3 that include enzymes involved in acetylation and ubiquitination. We show that acetylation of two key lysine residues on TULP3 by p300 increases TULP3 protein abundance and that deacetylation of these sites by HDAC1 decreases protein levels. Furthermore, we show that one of these sites is ubiquitinated in the absence of acetylation and that acetylation inversely correlates with ubiquitination of TULP3. This mechanism is evidently conserved across species and is active in zebrafish during development. Finally, we identify this same regulatory module in TULP1, TULP2, and TULP4 and demonstrate that the stability of these proteins is similarly modulated by an acetylation switch. This study unveils a signaling pathway that links nuclear enzymes to ciliary membrane receptors via TULP3, describes a dynamic mechanism for the regulation of all tubby-like proteins, and explores how to exploit it pharmacologically using drugs.

With marketing approval of the first ocular gene therapy, and other gene therapies in clinical trial, treatments for inherited retinal degenerations (IRDs) have become a reality. Biallelic mutations in the tubby like protein 1 gene (TULP1) are causative of IRDs in humans; a mouse knock-out model (Tulp1-/-) is characterized by a similar disease phenotype. We developed a Tulp1 supplementation therapy for Tulp1-/- mice. Utilizing subretinal AAV2/5 delivery at postnatal day (p)2-3 and rhodopsin-kinase promoter (GRK1P) we targeted Tulp1 to photoreceptor cells exploring three doses, 2.2E9, 3.7E8, and 1.2E8 vgs. Tulp1 mRNA and TULP1 protein were assessed by RT-qPCR, western blot and immunocytochemistry, and visual function by electroretinography. Our results indicate that TULP1 was expressed in photoreceptors; achieved levels of Tulp1 mRNA and protein were similar to wild type levels at p20. However, the thickness of the outer nuclear layer (ONL) did not improve in treated Tulp1-/- mice. There was a small and transient electroretinography benefit in the treated retinas at 4 weeks of age (not observed by 6 weeks) when using 3.7E8 vg dose. Dark-adapted mixed rod and cone a- and b-wave amplitudes were 24.3 ± 13.5 μV and 52.2 ± 31.7 μV in treated Tulp1-/- mice, which were significantly different (p < 0.001, t-test), from those detected in untreated eyes (7.1 ± 7.0 μV and 9.4 ± 15.1 μV, respectively). Our results indicate that Tulp1 supplementation in photoreceptors may not be sufficient to provide robust benefit in Tulp1-/- mice. As such, further studies are required to fine tune the Tulp1 supplementation therapy, which, in principle, should rescue the Tulp1-/- phenotype.

Mutations in tubby like protein 1 gene (TULP1) are causative of early-onset recessive inherited retinal degenerations (IRDs); similarly, the Tulp1-/- mouse is also characterized by a rapid IRD. Tulp1 mRNA and protein expression was analyzed in wild type mouse retinas and expression data sets (NCBI) during early postnatal development. Comparative histology was undertaken in Tulp1-/-, rhodopsin-/- (Rho-/-) and retinal degeneration slow-/- (Rds-/-) mouse retinas. Bioinformatic analysis of predicted TULP1 interactors and IRD genes was performed. Peak expression of Tulp1 in healthy mouse retinas was detected at p8; of note, TULP1 was detected in both the outer and inner retina. Bioinformatic analysis indicated Tulp1 expression in retinal progenitor, photoreceptor and non-photoreceptor cells. While common features of photoreceptor degeneration were detected in Tulp1-/-, Rho-/-, and Rds-/- retinas, other alterations in bipolar, amacrine and ganglion cells were specific to Tulp1-/- mice. Additionally, predicted TULP1 interactors differed in various retinal cell types and new functions for TULP1 were suggested. A pilot bioinformatic analysis indicated that in a similar fashion to Tulp1, many other IRD genes were expressed in both inner and outer retinal cells at p4-p7. Our data indicate that expression of Tulp1 extends to multiple retinal cell types; lack of TULP1 may lead to primary degeneration not only of photoreceptor but also non-photoreceptor cells. Predicted interactors suggest widespread retinal functions for TULP1. Early and widespread expression of TULP1 and some other IRD genes in both the inner and outer retina highlights potential hurdles in the development of treatments for these IRDs.

Due to improved phenotyping and genetic characterization, the field of \'incurable\' and \'blinding\' inherited retinal diseases (IRDs) has moved substantially forward. Decades of ascertainment of IRD patient data from Philadelphia and Toronto centers illustrate the progress from Mendelian genetic types to molecular diagnoses. Molecular genetics have been used not only to clarify diagnoses and to direct counseling but also to enable the first clinical trials of gene-based treatment in these diseases. An overview of the recent reports of gene augmentation clinical trials by subretinal injections is used to reflect on the reasons why there has been limited success in this early venture into therapy. These first-in human experiences have taught that there is a need for advancing the techniques of delivery of the gene products - not only for refining further subretinal trials, but also for evaluating intravitreal delivery. Candidate IRDs for intravitreal gene delivery are then suggested to illustrate some of the disorders that may be amenable to improvement of remaining central vision with the least photoreceptor trauma. A more detailed understanding of the human IRDs to be considered for therapy and the calculated potential for efficacy should be among the routine prerequisites for initiating a clinical trial.

Early-onset photoreceptor dystrophies are a major cause of irreversible visual impairment in children and young adults. This clinically heterogeneous group of disorders can be caused by mutations in many genes. Nevertheless, to date, 30%-40% of cases remain genetically unexplained. In view of expanding therapeutic options, it is essential to obtain a molecular diagnosis in these patients as well. In this study, we aimed to identify the genetic cause in two siblings with genetically unexplained retinal disease.
Whole exome sequencing was performed to identify the causative variants in two siblings in whom a single pathogenic variant in TULP1 was found previously. Patients were clinically evaluated, including assessment of the medical history, slit-lamp biomicroscopy, and ophthalmoscopy. In addition, a functional analysis of the putative splice variant in TULP1 was performed using a midigene assay.
Clinical assessment showed a typical early-onset photoreceptor dystrophy in both the patients. Whole exome sequencing identified two pathogenic variants in TULP1, a c.1445G>A (p.(Arg482Gln)) missense mutation and an intronic c.718+23G>A variant. Segregation analysis confirmed that both siblings were compound heterozygous for the TULP1 c.718+23G>A and c.1445G>A variants, while the unaffected parents were heterozygous. The midigene assay for the c.718+23G>A variant confirmed an elongation of exon 7 leading to a frameshift.
Here, we report the first near-exon RNA splice variant that is not present in a consensus splice site sequence in TULP1, which was found in a compound heterozygous manner with a previously described pathogenic TULP1 variant in two patients with an early-onset photoreceptor dystrophy. We provide proof of pathogenicity for this splice variant by performing an in vitro midigene splice assay, and highlight the importance of analysis of noncoding regions beyond the noncanonical splice sites in patients with inherited retinal diseases.

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy presenting remarkable genetic heterogeneity. Genetic annotations would help with better clinical assessments and benefit gene therapy, and therefore should be recommended for RP patients. This report reveals the disease causing mutations in two RP pedigrees with confusing inheritance patterns using whole exome sequencing (WES).
Twenty-five participants including eight patients from two families were recruited and received comprehensive ophthalmic evaluations. WES was applied for mutation identification. Bioinformatics annotations, intrafamilial co-segregation tests, and in silico analyses were subsequently conducted for mutation verification.
All patients were clinically diagnosed with RP. The first family included two siblings born to parents with consanguineous marriage; however, no potential pathogenic variant was found shared by both patients. Further analysis revealed that the female patient carried a recurrent homozygous C8ORF37 p.W185*, while the male patient had hemizygous OFD1 p.T120A. The second family was found to segregate mutations in two genes, TULP1 and RP1. Two patients born to consanguineous marriage carried homozygous TULP1 p.R419W, while a recurrent heterozygous RP1 p.L762Yfs*17 was found in another four patients presenting an autosomal dominant inheritance pattern. Crystal structural analysis further indicated that the substitution from arginine to tryptophan at the highly conserved residue 419 of TULP1 could lead to the elimination of two hydrogen bonds between residue 419 and residues V488 and S534. All four genes, including C8ORF37, OFD1, TULP1 and RP1, have been previously implicated in RP etiology.
Our study demonstrates the coexistence of diverse inheritance modes and mutations affecting distinct disease causing genes in two RP families with consanguineous marriage. Our data provide novel insights into assessments of complicated pedigrees, reinforce the genetic complexity of RP, and highlight the need for extensive molecular evaluations in such challenging families with diverse inheritance modes and mutations.