ExperimentName: Murine model of PDE6A loss-of-function (Pde6Anmf363/nmf363)

NonHumanModel: Mus musculus

GeneAlteration: Mice were found to develop retinal degeneration due to a homozygous missense variant (2159G>A) at codon 670 substituting glycine for aspartic acid.

ModelPhenotype: HP:0030609 (Photoreceptor layer loss on macular OCT), HP:0030478 (Abnormal amplitude of dark-adapted bright flash electroretinogram)

HumanPhenotype: HP:0011463 (Childhood onset), HP:0000662 (Nyctalopia), HP:0007994 (Peripheral visual field loss), HP:0000543 (Optic disc pallor), HP:0007843 (Attenuation of retinal blood vessels), HP:0007737 (Bone spicule pigmentation of the retina), HP:0000654 (Decreased light- and dark-adapted electroretinogram amplitude), HP:0007984 (Electronegative electroretinogram)

ModelEvidence: This mutation caused photoreceptor outer nuclear layer outer segment thinning (Figures 2C, 2D, 7), decreased dark-adapted bright flash ERG amplitude (Figure 2E), and decreased cGMP/PDE activity (Figure 3C).

ModelVariant: The model does not harbor a variant equivalent to one from the human patients, but rather has a mutagenesis-induced missense variant disrupting Pde6a leading to partial loss-of-function.

ExperimentName: Targeted disruption of Lrp5 in mice

NonHumanModel: Mus musculus

GeneAlteration: Model animals were subjected to targeted disruption of Lrp5 by inserting an IRES-LacZ-Neomycin cassette to replace the region encoding the extracellar domain, starting at amino acid 373.

ModelPhenotype: HP:0004349 (Reduced bone mineral density), HP:0410280 (Pediatric onset), decreased osteoblast proliferation, HP:0007968 (Remnants of the hyaloid vascular system), failure of macrophage-mediated endothelial cell apoptosis

HumanPhenotype: HP:0000939 (Osteoporosis), HP:0011463 (Childhood onset), HP:0000541 (Retinal detachment), HP:0007968 (Remnants of the hyaloid vascular system), HP:0008046 (Abnormal retinal vascular morphology), HP:0000618 (Blindness), HP:0001249 (Intellectual disability), HP:0020110 (Bone fracture), HP:0000938 (Osteopenia), HP:0000926 (Platyspondyly)

ModelEvidence: Model animals subjected to targeted disruption of Lrp5 resembled human patients in that they exhibited reduced bone mineral density in the post-natal stage (Figs. S2, 3A), decreased osteoblast proliferation (Fig. 4), delayed ossification (Fig. 5), and remnants of the hyaloid vascular system due to failure of macrophage-mediated endothelial cell apoptosis (Fig. 8).

ModelVariant: The model features disruption of the wild-type Lrp5 gene, but not a mutation equivalent to a human variant.

Selection A - Gene is expressed in tissues relevant to disease:

ExperimentName: RPE65 expression is highly specific to the retinal pigment epithelium

TissueExpression: pigmented layer of retina (also known as retinal pigment epithelium, UBERON:0001782)

ExpressionEvidence: RPE65 protein was first detected using an RPE-specific monocolonal antibody raised against human retinal pigment epithelium (RPE) cells. Retina-specific gene expression data from a separate publication (PMID: 30239781, https://eyeintegration.nei.nih.gov/) confirm that RPE65 expression is highly specific to the retina relative to other tissues within the eye or the human body in general. Note where the evidence is found in the article.

ExperimentName: GPR143 over-expression in neuroendocrine tumor cells

NonPatientCell: CL:0000336 (adrenal medulla chromaffin cell, specifically the PC-12 cell line)

GeneFunction: GO:0031175 (neuron projection development)

GeneAlteration: Over-expression of GPR143 was accomplished by stable transfection of PC-12 cells with mCherry-tagged wild-type or mutant GPR143.

FunctionalEvidence: Figure 4C indicates that GPR143 over-expression decreases the # of neurites longer than 100 microns, and that this occurs through a mechanism that is dependent on the G-protein Galpha13. Figure 5E shows that this effect of wild-type GPR143 is particularly pronounced for neurites longer than 150 microns, and is not mimicked by GPR143 missense variants reported in patients.

FAVariants: Patient-associated variants were p.Asp78Asn (ClinVar: 98620), p.Trp133Arg (ClinVar: 10516 or 10519), p.Ile261Asn (CAID: CA411996177), and p.Trp292Gly (ClinVar: 98647).

Selection B - Gene function is consistent with phenotype.

GOterm: GO:0007168 (receptor guanylyl cyclase signaling pathway)

FunctionalEvidence: Exogenous expression of the gene product in 293 cells exhibits a high level of cGMP production activity. When combined with the in situ hybridization evidence showing its expression along the retinal outer nuclear layer (coinciding with the rod and cone photoreceptor cell nuclei), this biochemical evidence indicates a function in synthesizing cGMP to promote recovery of the dark state following phototransduction.

FunctionalHPO: HP:0000662 (Nyctalopia), HP:0007663 (Reduced visual acuity), HP:0007688 (Undetectable light- and dark-adapted electroretinogram), HP:0007703 (Abnormality of retinal pigmentation)

PhenotypeEvidence: The ability of the exogenously expressed enzyme to efficiently catalyze cGMP production (Figure) is consistent with the loss of photoreceptor function that is characteristic of the disease, as abnormalities in cGMP levels have long been associated with photoreceptor degeneration (PMID: 4369896).

ExperimentName: Retinal dystrophy in dogs is caused by Rpe65 loss of function.

NonHumanModel: Canis familiaris

GeneAlteration: Swedish Briard / Briard Beagle dogs exhibit a biallelic 485delAAGA variant in the RPE65 locus that triggers a frameshift predicted to disrupt ~71% of the protein product.

ModelPhenotype: HP:0007642 (Congenital stationary night blindness), HP:0007663 (Reduced visual acuity), HP:0000618 (Blindness), HP:0007703 (Abnormality of retinal pigmentation), HP:0000556 (Retinal dystrophy), HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), HP:0007984 (Electronegative electroretinogram), HP:0030479 (Abnormal amplitude of light-adapted flicker electroretinogram)

HumanPhenotype: HP:0000662 (Nyctalopia), HP:0000529 (Progressive visual loss), HP:0000618 (Blindness), HP:0030479 (Abnormal amplitude of light-adapted flicker electroretinogram), HP:0030474 (Undetectable dark-adapted electroretinogram)

ModelEvidence: The model system recapitulates the human phenotype in that the dogs exhibit severe vision loss (Table 1 in PMID: 2804031, describing the same animal model), structural abnormalities of the RPE (PMID: 7851218, studying the same animal model), and similar ERG abnormalities (Figures 2 and 3 in PMID: 2804031). Similar to the human patient families, the heterozygous individuals are unaffected (Figure 1 in this publication).

ModelVariant: The dogs harbor a spontaneously occurring frameshift variant in exon 5 of 14 (485delAAGA), rather than a targeted mutation equivalent to a particular human patient variant.

ExperimentName: Rpe65 homozygous knockout abolishes rod photoreceptor function and disrupts RPE metabolism in mice.

NonHumanModel: Mus musculus

GeneAlteration: Rpe65 homozygous knockout was generated by replacing the 5' flanking region and the first three exons of Rpe65 with a PGK-Neo cassette.

ModelPhenotype: HP:0000479 (Abnormal retinal morphology), HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), HP:0007984 (Electronegative electroretinogram)

HumanPhenotype: HP:0000662 (Nyctalopia), HP:0007688 (Undetectable light- and dark-adapted electroretinogram), HP:0001098 (Abnormal fundus morphology), HP:0000529 (Progressive visual loss)

ModelEvidence: The model system recapitulates the human phenotype in that the mice exhibit progressive onset of abnormalities in retinal morphology (specifically photoreceptor outer segment loss, Figures 2a-2e), accompanied by reduced responses in dark-adapted (Figure 4) but not light-adapted (Figure 5) ERG. These ERG results are characteristic of abolished rod function. The mice also recapitulate the molecular features of the human patients in that retinal esters accumulate in the retinal pigment epithelium of the mice (HPLC data in Figures 8a-8c).

ModelVariant: The model disrupts the entire Rpe65 locus rather than introducing a mutation equivalent to a human patient variant.

ExperimentName: PRPF4 physically interacts with PRPF3.

InteractingGene: PRPF3 (HGNC:17348)

InteractionType: Physical

InteractionMethod: Co-immunoprecipitation

InteractionEvidence: Immunoprecipitation of endogenous PRPF3 from HeLa cell nuclear extracts pulled down PRPF4 (Figures 5 and 6).

Selection B- Gene function is consistent with phenotype.

ExperimentName: OPN1LW encodes a long-wave-sensitive opsin and can rescue cone-mediated function when subretinally injected in Opn1mw−/−/Opn1sw−/− mice.

GOterm: GO:0007602 (phototransduction)

FunctionalEvidence: Subretinal injection of AAV carrying the OPN1LW gene encoding human L-opsin into mice at age 2 months or younger promoted cone outer segment regeneration () and rescued cone functions such as the b-wave amplitude of the long wavelength-mediated electroretinogram response (Figures 2A, 2B, 4D) and visually guided behavior (Figure 4E).

FunctionalHPO: Protanopia HP:0011522

ExperimentName: "Prpf4 silencing in zebrafish causes defects in visual function."

NonHumanModel: Danio rerio

GeneAlteration: The model animals have been subjected to morpholino-based knockdown of Prpf4, the zebrafish ortholog of human PRPF4.

ModelPhenotype: Nystagmus HP:0000639, Abnormality of ocular smooth pursuit HP:0000617

HumanPhenotype: Constriction of peripheral visual field HP:0001133, Reduced visual acuity HP:0007663,, Decreased light- and dark-adapted electroretinogram amplitude HP:0000654. Retinal degeneration HP:0000546, Macular degeneration HP:0000608, Optic disc pallor HP:0000543, Attenuation of retinal blood vessels HP:0007843, Pigmentary retinopathy HP:0000580

ModelEvidence: Morpholino-based incomplete knockdown of the Prpf4 gene circumvents the lethality of complete silencing and causes optokinetic nystagmus with a significantly reduced number of saccades (Fig. 5B, 5C, 5D). Immunohistochemistry showed that retinal morphology showed mostly normal retinal layering but decreased rhodopsin staining in the photoreceptor cell layer (Figs. 5E–5H). Photoreceptor outer segments were primarily affected, but with unaffected cell body morphology (Figs. 5I and 5J). This phenotype was a complete match for the zebrafish with Prpf31 loss/. Semi-quantitative RT–PCR analysis of the RP-relevant transcripts opn1lw1, opn1mw1, gnat2, irx6a, rx3, crx and rhodopsin as well as the controls gapdh and lsm7 showed a very similar pattern of down-regulation in both Prpf31- and Prpf4-deficient animals (Fig. 5W). The phenotype was dose-dependent and could be rescued by co-injection of Prpf4 mRNA (Fig. S2).

ModelVariant: This model did not introduce a disease-associated variant known from an affected human patient, but rather studied the effects of heterozygous loss of the gene product.

ExperimentName: Rescue of mouse Gucy2e homozygous knockout model by AAV-borne human GUCY2D transgene

NonHumanModel: Mus musculus

GeneAlteration: The mouse Gucy2e gene was disrupted by the introduction of a neomycin-resistance gene cassette into exon 5, resulting in a truncated protein lacking its transmembrane domain (PMID: 10407028). The effects of this knockout were studied in the homozygous state.

RescueVariant: Rescue was performed with the wild-type human GUCY2D and mouse Gucy2e genes but not with a patient-derived variant.

RescuePhenotype: HP:0030465 (Undetectable light-adapted electroretinogram), HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0000479 (Abnormal retinal morphology), HP:0008020 (Cone dystrophy)

RescueMethod: Human GUCY2D or mouse Gucy2e was cloned into an overexpression construct under the control of the human rhodopsin kinase promoter and packaged into adeno-associated (AAV2/8) viral particles for 2 subretinal injections on postnatal day 10. Efficacy was measured by ERG recordings every two weeks.

RescueEvidence: Treatment with the human GUCY2D gene restored proper localization of alpha-transducin (Figs. 3a-3b), which was first reported by PMID: 15623748 to be abnormal in this model. Treatment corrected the defect in scotopic b-wave amplitude by 65-100% (Figs. 4a-4b). Cone amplitude was corrected from 20% of wild-type to 65% of wild-type (Fig. 4c). Similar results were obtained with the mouse Gucy2e transgene (Fig. 5). Head-tracking behavior (measure of visual acuity and contrast sensitivity) was fully corrected by treatment (Fig. 6). Moderate cone preservation was also demonstrated by normalization of alpha-transducin staining, showing a 20% increase in cone numbers relative to the untreated control (Fig. 7r).

ModelVariant: The mouse model disrupts the expression of the endogenous GC1 protein product but does not feature knock-in of a variant from a human patient.

ExperimentName: Knockout mouse model of Impg1

NonHumanModel: Mus musculus

GeneAlteration: The mouse model harbors a deletion of exon 6, resulting in disruption of reading frame and loss of Impg1 protein either in the heterozygous or homozygous state.

ModelPhenotype: HP:0025158 (Hyperautofluorescent retinal lesion), HP:0000512 (Abnormal electroretinogram, specifically attenuated responses), HP:0030609 (Photoreceptor layer loss on macular OCT, described as degeneration with loss of 11-cis-retinal signal), HP:0007703 (Abnormality of retinal pigmentation, specifically disruption of RPE cells and the IPM)

HumanPhenotype: HP:0007754 (Macular dystrophy), HP:0007769 (Peripheral retinal degeneration), HP:0032037 (Mildly reduced visual acuity, progressive), HP:0008179 (Decreased Arden ratio of electrooculogram), HP:0030505 (Nummular pigmentation of the fundus)

ModelEvidence: The model exhibits phenotypes consistent with a critical role of Impg1 in photoreceptor maintenance. Phenotypes included hyperpigmented and sometimes autofluorescent subretinal deposits that were present at 9 months of age (Figure 2B) and increased by 14 months of age (Figure 2C) that were localized between the photoreceptor outer segment and the RPE (Figure 2D). Some images showed nummular pigmentation of the fundus (Figure 2E). Interestingly, attenuation of ERG response was first noted at 9 months and pronounced at 14 months (Figure 3A), which is consistent with the findings from an independent mouse model that showed less dramatic findings at earlier 5 month and 8 month time points (PMID: 32265257). Scotopic ERG was more affected than photopic ERG (Figure 3B). Retinal thickness was also reduced in the outer layer relative to the inner layer (Figure 3C). Retinoid generation was also progressively abnormal (Figure 4), indicating that photoreceptors might be lost. The IPM was also disorganized at 14 months (Figure 5), with cellular material accumulating in the IPM consistent with cell death (Figure 6).

ModelVariant: The model harbors a knockout allele disrupting Impg1, rather than a specific variant from a human patient.

ExperimentName: BBS7 interacts with other components of the BBSome.

InteractingGene: BBS4 (HGNC:969)

InteractionType: Physical

InteractionMethod: co-immunoprecipitation

InteractionEvidence: Antibody-based isolation of BBS4 results in co-purification of BBS7, as well as BBS1, BBS2, BBS5, BBS8, and BBS9 in stoichiometric quantities. The co-purifying proteins are shown separated by gel electrophoresis in Figure 1F and were identified and quantified by mass spectrometry in Table S1. All 8 proteins are encoded by genes linked to Bardet-Biedl syndrome.

Selection A- Gene is expressed in tissues relevant to disease:

ExperimentName: Wild-type Prph2 expression in the developing mouse retina begins on day postnatal day 6.

TissueExpression: photoreceptor outer segment layer (UBERON:0003926)

ExpressionEvidence: Immunohistochemistry indicates that wild-type Prph2 expression in the developing mouse retina begins on day postnatal day 6 (Figure 3C). This time point coincides with the timing of the formation of the photoreceptor outer segment discs, and is consistent with a proposed function of PRPH2 as an adhesion molecule that acts to stabilize the outer segment discs.

Selection B - Gene function is consistent with phenotype.

Title: LRP5 is a canonical Wnt signaling pathway co-receptor.

GOterm: GO:0060070 (canonical Wnt signaling pathway)

FunctionalEvidence: Exogenous LRP5 expression in NIH3T3 cells increases the activity of the canonical Wnt signaling transcription factor LEF1 only when combined with exogenous expression of the Wnt-1 ligand in a separate population of cells grown in co-culture (Figure 1). This function is related to the phenotype in that canonical Wnt signaling is known to mediate bone formation.

FunctionalHPO: HP:0004349 (reduced bone mineral density), osteoporosis (HP:0000939), HP:0020110 (bone fracture)

ExperimentName: CRISPR/Cas9-based rescue of patient variant in the mouse RPE

RescueModel: Mus musculus

GeneAlteration: The Kcnj13W53X/+ΔR model has been generated from Kcnj13W53X/+ mice by retinal pigment epithelium-specific introduction of the variant encoding the p.Trp53Ter nonsense variant.

RescueVariant: Rescue was performed by editing the variant to restore wild-type KCNJ13, not by introducing an additional human variant.

RescuePhenotype: The rescued phenotypes are full-field electroretinogram responses measured at week 10 post-injection and retinal thickness by optical coherence tomography.

RescueMethod: This rescue model has subretinally injected sgRNA to edit the W53X allele and restore the wild-type by CRISPR/Cas9 base editing.

RescueEvidence: The ERG c-wave from the RPE layer shows 55% higher amplitude for the edited/targeted retina relative to the non-targeted control (Figures 6J and 6K) and is accompanied by intact retinal structure by OCT (Figures 6H and 6I).

ModelVariant: NM_002242.4(KCNJ13):c.158G>A (p.Trp53Ter), ClinVarID: 224857

ExperimentName: Homozygous disruption of the chicken ortholog of GUCY2D

NonHumanModel: Gallus gallus domesticus

GeneAlteration: The null allele of the chicken Rd gene (orthologous to human GUCY2D) exhibits a rearrangement in which a 723-bp region corresponding to exons 4-7 has been replaced by an in-frame insertion of 81-bp fragment likely derived from exon 9. This deletes the transmembrane domain and is predicted to result in complete inactivation of the enzyme. This variant has been studied in the homozygous state.

ModelPhenotype: HP:0007875 (Congenital blindness), HP:0000654 (Decreased light- and dark-adapted electroretinogram amplitude), Optokinetic reflex,

HumanPhenotype: HP:0007663 (Reduced visual acuity), HP:0007688 (Undetectable light- and dark-adapted electroretinogram), HP:0007703 (Abnormality of retinal pigmentation)

ModelEvidence: Chickens harboring the null variant in a homozygous state have dramatically lower levels of cGMP in the retina (Figure 1) and absent expression of GC1, the GUCY2D ortholog (Figure 2). Rod and cone photoreceptor cells develop normally, but have little to no function according to ERG. This is similar to the normal fundus appearance in LCA patients, despite severe early vision loss.

ModelVariant: The mouse model disrupts the expression of the endogenous GC1 protein product but does not feature knock-in of a variant from a human patient.

ExperimentName: Updated phenotypes from homozygous Gucy2e mouse knockout model

NonHumanModel: Mus musculus

GeneAlteration: The mouse Gucy2e gene was disrupted by the introduction of a neomycin-resistance gene cassette into exon 5, resulting in a truncated protein lacking its transmembrane domain (PMID: 10407028). The effects of this knockout were studied in both heterozygous and homozygous states.

ModelPhenotype: HP:0030465 (Undetectable light-adapted electroretinogram), HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0000479 (Abnormal retinal morphology)

HumanPhenotype: HP:0007663 (Reduced visual acuity), HP:0007688 (Undetectable light- and dark-adapted electroretinogram), HP:0007703 (Abnormality of retinal pigmentation)

ModelEvidence: This publication confirms previously-reported ERG abnormalities of rod and especially cone photoreceptors. The abnormal retinal morphology HPO term refers to abnormalities in the ultrastructure of the photoreceptor outer segment, which exhibited disorganization (Figures S2B, S2F, 8C, and 8E) and low GCAP1 and GCAP2 expression levels (Figures 5J and 5N), providing early signs of instability and degeneration.

ModelVariant: The mouse model disrupts the expression of the endogenous GC1 protein product but does not feature knock-in of a variant from a human patient.

ExperimentName: This macaque model harbors a homozygous loss-of-function variant in PDE6C.

NonHumanModel: Macaca mulatta

GeneAlteration: The model animals are homozygous for a missense variant, p.Arg565Gln, which is shown to cause a decrease in enzymatic activity.

ModelPhenotype: HP:0007663 (Reduced visual acuity, particularly in well illuminated settings where unaffected animals led them by the hand), HP:0001483 (Eye poking), HP:0030465 (Undetectable light-adapted electroretinogram), normal dark-adapted ERG, HP:0007401 (Macular atrophy), HP:0011504 (Bull's eye maculopathy), HP:0030619 (Reduced OCT-measured foveal thickness), HP:0030610 (Photoreceptor outer segment loss on macular OCT), HP:0030609 (Photoreceptor layer loss on macular OCT)

HumanPhenotype: HP:0030465 (Undetectable light-adapted electroretinogram), HP:0030534 (Abnormal best corrected visual acuity test), HP:0000551 (Color vision defect), HP:0000613 (Photophobia), HP:0000639 (Nystagmus), HP:0007401 (Macular atrophy), HP:0000545 (Myopia)

ModelEvidence: This is a naturally occurring macaque model that matches the autosomal recessive mode of inheritance from the human patients (Figure 7) and exhibits reduced visual acuity (Supplemental Video 1), lack of light-adapted ERG responses and normal dark-adapted ERG responses (Figures 1A-1E, Figure 2). The affected animals also exhibit progressive macular atrophy consistent with achromatopsia (Figure 3). Reduced foveal thickness was also observed (Figures 4A, 4B), as well as thinning of the outer nuclear layer and photoreceptor outer segments (Figures 5A, 5B).

ModelVariant: This model did not introduce a disease-associated variant known from an affected human patient, but rather studied a naturally occurring variant disrupting the PDE6C locus.

ExperimentName: CRISPR/Cas9-based disruption of mouse Impg2

NonHumanModel: Mus musculus

GeneAlteration: Model animals were subjected to CRISPR/Cas9-based deletion of 7bp from exon 3 of Impg2, and analyzed in the homozygous state. Absence of Impg2 signal was confirmed by immunofluorescent staining. Mice were studied at ages 5 months and 8 months.

ModelPhenotype: HP:0030612 (Abnormal retinal morphology, specifically mislocalization / scattering of IMPG1 throughout the outer segment and at the boundary with the RPE), HP:0030643 (Vitelliform-like subretinal lesions), HP:0000654 (Decreased light- and dark-adapted electroretinogram amplitude)

HumanPhenotype: HP:0000529 (Progressive visual loss), HP:0000662 (Nyctalopia), HP:0000551 (Color vision defect), HP:0008002 (Abnormality of macular pigmentation), HP:0000543 (Optic disc pallor), HP:0007843 (Attenuation of retinal blood vessels), HP:0000533 (Chorioretinal atrophy), HP:0000518 (Cataract), HP:0008323 (Abnormal light- and dark-adapted electroretinogram), HP:0007737 (Bone spicule pigmentation of the retina), HP:0007994 (Peripheral visual field loss), HP:0000603 (Central scotoma)

ModelEvidence: This is the first mouse model of Impg2 disruption, with confirmed absence of Impg2 in the homozygous animals. Abnormalities included mislocalization of Impg1 and the cone-specific marker Pna (Figure 2), accumulation of anomalous material between the photoreceptor outer segment and RPE (Figure 3), development of subretinal hyper-reflective vitelliform lesions (Figure 4), and reduction in the amplitudes of ERG responses from both rods (Figure 5A) and cones (Figure 5B).

ModelVariant: The model does not introduce a variant previously observed in human patients, but rather disrupts the expression of the Impg2 ortholog.

ExperimentName: CRISPR/Cas9-based disruption of mouse Impg1

NonHumanModel: Mus musculus

GeneAlteration: Model animals were subjected to CRISPR/Cas9-based deletion of 10bp from exon 2 of Impg1, and analyzed in the homozygous state. Absence of Impg1 signal was confirmed by immunofluorescent staining. Mice were studied at ages 5 months and 8 months.

ModelPhenotype: HP:0030612 (Abnormal retinal morphology, specifically mislocalization of chondroitin sulfate in the IPM, with none in the outer segment and more in the inner segment)

HumanPhenotype: HP:0007754 (Macular dystrophy), HP:0007769 (Peripheral retinal degeneration), HP:0032037 (Mildly reduced visual acuity, progressive), HP:0008179 (Decreased Arden ratio of electrooculogram)

ModelEvidence: This is the first mouse model of Impg1 disruption, with confirmed absence of Impg1 in the homozygous animals. The only observed abnormality is mislocalization of chondroitin sulfate in the IPM, with none in the outer segment and more in the inner segment (Figure 7). A mild disruption was observed in the localization of PNA, a cone-specific marker, to the outer segment (Fig. 2F). Impg2 appears to be normally localized (Figure 2C). ERG responses were normal (data not shown). Please note that an independent study generating a different model of homozygous Impg1 disruption reported different findings (PMID: 36140676).

ModelVariant: The model does not introduce a variant previously observed in human patients, but rather disrupts the expression of the Impg1 ortholog.

Selection A - Gene is expressed in tissues relevant to disease:

ExperimentName: High GUCY2D mRNA expression in human retina relative to other tissue types

TissueExpression: Retina (UBERON:0000966)

ExpressionEvidence: Evidence can be accessed on the publication-associated website (https://eyeintegration.nei.nih.gov/) through the Pan-Tissue Plots feature.

ExperimentName: Colorblind squirrel monkeys were rescued by human OPN1LW.

NonHumanModel: Saimiri sciureus

GeneAlteration: Male squirrel monkeys were missing OPN1LW gene. Controls with the intact gene were not part of the study.

RescueVariant: Rescue was performed by subretinal injection of recombinant adeno-associated virus containing human OPN1LW under control of the appropriate enhancer and promoter and featured the wild-type gene rather than a variant.

RescuePhenotype: The rescued phenotypes are inability to discriminate between blue and green wavelengths to obtain a food reward and defects in performance on a computer-administered Cambridge colour test.

RescueMethod: This rescue model has injection of recombinant adeno-associated virus containing human wild-type OPN1LW under control of the appropriate enhancer and promoter.

RescueEvidence: Squirrel monkeys gained the ability to discriminate between blue and green wavelengths to obtain a food reward (data not shown), as well as significant improvement in ability to distinguish colors occurring at ~20 weeks post-injection (Figure 3A). Gaps in the ability to detect certain wavelengths pre-injection (solid lines) were filled in post-injection (dotted lines) in monkey 1 (Panel 3B) and monkey 2 (Panel 3C). These confirmed that monkeys had become trichomatic and matched the new ability to access food rewards.

ModelVariant: The model does not include any human OPN1LW variant, only the wild-type gene.

Selection A- Gene with same function implicated in disease

ExperimentName: PRPF4 is a U4/U6 small nuclear ribonucleoprotein component that functions in mRNA splicing.

GOterm: GO:0000398 (mRNA splicing, via spliceosome)

FunctionalEvidence: PRPF4, PRPF3, PRPF31, and PRPF8 are all splicing factors involved in mRNA processing and are encoded by genes that harbor variants associated with retinitis pigmentosa.

HGNC: HGNC:17348 (PRPF3, retinitis pigmentosa 18), HGNC:15446 (PRPF31, retinitis pigmentosa 11), HGNC:17340 (PRPF8, retinitis pigmentosa 13)

GeneEvidence: PRPF3 harbors variants that cause retinitis pigmentosa 18 (PMID: 11773002), PRPF31 harbors variants that cause retinitis pigmentosa 11 (PMID: 12444105), and PRPF8 harbors variants that cause retinitis pigmentosa 13 (PMID: 7951236).

SharedGeneImplication: PRPF4, PRPF3, PRPF31, and PRPF8 are encode splicing factors involved in mRNA processing.

PRPF4, PRPF3, PRPF31, and PRPF8 are all splicing factors involved in mRNA processing and are encoded by genes that harbor variants associated with retinitis pigmentosa.

ExperimentName: Transgenic mouse expressing human GUCY2D R838S

NonHumanModel: Mus musculus

GeneAlteration: A human GUCY2D transgene encoding the R838S substitution was placed under the control of the rod opsin promoter and injected into mouse eggs, creating a mouse model with rod-specific expression of the variant (and only inefficient expression in cones).

ModelPhenotype: HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), HP:0030478 (Abnormal amplitude of dark-adapted bright flash electroretinogram), HP:0030609 (Photoreceptor layer loss on macular OCT), HP:0410280 (Pediatric onset), HP:0000546 (Retinal degeneration)

HumanPhenotype: HP:0000548 (Cone/cone-rod dystrophy), HP:0007663 (Reduced visual acuity), HP:0007994 (Peripheral visual field loss), HP:0000613 (Photophobia), HP:0011504 (Bull's eye maculopathy), HP:0030465 (Undetectable light-adapted electroretinogram), HP:0030469 (Abnormal dark-adapted electroretinogram)

ModelEvidence: The model system approximately recapitulates the mode of inheritance (autosomal dominant) of the human disease state. At the molecular level, it increases the sensitivity of cGMP synthesis to calcium ion concentration (Figures 2B, 7A). At the ocular level, rod response (dark-adapted ERG) amplitudes were diminished starting at one month of age (Figure 4A, 4B), particularly by 6 months (Figure 5A, 5B), while cones lacking transgene expression remained functional (Figure 5A, 5C). Retinal morphology between 3 and 5 months of age showed outer nuclear layer reduction (Figures 6A, 7C) linked to photoreceptor loss (Figures 6B, 7D).

ModelVariant: NM_000180.4(GUCY2D):c.2512C>A (p.Arg838Ser) (ClinVar: 98568)

ExperimentName: Higher GUCY2D activity in the R838S variant

NonPatientCell: HEK293 cells (EFO:0001182)

GeneFunction: GO:0006182 (cGMP biosynthetic process)

GeneAlteration: Site-directed mutagenesis was used to introduce the substitution encoding the R838S substitution into the gene expression construct (PMID: 15504042).

FunctionalEvidence: The variant exhibits higher activity than the wild-type in HEK293 cells, with enhanced Ca2+ sensitivity (Figure 1A), higher GCAP1 affinity, (Figure 1B), and reduced sensitivity to RD3 inhibition (Figure 1C).

FAVariants: NM_000180.4(GUCY2D):c.2512C>A (p.Arg838Ser) (ClinVar: 98568)

ExperimentName: Genotyping of 4 Australian Cattle Dogs affected with NCL

NonHumanModel: Canis familiaris

GeneAlteration: While the model does not have an engineered gene alteration, all four affected animals are homozygous for the CLN5:c.619C>T variant. The unaffected parents of a fifth affected dog were both heterozygotes for this variant.

ModelPhenotype: HP:0001250 (Seizure), HP:0000572 (Visual loss), HP:0000708 (Behavioral abnormality), HP:0002370 (Poor coordination), HP:0002312 (Clumsiness), HP:0001251 (Ataxia), HP:0100299 (Muscle fiber inclusion bodies), HP:0002283 (Global brain atrophy), HP:0002119 (Ventriculomegaly), Note: inclusion bodies are membrane-associated and autofluorescent. Onset of phenotypes was between 12-18 months, and the condition was rapidly progressive, with euthanasia occurring between 20-27 months of age.

HumanPhenotype: HP:0003208 (Fingerprint intracellular accumulation of autofluorescent lipopigment storage material), HP:0011463 (Childhood onset), HP:0001268 (Mental deterioration), HP:0000572 (Visual loss), HP:0001251 (Ataxia), HP:0002540 (Inability to walk), HP:0001250 (Seizure), HP:0100952 (Enlarged sylvian cistern), HP:0002059 (Cerebral atrophy), HP:0001272 (Cerebellar atrophy)

ModelEvidence: The model phenotypes closely match the human patient phenotypes in the timing of onset and rapid progression (Table 3), neurological details, vision problems, loss of coordination, and microscopic accumulation of storage/inclusion bodies (Figure 4, Figure 5).

ModelVariant: CLN5:c.619C>T p.Gln207Ter

ExperimentName: Recombinase-based variant knock-in to the mouse Rpe65 locus

NonHumanModel: Mus musculus

GeneAlteration: A substitution equivalent to the human NM_000329.3(RPE65):c.1430A>G (p.Asp477Gly) variant was knocked into the mouse Rpe65 locus by Flp-recombinase, which allowed the selection cassette to be subsequently excised.

ModelPhenotype: HP:0030602 (Abnormal fundus autofluorescence imaging), HP:0030329 (Retinal thinning), HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), HP:0008275 (Abnormal light-adapted electroretinogram)

HumanPhenotype: HP:0000662 (Nyctalopia), HP:0007688 (Undetectable light- and dark-adapted electroretinogram), HP:0001098 (Abnormal fundus morphology), HP:0000529 (Progressive visual loss)

ModelEvidence: The mice exhibit reduced levels of RPE65 protein (Figure 3A) and enhanced ubiquitination (Figure 3B), indicating higher susceptibility to proteolysis. Despite normal 11-cis retinal levels (Figure 7C), the heterozygous knock-in animals exhibit abnormal accumulation of retinyl esters (Figure 7B), recapitulating a key molecular defect of the human patients. Photobleaching revealed defects in regeneration of 11-cis-retinal (Figure 7F) and accumulation of retinyl esters (Figure 7G). These defects were observable in both the heterozygous knock-in (mild) and homozygous knock-in (more severe). Accumulation of autofluorescent spots in the fundus (Figure 4), progressive thinning of the outer nuclear layer (Figure 5), and abnormal ERG responses (Figure 6) were seen in heterozygous knock-in and especially homozygous knock-in mice with age. Similar phenotypic results were observed with a third, similar knock-in mouse model (PMID: 28041994).

ModelVariant: The model knocks the NM_000329.3(RPE65):c.1430A>G (p.Asp477Gly) variant (ClinVar:750796) into the endogenous mouse Rpe65 locus.

Selection A- Gene is expressed in tissues relevant to disease:

ExperimentName: Immunohistochemical localization of TIMP3 expression to Bruch's membrane and drusen deposits

TissueExpression: Strong expression was observed in Bruch's membrane (UBERON:0003957) and associated drusen, while weak expression was observed in retinal blood vessels (UBERON:0003501).

ExpressionEvidence: TIMP3 was detected in human eye sections (from patients aged 24 to 85 years) using a monoclonal antibody and a TIMP-3 synthetic peptide for specificity. Bruch's membrane was specifically labeled by the antibody (Figure 2B), with strong labeling observed from the RPE to the elastin sublayer. Drusen associated with Bruch's membrane (particularly smaller deposits) also stained strongly for TIMP3 (Figure 2D), while retinal blood vessels showed weak staining as well. Overall, staining was particularly intense in sections from older patients.

ExperimentName: Rescue in patient-derived IPSC cells harboring IFT140 variants

RescueProband: Forbes_2018_Proband_1 has been diagnosed with retinitis pigmentosa (age 1), end-stage kidney disease (age 6), and Mainzer-Saldino syndrome (at the time of this study). The proband and her non-consanguineous parents were genotyped by Trio whole exome sequencing, identifying compound heterozygous (paternal and maternal) IFT140 variants in the proband.

GeneAlteration: The proband harbors the NM_014714.4(IFT140):c.634G>A (p.Gly212Arg) and NM_014714.4(IFT140):c.2176C>G (p.Pro726Ala) variants in trans. The former variant has been shown to mislocalize when exogenously expressed as a cDNA, and is predicted to disrupt a donor splice site (SpliceAI change score of 0.61).

RescueVariant: The variant rescued by CRISPR/Cas9-based gene editing was NM_014714.4(IFT140):c.634G>A (p.Gly212Arg), (ClinVar ID: 31683). This variant was corrected at the same time as a synonymous 3 bp change was introduced to facilitate the detection of the editing event.

RescuedPhenotype: Patient IPSC-derived organoids exhibit Short / clubbed cilia with swollen sips (Figure 3A-3C), Approximately 50% expression of IFT140 at both the mRNA and protein levels (Figures 2D, 2E), and Disturbed apicobasal polarity compromising the ability of iPSC cells to establish polarized spheroids (Figures 5C, 5D).

RescueMethod: CRISPR/Cas9-based gene editing was used to correct one of the proband's two variants (NM_014714.4(IFT140):c.634G>A (p.Gly212Arg), (ClinVar ID: 31683)). A synonymous 3 bp change was simultaneously introduced to facilitate the detection of the editing event.

RescueEvidence: Gene-corrected cells had longer ciliary length with reduced clubbing (Figure 3), approximately twice the expression of IFT140 at the mRNA and protein levels (Figures 2D, 2E), and a higher percentage of polarized spheroids established in organoid culture (indicating recovery of apicobasal polarity).

ExperimentName: CRISPR/Cas9-based disruption of mouse Impg2

NonHumanModel: Mus musculus

GeneAlteration: Model animals were subjected to CRISPR/Cas9-based deletion of 166bp from exon 2 of Impg2, and analyzed in the homozygous state. Absence of Impg2 signal was confirmed by RT-PCR. Mice were studied at ages 3, 6, and 13 months.

ModelPhenotype: HP:0000654 (Decreased light- and dark-adapted electroretinogram amplitude), HP:0000580 (Pigmentary retinopathy), HP:0030609 (Photoreceptor layer loss on macular OCT)

HumanPhenotype: HP:0000529 (Progressive visual loss), HP:0000662 (Nyctalopia), HP:0000551 (Color vision defect), HP:0008002 (Abnormality of macular pigmentation), HP:0000543 (Optic disc pallor), HP:0007843 (Attenuation of retinal blood vessels), HP:0000533 (Chorioretinal atrophy), HP:0000518 (Cataract), HP:0008323 (Abnormal light- and dark-adapted electroretinogram), HP:0007737 (Bone spicule pigmentation of the retina), HP:0007994 (Peripheral visual field loss), HP:0000603 (Central scotoma)

ModelEvidence: Abnormalities of the homozygous Impg2 deletion mice included reduction in the amplitudes of ERG responses from both rods and cones at age 6 months (Figure 2). Pigmentary retinopathy was also observed (Figure 2C), as well as progressive photoreceptor layer loss (Figure 3). Cone markers such as PNA and M-opsin were mislocalized (Figure 4), indicating cone cell disorganization. Enhanced apoptosis was also observed in the photoreceptor cell segments (Figure 5), consistent with the decreased outer nuclei layer thickness.

ModelVariant: The model does not introduce a variant previously observed in human patients, but rather disrupts the expression of the Impg2 ortholog.

ExperimentName: GPR143 silencing in melanoma cells

NonPatientCell: CL:1000458 (melanocyte of skin, specifically the highly pigmented MNT-1 human melanoma cell line)

GeneFunction: GO:1903232 (melanosome assembly)

GeneAlteration: GPR143 silencing was accomplished by transient transfection of MNT-1 cells with specific siRNA, alongside a non-targeting siRNA control.

FunctionalEvidence: siRNA-treated cells exhibited 50% reduction in mature melanosomes and melanin-containing cells by immunofluorescence (Figures 2E, 2F, 2G). This was confirmed by electron microscopy (Figures 2H, 2I, 2J, 2K). GPR143 silencing also resulted in a combination of normal sized melanosomes and enlarged macromelanosome precursors (Figures 2I, S2A). These cellular defects are an approximate match to the giant melanosomes in the melanocytes of the human patients as well as the hypopigmentation of the fundus.

FAVariants: GPR143 disruption was siRNA-based, and patient-associated variants were not studied.

ExperimentName: Morpholino-based disruption of Impg2 in medaka fish

NonHumanModel: Oryzias latipes

GeneAlteration: Model animals were subjected to targeted disruption of Impg2 by morpholino-based silencing.

ModelPhenotype: HP:0030610 (Photoreceptor outer segment loss on macular OCT).

HumanPhenotype: HP:0000529 (Progressive visual loss), HP:0000662 (Nyctalopia), HP:0000551 (Color vision defect), HP:0008002 (Abnormality of macular pigmentation), HP:0000543 (Optic disc pallor), HP:0007843 (Attenuation of retinal blood vessels), HP:0000533 (Chorioretinal atrophy), HP:0000518 (Cataract), HP:0008323 (Abnormal light- and dark-adapted electroretinogram), HP:0007737 (Bone spicule pigmentation of the retina), HP:0007994 (Peripheral visual field loss), HP:0000603 (Central scotoma)

ModelEvidence: Morpholino-injected animals exhibit decreased photoreceptor outer segment length, especially for cones (Figures 4I, 4J).

ModelVariant: The model does not introduce a variant previously observed in human patients, but rather disrupts the expression of the Impg2 ortholog using a morpholino.

ExperimentName: Morpholino-based disruption of Impg1 in medaka fish

NonHumanModel: Oryzias latipes

GeneAlteration: Model animals were subjected to targeted disruption of Impg1 by morpholino-based silencing.

ModelPhenotype: HP:0030610 (Photoreceptor outer segment loss on macular OCT).

HumanPhenotype: HP:0007754 (Macular dystrophy), HP:0007769 (Peripheral retinal degeneration), HP:0032037 (Mildly reduced visual acuity, progressive), HP:0008179 (Decreased Arden ratio of electrooculogram)

ModelEvidence: Morpholino-injected animals exhibit decreased photoreceptor outer segment length, especially for cones (Figures 4I, 4J).

ModelVariant: The model does not introduce a variant previously observed in human patients, but rather disrupts the expression of the Impg1 ortholog using a morpholino.

ExperimentName: Phenotypic characterization of the heterozygous Timp3Ser179Cys Knock-In Mouse

NonHumanModel: Mus musculus (129/Sv and hybrid C57BL/6 × 129/SvJ background)

GeneAlteration: The mouse Timp3 ortholog was subjected to site-directed mutagenesis to introduce a change equivalent to the human variant encoding the Ser179Cys substitution associated with Sorsby Fundus Dystrophy (PMID:8634721). This variant was introduced into embryonic stem cells by homologous recombination, generating animals for germline transmission of the Timp3Ser179Cys knock-in allele of interest.

ModelPhenotype: HP:0007703 (Abnormality of retinal pigmentation), Abnormalities of the inner aspect of Bruch's membrane

HumanPhenotype: HP:0007703 (Abnormality of retinal pigmentation, PMID:27601084, PMID:16989765), thickening of the inner part of Bruch's membrane

ModelEvidence: While the model system phenotype does not recapitulate all of the human SFD phenotypes, most of which represent the later stages of disease progression, it does recapitulate some early events from the human phenotype, such as abnormalities of Bruch's membrane and the retinal pigment epithelium. The retinal pigment epithelium exhibits structural abnormalities in mutant animals relative to wild-type controls, at both 8 months and 30 months. At 8 months specifically, 15%–20% of RPE areas analyzed showed loss of vertical orientation and increased distances between processes at the cellular bases (Fig. 3) . Patchy thinning of basal microvilli was observed adjacent to Bruch’s membrane, with disrupted packing of microvilli and caverns extending from the basal labyrinth (Figure 3). Timp3 western blots of RPE-choroid ECM extracts from knock-in mice also accumulated higher-molecular weight complexes of Timp3 (Figure 5), similar to the exogenously expressed human variant (PMID:10854443).

ModelVariant: The model variant is a knock-in modification to the mouse Timp3 ortholog that is equivalent to the human NM_000362.5(TIMP3):c.536C>G, (p.Ser179Cys) missense variant (ClinVar:12678). While the article refers to the variant as "Ser156Cys", the updated nomenclature should be Ser179Cys (including the 23-amino acid N-terminal signal peptide).

ExperimentName: Morpholino-based knockdown of zbbs1 disrupts ciliary beating pattern.

NonHumanModel: Danio rerio

GeneAlteration: The model animals have been subjected to morpholino-based knockdown of zbbs1, the zebrafish ortholog of human BBS1.

ModelPhenotype: HP:0012262 (Abnormal ciliary motility), HP:0000107 (Renal cyst)

HumanPhenotype: HP:0000639 (Nystagmus), HP:0000510 (Rod-cone dystrophy), HP:0007663 (Reduced visual acuity), HP:0000662 (Nyctalopia), HP:0000518 (Cataract), HP:0008323 (Abnormal light- and dark-adapted electroretinogram), HP:0000107 (Renal cyst), HP:0001513 (Obesity), HP:0010442 (Polydactyly), HP:0002463 (Language impairment), HP:0000026 (Male hypogonadism)

ModelEvidence: Morpholino-based knockdown of the zbbs1 gene causes hydrocephalus (Figure 3B), formation of pronephritic cysts (Figure 3G), embyronic body axis abnormalities (Figure 4B), heart looping defects (Figure 5B), longer cilia (Figure 7F), and normal ciliary ultrastructure (Figure 7I), and aberrant ciliary motility in the pronephritic tubule and nasal pit (Supplementary Movies).

ModelVariant: This model did not introduce a disease-associated variant known from an affected human patient, but rather studied the effects of silencing the gene product.

ExperimentName: PDE6A forms a complex with PDE6B and PDE6G

InteractingGene: HGNC:8786 (PDE6B, implicated in retinitis pigmentosa 40) and HGNC:8789 (PDE6G, implicated in retinitis pigmentosa 57)

InteractionType: physical

InteractionMethod: size exclusion chromatography

InteractionEvidence: Purification of the cGMP phosphodiesterase complex from bovine rod outer segments yields a subunit of 88 kDa (PDE6A), a subunit of 84 kDa (PDE6B), and a subunit of 13 kDa (PDE6G), (Figure 2). This article shows that PDE6A interacts with and co-purifies with PDE6B and PDE6G in a single complex isolated from bovine retina. A subsequent article by Pittler et al. later established the position of PDE6A on chromosome 5 (PMID: 2155175).

ExperimentName: Intracellular localization of exogenously expressed wild-type and variant CLN5

NonPatientCell: BKH-21 cells (fibroblast-derived cell line, EFO:0002009)

GeneFunction: Localization to lysosome (GO:0005764)

GeneAlteration: The variant was introduced into the construct encoding CLN5 using site-directed mutagenesis.

FunctionalEvidence: Localization studies were performed in BHK-21 cells transiently transfected with wild-type or variant CLN5. The wild-type co-localized with lysosome and late endosome markers, while the variant co-localized with a Golgi marker (Fig. 3D–F) and showed some ER staining and vesicular-like staining. The stability of the variant following cyclohexamide treatment was also shorter than the wild-type. (Fig. 4G-4H).

FAVariants: The variant used was NM_006493.4(CLN5):c.1028_1029del (p.Thr342_Tyr343insTer) (ClinVar:2564).

ExperimentName: Prph2 interacts with Rom1

InteractingGene: ROM1 (HGNC:10254)

InteractionType: Physical

InteractionMethod: Co-immunoprecipitation

InteractionEvidence: Immunoprecipitation of endogenous Prph2 from mouse retinal extracts pulled down Rom1, and vice versa (Fig. 6C).

ExperimentName: Knockin of the p.Tyr141Cys variant into mouse Prph2 results in retinal defects.

NonHumanModel: Mus musculus

GeneAlteration: The model animals are heterozygous for knock-in of the p.Tyr141Cys variant into the Prph2 locus.

ModelPhenotype: HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0000546 (Retinal degeneration), HP:0030610 (Photoreceptor outer segment loss on macular OCT), HP:0012045 (Retinal flecks)

HumanPhenotype: HP:0030643 (Vitelliform-like retinal lesions), HP:0000533 (Chorioretinal atrophy), HP:0001105 (Retinal atrophy), HP:0007663 (Reduced visual acuity), HP:0000580 (Pigmentary retinopathy), HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0008275 (Abnormal light-adapted electroretinogram), HP:0007843 (Attenuation of retinal blood vessels), HP:0011506 (Choroidal neovascularization), HP:0030499 (Macular drusen)

ModelEvidence: This knock-in mouse model has normal expression and localization of Prph2 (Fig. 1), but exhibits phenotypes including photoreceptor outer segment loss (Fig. 2), decreased rod ERG (Fig. 3A), and photoreceptor layer degeneration (Fig. 4). Homozygous mice showed decreased cone ERG as well (Figs. 3B, 3C). Yellow retinal flecks were observed in the fundus (Fig. 5). Abnormal high molecular weight complexes containing Prph2 were also observed in western blotting, along with the absence of normal monomers and dimers in the homozygous animals (Figs. 6 and 7).

ModelVariant: This mouse model has introduced a disease-associated variant known from an affected human patient, NM_000322.5(PRPH2):c.422A>G (p.Tyr141Cys), ClinVar ID: 98666.

Wild-type Prph2 (referred to as RDS) co-localizes with ROM-1 protein within the rim regions of outer segment discs.

Selection B- Gene function is consistent with phenotype.

GOterm: GO:0047555 (3',5'-cyclic-GMP phosphodiesterase activity)

FunctionalEvidence: Purified PDE6A fusion protein tagged with EGFP and fused with sequences from PDE6C catalyzes cGMP hydrolysis comparably to the known phosphodiesterase enzymes PDE6B and PDE6C (Fig. 4), confirming that PDE6a is a rod-associated phosphodiesterase that hydrolyzes cGMP.

FunctionalHPO: HP:0000662 (Nyctalopia), HP:0007994 (Peripheral visual field loss), HP:0000543 (Optic disc pallor), HP:0007843 (Attenuation of retinal blood vessels), HP:0007737 (Bone spicule pigmentation of the retina), HP:0000654 (Decreased light- and dark-adapted electroretinogram amplitude)

ExperimentName: Exogenous expression of the RPE65 D477G variant in HEK293-F cells

NonPatientCell: HEK293-F cell line (EFO:0001182)

GeneFunction: all-trans-retinyl-ester hydrolase, 11-cis retinol forming activity (GO:0052885)

GeneAlteration: A substitution equivalent to the human NM_000329.3(RPE65):c.1430A>G (p.Asp477Gly) variant was introduced by site-directed mutagenesis into canine RPE65 and transiently transfected into HEK293-F cells.

FunctionalEvidence: The variant triggered a moderate decrease in retinoid isomerase activity / 11-cis retinol formation that appeared to be balanced out by the presence of the wild-type allele, rather than acting in a dominant negative fashion (Figure 1B). This loss of function was less dramatic than other hypomorphs known to be associated with autosomal recessive RPE65 retinopathy. Thus, loss of enzymatic function is unlikely to be the primary mechanism by which the variant contributes to autosomal dominantly inherited disease.

FAVariants: NM_000329.3(RPE65):c.1430A>G (p.Asp477Gly) variant (ClinVar:750796)

ExperimentName: Variant knock-in to the mouse Rpe65 locus triggers aberrant mRNA splicing.

NonHumanModel: Mus musculus

GeneAlteration: A substitution equivalent to the human NM_000329.3(RPE65):c.1430A>G (p.Asp477Gly) variant was knocked into the mouse Rpe65 locus by CRISPR/Cas9-based editing.

ModelPhenotype: HP:0000479 (Abnormal retinal morphology), HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), Retinal thinning HP:0030329

HumanPhenotype: HP:0000662 (Nyctalopia), HP:0007688 (Undetectable light- and dark-adapted electroretinogram), HP:0001098 (Abnormal fundus morphology), HP:0000529 (Progressive visual loss)

ModelEvidence: Despite normal 11-cis retinal levels (Figure 3E), the heterozygous knock-in animals exhibit abnormal accumulation of retinyl esters (Figure 3F), recapitulating a key molecular defect of the human patients. The homozygous (but not heterozygous) knock-in animals also exhibit vacuole formation in the outer segment adjacent to the retinal pigment epithelium (Figure 3B) and abnormalities on dark-adapted ERG indicative of slower recovery of rod function (Figures 4A, 4B) and cone function (Figure 5D) as well as reduced retinal thickness (Figure 5A) after long-term chronic visual pigment bleaching.

ModelVariant: The model knocks the NM_000329.3(RPE65):c.1430A>G (p.Asp477Gly) variant (ClinVar:750796) into the mouse Rpe65 locus.

ExperimentName: Conditional Kcnj13 knockout in mice

NonHumanModel: Mus musculus

GeneAlteration: Conditional knockout was generated by inserting a cassette into exon 2 of 3 in the presence of Cre driven by the VMD2 (RPE-specific) promoter.

ModelPhenotype: Photoreceptor layer loss on macular OCT HP:0030609, Retinal degeneration HP:0000546, Abnormality of retinal pigmentation HP:0007703, Attenuation of retinal blood vessels HP:0007843, Decreased light- and dark-adapted electroretinogram amplitude HP:0000654

HumanPhenotype: Reduced visual acuity HP:0007663, Constriction of peripheral visual field HP:00011, Infantile onset HP:0003593, Strabismus HP:0000486, Nystagmus HP:0000639, Progressive night blindness HP:0007675, Moderate myopia HP:0031624, Decreased light- and dark-adapted electroretinogram amplitude HP:0000654, Presenile cataracts HP:0007819, Nummular pigmentation of the fundus HP:0030505, Retinal pigment epithelial loss on macular OCT HP:0030611

ModelEvidence: The 6-month-old mouse with induced Cre recombinase shows loss of photoreceptor cell layers (Figure 4F) and severe retinal degeneration (Figure 4G) relative to the control lacking Cre (Figure 4E), but with relative preservation of retinal morphology near the optic nerve. The RPE layer in the Cre-positive knockout cells shows irregular and flattened cell shapes with abnormal nuclear and cytoskeletal morphology relative to the control (Figure 5D). The knockout mouse fundus shows distinct inclusions, especially in the outer segment, as well as some attenuation of retinal blood vessels (Figure 6). ERG a-waves and b-waves are also extinguished (Figure 7A), consistent with loss of the entire photoreceptor layer (Figures 4F, 5B). There was also low amplitude of c-waves originating from the RPE cells (Figure 7C) that could be restored by lentiviral transduction of the functional KCNJ13 gene (Figures 7C, 7D).

ModelVariant: The model features knockout of the endogenous mouse Kcnj13 rather than knock-in of a mutation equivalent to a variant from a human patient.

ExperimentName: The Nr2e3rd7/rd7 mouse has aberrant proliferation of mitotic progenitors caused by aberrant expression of rod-specific and cone-specific genes such as blue opsin, resulting from failure to suppress the cone generation program and resulting aberrant expression of rod-specific and cone-specific genes.

NonHumanModel: Mus musculus

GeneAlteration: The Rd7 mouse model has been previously described in PMID: 10805811 and PMID: 11487564, and harbors a large out-of-frame deletion within the Nr2e3 locus in the homozygous state.

ModelPhenotype: Abnormal retinal morphology on macular (whorls and rosettes in outer nuclear layer) OCT HP:0030612, Retinal degeneration (late onset) HP:0000546, Retinal thinning HP:0030329

HumanPhenotype: Retinal degeneration HP:0000546, Sensory hypersensitivity (enhanced blue cone sensitivity) HP:5200058

ModelEvidence: The model has been previously published and exhibits ~30x enhanced blue cone sensitivity (PMID: 11487564), retinal spotting throughout the fundus including whorls in the outer nuclear layer (PMID: 11487564), and late onset retinal degeneration characterized by non-apoptotic cell death reduction of outer nuclear layer thickness (PMID: 34561487), and over-proliferation of ectopic retinal progenitors (PMID: 17266784), as well as increased expression of cone-associated genes (Figure 1).

ModelVariant: The model harbors a deletion of 380bp spanning intronic and coding regions, rather than a specific variant from a human patient.

ExperimentName: Naturally occurring mouse model of Nyx loss-of-function

NonHumanModel: Mus musculus

GeneAlteration: This naturally occurring model, previously known to exhibit the absence of b-wave in ERG recordings, harbors an 85-bp deletion in the final exon of the Nyx gene that shifts the frame and replaces the C-terminal 61% of the protein product with 170 novel amino acids. The predicted effect is loss-of-function.

ModelPhenotype: HP:0007984 (Electronegative electroretinogram), HP:0000649 (Abnormality of visual evoked potentials), HP:0007663 (Reduced visual acuity), HP:0001419 (X-linked recessive inheritance)

HumanPhenotype: HP:0030640 (Complete congenital stationary night blindness), HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0007642 (Congenital stationary night blindness), HP:0007663 (Reduced visual acuity), HP:0031624 (Moderate myopia), HP:0007984 (Electronegative electroretinogram)

ModelEvidence: The model exhibited reduced amplitude of dark-adapted bright flash electroretinogram b-wave (electronegative ERG) but normal amplitude of the a-wave (PMID: 9804152). Visually-driven cortical activity was also abnormal (9804152). Visual acuity was also reduced (Figure 4). Inheritance was X-linked recessive (Figure 3), which also matches the human families.

ModelVariant: The model is naturally occurring, and so the affected animals harbor a variant that does not have an exact human patient equivalent.

ExperimentName: RP1L1 physical interaction with RP1

InteractingGene: HGNC:10263 (RP1)

InteractionType: Physical interaction

InteractionMethod: Co-immunoprecipitation

InteractionEvidence: Immunoprecipitation of Rp1 or Rp1l1 from transfected 293T cells successfully led to co-immunoprecipitation of the other, indicating their presence in a complex (Figure 7).

Selection B- Gene function is consistent with phenotype.

ExperimentName: RP1L1 protein localizes to the axoneme of the outer segment of rod photoreceptor cells, but not to the connecting cilium, inner segment, or basal bodies (and from cone cells altogether).

GOterm: GO:0120200 (rod photoreceptor outer segment)

FunctionalEvidence: Immunofluorescent staining of Rp1l1 was performed in retinal sections, with staining of rhodopsin, Rp1, and gamma-tubulin for comparison (Figures 1E, 2A, 2B, 2C).

FunctionalHPO: Macular dystrophy HP:0007754, Reduced visual acuity HP:0007663

PMID: 19687512

Gene: VWF

HGNCID: 12726

RescueModel Mus Musculus

GeneAlteration knock-in strain was generated by homologous recombination of the targeting vector with 129Sv/J embryonic stem cells genome. A 450-bp DNA segment encompassing 5′ UTR and exon 1 of the CASQ2 was used to screen RPCI-21 PAC mouse genomic library. Positive clones were sequenced to define the structure of an 8.5-Kb region encompassing promoter, exon 1, and part of intron 1 of mouse CASQ2. This region was cloned in 3 parts and assembled into the targeting vector pFlrt (supplemental Figure I). The linearized targeting vector was electroporated into 129Sv/J embryonic stem cells. The clone selected with G418 and gancyclovir was injected into C57BL/6NCrL blastocyts and transferred to pseudopregnant CD-1 females. Genotype was determined by sequencing of DNA extracted from tail biopsy specimens (DNasy Tissue Kit, Qiagen).

RescueVariant murine cardiac CASQ2 , WT?

RescuePhenotype HP:0004756

RescueMethod The AAV recombinant virus (AAV9-CASQ2) and the empty virus (AAV9-GFP) were produced by the AAV Vector Core of the Telethon Institute of Genetics and Medicine (Napoli, Italy) according to a previously published protocol.18 The viral titer was 2.6×1012 genome copies per milliliter for AAV9-CASQ2 and 4.3×1012 genome copies per milliliter for AAV9-GFP. The infection was performed by intraperitoneal injection in pups on postnatal day 3 (100 μL), which recovered until euthanasia was performed at 6, 9, or 12 months. Three-month-old mice were anesthetized with Avertin (0.025 mg/kg), and viral particles (200 μL) were injected into the tail vein and studied 2 months later. ECG recordings in mice of either sex were performed with implantable loop recorders (Data Sciences International).

RescueEvidence (Fig.5&8) Epinephrine/Isoprenol administration had lead to diminished incidence of VT in CASQ infected mice vs R33Q mice.

ModelVariant NM_001232.3(CASQ2):c.97C>T (p.Arg33Ter), ClinVar: 41043

NonPatientCell Sarcoplasmic reticulum of cardiac muscle cell, FMA:62390

GeneAlteration The D307H mutant was generated using Quick Change (Life Sciences). Substitution of the CAC codon with GAC resulted in a BamH1 restriction site that was used along with complete sequencing of the DNA inserts to verify sequence.

FunctionalEvidence (fig. 3) Diminished sensitivity to Ca2+ leading to more intercellular Ca2+

FAVariant NM_001232.3(CASQ2):c.919G>C (p.Asp307His), 17610

CaseAddInfo Fig 4&5 show evidence for the conformation change responsible for the sensitivity difference.

gnomAD 0.00003249 link

NonPatientCell Mouse cell line, EFO:0002887 "Myotubes of the homozygous CaVα1S-null (dysgenic) mouse cell line GLT were cultured on 35-mm culture dishes as described previously (32)."

GeneAlteration Mouse GLT line myotubes were transfected with GFP-tagged α1S cDNA constructs at the onset of myoblast fusion using FuGENE 6 transfection reagent according to the manufacturer's protocols (Roche Diagnostics).

FunctionalEvidence Fig 3: faster voltage-gated activation than WT. Fig 6: increased release of calcium ions in the presence of caffeine vs WT.

FAVariants NM_000069.2(CACNA1S):c.4060A>T (p.Thr1354Ser), ClinVar ID: 161208

PatientCell CL:0000057 (Fibroblast)

GeneFunction GO:0032006, (regulation of TOR signaling)

GeneAlteration n/a

FunctionalEvidence Western blot of PS6 isoforms, a product of mTOR signalling, were 74% reduced in patient 2-1 fibroblasts compared to control (Fig 3).

Western blot of PS6 in subject 1-1&1-2 fibroblasts also demonstrated 78% reduced PS6 compared to controls (Fig 2b).

As S6 is phosphorylated during normal mTOR activation, reduced PS6 suggests reduced mTOR signalling.

29283439 (fig 3) suggests that the p.Arg126Ter variant in patient fibroblasts causes increased autophagy compared to controls due to the increase in autophagiosomes. (fig 5) the same variant also leads to excess oligosaccharides as a downstream effect of increased autophagy as show using mass spec.

FAVariants (Fig 3) NM_001163435.2(TBCK):c.831_832insTA (p.Pro278Tyrfs), ClinVar: 225238, (Fig 2) NM_033115.4(TBCK):c.1708+1G>A, ClinVar: 183338

NonHumanModel Mus musculus

GeneAlteration oligonucleotide-directed mutagenesis of lambda phage to make a p.T494M vector to inject mice.

ModelPhenotype HP:0031531

HumanPhenotype HP:0000572

ModelEvidence Electron microscopy of retina showed degeneration of RPE indicated by abnormal basal morphology (fig. 5)

ModelVariant NM_004698.3(PRPF3):c.1481C>T (p.Thr494Met), 3352

Selection B- Gene function is consistent with phenotype.

GOterm: GO:0047555 (3',5'-cyclic-GMP phosphodiesterase activity)

FunctionalEvidence: Purified PDE6C hydrolyzed cGMP with a Km and kcat slightly lower than trypsin-activated bovine rod PDE6 (Figure 7A).

FunctionalHPO: HP:0000551 (Color vision defect), HP:0030465 (Undetectable light-adapted electroretinogram)

ExperimentName: "Variant overexpression in zebrafish causes defects in visual function."

NonHumanModel: Danio rerio

GeneAlteration: The model animals have been subjected to microinjection of PRPF4 mRNA (either wild-type or harboring the p.Pro315Leu variant), with expression levels comparable to the endogenous gene.

ModelPhenotype: Photoreceptor layer loss on macular OCT HP:0030609, Inner retinal layer loss on macular OCT HP:0030620, Photoreceptor outer segment loss on macular OCT HP:0030610

HumanPhenotype: Constriction of peripheral visual field HP:0001133, Reduced visual acuity HP:0007663,, Decreased light- and dark-adapted electroretinogram amplitude HP:0000654. Retinal degeneration HP:0000546, Macular degeneration HP:0000608, Optic disc pallor HP:0000543, Attenuation of retinal blood vessels HP:0007843, Pigmentary retinopathy HP:0000580

ModelEvidence: The variant causes severely disrupted photoreceptors in the retina (Figure 5D), reduced rhodopsin staining (rod marker) in the IS/OS layer (Figures 5E-5J) and other deformities such as malformed brains, short trunks, cardiac edema and curved body axis, at higher levels than in the wild-type control (Figures 5B and 5C). The retinal transcripts opn1lw1, gnat2, rs1, snrpg, prph2b, gnb3, and rho were decreased in the variant-expressing zebrafish (Figure 5K), while the splicing factor transcripts prpf4, prpf3, prpf31, prpf6, prpf8, snrnp200, eftud2, and sart1 were increased (Figures 5A, 5L).

ModelVariant: This model introduced the disease-associated variant NM_001244926.2(PRPF4):c.941C>T (p.Pro314Leu) in the heterozygous state (ClinVar ID: 143057).

ExperimentName: Altered pattern of splicing speckles in patient fibroblasts harboring a heterozygous p.Pro315Leu variant in PRPF4

PatientCell: CL:0000057 (fibroblast)

GeneFunction: GO:0000398 (mRNA splicing, via spliceosome)

GeneAlteration: Cells harbor a heterozygous p.Pro315Leu variant in PRPF4.

FunctionalEvidence: Patient cells harboring the variant exhibit a much higher level of endogenous marker of splicing speckles (SC-35) with a diffused distribution rather than a pattern of distinct speckles (Figures 4B and 4C). RT-PCR showed increased expression of SRSF1 and SRSF2 as well (Figure 4A). Collectively, the patient cells harboring the variant show molecular evidence of disrupted splicing.

FAVariants: The model harbors the NM_001244926.2(PRPF4):c.941C>T (p.Pro314Leu) variant in the heterozygous state (ClinVar ID: 143057).

ExperimentName: The rds mouse harbors loss-of-function variants in the Prph2 gene.

NonHumanModel: Mus musculus

GeneAlteration: The model animals are homozygous for a loss-of-function variant in the Prph2 locus known as retinal degeneration slow (rds). The causal variant appears to encode a p.Gln340Pro missense change that results in the inability to detect the Prph2 protein by antibody.

ModelPhenotype: HP:0000546 (Retinal degeneration), HP:0030610 (Photoreceptor outer segment loss on macular OCT)

HumanPhenotype: HP:0030643 (Vitelliform-like retinal lesions), HP:0000533 (Chorioretinal atrophy), HP:0001105 (Retinal atrophy), HP:0007663 (Reduced visual acuity), HP:0000580 (Pigmentary retinopathy), HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0008275 (Abnormal light-adapted electroretinogram), HP:0007843 (Attenuation of retinal blood vessels), HP:0011506 (Choroidal neovascularization), HP:0030499 (Macular drusen)

ModelEvidence: This is a naturally occurring mouse model with homozygous disruption of the Prph2 locus. Phenotypes include progressive degeneration of retina, absence of rods at age 7-10 months, and reduction of the number of rows of photosensitive cell nuclei to only 1-4 rows (described in previous publications PMID: 705766 and PMID: 419532). Other previous publications have reported that the mice exhibit abnormal development of photoreceptor outer segments, as well as subsequent gradual photoreceptor degeneration of photoreceptors that is complete by age 1 year (PMID: 2918924).

ModelVariant: This model did not introduce a disease-associated variant known from an affected human patient, but rather studied a naturally occurring p.Gln340Pro missense variant disrupting the Prph2 locus.

Selection A- Gene is expressed in tissues relevant to disease:

ExperimentName: Wild-type Prph2 localizes to mouse rod outer segments.

TissueExpression: photoreceptor outer segment layer (UBERON:0003926)

ExpressionEvidence: Western blotting with an anti-Prph2 antibody confirms that Prph2 is present in retinal outer segments and exists a a dimer (Fig. 2), although other control tissues are absent.

ExperimentName: Oxidative stress sensitivity in the homozygous Timp3Ser179Cys Knock-In Mouse

NonHumanModel: Mus musculus (C57Bl/6J background)

GeneAlteration: The gene alteration was constructed in a previous paper (PMID:12147610), in which the mouse Timp3 ortholog was subjected to site-directed mutagenesis to introduce a change equivalent to the human variant encoding the Ser179Cys substitution associated with Sorsby Fundus Dystrophy (PMID:8634721). This variant was introduced into embryonic stem cells by homologous recombination, generating animals for germline transmission of the Timp3Ser179Cys knock-in allele of interest.

ModelPhenotype: HP:0000580 (Pigmentary retinopathy), HP:0007703 (Abnormality of retinal pigmentation)

HumanPhenotype: HP:0007703 (Abnormality of retinal pigmentation)

ModelEvidence: The phenotypes of this model system recapitulate some of the degenerative features of the human model. Sensitivity to oxidative stress was revealed when low-dose sodium iodate triggered RPE degeneration and disorganization in the Timp3 S179C/S179C mice but not the wild-type controls (Figure 4). Rhodamine-phalloidin staining also exhibited an increased area of degeneration in the central region of the RPE in mutant mice but not in the wild-type controls (Figure 5). It is important to point out the caveat that the mice were homozygous for Timp3 Ser179Cys, whereas human patients are heterozygous. This difference in genotype introduces the possibility of loss-of-function effects that are specific to the mouse but absent from the human patients.

ModelVariant: The model variant is a knock-in modification to the mouse Timp3 ortholog that is equivalent to the human NM_000362.5(TIMP3):c.536C>G, (p.Ser179Cys) missense variant (ClinVar:12678).

ExperimentName: Phenotypic characterization of the homozygous Timp3-/- Knock-Out Mouse

NonHumanModel: Mus musculus (C57/Bl6 or CD1 background)

GeneAlteration: A neomycin resistance expression cassette was cloned into plasmid-borne exon 3 of mouse Timp3. The linearized construct was transfected into embryonic stem cells and injected into blastocysts. Animals that had undergone homologous recombination were identified by locus-specific genomic PCR reactions. Predicted loss of Timp3 due to a premature stop codon and nonfunctional mRNA was confirmed at the protein level.

ModelPhenotype: HP:0030605 (Abnormal indocyanine green angiography), HP:0025568 (Abnormal morphology of the choroidal vasculature), HP:0030979 (Dilatation of large choroidal vessels)

HumanPhenotype: HP:0011506 (Choroidal neovascularization)

ModelEvidence: The model showed dilatation of the large choroid vessels, which appeared large and hyperfluorescent during ICG angiography, with balloon-like spots (Figure 3A-3B). While wild-type controls exhibited normal choroidal blood vessel network, Timp3-/- animals had abnormal vessel formations that were strongly dilated or fused (Figures 3C-3H). During in situ zymography, higher local fluorescence intensity indicated increased MMP2 and MMP9 activity in the knockout animals, as expected. Aortic explants from knockout mice showed higher spontaneous microvessel outgrowth than their counterparts from wild-type mice (Figure 6). Rescue assays suggest that the antiangiogenic activity requires the C-terminal region of TIMP3 (Figure 7).

ModelVariant: N/A (Timp3 knockout)

ExperimentName: Knock-in of the p.M390R variant into the mouse Bbs1 gene.

NonHumanModel: Mus musculus

GeneAlteration: The model animals have been subjected to homologous recombination-based knock-in of the p.M390R variant into exon 12 of the endogenous mouse Bbs1 gene. The variant has been studied in the homozygous state.

ModelPhenotype: HP:0032558 (Absent sperm flagella), HP:0010633 (Partial anosmia), HP:0012433 (Abnormal social behavior), HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0030610 (Photoreceptor outer segment loss on macular OCT), HP:0030609 (Photoreceptor layer loss on macular OCT), HP:0001513 (Obesity), HP:0002119 (Ventriculomegaly),

HumanPhenotype: HP:0000639 (Nystagmus), HP:0000510 (Rod-cone dystrophy), HP:0007663 (Reduced visual acuity), HP:0000662 (Nyctalopia), HP:0000518 (Cataract), HP:0008323 (Abnormal light- and dark-adapted electroretinogram), HP:0000107 (Renal cyst), HP:0001513 (Obesity), HP:0010442 (Polydactyly), HP:0002463 (Language impairment), HP:0000026 (Male hypogonadism)

ModelEvidence: Sperm flagella were absent (Figures 2A, 2B). Loss of the outer nuclear layer and degeneration of the photoreceptor inner and outer segments were observed in older mice (Figures 2C, 2D). Photoreceptor morphology in the outer segment was also disrupted in TEM images (Figure 2E). Scotopic responses showed a- and b-wave attenuation (Figure 2G), while photopic responses were not significantly different (Figure 2H). Homozygous mice showed obesity relative to wild-type and heterozygous mice (Figure 3). Brain sections showed neuroanatomical defects, especially ventriculomegaly (Figure 4). Brain cilia were morphologically abnormal (Figure 5). Renal cysts and polydactyly were investigated but not observed.

ModelVariant: This model introduced a disease-associated variant known from affected human patients (NM_024649.5(BBS1):c.1169T>G (p.Met390Arg), ClinVar ID: 12143).

ExperimentName: Horse model of TRPM1 loss-of-function

NonHumanModel: Equus ferus caballus

GeneAlteration: The horse has homozygous disruption of the TRPM1 gene by a naturally occurring insertion of a 1378 bp transposon in intron 1 (g. 108,297,929_108,297,930 ins1378). A previous publication by the same group has shown that TRPM1 expression is absent from the skin and retina of these horses (PMID: 18660533).

ModelPhenotype: HP:0001053 (Hypopigmented skin patches), HP:0000591 (Abnormal sclera morphology), HP:0000662 (Nyctalopia), HP:0007984 (Electronegative electroretinogram),

HumanPhenotype: HP:0000662 (Nyctalopia), HP:0000545 (Myopia), HP:0000572 (Visual loss), HP:0007984 (Electronegative electroretinogram), HP:0000958 (Dry skin), HP:0000964 (Eczema)

ModelEvidence: The affected horses exhibit night blindness and abnormal dark-adapted ERG with absent b-wave and increased amplitude of a-wave (Figure 1). Both phenotypes are similar to the affected human patients. Melanocyte dysfunction is also shown by unpigmented areas of the skin (Figures 1 and 2) and the sclera of the eye (Figure 2A). These pigment phenotypes do not have a human equivalent, although some affected patients have been reported to have dry skin or a history of eczema (PMID: 19878917).

ModelVariant: The model harbors a naturally occurring variant that does not have an equivalent / orthologous variant reported in human patients.

ExperimentName: Mouse model of Adam9 loss-of-function

NonHumanModel: Mus musculus

GeneAlteration: The mice harbor homozygous knockout of the Adam9 gene by an insertion of a neomycin selection cassette in to the exon encoding amino acids 32 to 66 of Adam9 (PMID: 11839819).

ModelPhenotype: HP:0000546 (Retinal degeneration), HP:0007703 (Abnormality of retinal pigmentation), HP:0030610 (Photoreceptor outer segment loss on macular OCT), HP:0030483 (Reduced amplitude of dark-adapted bright flash electroretinogram a-wave), HP:0007984 (Electronegative electroretinogram), HP:0008323 (Abnormal light- and dark-adapted electroretinogram)

HumanPhenotype: HP:0410280 (Pediatric onset), HP:0007663 (Reduced visual acuity), HP:0007401 (Macular atrophy), HP:0000580 (Pigmentary retinopathy), HP:0007769 (Peripheral retinal degeneration), HP:0008323 (Abnormal light- and dark-adapted electroretinogram), HP:0000548 (Cone/cone-rod dystrophy), HP:0007994 (Peripheral visual field loss), HP:0000545 (Myopia)

ModelEvidence: The affected mice exhibit an abnormal gap between the RPE and the photoreceptor outer segment, which would normally be in contact (Figure 5B). The gap contains macrophages (Figure 5L). Outer nuclear layer thinning is observed (Figure 5D), as well as material deposited between the RPE and Bruch's membrane, which may be analogous to drusen deposits in humans (Figure 5K). These findings collectively show an adhesion defect at the interface between the photoreceptor outer segment and the RPE. This evidence of retinal degeneration was complemented by abnormalities in both rod- and cone-driven ERG responses (Figure 4), matching a feature of the human patients.

ModelVariant: The model harbors a targeted disruption that does not have an equivalent / orthologous variant reported in human patients.

Zebrafish harboring an engineered mutation in the prpf4 gene exhibit enhanced brain and spinal cord apoptosis. The mechanism is loss-of-function. Matches the patients?

ExperimentName: Phenotypes from homozygous Gucy2e mouse knockout model

NonHumanModel: Mus musculus

GeneAlteration: The mouse Gucy2e gene was disrupted by the introduction of a neomycin-resistance gene cassette into exon 5, resulting in a truncated protein lacking its transmembrane domain (PMID: 10407028). The effects of this knockout were studied in both heterozygous and homozygous states.

ModelPhenotype: HP:0030469 (Abnormal dark-adapted electroretinogram), HP:0030465 (Undetectable light-adapted electroretinogram), HP:0008020 (Cone dystrophy)

HumanPhenotype: HP:0007663 (Reduced visual acuity), HP:0007688 (Undetectable light- and dark-adapted electroretinogram), HP:0007703 (Abnormality of retinal pigmentation)

ModelEvidence: By 5 weeks of age, Gucy2e-deficient animals exhibit dramatic reduction/degeneration of cones in the outer nuclear layer (Fig. 3a–d). This was confirmed by disappearance of labeling of cone cell nuclei by the lectin PNA (Fig. 3g-h). ERG showed that rod a-wave and b-wave were markedly reduced by 1 month of age, while cone responses were nondetectable as early as 2 months of age (Fig. 4a-b). A proposed explanation for the absence of more severe rod defects is the continued presence of the Gucy2f paralog in this mouse model, which may compensate for the absence of Gucy2e function in rod cells specifically. This hypothesis has been confirmed in a subsequent double-knockout model (PMID: 17255100)

ModelVariant: The mouse model disrupts the expression of the endogenous GC1 protein product but does not feature knock-in of a variant from a human patient.

NonPatientCell: COS-7 cells (EFO:0001643, CLO:0002597)

GeneFunction: GO:0008191 (metalloendopeptidase inhibitor activity), GO:0046872 (metal ion binding), GO:0002020 (protease binding), GO:0005615 (active in extracellular space), GO:0031012 (active in extracellular matrix)

GeneAlteration: TIMP3 expression constructs encoding the "Ser181Cys", "Ser156Cys", "Gly166Cys", and "Glu139X" variants (officially referred to as the Ser204Cys, Ser179Cys, Gly189Cys, and Glu162X variants) were generated by PCR-based mutagenesis of the wild-type TIMP-3-pCI.neo construct.

FunctionalEvidence: The TIMP3 variants were exogenously expressed in COS-7 cells alongside the wild-type control. Reverse zymography of extracellular matrix from these cells revealed the presence of dimeric species with metalloproteinase inhibitory activity in the variant-transfected conditions, but not in the wild-type control (Figure 3). These bands were confirmed to be TIMP3 by immunoblotting the extracellular matrix from these cells under non-reducing conditions, as they disappeared under reducing conditions (Figure 4). Under reducing conditions, the variant-transfected cells show higher levels of monomeric TIMP3 relative to the wild-type control (Figure 4b), but it should be noted that the wild-type control is depicted on a separate blot / exposure. The dimerized Glu162X variant is difficult to distinguish from other full-length monomeric species, but appears to be present as well (Figure 5).

FAVariants: Variants used were equivalent to NM_000362.5(TIMP3):c.610A>T (p.Ser204Cys) (ClinVarID:12676), NM_000362.5(TIMP3):c.536C>G, (p.Ser179Cys) (ClinVarID:12678), NM_000362.5(TIMP3):c.565G>T (p.Gly189Cys) (ClinVarID:12679), and NM_000362.5(TIMP3):c.484G>T (p.Glu162X) (ClinVarID:12680).

This article studies mice known as rds with homozygous disruption of the Prph2 locus. Phenotypes include progressive degeneration of retina, absence of rods at age 7-10 months, and reduction of the number of rows of photosensitive cell nuclei to only 1-4 rows. The mouse has been previously published (PMID: 705766).

ExperimentName: Genetically engineered Bbs7 homozygous knockout mouse

NonHumanModel: Mus musculus

GeneAlteration: The model animals harbor a homozygous replacement of Bbs7 exon 5 with a neomycin cassette, resulting in a frameshifted allele confirmed by western blotting to result in a null effect on the gene product.

ModelPhenotype: HP:0000546 (Retinal degeneration), HP:0032558 (Absent sperm flagella), HP:0001513 (Obesity), HP:0002119 (Ventriculomegaly), HP:0003251 (Male infertility), HP:0030609 (Photoreceptor layer loss on macular OCT), HP:0012258 (Abnormal axonemal organization of respiratory motile cilia), HP:0033524 (Abnormal sperm axoneme morphology)

HumanPhenotype: HP:0001956 (Truncal obesity), HP:0000618 (Blindness), HP:0001249 (Intellectual disability), HP:0001631 (Atrial septal defect), HP:0001640 (Cardiomegaly), HP:0100598 (Pulmonary edema), HP:0012584 (Bilateral renal hypoplasia), HP:0004719 (Hyperechogenic kidneys), HP:0003774 (Stage 5 chronic kidney disease), HP:0000580 (Pigmentary retinopathy), HP:0000648 (Optic atrophy), HP:0008043 (Retinal arteriolar constriction), HP:0007737 (Bone spicule pigmentation of the retina), HP:0007754 (Macular dystrophy)

ModelEvidence: This is a genetically engineered mouse model of biallelic Bbs7 disruption. The mutant animals with biallelic Bbs7 disrupion exhibit photoreceptor inner and outer segment loss (Figure 1), ventriculomegaly (Figure 1), reduced number of cilia and shortened cilia in brain tissue (Figure 2), abnormal axonemal structure / microtubule number abnormality in brain cilia (Figures 2F, 2G), lack of sperm flagella (Figure 3), Abnormal sperm axoneme microtubule organization (Figure 3), destabilization /absence of BBS2 in testes (Figure 4), and abnormal accumulation of some ciliary membrane proteins (dopamine D1 receptor, Figure 6) but not others (polycystin-1 or polycystin-2, Figure 5). Kidney cysts and polydactyly were looked for and found to be absent.

ModelVariant: This model did not introduce a disease-associated variant known from an affected human patient, but rather studied a genetically engineered null variant disrupting Bbs7 expression.

ExperimentName: IFT140 variant rescue in Ift140 knockout cells

RescueCellLine: Mouse Inner Medullary Collecting Duct 3 (mIMCD3) cells

RescueCellEFO: EFO:0002887 (mouse cell line), EFO:0001643 (kidney-derived cell line)

GeneAlteration: The cell line was subjected to CRISPR-based knockout of endogenous IFT140 (targeting exon 8).

RescueCellPhenotype: Ift140 knockout cells exhibited a complete inability to form cilia relative to the control rescued by overexpression of wild-type Ift140 (Figure 3b) and aberrant accumulation of the IFT-B protein IFT88 at the ciliary tip (Figure 3d).

RescueMethod: Transient transfection and over-expression of wild-type or variant IFT140 was used to rescue the cell phenotypes.

RescueEvidence: Wild-type IFT140 restored ciliogenesis in 11% of cells, while either variant restored ciliogenesis to 6%, despite the surprising continued absence of the frameshift variant at the protein level (Figure 3b). Wild-type IFT140 reduced the proportion of cells exhibiting IFT88 accumulation at the ciliary tip to 23%, while either variant partially reduced IFT88 localization to the ciliary tip to 50% or 56% (Figure 3d).

RescueCellVariant: The variants used for the rescue experiment were NM_014714.3(IFT140):c.992_993dup (p.Tyr332Valfs*18) (cannot be registered manually) and NM_014714.3:c.2767T>G (p.Tyr923Asp) (CA394227092).

ExperimentName: Mouse ciliated cell model of Ift140 loss-of-function

CellLine: Mouse Inner Medullary Collecting Duct 3 (mIMCD3) cells

CellEFO: EFO:0002887 (mouse cell line), EFO:0001643 (kidney-derived cell line)

GeneAlteration: The cell line was subjected to CRISPR-based knockout of endogenous IFT140.

CellPhenotype: Ift140 knockout cells exhibited a complete inability to form cilia relative to the control rescued by overexpression of wild-type Ift140 (Figure 3b).

CellEvidence: The defect in ciliary formation induced by Ift140 knockout (Figure 3b) recapitulates one of the cellular features of human patients harboring IFT140 variants (absence of cilia from a high proportion of fibroblasts). This also matches the Chlamydomonas model of IFT140 loss described in PMID: 28207750, albeit with greater severity.

CellVariant: CRISPR-based knockout was performed in this study to mimic IFT140 loss of function, while the human IFT140 variants were tested in the related rescue experiment instead.

Selection A - Gene is expressed in tissues relevant to disease:

ExperimentName: Nyx localization to the post-synaptic side of both synaptic layers of the zebrafish retina

TissueExpression: UBERON:0001790 (Outer plexiform layer of retina), UBERON:0001795 (Inner plexiform layer of retina

ExpressionEvidence: Nyx was detected by antibody staining of zebrafish retinal sections and was found to localize strongly to both synaptic layers (the outer plexiform layer and inner plexiform layer) but not the outer nuclear layer or inner nuclear layer (Fig. 5A). Lack of co-localization between Nyx and either SV2 or PSD-95 (two presynaptic markers) confirmed that Nyx is specifically post-synaptically localized (Figs. 5C, 6A). This was further confirmed by the detection of Nyx-labeled second-order neuron dendrites invaginating into presynaptic cone pedicles (Figs. 6B and 6C). This data is highly compatible with the findings of a separate mouse experiment (PMID: 17881478) showing that Nyx transgene expression in the dendritic tips of retinal depolarizing bipolar cells rescues the phenotype of Nyx-deficient mice.

Selection B- Gene function is consistent with phenotype.

GOterm: GO:0008191 (metalloendoproteinase inhibitor activity)

FunctionalEvidence: The functional evidence consists of an electrophoretic assay in which the 21-kDa chicken ortholog of human TIMP3 shows inhibitory activity towards a metalloproteinase.

FunctionalHPO: Abnormal extracellular matrix turnover in the inner aspect of Bruch's membrane

PhenotypeEvidence: This metalloproteinase inhibitor activity and ECM localization of the gene product indicate that it functions in ECM remodeling, balancing the activity of matrix metalloproteinases. This is consistent with the disease phenotype of abnormal thickening of the inner aspect of Bruch's membrane (PMID:11879143). The inhibitor evidence is shown in Figure 2B, where a 21-kDa band protects a gelatin substrate from enzymatic metalloproteinase activity. The purification of the gene product from the extracellular matrix is depicted in Figure 1. A later study showed that the human ortholog of this chicken gene is the TIMP3 gene, mapped to the region on chromosome 22 between q12.1-q13.2 (PMID:8188246).

ExperimentName: Patient iPSC-derived retinal organoids exhibit severe outer segment defects that can be rescued by CRISPR/Cas9-based editing.

RescuePatientCell: EFO:0004905 (induced pluripotent stem cell, differentiated into a retinal organoid)

GeneAlteration: The patient cells have inherited compound heterozygous variants in IMPG2, encoding either p.Tyr254Cys or a frameshift variant starting at codon 805.

RescuePhenotype: Patient iPSC-derived retinal organoids harboring these two variants exhibited almost complete failure to form an outer segment or an interphotoreceptor matrix during a 200-day period (Figure 2A), relative to control retinal organoids with CRISPR/Cas9-based editing / rescue of both variants (Figure 2D).

RescueMethod: CRISPR/Cas9-based editing was used to correct both variants.

RescueEvidence: Control retinal organoids with CRISPR/Cas9-based editing / rescue of both variants (Figure 2D) showed development of both the IPM and the outer segment, relative to the uncorrected retinal organoids (Figure 2A). This rescue argues for a role of IMPG2 in photoreceptor cell outer segment development / maintenance.

RescueVariants: NM_016247.4:c.761A>G (p.Tyr254Cys, CA353867908) and an undefined frameshift variant at codon 805, either NM_016247.4:c.2413del (p.Ala805LeufsTer8) or NM_016247.4:c.2414del (p.Ala805ValfsTer8).

ExperimentName: Mouse model of Trpm1 loss-of-function

NonHumanModel: Mus musculus

GeneAlteration: The mouse has homozygous disruption of the Trpm1 gene by targeted deletion of regions from exons 3, 4, and 5 (PMID: 19439586).

ModelPhenotype: HP:0007984 (Electronegative electroretinogram)

HumanPhenotype: HP:0000662 (Nyctalopia), HP:0000545 (Myopia), HP:0000572 (Visual loss), HP:0007984 (Electronegative electroretinogram)

ModelEvidence: The affected mice exhibit abnormal dark-adapted ERG with absent b-wave and normal a-wave (Figure 3), similar to the affected human patients. Retinal slices from these mice exhibit defects in chemically stimulated light responses (Figure 4), indicating that Trpm1 is required for generating the depolarizing light response in ON-bipolar cells.

ModelVariant: The model harbors a targeted deletion that does not have an equivalent / orthologous variant reported in human patients.

ExperimentName: Canine model of PDE6A loss-of-function

NonHumanModel: Canis lupus familiaris

GeneAlteration: Cardigan Welsh Corgi animals were found to develop retinal degeneration due to a homozygous 1-bp deletion variant at codon 616 that results in a frameshift and a premature stop codon (genotype previously reported in PMID: 10393029).

ModelPhenotype: HP:0030474 (Undetectable dark-adapted electroretinogram), HP:0000505 (Visual impairment), HP:0007843 (Attenuation of retinal blood vessels), HP:0030610 (Photoreceptor outer segment loss on macular OCT)

HumanPhenotype: HP:0011463 (Childhood onset), HP:0000662 (Nyctalopia), HP:0007994 (Peripheral visual field loss), HP:0000543 (Optic disc pallor), HP:0007843 (Attenuation of retinal blood vessels), HP:0007737 (Bone spicule pigmentation of the retina), HP:0000654 (Decreased light- and dark-adapted electroretinogram amplitude), HP:0007984 (Electronegative electroretinogram)

ModelEvidence: This mutation caused an Electronegative electroretinogram (HP:0007984) and Abnormal light-adapted electroretinogram (HP:0008275), (Figures 2, 3, and 4). Retinal degeneration (HP:0000546) was observed with outer nuclear photoreceptor layer thinning (HP:0030610, Figure 5). Photoreceptor outer segment loss is visible on macular OCT (Figure 6, Figure 7), particularly loss of rod photoreceptor cells.

ModelVariant: The model does not harbor a variant equivalent to one from the human patients, but rather has a naturally occurring frameshift variant disrupting Pde6a leading to full loss-of-function.

Selection B- Gene function is consistent with phenotype.

Title: TRPM1 (MLSN) is a plasma membrane protein that mediates the cellular influx of calcium and other divalent cations, leading to depolarization, as part of the depolarizing photoresponse within ON-bipolar cells of the retina.

GOterm: GO:0005261 (cation channel activity), GO:0005262 (calcium channel activity)

FunctionalEvidence: Exogenous expression of TRPM1 (MLSN) in HEK293 cells conferred the ability to allow entry of calcium, barium, and strontium ions, unlike the pYFP or empty vector controls (Figures 4A, 4B, 3C). Confocal microscopy also showed localization to the plasma membrane (Figures 4C, 4D).

FunctionalHPO: HP:0000662 (Nyctalopia), HP:0000545 (Myopia), HP:0000572 (Visual loss), HP:0007984 (Electronegative electroretinogram)

PhenotypeEvidence: This evidence is consistent with the view of congenital stationary night blindness 1C as a channelopathy caused by a defect in divalent cation influx, signal transduction, and the polarization / depolarization of ON-bipolar cells.

ExperimentName: Rpe65 homozygous knockout triggers loss of cone response in certain mouse models.

NonHumanModel: Mus musculus

GeneAlteration: Rpe65 homozygous knockout was generated by replacing the 5' flanking region and the first three exons of Rpe65 with a PGK-Neo cassette (described in PMID: 9843205).

ModelPhenotype: HP:0008275 (Abnormal light-adapted electroretinogram), HP:0030465 (Undetectable light-adapted electroretinogram)

HumanPhenotype: HP:0030479 (Abnormal amplitude of light-adapted flicker electroretinogram)

ModelEvidence: The human phenotype includes both dark-adapted (rod) and light-adapted (cone) ERG defects. The Rpe65 homozygous knockout mouse model was previously found to have dark-adapted but not light-adapted response defects (PMID: 9843205). This study shows that in mice harboring homozygous knockout of Nrl (homozygous knockout) , Rpe65 homozygous knockout triggers light-adapted (cone) ERG defects as well (Figures 6A and 6B).

ModelVariant: The model disrupts the entire Rpe65 locus rather than introducing a mutation equivalent to a human patient variant.

Dog rescue RPGR.

Selection B- Gene function is consistent with phenotype.

GOterm: GO:0016525 (negative regulation of angiogenesis)

FunctionalEvidence: The previously known ability of TIMP3 protein to block the binding of VEGF to the VEGFR-2 receptor (PMID:12652295, PMID:19478078) is mapped to the C-terminal region of TIMP3 consisting of amino acids 160-211 (Figures 1c, 2b). This region is relevant to disease in that nonsense variants (PMID:10854443) and splice site disrupting variants (PMID:9760202) of TIMP3 are predicted to remove most or all of this domain. This region is not only necessary and sufficient to block VEGF binding to VEGFR-2 in ELISA assays (Figure 2b), but also can block VEGF-stimulated endothelial migration (Figure 4) and proliferation (Figure 6). In an animal model of choroidal neovascularization (CNV) with lesions induced by laser, the injection of C-terminal TIMP3 peptides can limit CNV-based lesion size (Figure 7). This introduces the possibility that some phenotypes of Sorsby fundus dystrophy such as CNV may be mediated by loss of normal TIMP3 function rather than gain of an abnormal TIMP3 function.

FunctionalHPO: HP:0011506 (Choroidal neovascularization)

Selection B Expression altered in patients

ExperimentName: Immunohistochemical localization of TIMP3 expression in eye sections from SFD and unaffected human patients

TissueExpression: In both unaffected and SFD patient tissues, strong TIMP3 expression was observed in Bruch's membrane (UBERON:0003957) and associated drusen (Figure 1).

ExpressionEvidence: As previously published (PMID:9006347), strong TIMP3 expression was observed in Bruch's membrane and drusen in both normal and SFD samples (Figure 1). In SFD patient tissues, thickened Bruch's membrane stained positively for TIMP3 as well, but only in areas where the retinal pigment epithelial cells were still intact (not in areas exhibiting RPE loss). Numerous extracellular deposits that stained positively with Periodic acid-Schiff (presumably drusen) were also TIMP3-positive. In some areas, retinal blood vessels were occluded by TIMP3-positive deposits as well. These findings clearly localize TIMP3 to the sites closely associated with pathogenesis within the SFD retina. Interestingly, SFD-like deposits in retinitis pigmentosa tissues were similarly TIMP3-positive (Figure 2).

ExperimentName: Rescue of Nyx loss-of-function mouse by transgene expression in depolarizing bipolar cells of the retina

RescueModel: Mus musculus

GeneAlteration: This naturally occurring model, previously known to exhibit the absence of b-wave in ERG recordings, harbors an 85-bp deletion in the final exon of the Nyx gene that shifts the frame and replaces the C-terminal 61% of the protein product with 170 novel amino acids. The predicted effect is loss-of-function.

RescueVariant: Rescue was performed with a wild-type Nyx transgene, rather than a variant.

RescuePhenotype: HP:0007984 (Electronegative electroretinogram), HP:0000649 (Abnormality of visual evoked potentials), HP:0007663 (Reduced visual acuity)

RescueMethod: An EYFP-tagged mouse Nyx gene was placed under the control of the mouse GABAcp1 promoter for tissue-specific expression in bipolar cells of the retina, including rod and cone depolarizing and hyperpolarizing bipolar cells. The transgene was introduced by pronuclear injection to generate transgenic lines, then crossed to homozygous Nyx mice to yield transgenic male offspring with Nyx disruption.

RescueEvidence: The EYFP-Nyx transgene restores both the ERG b-wave (Figure 5B) and the spontaneous and light-evoked responses of retinal ganglion cells (Figure 6) in Nyx-deficient mice.

ModelVariant: The model is naturally occurring, and so the affected animals harbor a variant that does not have an exact human patient equivalent.