NonPatientCell: ARPE-19 (human retinal pigment epithelial) cell line (CLO:0001749, CL:0009004)

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: A TIMP3 expression construct encoding the Glu162Lys variant (referred to in the paper as "Glu139Lys") was generated by PCR-based mutagenesis of the wild-type pCDNA3-TIMP3.

FunctionalEvidence: The TIMP3 variant was exogenously expressed in ARPE-19 cells alongside a mock transfected control (not a wild-type control). Immunoblotting of non-reduced samples detected both monomeric (24 kDa) and dimeric (48 kDa) forms of the variant (Figure 8). Reverse zymographs of extracellular matrix from both wild-type TIMP3- and variant TIMP3-transfected cells revealed the presence of the dimeric species for the variant but not the wild-type, and confirmed its intact metalloproteinase inhibitory activity (Figure 9). This indicated that the free cysteine present in the variant may not have compromised metalloproteinase inhibitory activity, but likely facilitated an intramolecular disfulfide bonding interaction with another TIMP3 molecule. This gain-of-function interpretation of the variant effect would be consistent with the autosomal dominant mechanism of inheritance.

FAVariants: Variant used was NM_000362.5(TIMP3):c.484G>A (p.Glu162Lys) (CAID:CA411540222). The variant is referred to in this publication as "Glu139Lys" (due to the removal of a 23-amino acid N-terminal signal peptide from the polypeptide sequence) but encodes Glu162Lys in the full/updated polypeptide sequence.

ExperimentName: LRP5 inhibition using a dominant negative construct blocks bone accrual in mouse calvarial explant culture.

NonPatientCell: CL:0001035 (bone cell, specifically mouse calvarial explant cultures)

GeneFunction: GO:0098868 (bone growth)

GeneAlteration: Calvarial explant cultures from mice were treated with conditioned media from cells exogenously secreting either wild-type LRP5 or dominant negative LRP5deltaTM.

FunctionalEvidence: Bone accrual / thickness decreases significantly at multiple points in mouse calvarial (skull) explants treated with LRP5deltaTM relative to LRP5-WT (Figure 7).

FAVariants: LRP5 function was attenuated using an artificial dominant negative mutant while a human variant was not introduced into the LRP5 gene.

ExperimentName: OPN1LW variants identified in myopic patients were expressed exogenously in HEK293T cells and exhibited reduced expression at the mRNA level.

NonPatientCell: HEK293T cells (EFO:0001184)

GeneFunction: GO:0007602 (phototransduction)

GeneAlteration: Site-directed mutagenesis was used to introduce the variants into minigenes encoding portions of the wlid-type OPN1LW gene.

FunctionalEvidence: Three OPN1LW variants identified in myopic patients were expressed exogenously in HEK293T cells and two out of three exhibited reduced expression at the mRNA level (Figures 1A and 1B).

FAVariants: NM_020061.6(OPN1LW):c.973A>G (p.Met325Val), CA415309107, NM_020061.6(OPN1LW):c.970T>A (p.Phe324Ile), CA415309100, NM_020061.6(OPN1LW):c.971T>A (p.Phe324Tyr), CA415309101

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: Functional testing of Ser179Cys, Gly190Cys, Tyr191Cys and Ser204Cys variants of TIMP3 in baby hamster kidney (BHK) cells

NonPatientCell: Baby hamster kidney (BHK) cells, EFO:0001643 (kidney-derived cell line), CLO:0000696 (immortal hamster kidney-derived cell line), CLO:0001965 (BHK-21 cell line)

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 Ser179Cys, Gly190Cys, Tyr191Cys, and Ser204Cys variants (referred to in the paper as "Ser156Cys", "Gly167Cys", "Tyr166Cys", and "Ser181Cys") were generated by PCR-based mutagenesis of wild-type TIMP3 and cloned into the pNUT expression vector.

FunctionalEvidence: BHK cells were stably transfected with each of four variants or wild-type TIMP3. All variants expressed and localized successfully to the extracellular matrix (western blotting), Both variants tested exhibited intact metalloproteinase inhibitor activity in ECM fractions (reverse zymography). All four of the variants accumulated as higher molecular weight species (Figure 1A, Figure 2), with Ser179Cys and Ser204Cys in particular accumulating to a much greater degree than the other variants or the wild-type control (Figure 2). Cell morphology and adherence were affected within the variant-expressing cells. While wild-type-transfected cells were more adherent to the ECM than the untransfected controls were, both variant-transfected cells (Ser179Cys, Ser204Cys) were even more ECM-adherent (Figure 5). Association constants were measured for TIMP3 interaction with a series of metalloproteinases, calculating their values for wild-type TIMP3 and for the Ser156Cys variant, which exhibited a 2- or 3-fold loss of reduction, depending on the substrate (Table 2).

FAVariants: The four variants used were NM_000362.5(TIMP3):c.536C>G, p.Ser179Cys (ClinVar:12678), NM_000362.5(TIMP3):c.568G>T, p.Gly190Cys (CA411540415), NM_000362.5(TIMP3):c.572A>G, p.Tyr191Cys (ClinVar:12677), and NM_000362.5(TIMP3):c.610A>T, p.Ser204Cys (ClinVar:12676).

ExperimentName: Blocking IMPG1 in retinal organoids disrupts early development of IPM, photoreceptors and inner/outer segments, and connecting cilia.

NonPatientCell: UBERON:0000966 (retina, specifically IPSC-derived retinal organoids) or CL:0009004 (retinal cell)

GeneFunction: GO:0033165 (interphotoreceptor matrix)

GeneAlteration: Alteration was not performed at the genetic level, but rather a blocking antibody to IMPG1 protein was added to the culture media of the developing retinal organoid at days 90 and 150 of the differentiation process. After incubation, the effects were assessed by IHC and qRT-PCR detecting key markers of differentiation (Figure 6, Figures S12-S15).

FunctionalEvidence: Expression of RCVRN (a marker of photoreceptor and bipolar cells) and proportion of recoverin-positive cells were reduced (Figs. 6B, S12B, S14B, S14C, S15B). Expression of ARL13B (marker of photoreceptor connecting cilia) was reduced (Figs. 6B, S15B). Expression of RBP3 (marker of IPM formation) was changed only at the later time point (Figs. 6B, S12B, S15B). Expression of OPN1LW/OPN1MW (cone marker) was down-regulated (Fig. S14B). Expression of RHO (rod cell marker) and proportion of rhodopsin-positive cells were reduced, and the typical pattern of staining was replaced with a punctate pattern at the surface of the organoids (Fig. 6B, 6C). Staining for WGA (rod inner/outer segment marker) was reduced, indicating disrupted development of these structures (Fig. 6B, 6C). The authors conclude that IMPG1 is important for development of photoreceptors, inner and outer segments, connecting cilia, and IPM.

FAVariants: No variant in IMPG1 was used, but rather an inhibitory antibody.

NonPatientCell: EFO:0001182 (HEK293 Cells)

GeneFunction: GO:0008233 (Peptidase activity)

GeneAlteration: HEK293 cells were grown and 8 missense mutations were identified and generated by PCR-mutagenesis. The human mutant cDNAs were transfected into the cells using the calcuim-phosphate coprecipitation method and examined.

FunctionalEvidence: When HEK293 cells were transfected with wild-type and mutant human cDNA for PHEX, the resulting cells were subject to immunoblotting to determine the protein levels in both the cell lysate and the media of cells. All the variants and the wild-type showed presence in the lysate at various levels, but C85R, G579R, G579V, S711R, and A720T were all devoid of any secreted protein in the media. Ananlysis of these cells also showed possible rescue by growing transfected cells at 26 C, thus glycosylating and maturing the proteins. The endopeptidase activity of the wild-type and variants were also compared, with D237G and Y317F mutant proteins exhibiting 50–60% of wild-type activity; E581V and S711R also displayed nearly undetectable activity. This displayed that the disease causing mechanism could be related to loss of function of peptidase activity or cellular trafficking. Evidence is found under "Results".

FAVariant1: NM_000444.5:c.253T>C (p.Cys85Arg)

ClinVar: 438549 https://www.ncbi.nlm.nih.gov/clinvar/variation/438549/

FAVariant2: NM_000444.5:c.1735G>A (p.Gly579Arg)

ClinVar: 226119 https://www.ncbi.nlm.nih.gov/clinvar/variation/226119/

FAVariant3: NM_000444.5:c.1736G>T (p.Gly579Val)

ClinVar: 372777 https://www.ncbi.nlm.nih.gov/clinvar/variation/372777/

FAVariant4: NM_000444.5:c.2131A>C (p.Ser711Arg)

CAID: CA412575294 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA412575294

FAVariant5: NM_000444.5:c.2158G>A (p.Ala720Thr)

CAID: CA412575364 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA412575364

NonPatientCell: EFO:0001182 (HEK293 Cells)

GeneFunction: GO:0008233 (Peptidase activity)

GeneAlteration: HEK293 cells were grown and 8 missense mutations were identified and generated by PCR-mutagenesis. The human mutant cDNAs were transfected into the cells using the calcuim-phosphate coprecipitation method and examined.

FunctionalEvidence: When HEK293 cells were transfected with wild-type and mutant human cDNA for PHEX, the resulting cells were subject to immunoblotting to determine the protein levels in both the cell lysate and the media of cells. All the variants and the wild-type showed presence in the lysate at various levels, but C85R, G579R, G579V, S711R, and A720T were all devoid of any secreted protein in the media. Ananlysis of these cells also showed possible rescue by growing transfected cells at 26 C, thus glycosylating and maturing the proteins. The endopeptidase activity of the wild-type and variants were also compared, with D237G and Y317F mutant proteins exhibiting 50–60% of wild-type activity; E581V and S711R also displayed nearly undetectable activity. This displayed that the disease causing mechanism could be related to loss of function of peptidase activity or cellular trafficking. Evidence is found under "Results".

FAVariant1: NM_000444.5:c.253T>C (p.Cys85Arg)

ClinVar: 438549 https://www.ncbi.nlm.nih.gov/clinvar/variation/438549/

FAVariant2: NM_000444.5:c.1735G>A (p.Gly579Arg)

ClinVar: 226119 https://www.ncbi.nlm.nih.gov/clinvar/variation/226119/

FAVariant3: NM_000444.5:c.1736G>T (p.Gly579Val)

ClinVar: 372777 https://www.ncbi.nlm.nih.gov/clinvar/variation/372777/

FAVariant4: NM_000444.5:c.2131A>C (p.Ser711Arg)

CAID: CA412575294 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA412575294

FAVariant5: NM_000444.5:c.2158G>A (p.Ala720Thr)

CAID: CA412575364 https://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA412575364

NonPatientCell UBERON:0005424

GOterm GO:0042802

GeneAlteration shRNAs cloned into pCAG-mir30 vector that contained a puromycin resistance gene.

FunctionalEvidence electron microscopy of RPE in 1-2 yo mice showed 37-48% reduced phagocytosis in mutants. (fig. 1) Assayed cryosection of mouse RPE showed mislocalization of adhesion/phagocytosis markers in RPE (fig. 4)

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

NonPatientCell UBERON:0005424

GOterm GO:0042802

GeneAlteration shRNAs cloned into pCAG-mir30 vector that contained a puromycin resistance gene.

FunctionalEvidence electron microscopy of RPE in 1-2 yo mice showed 37-48% reduced phagocytosis in mutants. (fig. 1) Assayed cryosection of mouse RPE showed mislocalization of adhesion/phagocytosis markers in RPE (fig. 4)

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

NonPatientCell Mouse cell line, EFO:0002887 "Myotubes generated from the homozygous dysgenic (mdg/mdg) mouse cell line GLT were cultured as described previously (44)."

GeneAlteration GFP tagged point mutation transfected into mouse cells

FunctionalEvidence Variant increased channel sensitivity to caffeine/voltage (fig. 2)

FAVariants NM_000069.2(CACNA1S):c.3257G>A (p.Arg1086His), ClinVar ID: 17626

NM_000445.4(PLEC):c.3719G>A (p.Arg1240His), ClinVar ID: 448061

NonPatientCell Mouse cell line, EFO:0002887 "Myotubes generated from the homozygous dysgenic (mdg/mdg) mouse cell line GLT were cultured as described previously (44)."

GeneAlteration GFP tagged point mutation transfected into mouse cells

FunctionalEvidence Variant increased channel sensitivity to caffeine/voltage (fig. 2)

FAVariants NM_000069.2(CACNA1S):c.3257G>A (p.Arg1086His), ClinVar ID: 17626

NM_000445.4(PLEC):c.3719G>A (p.Arg1240His), ClinVar ID: 448061