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).

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: 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: 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)

NonPatientCell: E-Coli (DH5α)

GeneFunction: GO:0006559

GeneAlteration: PAH cDNA was cloned into a pMAL-c2E expression vector encoding an N-terminal MBP tag and an enterokinase cleavage site. PAH mutants were constructed using site-directed mutagenesis and confirmed using DNA sequencing. Both the wild-type and the 10 mutant vectors were transformed into E. coli DH5α via overexpresion of fusion proteins at midexponential phase. The proteins were purified and concentration was determined.

FunctionalEvidence: Expression of the 10 mutant proteins compared to the wild-type demonstrated that several of these mutants induce protein destabilization and subsequent loss of function. Upon deconvolution analysis, five variants showed significanly distrubed oligomerization and a preponderance to misfold and form high-molecular-weight protein aggregates. Enzyme kinetic parameter analysis demonstrated moderate reduction in PAH activity and severely affected allostery in all but one mutation, indicating the strong possibility of global confirmational changes. Protein confirmation was tested by limited proteolysis with proteinase K with six out of eight tested mutants decreasing the half-life of the protein by >50% and decreased stability in five out of eight. Thermal protein denaturation was analyzed by fluorescence and demonstrated that all of the mutations led to substantial distortion of the protein's confirmation and/or loss of structural integrity compared to the wild-type, especially those in the regulatory domain. Finally, amino acid replacements were shown to disrupt local networks of amino acid interactions and alter the normal oligomeric state and therfore the function of the protein.

FAVariant1: NM_000277.3(PAH):c.1222C>T (p.Arg408Trp)

ClinVar: 577 https://www.ncbi.nlm.nih.gov/clinvar/variation/577/

FAVariant2: NM_000277.3(PAH):c.898G>T (p.Ala300Ser)

ClinVar: 92751 https://www.ncbi.nlm.nih.gov/clinvar/variation/92751/

FAVariant3: NM_000277.3(PAH):c.165T>G (p.Phe55Leu)

ClinVar: 92734 https://www.ncbi.nlm.nih.gov/clinvar/variation/92734/

FAVariant4: NM_000277.3(PAH):c.1249T>C (p.Tyr417His)

ClinVar: 102579 https://www.ncbi.nlm.nih.gov/clinvar/variation/102579/

FAVariant5: NM_000277.3(PAH):c.940C>T (p.Pro314Ser)

ClinVar: 102905 https://www.ncbi.nlm.nih.gov/clinvar/variation/102905/

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

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).

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: TIMP3 expression constructs encoding the "Ser181Cys", "Ser156Cys", and "Glu139X" variants (officially referred to as the Ser204Cys, Ser179Cys, and Glu162X variants) were generated by PCR-based mutagenesis of the wild-type TIMP-3-pCI.neo construct (previously described in PMID:10854443).

FunctionalEvidence: TIMP3 variants were exogenously expressed in ARPE-19 cells alongside wild-type and empty vector controls. The percentage of exogenously expressed wild-type TIMP3 protein remaining associated with the extracellular matrix after separation of transfected cells from ECM was 20% (after 1 day) or <1% (after 4 days). All three variants were turned over at a slower rate, with 35-50% remaining after 1 day, and 20-25% remaining after 4 days (Figure 2). On the other hand, while the Ser179Cys and Glu162X variants showed reduced ability to inhibit cell surface activation of Pro-MMP2, Ser204Cys did not (Figure 3). As a result, the key, consistent function of TIMP3 variants in SFD may be (toxic) aggregation rather than loss of inhibitory activity.

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), and NM_000362.5(TIMP3):c.484G>T (p.Glu162X) (ClinVarID:12680).

ExperimentName: Factor VIII binding assays for variants exogenously expressed in COS-7 cells

NonPatientCell: COS-7 cell line (CLO:0002597, EFO:0002009)

GeneFunction: GO:0005515 (protein binding, specifically to Factor VIII)

GeneAlteration: Site-directed mutagenesis was used to introduce a mutation encoding "His54Gln" (corresponding to the p.His817Gln substitution from the variant) into the mature (cleaved) VWF protein.

FunctionalEvidence: The variant did not disrupt multimer formation (Figure 5) but reduced Factor VIII binding to 75% of wild-type levels (Figure 6). In combination with another missense mutation in cis that reduced Factor VIII binding by an additional 21-23%, and a frameshift mutation in trans predicted to cause the absence of a gene product, the variant appears likely to be contributing to pathogenicity.

FAVariants: A construct equivalent to the c.2451T>A (p.His817Gln) variant was used (ClinVar:100224), although it expressed the proteolytically processed version of the protein product and referred to the variant as "His54Gln".

NonPatientCell: EFO:0002529 (INS-1), EFO:0000856 (islet of Langerhans), CL:0000182 (Hepatocyte)

GeneFunction: GO:0004353 (glutamate dehydrogenase [NAD(P)+] activity)

GeneAlteration: Recombinant adenoviruses encoding WT human GLUD1 and the S498L variant were generated via an expression system, with the human GLUD1 gene being amplified and sub-cloned into a plasmid with a promoter. Digestion using EcoRV was used to discriminate between the wild-type and the mutant with sequencing verifying each step of the cloning. This adenovirus was amplified in HEK-293 cells and INS-1E β-cells were transduced with it. Mouse and human hepatocytes were isolated and seeded before being transduced with the same adenoviruses. The transfection efficency was confirmed via immunoblotting.

FunctionalEvidence: The enzymatic activity was evaluated in both wild-type and mutant mice, with both showing very low activity without glutamate concentrations and the mutant showing higher activity when glutamate was increased. The reductive direction using α-ketoglutarate as substrate was also tested with similar increased results for the mutant. Sensitivity of both lines to the reaction activator (ADP) and inhibitor (GTP) levels were tested, showing that the mutant was more sensitive to the ADP activator and slightly reduced for the GTP inhibitor (particularly in the presence of ADP). Mitochondiral function in wid-type and mutant cells was assessed, demonstrating that glucose response from mutant and WT cells was preserved but that glutamine stimulation abnormally generates ATP in mutant cells.Calcium stimulation was also activated by both glutamine and glucose in mutant cells while only being affected by glucose in WT. Studies in mutant and WT-transfected mouse and human islets showed increased insulin and ammonia production in mutants from stimulation through this responsive glutamine sensitivity. All of this supports GLUD1's pathogenic mechanism, specifically causing a hyperctive mutant protein that responds to glutamine levels rather than simply glucose.

FAVariants: NM_005271.5(GLUD1):c.1493C>T (p.Ser498Leu)

ClinVar: 16122, https://www.ncbi.nlm.nih.gov/clinvar/variation/16122/

ExperimentName: Reduced E2-lipoylation in LIPT2 patient cells

PatientCell: Fibroblast (not specified where it was derived)

GeneFunction: GO:0009249

GeneAlteration: Patient fibroblasts from P1 harboring the LIPT2 c.89T>C(p.Leu30Pro) and c.377T>G(Leu126Arg) and fibrobalsts from P2 harboring the c.377T>G(Leu126Arg) and c.314T>G(p.Leu105Pro) were used in this assay.

FunctionalEvidence: Patient fibroblasts both from P1 and P2, both with compound heterozygous mutation in LIPT2 that represent 3 different variants (one overlaps), were assayed for E2 lipoylation in PDHc and alpha-KGDHc (Figure 3), compared to control cells. The assay indicates reduced lipoylation of both PDHc and alpha KGSHc compared to controls both by western blot (Figure 3A) and immunocytochemistry (figure 3B). the reduction in lipoylation was consistent with that observed in patients with LIPT1 mutations, indicating a role for LIPT2 in lipoylation.

FAVariants:<br /> 1) CA224979286 (NM_001144869.1:c.377T>G(p. Leu126Arg))

2) CA224979330 (NM_001144869.1:c.89T>C(p.Leu30Pro))

3) CA381976642 (NM_001144869.1:c.314T>C(p.Leu105Pro)

NonPatientCell myocardium, UBERON:0002349

GeneAlteration isolated adult rat ventricular myocytes infected with adenoviruses for expression of either the wild-type (WT) or mutant forms of CASQ2. Cytosolic and intra-SR [Ca2+]

FunctionalEvidence Causes leakage of Ca2+ transients from SR (fig. 5)

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

ExperimentName: Ojala 2016 hiPSC-derived CMs

PatientCell: CL_0002620 (skin fibroblasts)

GeneFunction: GO:0008016 (regulation of heart contraction)

GeneAlteration: hiPSC-derived cardiomyocytes (CMs) generated from skin fibroblasts collected from patients carrying a TPM1-Asp175Asn mutation

FunctionalEvidence: Figure 2 shows enlarged cardiomyocytes (CMs) (a-b), an increase in multinucleated CMs (c), and calcium handling abnormalities (d) for the HCM-TPM1 variant CMs compared to WT CMs.

FAVariants: NM_001018005.2(TPM1):c.523G>A (p.Asp175Asn), CA018106

ExperimentName: Defective lysosomal sorting receptor degradation in CLN5-depleted HeLa cells

NonPatientCell: HeLa cells (EFO:0001185)

GeneFunction: Lysosomal organization (GO:0007040)

GeneAlteration: CLN5 (or the CLN1 control) was depleted by transient transfection of small interfering RNA (siRNA) into HeLa cells, 48 hrs. before assays were performed.

FunctionalEvidence: CLN5-depleted cells have reduced immunofluorescent signal (Fig. 2K) and reduced expression (Fig. 3A) of two lysosomal sorting receptors; cation-independent mannose 6-phosphate receptor (CI-MPR) and sortilin (Fig. 2K). CLN5-depleted cells also have fragmented TGN46 signal (Fig. 2Q) and increased precursor and intermediate forms of the lysosomal cargo protein cathepsin D (Fig. 3C). These findings are consistent with CI-MPR and sortilin failing to correctly recycle to the Golgi and being degraded in lysosomes instead, possibly due to a defect in a complex called retromer, which returns membrane-associated proteins from the endosome to the Golgi or other cellular compartments. Endosomal signal for the retromer subunit Vps26 is also decreased (Fig. 4E), and both Vps26 and Vps35 are increasingly cytosolic rather than membrane-bound in CLN5-depleted cells (Fig. 6A). These defects are reversed by exogenously expressing CLN5 (Fig. 7B). Rab7 recruitment to the endosome is also defective in CLN5-depleted cells (Figs. 8B,8M). Considering that CLN5 interacts with Rab5 and Rab7 (Fig. 9A), it may serve as a scaffold recruiting Rab7 and the retromer complex to the endosomal membrane, for the purpose of recycling membrane-associated receptors such as sortilin and CI-MPR out of the endosome. Thus, patients with CLN5 loss-of-function variants may have defective recruitment of the retromer complex to the lysosome / endosome and disordered trafficking of membrane proteins out of these compartments as a result.

FAVariants: N/A

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: A TIMP3 expression construct encoding the "Ser181Cys" variant (more accurately referred to as a Ser204Cys variant) was generated by PCR using a 3' primer that contained the desired substitution.

FunctionalEvidence: The TIMP3 variant was exogenously expressed in COS-7 cells alongside a mock transfected control (not a wild-type control). Reverse zymography of extracellular matrix from these cells revealed the presence of both a monomeric and a dimeric species with metalloproteinase inhibitory activity (Figure 4). This was confirmed by immunoblotting to be TIMP3, and the dimeric species appeared under non-reducing conditions but disappeared under reducing conditions (Figure 5). 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 its inheritance. The wild-type control was absent from this figure. It is not clear whether a separate but similar figure showing the wild-type control was performed under reducing or non-reducing conditions.

FAVariants: Variant used was equivalent to NM_000362.5(TIMP3):c.610A>T (p.Ser204Cys) (ClinVarID:12676). The variant is referred to in this publication as "Ser181Cys" (due to the removal of a 23-amino acid N-terminal signal peptide from the polypeptide sequence) but encodes Ser204Cys in the full/updated polypeptide sequence.

MM is a patient with replacement of the entire OPN1LW locus by an additional copy of OPN1MW. This proband cannot be scored due to the variant type, but might be used for Functional Alteration in Patient Cells?

ExperimentName: Functional characterization of patient-derived hiPSC-RPE cells

PatientCell: CL:0002586, or retinal pigment epithelial cell (retinal pigmented epithelium generated from patient-derived induced pluripotent stem cells, or hiPSC-RPE)

GeneFunction: GO:0008191 (metalloendopeptidase inhibitor activity), GO:0051045 (negative regulation of membrane protein ectodomain proteolysis), GO:0016525 (negative regulation of angiogenesis)

GeneAlteration:** Patient cells were heterozygous carriers of the NM_000362.5(TIMP3):c.610A>T p.Ser204Cys variant, or unaffected controls that were homozygous for the non-variant allele.

FunctionalEvidence: hiPSC-RPE cells from SFD patients exhibit altered function relative to equivalent cells from unaffected individuals. They secrete a decreased quantity of TIMP3 (Figure 4C), indicating accumulation and retention of TIMP3 either within the cells or in the associated extracellular matrix. Finally, they express abnormal proteomic profiles with elevated levels of the known TIMP3 binding partner EFEMP1 and the known drusen component apolipoprotein E (Figure 5). The epithelial layer formed by these patient-derived cells exhibits reduced transepithelial electrical resistance (Figure 2C). Other known functions like metalloproteinase inhibition are retained by the variant-harboring cells (Figure 4D). These findings are consistent with a gain-of-function mechanism.

FAVariants: This experiment studied patient cells harboring the NM_000362.5(TIMP3):c.610A>T p.Ser204Cys variant (ClinVarID 12676).

ExperimentName: Transient expression of VWD type 2N variants in 293T cells

NonPatientCell: human embryonic kidney 293T cells (EFO:0001082)

GeneFunction: GO:0005515 (protein binding, specifically to Factor VIII)

GeneAlteration: A mutation encoding p.His817Gln was introduced into a construct for exogenous expression of the VWF protein.

FunctionalEvidence: The variant protein exhibited a mild defect in FVIII binding relative to the wild-type (less severe than other VWD type 2N variants) (Figure 1C). Because of the defect in FVIII interaction, the variant also exhibits mildly reduced cleavage by ADAMTS13 (Figures 1F, 1G, and 1H).

FAVariants: A construct equivalent to the c.2451T>A (p.His817Gln) variant was used (ClinVar:100224).

ExperimentName: TAZ Function in Patient Fibroblasts

PatientCell: CL:0000057 (Fibroblast)

GeneFunction: GO:0003841 (1-acylglycerol-3-phosphate O-acyltransferase activity)

GeneAlteration: Fibroblasts were utilized from frozen tissue samples that were taken from 14 Barth Syndrome probands and compared to age-matched controls.

FunctionalEvidence: Several relevant tafazzin slice variants (full transcript, -ex5, and -ex7) were expressed in human BTHS fibroblasts and the MLCL/CL ratio was compared to age-matched controls and the previous results from the fibroblasts themselves. This ratio is important as the amount of cardiolipin (CL) conversion by tafazzin is crucial to the disease progress and observable phenotypes. Overall, the ratio observed in fibroblasts alone was significantly higher than controls (~1.1, reference 0.12). The expression of tafazzin splice variants did rescue some of the ratio, although only the preumed active variant lacking exon 5 showed significant decrease of the ratio (~0.5).

FAVariant1: NM_000116.5(TAZ):c.153C>G (p.Tyr51Ter)

ClinVar: 11101, https://www.ncbi.nlm.nih.gov/clinvar/variation/11101/

FAVariant2: NM_000116.4:c.170G>T (p.Arg57Leu)

CAID: CA415179752, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA415179752

FAVariant3: NM_000116.4:c.590G>T (p.Gly197Val)

CAID: CA415184091, http://reg.clinicalgenome.org/redmine/projects/registry/genboree_registry/by_caid?caid=CA415184091

FAVariant4: NM_000116.5(TAZ):c.280C>T (p.Arg94Cys)

ClinVar: 202092, https://www.ncbi.nlm.nih.gov/clinvar/variation/202092/

NonPatientCell: EFO:0005214

GOterm: GO:0016192

GeneAlteration: Undifferentiated N2A cells (murine neuroblastoma cell line) were infected with shRNAs to downregulate endogenous Rab39b expression by 45%.

FunctionalEvidence: N2A cells 4d after treatment with shRNA showed a change in morphology. Clumps of actin and reduced and/or altered membrane protrusions were observed. Rescue with WT RAB39B was able to restore the normal actin and cytoskeletal morphology, indicating that Rab39b may alter intracellular vesicular trafficking steps essential for membrane remodeling.

FAVariants:

NonPatientCell: CL:0002608

GOterm: GO:0016192

GeneAlteration: Rab39b expression was knocked down in mouse primary hippocampal neurons using shRNA

FunctionalEvidence: Expression of Rab39b was reduced by 40% as determined by RT-PCR. Morphological analysis showed significant alteration and the number of growth cones was decreased at each neurite terminal. Authors also show that RAB39B was localized to growth cones and could be involved in intracellular trafficking related to membrane recycling of Golgi-derived vesicles. Therefore RAB39B gene alterations can lead to impaired trafficking and lead to accumulation in membrane content and disorganized growth of growth cones.

FAVariants:

NonPatientCell HL-1 atrial cell line, BTO:0003264

GeneFunction nuclear membrane organization, GO:0071763

GeneAlteration constructs consisting of full length wild-type and mutant (p.S358L) human TMEM43 tagged at the C-terminus with GFP were used to generate stably transfected HL-1 cells.

CellPhenotype HP:0011675 Arrhythmia

CellEvidence 1.2 cm/s slower electrical conduction velocity than WT and control (40% reduction)

FAVariants NM_024334.2(TMEM43):c.1073C>T, CA ID: 734

NonPatientCell myocardium of ventricle, UBERON:0001083

GeneAlteration The D307H mutation was introduced into the coding sequence of canine CASQ2 by site-directed mutagenesis. The canine CASQ2D307H coding sequence was inserted into an adenoviral vector to generate Ad-CASQ2D307H for subsequent gene transfer into adult rat ventricular myocytes.

FunctionalEvidence (fig. 2) More intracellular Ca2+ due to reduced ability of SR to store Ca2+

Add Fig. 4 Increased transients through Ca2+ sparks

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

NonPatientCell UBERON:0005424, "ARPE19 cells"

GOterm GO:0042802

GeneAlteration HD-Ad-F3iT494M was constructed by introducing the HA-tagged PRPF3 coding sequence carrying T493M mutation, under control of the Ubiquitin C promoter and followed by the bovine growth hormone poly(A) signal, into HD-Ad-F3i.

FunctionalEvidence T494M phosphorylates less readily than WT

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

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).

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 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

PMID: 1522213

Gene: GALNS

Disease: Mucopolysaccharidosis IVA

MonDO: MONDO:0009659

InheritancePattern: AR

ExperimentName: Expression of variant carrying cDNA in GALNS deficient fibroblasts

FunctionalEvidence: cDNA encoding each of the 15 variants identified in the study were transfected into a GALNS deficient fibroblast cell line. 14 of the 15 altered cDNA's showed no increase in GALNS activity when the cells were assayed, confirming a lack of enzymatic activity.

HGNC:

GeneEvidence:

SharedGeneImplication:

CellLine: Human Mononuclear Myoblast

CellEFO: CL:0000056

GeneFunction: GO:0007015 (Actin Filament Organization)

GeneAlteration: A wild-type C-terminal EGFP-tagged β-TM fusion gene was generated by amplifying TPM2 and cloning it into a vector while introducing XhoI and EcoRI restriction sites. All mutant constructs were formed by site-directed mutagenesis and cloning into E. coli. These constructs were sequenced to verify the correct coding sequence and introduction of the mutations. They were then introduced to human myoblast cells and mouse C2C12 cells via transfection.

FunctionalEvidence: The expression of these TPM2 mutants in cultured cells demonstrated several morphological abnormalities reminiscent of those seen in nemaline myopathy patients. In the human myoblasts/differentiated cells, these included various types of aggregates including nuclear, cytoplasmic, endogenous, and perinuclear. Large accumulations of actin along with clouds around the nucleus and cytoplasm with unorganized structure were also present. Poor incorporation of stress fibers, what appeared to be disorganized sarcomeric structure, and rod-like structures both intranuclear and in the myotubes in particular all demonstrate clear similarities to those phenotypes seen in nemaline myopathy, namely the presence of nemaline rods, disorganized sarcomeres, and z-disc abnormalities. These two groups of pathologies also seem to share the same mechanism, specifically the TPM2 mutations destabilizing the actin-myosin structures and causing accumulation and abnormal structures to form. Similarly, the modified C2C12 myoblasts/myotubes also displayed peripheral and perinuclear aggregates in both stages of development as well as diffuse cytoplasmic labeling of stress fibers. They had difficulty differentiating, as they appeared to be fused rather than elongated and multinucleated. This further supports the connection between the gene disruption and the pathologies that are demonstrated here.

CellVariant1: NM_003289.3:c.121G>A (p.Glu41Lys)

ClinVar: 12464 http://www.ncbi.nlm.nih.gov/clinvar/variation/12464

CellVariant2: NM_003289.3:c.142_144delAAG (p.Lys49del)

ClinVar: 12466 https://www.ncbi.nlm.nih.gov/clinvar/variation/12466/

CellVariant3: NM_003289.3:c.154_156dup (p.Gly52dup)

ClinVar: 140485 https://www.ncbi.nlm.nih.gov/clinvar/variation/140485/

CellVariant4: NM_003289.3:c.364G>A (p.Glu122Lys)

CAID: CA373367963 http://reg.genome.network/redmine/projects/registry/genboree_registry/by_caid?caid=CA373367963

CellVariant5: NM_003289.3:c.606C>G (p.Asn202Lys)

ClinVar: 12467 https://www.ncbi.nlm.nih.gov/clinvar/variation/12467/

PatientCell: CL:0000187 (Muscle Cell)

GeneFunction: Muscle Cell Apoptotic Process (GO:0010657)

GeneAlteration: Samples were collected from deltoid muscle biopsy of control individuals and LGMD2C individuals with the 525delT founder mutation. No further alteration necessary.

FunctionalEvidence: TUNEL staining was perfomed on muscle samples from LGMD2C patients and healthy controls to test for cell apoptosis. No positive staining was detected in any control individuals while it was commonly found in the patient muscle. This indicates that a result of SGCG disruption is increased cellular apoptosis likely contributing to the myofiber degeration and muscle atrophy seen commonly in LGMD patients.

FAVariant: NM_000231.2(SGCG):c.525del (p.Phe175fs)

ClinVarID: 189243 https://www.ncbi.nlm.nih.gov/clinvar/variation/189243/

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

PatientCell CL:0000057 (fibroblast)

GeneFunction GO:0070131 (positive regulation of mitochondrial translation)

GeneAlteration n/a

FunctionalEvidence (Figure 2D) Labelling patient fibroblast proteins with EasyTag EXPRESS35S Protein Labeling Mix showed decreased assembly of complex I (NDUFB8) and IV (COXI) which corroborates the reduced mean enzyme activity and mitochondrial translation in Table 1.

FAVariants NM_017819.3(TRMT10C):c.814A>G (p.Thr272Ala)

ClinVar: 224317

and

NM_017819.3(TRMT10C):c.542G>T (p.Arg181Leu)

ClinVar: 224316