RescuePatientCell: Primary fibroblasts from a SLC6A8-deficient patient.

GeneAlteration: c.1540C>T (p.Arg514X) (previously described by Salomons et al, 2001, Am J Hum Genet, 68: 1497-1500)

RescuePhenotype: Ability to transport creatine into the cell.

RescueMethod: The wild type SLC6A8 open reading frame was cloned into an expression vector with EGFP. This construct was stably transfected into primary fibroblasts from patient with creatine transporter deficiency due to a nonsense variant, c.1540C>T (p.Arg514X) in SLC6A8 (previously described in PMD 11326334). Expression of the SLC6A8 fusion protein or EGFP protein (empty vector) was confirmed by the presence of green fluorescence. Expression was also confirmed by RT-PCR. Transfected cells were incubated for 24 hours with 25 umol/L creatine, 500 umol/L creatine, or 500 umol/L creatine plus 500 umol/L guanidinoproprionate (creatine transporter inhibitor). Intracellular creatine level was measured by GC-MS.

RescueEvidence: When the wild type SLC6A8 open reading frame was expressed in primary fibroblasts from patient with creatine tarnsporter deficiency, due to a nonsense variant, c.1540C>T (p.Arg514X) in SLC6A8 (previously described in PMID 11326334), creatine uptake was restored to a level comparable with controls.

RescueVariants: ClinVarID: 11696

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: 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).

RescuePatientCell CL:0000057

GeneAlteration Patient fibroblasts transfected with EBO vector containing either p.Tyr438Asn or WT cDNA.

RescuePhenotype HP:0008344

RescueMethod Measured decarboxylation rate of patient cells with various EBO vectors(empty, WT, or with variant) in comparison to an untransfected normal cell.

RescueEvidence Patient fibroblasts with the variant, empty vector, and vector containing the variant all showed virtually no decarboxylation activity. Vector containing WT restores decarboxylation rate to that of the normal cell (Figure 6). Figure 7 western blot shows that E1b subunit levels are also rescued.

RescueVariants NM_000709.3(BCKDHA):c.1312T>A (p.Tyr438Asn)

RescuePatientCell CL:0000057

GeneAlteration NM_017819.3(TRMT10C):c.814A>G (p.Thr272Ala) and/or NM_017819.3(TRMT10C):c.542G>T (p.Arg181Leu)

RescuePhenotype defective oxidative phosphorylation and tRNA 5' processing affecting high energy demand tissues: HP:0001508 (Failure to thrive)

RescueMethod Fibroblasts transfected with lentiviral vector containing single copy of WT TRMT10C.

RescueEvidence Transfected patient cells had increased MRPP1 expression, restored mitochondrial translation, normalized levels of respiratory chain complexes and mt-tRNA precursors which rescues the reduced cellular oxidative phosphorylation and slowed growth. 21857155 demonstrates both the restriction of translation and reduction of the rate of oxidative phosphorylation during MRPP1 knockdown in HeLa cells (Fig 5). Those result corroborate the gap between reduced MRPP1 expression and reduction in translation of oxidative phosphorylation machinery.

RescueVariants n/a