Journal: Cold Spring Harbor Molecular Case Studies

Article Title: A uniparental isodisomy event introducing homozygous pathogenic variants drives a multisystem metabolic disorder

doi: 10.1101/mcs.a004457

Figure Lengend Snippet: Identified homozygous variant c.1122C > G reduces PUS1 mRNA and protein levels. ( A ) The percentage of variant reads for all variants on Chromosome 12 identified by whole-exome sequencing ( left ). This demonstrates an absence of heterozygous variants, which was not seen for other chromosomes—for example, Chromosome 13 ( right ). ( B ) Pedigree of the affected individual illustrating paternal isodisomy. ( C ) Sanger sequencing of fibroblasts from the affected individual illustrating the homozygous C > G point mutation introducing a nonsense stop codon. ( D ) Quantitative polymerase chain reaction (qPCR) measuring mRNA expression of PUS1 in healthy control (black) and the affected individual (gray) in fibroblasts, normalized to β-actin mRNA. Data are represented as the mean ( N = 3), (**) P < 0.005 as calculated by a two-tailed Student's t -test. ( E ) Western blot showing the reduced PUS1 protein levels in fibroblasts derived from the affected individual compared to a healthy control. All western blots shown are representative of at least three independent experiments. ( F ) Confocal microscopy of fibroblasts illustrating the localization of PUS1 (green), mitochondria demarked by TOMM20 (red), and an overlay (yellow) including DAPI (blue) staining.

Article Snippet: The primary antibodies were PUS1 (NBP1-32902, Novus Biologicals), PUS1 (sc-376443, Santa Cruz), MTCO1 (ab14705, Abcam), SDHA (ab14715, Abcam), TP53 (sc-6243, Santa Cruz), BCL-2 (ab692-500, Abcam), BAX (#2772, Cell Signaling), and β-actin (A5441, Sigma-Aldrich).

Techniques: Variant Assay, Sequencing, Mutagenesis, Real-time Polymerase Chain Reaction, Expressing, Control, Two Tailed Test, Western Blot, Derivative Assay, Confocal Microscopy, Staining

Journal: Cold Spring Harbor Molecular Case Studies

Article Title: A uniparental isodisomy event introducing homozygous pathogenic variants drives a multisystem metabolic disorder

doi: 10.1101/mcs.a004457

Figure Lengend Snippet: Description of variants described in this study

Article Snippet: The primary antibodies were PUS1 (NBP1-32902, Novus Biologicals), PUS1 (sc-376443, Santa Cruz), MTCO1 (ab14705, Abcam), SDHA (ab14715, Abcam), TP53 (sc-6243, Santa Cruz), BCL-2 (ab692-500, Abcam), BAX (#2772, Cell Signaling), and β-actin (A5441, Sigma-Aldrich).

Techniques: Variant Assay

Journal: Cold Spring Harbor Molecular Case Studies

Article Title: A uniparental isodisomy event introducing homozygous pathogenic variants drives a multisystem metabolic disorder

doi: 10.1101/mcs.a004457

Figure Lengend Snippet: The PUS1 variant specifically impairs mitochondrial translation. ( A ) Western blot showing the protein levels of the nuclear-encoded SDHA compared to the mitochondrial-encoded MTCO1 in fibroblasts carrying the PUS1 p.Tyr374* variant or derived from a healthy control. ( B ) Quantification of the results in A . The levels of SDHA ( left ) and MTCO1 ( right ) in fibroblasts from the affected individual (gray) and healthy control (black) normalized to β-actin. Protein levels of MTCO1 are significantly reduced in cells from affected individual compared to healthy control, whereas the SDHA protein levels remain stable. Data are represented as the mean ( N = 3), (****) P < 0.00005 as calculated by a two-tailed Student's t -test.

Article Snippet: The primary antibodies were PUS1 (NBP1-32902, Novus Biologicals), PUS1 (sc-376443, Santa Cruz), MTCO1 (ab14705, Abcam), SDHA (ab14715, Abcam), TP53 (sc-6243, Santa Cruz), BCL-2 (ab692-500, Abcam), BAX (#2772, Cell Signaling), and β-actin (A5441, Sigma-Aldrich).

Techniques: Variant Assay, Western Blot, Derivative Assay, Control, Two Tailed Test

Journal: Cold Spring Harbor Molecular Case Studies

Article Title: A uniparental isodisomy event introducing homozygous pathogenic variants drives a multisystem metabolic disorder

doi: 10.1101/mcs.a004457

Figure Lengend Snippet: Cells carrying the PUS1 p.Tyr374* variant stabilize TP53 and alter mitochondrial protein BCL-2 and BAX levels. ( A ) Western blot showing the protein levels of TP53, p21, BCL-2, and BAX in fibroblasts derived from a healthy control or the affected individual upon 50 J/m 2 UVC exposure after 24 h. ( B ) Protein levels of TP53 were significantly increased in cells from affected individual (gray) under basal conditions (−UV) and further increased upon UVC exposure (+UV) compared to control cells (black). ( C ) Protein levels of p21 were significantly lower in cells from the affected individual under both conditions; however, p21 was still induced upon UVC exposure. ( D ) BCL-2 and ( E ) BAX protein levels were reduced in cells from the affected individual under both conditions. ( F ) The ratio of BAX:BCL-2 is shown to remain stable in cells from affected individual and control cells under both conditions. Data are represented as the mean ( N = 3), (*) P < 0.05, (**) P < 0.005, (***) P < 0.0005 as calculated by a two-tailed Student's t -test.

Article Snippet: The primary antibodies were PUS1 (NBP1-32902, Novus Biologicals), PUS1 (sc-376443, Santa Cruz), MTCO1 (ab14705, Abcam), SDHA (ab14715, Abcam), TP53 (sc-6243, Santa Cruz), BCL-2 (ab692-500, Abcam), BAX (#2772, Cell Signaling), and β-actin (A5441, Sigma-Aldrich).

Techniques: Variant Assay, Western Blot, Derivative Assay, Control, Two Tailed Test

Journal: Cold Spring Harbor Molecular Case Studies

Article Title: A uniparental isodisomy event introducing homozygous pathogenic variants drives a multisystem metabolic disorder

doi: 10.1101/mcs.a004457

Figure Lengend Snippet: Electron microscopy of fibroblasts with and without UVC exposure. ( A ) Patient (PUS1 p.Tyr374*) ( lower panels) and healthy control ( upper panels) fibroblasts show no morphological difference at basal level ( left panels). Twenty-four hours after exposure to 50 J/m 2 UVC (+UV, right panels), note the increased fragmentation of mitochondria in the cells carrying the PUS1 variant in the dashed box outline labeled R. ( B ) Enlarged images of UVC-treated fibroblasts from a healthy control on the left (L) and carrying the PUS1 p.374Tyr* variant on the right (R). White arrowheads indicate swollen cristae.

Article Snippet: The primary antibodies were PUS1 (NBP1-32902, Novus Biologicals), PUS1 (sc-376443, Santa Cruz), MTCO1 (ab14705, Abcam), SDHA (ab14715, Abcam), TP53 (sc-6243, Santa Cruz), BCL-2 (ab692-500, Abcam), BAX (#2772, Cell Signaling), and β-actin (A5441, Sigma-Aldrich).

Techniques: Electron Microscopy, Control, Variant Assay, Labeling