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PMID244156742600304626319512
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DOI / linkdoi: 10.1002/humu.22511doi: 10.1007/s00438-015-1067-xDOI: 10.1016/j.gene.2015.08.045
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AuthorMercimek-MahmutogluDesroches Mercimek-Mahmutoglu
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Year201420152016
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Assay (general description)In vitro expression of cDNA constructs in GAMT-deficient cell line followed by assay of GAMT activity and Western blot (19 missense variants, 17 of which were found in patients with GAMT deficiency)In vitro expression of cDNA constructs in HeLa cells followed by assay of GAMT activity and Western blot (14 novel missense variants)In vitro expression of cDNA constructs in GAMT-deficient cell line followed by assay of GAMT activity and Western blot.
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Material used (patient cells, engineered variants, cell lines, animal model, etc.Missense variant were introduced into a pGAMT-EGFP plasmid by site-directed mutageneis (see PMID 26319512) and expressed in a primary GAMT-deficient human fibroblast cell line (homozygous for a frameshift variant; note - variant was not provided in the paper).Missense variant were introduced into a pGAMT-EGFP plasmid by site-directed mutageneis and expressed in HeLa cells. The fidelity of the insert was verified by sequencing.Missense variant were introduced into a pGAMT-EGFP plasmid by site-directed mutageneis and expressed in a primary GAMT-deficient human fibroblast cell line (same method as PMID 24415674).
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Readout type (qualitative/quantitative)Quantitative (enzyme assay), qualitative (Western blot)Quantitative for three variants with deficient activity (Fig 1), and qualitative for the remaining 6, which all have "normal activity" (enzyme assay).Qualitative ("similar levels as the wild type")
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Readout description% of recombinant wild-type GAMT activity% of recombinant wild-type GAMT activity% of recombinant wild-type GAMT activity
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Biological replicates (met/not met)met; Transfections were performed in triplicatemet; Transfections were performed in triplicatemet; Transfections were performed in triplicate
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Technical replicates (met/not met); descriptionnot met?not met?not met?
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Basic positive control (met/not met); descriptionmet; wild type pGAMT-EGFPN1; GFP signal indicates successful transfection; cells with normal GAMT activity also includedmet; wild type pGAMT-EGFPN1; GFP signal indicates successful transfection.met; wild type pGAMT-EGFPN1; GFP signal indicates successful transfection; cells with normal GAMT activity also included
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Basic negative control (met/not met); descriptionmet; 1) pEGFPN1 empty vector, 2) untransfected cellsmet; 1) pEGFPN1 empty vector, 2) untransfected cellsmet; 1) pEGFPN1 empty vector, 2) untransfected cells
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Validation controls P/LP (#)At least 2: One "known pathogenic missense variant" c.590T>C (p.Leu197Pro), one LP (without functional data included) based on the assessment of this VCEP. Four additional variants P/LP in ClinVar (need to be assessed without functional data - this will be done as part of the pilot study)One LP in ClinVar ((need to be assessed without functional data - this will be done as part of the pilot study)None
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Validation controls B/LB (#)One benign control: c.79T>C (p.Tyr27His) (MAF in gnomAD = 0.004706 (Latino), 0.004298 (European NF), 2 homozygotes in European NF; a patient who is homozygous for this variant has normal GAMT activity.None (based on MAF cut-offs for BA1 and BS1 assigned by this VCEP)None
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Statistical analysis (general description)Error bars show standard error of the mean for enzyme activity for triplicate transfections (Figure 1).Mean of biological triplicates measured as % of pGAMTvariant 1-EGFP-N1 wild type.not met
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Threshold for normal readout Not providedNot providedNot provided
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Threshold for abnormal readoutNot providedNot providedNot provided
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Approved assay (y/n)YesYesYes
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Proposed strengthSupportingSupportingSupporting
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