Journal: medRxiv

Article Title: Strategic validation of variants of uncertain significance in ECHS1 genetic testing

doi: 10.1101/2022.10.09.22280834

Figure Lengend Snippet: Gene structure (top) and corresponding amino acids (bottom) of ECHS1 . Variants registered r as pathogenic (red) and likely pathogenic (yellow) in ClinVar, and jMorp (square) are shown. The two underlined variants were identified from our genomic analysis study; no experimental VUS verification was performed.

Article Snippet: cDNA of ECHS1 (NM_00492) WT and VUS were synthetized and inserted into pCDNA3.1 Lifect-EGFP (Addgene 67303) with HindIII and XbaI sites by GENEWIZ/Azenta.

Techniques:

Journal: medRxiv

Article Title: Strategic validation of variants of uncertain significance in ECHS1 genetic testing

doi: 10.1101/2022.10.09.22280834

Figure Lengend Snippet: A. Genomic analysis of ECHS1 KO cells by Sanger sequencing. B. EHCS1 expression levels in WT and ECHS1 KO HEK293FT cells quantified by qRT-PCR. Whole cell extracts from WT and ECHS1 KO HEK293FT cells analyzed by immunoblotting using the indicated antibodies. Single and double asterisks indicate uncleaved and cleaved forms, respectively. C. ATP assay of WT and ECHS1 KO HEK293FT cells cultured in glucose or galactose medium. The graph shows the ATP level in galactose medium divided by galactose. D. ATP assay of WT and ECHS1 KO HEK293FT cells treated with the indicated L-valine concentrations. Bar graphs represent the average ATP level in each condition from three biological independent experiments. Error bars, ±SEM. Statistical analysis was performed using ANOVA followed by Dunnet’s test. RLU, relative luciferase unit.

Article Snippet: cDNA of ECHS1 (NM_00492) WT and VUS were synthetized and inserted into pCDNA3.1 Lifect-EGFP (Addgene 67303) with HindIII and XbaI sites by GENEWIZ/Azenta.

Techniques: Sequencing, Expressing, Quantitative RT-PCR, Western Blot, ATP Assay, Cell Culture, Luciferase

Journal: medRxiv

Article Title: Strategic validation of variants of uncertain significance in ECHS1 genetic testing

doi: 10.1101/2022.10.09.22280834

Figure Lengend Snippet: WT and ECHS1 KO HEK293FT cells transfected with the indicated expression vectors subjected to immunoblotting analysis (A) and ATP assay (B). A. Whole cell extracts were analyzed by immunoblotting using the indicated antibodies. Single and double asterisks indicate uncleaved and cleaved forms, respectively. B. ATP assay 4 days after treatment with L-valine (0.8 mM). Bar graphs represent the average ATP level in each condition from three biological independent experiments. Error bars, ±SEM. Red bars, pathogenic variants. Gray bars, VUS. RLU, relative luciferase unit. C. Statistical analysis of Figure B using ANOVA followed by Dunnet’s test. The color scale shows the P value compared with vector (vs Vec, statistically different red to blue) and WT ECHS1 (vs WT, statistically different blue to red).

Article Snippet: cDNA of ECHS1 (NM_00492) WT and VUS were synthetized and inserted into pCDNA3.1 Lifect-EGFP (Addgene 67303) with HindIII and XbaI sites by GENEWIZ/Azenta.

Techniques: Transfection, Expressing, Western Blot, ATP Assay, Luciferase, Plasmid Preparation

Journal: medRxiv

Article Title: Strategic validation of variants of uncertain significance in ECHS1 genetic testing

doi: 10.1101/2022.10.09.22280834

Figure Lengend Snippet: A. Whole cell extracts from WT and ECHS1 KO HEK293FT cells transfected with the indicated expression vectors analyzed by immunoblotting using the indicated antibodies. B. ATP assay of ECHS1 KO HEK293FT cells expressing the indicated expression vectors treated with additional L-valine (0.8 mM) for four days. Bar graphs represent the average ATP level in each condition from three biological independent experiments. Error bars, SEM. RLU, relative luciferase unit. C. Statistical analysis of Figure B using ANOVA followed by Dunnet’s test. The color scale shows the P value compared with vector (vs Vec, statistically different red to blue) and WT ECHS1 (vs WT, statistically different blue to red).

Article Snippet: cDNA of ECHS1 (NM_00492) WT and VUS were synthetized and inserted into pCDNA3.1 Lifect-EGFP (Addgene 67303) with HindIII and XbaI sites by GENEWIZ/Azenta.

Techniques: Transfection, Expressing, Western Blot, ATP Assay, Luciferase, Plasmid Preparation

Journal: medRxiv

Article Title: Strategic validation of variants of uncertain significance in ECHS1 genetic testing

doi: 10.1101/2022.10.09.22280834

Figure Lengend Snippet: A. RNA-seq data from two patients with c.489G>A (p.P163=) showing reads suggestive of splicing abnormalities in exons with c.489G>A. B. Read counts of the ECHS1 gene are plotted on the horizontal axis and the number of detected exon skipping is plotted on the vertical axis. Gene counts were calculated by STAR, and the number of exon skipping was extracted from the Sashimi plot data. C. Ratio of c.489G>A and c.796A>C and c.832G>A variants on the IGV viewer. In two cases, the allele expression with c.489G>A was decreased.

Article Snippet: cDNA of ECHS1 (NM_00492) WT and VUS were synthetized and inserted into pCDNA3.1 Lifect-EGFP (Addgene 67303) with HindIII and XbaI sites by GENEWIZ/Azenta.

Techniques: RNA Sequencing, Expressing

Journal: medRxiv

Article Title: Strategic validation of variants of uncertain significance in ECHS1 genetic testing

doi: 10.1101/2022.10.09.22280834

Figure Lengend Snippet: A. OUTRIDER analysis illustrating protein expression in a volcano plot. ECHS1 was detected as a protein with a large decrease in expression. B. Western blotting for ECHS1 in patients with ECHS1 mutations and controls. Compared to previously reported ECHS1 cases, case 1 showed significantly reduced ECHS1 expression. β-actin was detected as a loading control.

Article Snippet: cDNA of ECHS1 (NM_00492) WT and VUS were synthetized and inserted into pCDNA3.1 Lifect-EGFP (Addgene 67303) with HindIII and XbaI sites by GENEWIZ/Azenta.

Techniques: Expressing, Western Blot, Control