Journal: Human Genomics

Article Title: A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease

doi: 10.1186/s40246-025-00907-6

Figure Lengend Snippet: GATA4 VUS genetic variant and stem-cell gene editing. A Protein structure showing the patient mutation in the carboxyl zinc finger domain. B Patient Human Phenotype Ontology terms as described by physician. C CRISPR gene editing efficiency for single nucleotide variant introduction in KOLF2C iPSCs sequencing reads aligned to GATA4_WT and GATA4 VUS amplicon sequence. WT, sequencing reads aligned to WT sequence. Imp WT, imperfect sequencing reads aligned to WT with other mutations. HDR, reads aligned to the VUS sequence. Amb, sequencing reads that align to the GATA4 amplicon that have deleted the target site. D Bar graph showing variant representation in genomic DNA from healthy (WT) and heterozygous GATA4 p.Arg284His (VUS) clones, with clone identity. E Light microscopy images demonstrating normal stem-cell morphology. Scale bar, 100 micron

Article Snippet: Next, cells were permeabilised 15 min in 0.1% Triton-X-100 (Sigma-Aldrich), blocked with Intercept ® Blocking Buffer (LI-COR) for 1 h room temperature, and incubated at 4 °C overnight with rabbit anti-GATA4 antibody (1:400; D3A3M, Cell Signalling Technology).

Techniques: Variant Assay, Mutagenesis, CRISPR, Sequencing, Amplification, Clone Assay, Light Microscopy

Journal: Human Genomics

Article Title: A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease

doi: 10.1186/s40246-025-00907-6

Figure Lengend Snippet: GATA4 WT and GATA4 VUS iPSC cardiac disease modelling. A iPSC cardiomyocyte differentiation schematic. B Beating score for cardiomyocytes. C Flow cytometry gating. Cells were gated on forward and side scatter, single cells, and cell viability; with subsequent gating for OCT3 and NANOG expression, or TNNT2 and GATA4 expression. D Bar plots indicate OCT3 + NANOG + percentage frequency expression in GATA4 WT and GATA4 VUS cells at day 0 (iPSCs) and day 16 (cardiomyocytes, CM). E Bar plots indicate expression percentage frequency GATA4 + TNNT2 + in cardiomyocytes. F Bar plots indicate GATA4% frequency expression in iPSCs and cardiomyocytes, and GATA4 mean fluorescent intensity (MFI) in cardiomyocytes. G Fluorescent immunohistochemistry indicates that GATA4 WT and GATA4 VUS protein localizes to the nucleus. MFI, mean fluorescent intensity. Scale bar, 100 micron. Data represents two experiments with paired WT and VUS clones (GATA4_WT clone 6 and GATA4_VUS clone 2; GATA4_WT clone 4.9 and GATA4_VUS clone 4.5). *p ≤ 0.05, two-way ANOVA, with Bonferonni’s correction for multiple testing)

Article Snippet: Next, cells were permeabilised 15 min in 0.1% Triton-X-100 (Sigma-Aldrich), blocked with Intercept ® Blocking Buffer (LI-COR) for 1 h room temperature, and incubated at 4 °C overnight with rabbit anti-GATA4 antibody (1:400; D3A3M, Cell Signalling Technology).

Techniques: Flow Cytometry, Expressing, Immunohistochemistry, Clone Assay

Journal: Human Genomics

Article Title: A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease

doi: 10.1186/s40246-025-00907-6

Figure Lengend Snippet: Changes in GATA4 VUS cardiomyocytes and cardiac differentiation reflect patient disease phenotype. GATA4 WT and GATA4 VUS iPSCs were stimulated to form cardiomyocytes. A Bubble plot indicates GSEA pathway changes using the DisGeNET data set, for the difference between ventricular cardiomyocytes, and the differences in differentiation. B Alterations in calcium transients in GATA4 WT and GATA4 VUS atrial cardiomyocytes. C–E Graphs indicate changes in Amplitude, spontaneous firing, and FWHM for GATA4 WT and GATA4 VUS cardiomyocytes, as indicated. (Two independent experiments, Mann-Whitney test)

Article Snippet: Next, cells were permeabilised 15 min in 0.1% Triton-X-100 (Sigma-Aldrich), blocked with Intercept ® Blocking Buffer (LI-COR) for 1 h room temperature, and incubated at 4 °C overnight with rabbit anti-GATA4 antibody (1:400; D3A3M, Cell Signalling Technology).

Techniques: MANN-WHITNEY

Journal: Human Genomics

Article Title: A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease

doi: 10.1186/s40246-025-00907-6

Figure Lengend Snippet: Pathway changes due to GATA4 VUS genetic variant expression in cardiomyocyte function and differentiation. A Transcriptomics with GSEA and Gene Ontology database comparing GATA4 VUS to GATA4 WT for the differences between cardiomyocytes (∆CM), and the difference in differentiation (∆diffn) in GO databases -Biological Processes, Molecular Function, and Cellular Component. ( n = 4 experiments) B TNNT2 and a-actinin-2 staining of cardiomyocytes. Scale bar, 10 µM. C MYBPC3 and a-actinin-2 staining of cardiomyocytes. Scale bar, 10 micron

Article Snippet: Next, cells were permeabilised 15 min in 0.1% Triton-X-100 (Sigma-Aldrich), blocked with Intercept ® Blocking Buffer (LI-COR) for 1 h room temperature, and incubated at 4 °C overnight with rabbit anti-GATA4 antibody (1:400; D3A3M, Cell Signalling Technology).

Techniques: Variant Assay, Expressing, Staining

Journal: Human Genomics

Article Title: A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease

doi: 10.1186/s40246-025-00907-6

Figure Lengend Snippet: Changes in calcium pathways in GATA4 VUS genetic variant cardiomyocytes. Transcriptomics analysis with GSEA and KEGG database comparing GATA4 VUS to GATA4 WT for the differences between cardiomyocytes, pathway changes in Adrenergic signalling in cardiomyocytes and Cardiac muscle contraction, as indicated

Article Snippet: Next, cells were permeabilised 15 min in 0.1% Triton-X-100 (Sigma-Aldrich), blocked with Intercept ® Blocking Buffer (LI-COR) for 1 h room temperature, and incubated at 4 °C overnight with rabbit anti-GATA4 antibody (1:400; D3A3M, Cell Signalling Technology).

Techniques: Variant Assay