Journal: Genetics in Medicine Open

Article Title: Refining the interpretation of variants of uncertain significance in hereditary cancer screening through integrated RNA sequencing

doi: 10.1016/j.gimo.2024.101914

Figure Lengend Snippet: Summary of variants of uncertain significance (VUS) included in this analysis. A. Of the total VUS analyzed, 411 qualified for RNA sequencing, and these RNA-sequencing-analyzed VUS were found in both the intron and exon. B. Number of VUS analyzed by gene and stratified further by variant location and type.

Article Snippet: Data are available upon request from the corresponding author (Wenbo Xu, MD, PhD wxu@natera.com and Natera, Inc), except for primary sequencing data because the patient samples with the unique variants are impossible to guarantee anonymity for.

Techniques: RNA Sequencing, Variant Assay

Journal: Genetics in Medicine Open

Article Title: Refining the interpretation of variants of uncertain significance in hereditary cancer screening through integrated RNA sequencing

doi: 10.1016/j.gimo.2024.101914

Figure Lengend Snippet: Reclassification across all variants of uncertain significance (VUS). A. Sankey plot showing the outcomes of including RNA sequencing data into the evidence for variant interpretation and classification. B. Pie chart of the VUS that were upgraded and downgraded based on variant location and type. C. Classification of VUS based on gene.

Article Snippet: Data are available upon request from the corresponding author (Wenbo Xu, MD, PhD wxu@natera.com and Natera, Inc), except for primary sequencing data because the patient samples with the unique variants are impossible to guarantee anonymity for.

Techniques: RNA Sequencing, Variant Assay

Journal: Genetics in Medicine Open

Article Title: Refining the interpretation of variants of uncertain significance in hereditary cancer screening through integrated RNA sequencing

doi: 10.1016/j.gimo.2024.101914

Figure Lengend Snippet: Splicing effects of missense variants. A. Overall findings. Sankey plot showing the outcomes of including RNA sequencing data into the evidence for variant interpretation and classification. B. Sashimi plot demonstrating the change in splicing found at BRCA2 NM_000059.4:c.473C>T (p.S158L) that causes whole-exon skipping. C. Sashimi plot demonstrating the change in splicing found at MUTYH NM_001128425.2:c.32T>G (p.Leu11Arg) that causes partial-exon skipping. D. Sashimi plot demonstrating the change in splicing found at MSH3 NM_002439.5:c.909G>C (p.K303N) that causes partial-intron retention. VUS, variants of uncertain significance.

Article Snippet: Data are available upon request from the corresponding author (Wenbo Xu, MD, PhD wxu@natera.com and Natera, Inc), except for primary sequencing data because the patient samples with the unique variants are impossible to guarantee anonymity for.

Techniques: RNA Sequencing, Variant Assay

Journal: Genetics in Medicine Open

Article Title: Refining the interpretation of variants of uncertain significance in hereditary cancer screening through integrated RNA sequencing

doi: 10.1016/j.gimo.2024.101914

Figure Lengend Snippet: Effects of splice-altering synonymous variants. A. Overall findings. Sankey plot showing the outcomes of including RNA sequencing data into the evidence for variant interpretation and classification. B. Sashimi plot demonstrating the change in splicing as a result of ATM NM_000051.4:c.1695A>G (p.Glu565=) that results in partial exon skipping. C. Sashimi plot showing the change in splicing because of the ATM NM_000051.4:c.8010G>A (p.K2670=) that causes whole-exon skipping. VUS, variants of uncertain significance.

Article Snippet: Data are available upon request from the corresponding author (Wenbo Xu, MD, PhD wxu@natera.com and Natera, Inc), except for primary sequencing data because the patient samples with the unique variants are impossible to guarantee anonymity for.

Techniques: RNA Sequencing, Variant Assay

Journal: Genetics in Medicine Open

Article Title: Refining the interpretation of variants of uncertain significance in hereditary cancer screening through integrated RNA sequencing

doi: 10.1016/j.gimo.2024.101914

Figure Lengend Snippet: Intronic variants outside of the canonical splice site can affect splicing. A. Overall findings. Sankey plot showing the outcomes of including RNA sequencing data into the evidence for variant interpretation and classification. B. Sashimi plot showing the change in RNA splicing as a result of the BRIP1 NM_032043.3:c.1341-3C>G variant, causing a frameshift and whole-exon skipping. C. Sashimi plot demonstrating splicing changes associated with the LZTR1 NM_006767.4:c.263+3G>C variant that results in intron retention and introduction of a premature stop codon. D. Sashimi plot visualizing the 2 deleterious splicing events observed in a patient with the BARD1 NM_000465.4:c.1569-7T>G variant. VUS, variants of uncertain significance.

Article Snippet: Data are available upon request from the corresponding author (Wenbo Xu, MD, PhD wxu@natera.com and Natera, Inc), except for primary sequencing data because the patient samples with the unique variants are impossible to guarantee anonymity for.

Techniques: RNA Sequencing, Variant Assay

Journal: Nature

Article Title: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion

doi: 10.1038/s41586-019-1410-1

Figure Lengend Snippet: a, Flow cytometry analysis using NKG2D– Fc to determine percentages of NKG2DL− (red) and NKG2DL+ (blue) AML cells in 177 cases of AML (Supplementary Table 1). b–m, NKG2DL− and NKG2DL+ subpopulations of AML cells are sorted from the same patients, and analysed side-by-side using equal cell numbers. b, Representative examples of the gating strategy. c, Representative examples of forward and sideward scatter plots. d, Representative examples of May–Grünwald– Giemsa staining. e, f, Quantification of cell-to-nucleus size ratio (e) (n = 50 cells quantified for each subpopulation, n = 5 cases of AML; boxes represent median and 25th–75th percentiles, whiskers are minimum to maximum) and in vitro colony formation (f) (means of technical triplicates, n = 38 cases of AML). g–i, Long-term engraftment in NSG mice. Flow cytometry of mouse bone marrow (BM) (g; n = 18 cases of AML), and peripheral blood (PB) and organs (h; n = 10 cases of AML). Each dot represents one mouse. i, Representative bone marrow histopathology images. Left, haematoxylin and eosin (H&E); right, anti-CD33, 630× magnification, n = 5 cases of AML, n = 3 mice per group). j, Kaplan–Meier survival analyses. Transplanted mice per case of AML for NKG2DL− cells: 5 for no. 1, 6, 7, 8, 12 and 110; 4 for no. 34; 3 for no. 76, 111, 119, 133 and 168; 2 for no. 72. Transplanted mice per case of AML for NKG2DL+ cells: 7 for no. 76; 6 for no. 110; 5 for no. 1, 6, 7, 8 and 12; 3 for no. 72, 111, 133 and 168; 4 for no. 34 and 119. k, l, Quantification of NKG2DL− and NKG2DL+ AML cells from engrafted mice (post-transplantation) compared to corresponding patient-derived samples (pre-transplantation). Representative plots (k) and summarized results (l) (no. 1, 7 and 8, n = 3; no. 6, n = 4 mice per subpopulation). m, Percentage of CFSE-labelled human CD33+ AML cells that home to the bone marrow (each dot represents 1 mouse, n = 3 mice per subpopulation, n = 3 cases of AML). n, o, Mice engrafted with AML cells were treated with cytarabine (1 mg subcutaneously daily, for 2 to 4 days), and percentages of NKG2DL− and NKG2DL+ AML cells analysed in the mouse bone marrow before and after treatment. n, Exemplary results. o, n = 8 cases of AML. Connected dots depict percentages of NKG2DL− (left) and NKG2DL+ (right) AML cells before and after treatment in one mouse. p, Correlation of surface expression of NKG2DLs and overall survival in patients with AML (≤65 years old with ≥ 80 NKG2DL+ (n = 10) versus ≥ 80% NKG2DL− cells (n = 32)). q–s, LSC, 17-gene stemness signatures and leukaemia-specific genetic aberrations in NKG2DL− versus NKG2DL+ cells. q, Gene-expression array and GSEA analyses (no. 1, 6, 7, 8 and 12). r, Quantitative PCR with reverse transcription (qRT–PCR). s, Targeted next-generation sequencing. e–h, m, Mann–Whitney U test. j, p, log-rank (Mantel–Cox) test. o, Wilcoxon test. q, Nominal P value and normalized enrichment score (NES). Except in o, statistical tests were two-sided. Centre values in f–h and l–m represent mean, error bars represent s.d.

Article Snippet: 46 Analysis of external datasets RSEM normalised RNA-sequencing expression data of 179 primary AML samples from The Cancer Genome Atlas (TCGA) 47 were downloaded from the Broad Institute.

Techniques: Flow Cytometry, Staining, In Vitro, Histopathology, Transplantation Assay, Derivative Assay, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Reverse Transcription, Quantitative RT-PCR, Next-Generation Sequencing, MANN-WHITNEY

Journal: Nature

Article Title: Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion

doi: 10.1038/s41586-019-1410-1

Figure Lengend Snippet: a, Differentially regulated genes in NKG2DL− versus NKG2DL+ cells (gene-expression arrays, no. 1, 6, 7, 8 and 12; RNA-seq, no. 9, 10, 11, 33 and 34; colour-coded z-score). FAM60A is also known as SINHCAF; GPR56 is also known as ADGRG1; RLTPR is also known as CARMIL2. b, Immunoblots showing expression of PARP1 protein and loading control in NKG2DL− and corresponding NKG2DL+ subpopulations of AML cells. Cropped images; for additional patients and full images see Extended Data Fig. 8b, c, Supplementary Fig. 1. c–e, In vitro PARP1 inhibition. Quantifications of NKG2DL+ percentages after treatment with PARP1 siRNAs versus control siRNAs (c, n = 4 cases of AML), and AG-14361 (20 μM) versus DMSO (0.2%) treatment (d, representative staining, no. 42; e, summarized quantification, bulk cells from n = 18 cases of AML, n = 3 healthy CD34+ cord blood cells; mean of technical triplicates). HSPCs, haematopoietic stem and progenitor cells. f, g, Analysis of induction of NKG2DLs on sorted NKG2DL− AML cells after treatment with AG-14361 (20 μM), the PARP inhibitor veliparib (1 μM) or DMSO control (0.2%). Representative results (f, no. 35) and quantification (g, each dot represents the average of technical replicates; n = 5 cases of AML). h, Summarized relative NKG2DL mRNAs in PARP1-inhibited versus control-treated bulk AML cells (PARP1 versus control siRNAs: no. 4 and 151; AG-14361 (20 μM) versus DMSO (0.2%), 3 cases of AML). See Extended Data Fig. 9f for analyses of single NKG2DLs. i, Chromatin immunoprecipitation analysis illustrating direct recruitment of PARP1 to the MICA and MICB promoter (biological triplicates, n = 3 cases of AML). j, NSG mice transplanted with bulk AML cells (1.5 × 106 cells per mouse) were treated with AG-14361 or DMSO (days 1–5) ± pNKCs (1.5 × 107 cells per mouse on day 6) and investigated for long-term induction of leukaemia. Summarized flow cytometry data in bone marrow, peripheral blood and organs are shown. Each dot represents one mouse; n = 5 biological experiments with AML cells from no. 35 (triangles) and no. 42 (circles) and NK cells from n = 5 donors. For numbers, see table on the right. Centre values represent mean, error bars represent s.d. c, One-way analysis of variance (ANOVA). e, i, Two-sided Mann–Whitney U test. g, Two-sided Student’s t-test. j, Left, two-way ANOVA; right, Fisher’s exact test.

Article Snippet: 46 Analysis of external datasets RSEM normalised RNA-sequencing expression data of 179 primary AML samples from The Cancer Genome Atlas (TCGA) 47 were downloaded from the Broad Institute.

Techniques: Gene Expression, RNA Sequencing, Western Blot, Expressing, Control, In Vitro, Inhibition, Staining, Chromatin Immunoprecipitation, Flow Cytometry, MANN-WHITNEY