Fig. 3-. Somatic microglial clones with multiple and recurrent CBL and MAP-Kinase pathway activating variants.
(A) Pathway enrichment analysis for the genes target of P-SNVs using the panel of 716 genes as background set. Graph shows the most enriched pathways by: Reactome Gene Sets, GO Molecular Functions, Canonical Pathways and KEGG Pathway (see complete list in Supplementary Table 4). (B) Bar plot indicates the genes carrying P-SNV (y-axis) and the % of AD patients carrying P-SNV for each gene (x-axis). Genes are color-coded by pathway. (C) Representation of the classical MAPK pathway, the 6 genes mutated in AD patients are labeled in red, TEK is labeled in blue, and larger font size indicate reccurence of variants in a given gene. Violin plot shows distribution of P-SNV in genes from classical MAPK pathway per Mb sequenced and per sample in patients and controls, p-value: unpaired two-tailed Mann-Whitney U test. (D) Summary Table showing patients carrying P-SNV in the classical RTK/MAPK pathway and CML associated genes (see Supplementary Table 3) and indicating the detection of variants in blood, and their association with other variants in microglia. (E) Recurrent variants in the ring-like domain of CBL are indicated in red on the diagram structure of gene, above representative Western blot from cell lysates from HEK293T cells expressing WT, positive control (Y371H), or CBL variants alleles found in patients, and stimulated with EGF or control, probed with antibodies against Phospho-p44/42 MAPK (Erk 1/2, Thr202/Tyr204), total p44/42 MAPK (Erk1/2), HA-tag and tubulin (BOTTOM). Histogram (RIGHT) represents quantification of the increase of the Phospho-ERK1/2 /total ERK1/2 ratio in western blots in n=5 independent experiments, statistics: unpaired one-tailed t-test. (F) RIT1 M90I and F82L are represented on the 3D structure of the gene (pdb code: 4klz, F82 is within a segment whose structure was not resolved) and representative western blot from HEK293T cells expressing Flag-RIT1 (WT and mutants) and treated -/+ 20% FBS before harvesting. Lysates were probed with antibodies against Phospho-p44/42 MAPK (Erk 1/2, Thr202/Tyr204), total p44/42 MAPK (Erk1/2, (MAPK)), Flag, and tubulin. Histogram (RIGHT) represents quantification of the increase of the Phospho-ERK1/2 /total ERK1/2 ratio in western blots in n=4 independent experiments, statistics: unpaired one-tailed t-test. (G) Variant allelic frequency (VAF, %) for the BRAFV600E allele in PU.1+ nuclei from brain samples from histiocytosis patients (each dot represents a sample) and for P-SNVs in in PU.1+ nuclei from brain of AD patients (each dot represent a variant). Note: non-parametric tests were used when data did not follow a normal distribution (D’Agostino-Pearson normality test). (H) Percentage of P-SNVs detected by targeted deep sequencing (TDS) which were also detected by Whole-Exome-Sequencing (WES).