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Cited by 21Neuroinflammation and Neurodegeneration MechanismsAlzheimer's disease research and treatmentsInflammasome and immune disorders

A microglia clonal inflammatory disorder in Alzheimer’s disease

Open access
Vicario, Rocio, Fragkogianni, Stamatina, Weber, Leslie, Lazarov, Tomi, Hu, Yang, Hayashi, Samantha Y, Craddock, Barbara, Socci, Nicholas D +18 more·2025·eLife
DOI 10.7554/elife.96519.3PMC PMC10849735

Published version of a preprint

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Abstract

Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however, few studies have investigated its role in neurodegenerative processes such as Alzheimer’s disease (AD). Here, we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls. Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations. These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both in vitro and in patients. Although the natural history of AD-associated microglial clones is difficult to establish in humans, microglial expression of a MAPK pathway activating variant was previously shown to cause neurodegeneration in mice, suggesting that AD-associated neuroinflammatory microglial clones may contribute to the neurodegenerative process in patients.

AI snapshot

Written from the paper’s full text.

TL;DR

Microglia from a subset of Alzheimer’s disease patients are selectively enriched for somatic pathogenic clones, especially RTK/MAPK pathway variants such as recurrent CBL RING-domain mutations, compared with age-matched controls. These variants activate ERK and drive a neuroinflammatory/neurotoxic transcriptional program in microglia models and are detectable in patient microglia.

Key findings

  • Pathogenic somatic single-nucleotide variants (P-SNVs) were enriched in PU.1+ microglia from AD patients versus age-matched controls (p=0.0003); logistic regression adjusting for sex and age gave OR=7, p=0.0035, and mixed-effects modeling showed excess P-SNVs in AD independent of age (p=0.0215).
  • AD-associated microglial variants preferentially targeted the RTK/MAPK pathway: ~25% of AD patients tested had P-SNVs in classical RTK/MAPK genes (p=0.0145 vs age-matched controls), and 12/45 (~27%) carried one or several oncogenic RTK/MAPK-activating variants; 6 patients (13%) had recurrent CBL RING-domain variants, and 2 had RIT1 variants.
  • CBL and RIT1 variants increased MAPK/ERK phosphorylation in HEK293T cells; heterozygous CBL C404Y human iPSC-derived microglia-like cells showed increased ERK activation, slightly increased proliferation, and upregulated glycolysis, oxidative phosphorylation, mTORC1, TNF, JAK-STAT, complement, and inflammatory signatures, with increased TNF, IL-6, IFN-γ, IL-1β, C3, and complement factor H by ELISA.
  • Patient snRNAseq microglia clusters from variant carriers were enriched for inflammatory, TNF, mTOR, oxidative phosphorylation, and glycolysis signatures; KRAS A59G variant reads were detected in cluster 2/2B from patient AD52.

Methods

  • Post-mortem frozen brain and matched blood from 45 intermediate-onset sporadic AD patients and 44 controls, including 27 age/sex-matched controls; nuclei were sorted into PU.1+ microglia, NeuN+ neurons, double-negative glia/stromal cells, and blood, yielding 744 DNA samples.
  • Targeted deep sequencing used the BRAIN-PACT panel of 716 genes (3.43 Mb) at mean coverage ~1,100x, with variant calling by ShearwaterML and curated Mutect1, annotation by OncoKB/ClinVar, and ddPCR validation; selected samples also underwent whole-exome sequencing of PU.1+ nuclei at ~400x.
  • Functional validation used HEK293T cells, BV2 murine microglial cells, SV-U19-5-transformed mouse macrophage lines, and prime-edited hiPSC-derived CBL C404Y heterozygous and isogenic control microglia-like cells, with Western blotting, EdU proliferation assays, bulk RNAseq/GSEA, ELISA, and snRNAseq from 5 PU.1+ samples from 4 donors.

Limitations

  • The paper states that the natural history of AD-associated microglial clones is difficult to establish in humans: it is unknown whether they contribute early to neuroinflammation or expand later in the inflammatory AD brain; the cohort was relatively modest; WES had lower sensitivity than targeted deep sequencing; and the functional significance of DNA-repair and DNA/histone-methylation variants was not investigated.
  • The paper notes that overexpression of mutant alleles in mouse cell lines may not recapitulate heterozygous variants under physiological conditions, and the snRNAseq analysis involved a small number of samples/donors.
  • Evident from the text: targeted sequencing cannot identify variants outside the 716-gene BRAIN-PACT panel; half of AD patients had no microglia pathogenic variants identified; the design shows association rather than causation; and brain-region sampling was imbalanced, with hippocampus samples mostly from AD patients.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.7554/elife.96519.3

Cites

160 of 160 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 111 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In most cases, pathogenic variants in microglia from AD patients are absent from blood, glia, or neurons.

    “These pathogenic variants are absent from blood, glia or neurons in most cases.”
    Quote in the paperSubstantial claim
  • Findingown data

    Microglia-specific AD-associated variants preferentially target the MAPK pathway.

    “Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations.”
    Quote in the paperSubstantial claim
  • Findingown data

    Microglia-specific AD-associated variants include recurrent CBL ring-domain mutations.

    “Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations.”
    Quote in the paperSubstantial claim
  • Findingown data

    Microglia clones carrying pathogenic variants are selectively enriched in AD patients compared to age-matched controls.

    “Here we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls.”
    • Also stated in: § Introduction · plain
      “We found that microglia from AD patients were enriched for pathogenic variants in comparison to age-matched controls.”
    Quote in the paperSubstantial claim
  • Findingown data

    The AD-associated microglia variants drive a microglia transcriptional program characterized by a strong neuro-inflammatory response previously associated with neurotoxicity, including production of IL1 and TNF, both in vitro and in patients.

    “These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both in vitro and in patients.”
    • Also stated in: § Introduction · boosted
      “In addition, we showed that these variants drive a microglia transcriptional program characterized by a strong neuro-inflammatory response previously associated with neurotoxicity, including the production of IL1 and TNF, both in in vitro microglia models and in patients.”
    Quote in the paperSubstantial claim
  • May combine two claims. 52
  • Classified as probably not a substantial claim. 65
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    Hallmark and KEGG pathway analysis of RNAseq data from control and mutant lines showed increased RAS, TNF, IL6 and JAK STAT signaling, complement, inflammatory responses, and mTOR pathway activation signatures in mutants.

    “Hallmark and KEGG pathway analysis of RNAseq data from control and mutant lines showed increased RAS, TNF, IL6 and JAK STAT signaling, complement, inflammatory responses, and mTOR pathway activation signatures in mutants”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The P-SNV/Mb burden was similar between brain regions within each group, and therefore attributable to AD status rather than sampling bias.

    “Analysis of PU1 + P-SNV/Mb burden per brain region indicated that the P-SNV/Mb burden was similar between brain regions within each group (Fig. 2F and Supplementary Fig. 2C), and therefore attributable to AD status rather than sampling bias.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    A 12th patient carried a gain of function (GOF) U2AF1 (p.S34F) variant, which is not a classical MAPK gene but activates the MAPK pathway in myeloid malignancies.

    “a 12 th patient carried a gain of function (GOF) U2AF1 (p.S34F) variant 78 , which is not a 'classical MAPK gene' but activates the MAPK pathway in myeloid malignancies 79”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [78]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [79]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    Only 6/15 (40%) of the pathogenic SNVs previously identified by TDS and confirmed by ddPCR were detectable by WES in these samples, indicating a lower sensitivity of WES.

    “Only 6/15 (40%) of the pathogenic SNVs previously identified by TDS and confirmed by ddPCR were detectable by WES in these samples (Fig. 3H), indicating a lower sensitivity of WES.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CBL 404C/Y microglia-like cells also presented with signatures from the KEGG database associated with neurodegenerative disorders, and for the recently published human microglia AD scRNAseq signature, obtained by analysis of 24 sporadic AD patients and 24 controls.

    “CBL 404C/Y microglia-like cells also presented with signatures from the KEGG database associated with neurodegenerative disorders (Fig. 5A; Supplementary Table 8), and for the recently published human microglia AD scRNAseq signature, obtained by analysis of 24 sporadic AD patients and 24 controls 21”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    CBL 404C/Y cells were larger and presented with more lamellipodia, resulting in an amoeboid morphology less frequently observed in isogenic controls.

    “CBL 404C/Y cells were larger and presented with more lamellipodia, resulting in an amoeboid morphology less frequently observed in isogenic controls”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Pathogenic variants that did not involve the MAPK pathway included LOF variants in the DNA repair gene CHEK2 including CHEK2 c.319+1G>A and CHEK2 R346H, Mediator Complex gene MED12, Histone methyltransferases SETD2 and KMT2C/MLL3, the DNA methyltransferase DNMT3A, DNA demethylating enzymes TET2 and the Polycomb proteins ASXL1.

    “Pathogenic variants that did not involve the MAPK pathway included LOF variants in the DNA repair gene CHEK2 including CHEK2 c.319+1G>A 99 and CHEK2 R346H (Supplementary Fig. 3 and 4D), Mediator Complex gene MED12 100 , Histone methyltransferases SETD2 101 and KMT2C/MLL3, the DNA methyltransferase DNMT3A 86,102 , DNA demethylating enzymes TET2 and the Polycomb proteins ASXL1 86 .”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [99]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [100]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [101]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    Five patients also carried additional P-SNV targeting genes involved in DNA repair with tumor suppressor function, including loss of function variants in ATR (c.6318A>G) and SMC1A (p.X285_splice), and in DNA/histone methylation including TET2 (p.Q1627*), IDH2 (p.R140Q), and PBRM1 (c.996-7T>A).

    “Five patients also carried additional P-SNV targeting genes involved in DNA repair with tumor suppressor function 83,84 , including the loss of function variants in ATR (c.6318A>G) 85 and SMC1A (p.X285_splice) (Fig. 3D and Supplementary Fig. 3), and in DNA/histone methylation including TET2 (p.Q1627*) 63,86 , IDH2 (p.R140Q) 87 , and PBRM1 (c.996-7T>A) 88 )”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [83]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [84]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [85]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [63]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [86]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    Most (>90%) SNV and P-SNV were cell-type or tissue specific, with ~5% of SNV and ~8% of P-SNV shared between the blood and brain of individual donors.

    “A venn-diagram analysis of SNVs detected in PU.1 + , NeuN + , DN, and blood samples indicated that most (>90%) SNV and P-SNV were cell-type or tissue specific, with ~ 5% of SNV and ~ 8% of P-SNV shared between the blood and brain of individual donors”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The allelic frequency of APOE4 allele is lower in patients with pathogenic variants (16/46 alleles, 34%) than patients without detected variant (23/44 alleles, 53%) although the difference did not reach significance in this series.

    “we noted that the allelic frequency of APOE4 allele is lower in patients with pathogenic variants (16/46 alleles, 34%) than patients without detected variant (23/44 alleles, 53%) although the difference did not reach significance in this series.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    MAC lines expressing CBL, RIT1, KRAS and PTPN11 variants presented with increased ERK phosphorylation and/or increased proliferation in comparison to their WT controls, as measured by Western immunoblotting and EdU incorporation.

    “MAC lines expressing CBL, RIT1, KRAS and PTPN11 variants presented with increased ERK phosphorylation and/or increased proliferation in comparison to their WT controls, as measured by Western immunoblotting and EdU incorporation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Increased P-SNV burden was correlated not only with age, but also with the disease status (AD).

    “In contrast to the global SNV burden, increased P-SNV burden was correlated not only with age (Fig. 2D), but also with the disease status (AD) (Fig. 2E-H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Two patients carried 2 different MAPK activating variants: one patient carried an activating BRAF (p.L505H) variant in addition to loss of function (LOF) variant CBL (p.C416S), and another patient carried the NF1 (p.L2442*) LOF variant in addition to the activating RIT1 (p.M90I) variant.

    “Two patients carried 2 different MAPK activating variants: microglia from 1 patient carried an activating BRAF (p.L505H) variant 80 in addition to loss of function (LOF) variant CBL (p.C416S), and another patient carried the NF1 (p.L2442*) LOF variant 81,82 in addition to the activating RIT1 (p.M90I) variant”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [80]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [81]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [82]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingcitation

    The allelic frequencies of pathogenic variants found in AD patients is lower than values classically observed in solid tumors or leukemia, but within the range of the clonal frequency of pathogenic T cells observed in auto-immune diseases, and in the range of the allelic frequencies observed for the BRAF V600E variant in microglia in the brain of Histiocytosis patients.

    “The allelic frequencies of pathogenic variants found in AD patients is lower than values classically observed in solid tumors or leukemia, but within the range of the clonal frequency of pathogenic T cells observed in auto-immune diseases 119 , and we found that they were in the range of the allelic frequencies observed for the BRAF V600E variant in microglia in the brain of Histiocytosis patients”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [119]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingnone given

    Variants targeting the DNA-repair and DNA/histone methylation pathways are also enriched among AD patients, sometimes associated in the same patients, albeit their functional significance was not investigated here.

    “Variants targeting the DNA-repair and DNA/histone methylation pathways are also enriched among AD patients, sometimes associated in the same patients, albeit their functional significance was not investigated here.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    GSEA of RNAseq comparing CBL 404C/Y and isogenic CBL 404C/C microglia-like cells showed upregulation of Glycolysis, Oxidative Phosphorylation, and mTORC1 signatures, indicating increased metabolism and energy consumption by the mutant cells.

    “Gene Set Enrichment Analyses (GSEA) of RNAseq comparing CBL 404C/Y and isogenic CBL 404C/C microglia-like cells showed upregulation of Glycolysis, Oxidative Phosphorylation, and mTORC1 signatures, indicating increased metabolism and energy consumption by the mutant cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The pathogenic variants are found predominantly in the MAPK pathway and include recurrent variants (CBL RING domain variants in 6 patients), which promote microglial proliferation, activation, and expression of a neuroinflammatory/neurodegeneration-associated transcriptional programme in vitro and in vivo, and the production of neurotoxic cytokines IL1b, TNF, and IFNg.

    “They are found predominantly in the MAPK pathway and include reccurent variants (CBL RING domain variants in 6 patients), which promote microglial proliferation, activation, and expression of a neuroinflammatory/neurodegereration-associated transcriptional programme in vitro and in vivo, and the production of neurotoxic cytokines IL1b, TNF, and IFNg”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [115]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [116]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [117]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [118]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    The allelic frequencies at which MAPK activating variants are detected in brain samples from AD patients range from ~1-6% in microglia, which correspond to mutant clones representing 2 to 12% of all microglia in these samples, assuming heterozygosity.

    “The allelic frequencies at which MAPK activating variants are detected in brain samples from AD patients range from ~1-6% in microglia (Fig. 3G), which correspond to mutant clones representing 2 to 12% of all microglia in these samples, assuming heterozygosity.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: MAPK activating variants detected in microglia from AD patients are heterozygous.

  • May combine two claims.

    Findingown data

    Both age and AD are associated with a significant increase in SNV burden in the mixed-effects model (P<1x10^-4 and P=1x10^-4, respectively, by likelihood ratio test).

    “Both age and AD are associated with a significant increase in SNV burden in this model (P<1x10^-4 and P=1x10^-4, respectively, by likelihood ratio test).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    30% of the events (111 variants) that were called by MuTect1 were also called by ShearwaterML.

    “We compared the final mutant calls from Muetct1 and ShearwaterML and found that 30% of the events (111 variants) that were called by MuTect1 were also called by ShearwaterML.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    In the mixed-effects linear regression, the control trend line had P=0.0025 and R^2=0.13, and the NDD trend line had P=9.1x10^-16 and R^2=0.50 by Pearson's correlation.

    “Linear lines represent trend lines from mixedeffects linear regression that incorporates individual donor as a random effect (blue, control: P=0.0025, R^2=0.13;red, NDD: P=9.1x10^-16, R^2=0.50 by Pearson's correlation).”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    GSEA showed enrichment of the human AD microglia snRNAseq signature (MIC1) in differentially expressed genes between CBL 404Y/C microglial like cells and isogenic controls.

    “GSEA analysis for enrichment of the human ADmicroglia snRNAseq signature (MIC1) 21 in differentially expressed genes between CBL 404Y/C microglial like cells and isogenic controls.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    An average of 90 million paired reads was generated per sample.

    “An average of 90 million paired reads was generated per sample.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The filtering resulted in 509 SNVs.

    “This resulted in 509 SNVs.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Overall, 826 single-nucleotide variants (SNVs) were detected, resulting in 0.3 mutations/Mb sequenced.

    “Overall, a total of 826 single-nucleotide-variants (SNV) were detected resulting in 0.3 mutations/Mb sequenced.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The mean number of mutant reads was 29.1.

    “and a mean of 29.1 mutant reads (10% percentile: 4, 90% percentile: 52)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The mean coverage at the mutant site was 668.3X.

    “with a mean coverage at the mutant site of 668.3X (10% percentile: 276X, 90% percentile: 1181X)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The mean depth of ddPCR was ~5000x.

    “The mean depth of ddPCR was ~5000x”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingnone given

    Unique Molecular Identifiers (UMIs) per nucleus and gene count per nucleus were comparable between donors.

    “Unique Molecular Identifiers (UMIs) per nucleus and gene count per nucleus were comparable between donors.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: PU.1 is a marker that identifies microglia.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    84% of mutated variants were supported by at least 5 mutant reads.

    “with 84% of mutated supported by at least 5 mutant reads (Supplementary Table S3).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Around 15% (15/69) of the variants analyzed by ddPCR were called by ShearwaterML.

    “Around 15% (15/69) of the variants analyzed by ddPCR were called by ShearwaterML”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    One cluster was marked by a low number of cluster-enriched genes.

    “and was marked by a low number of cluster-enriched genes”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: PU.1 is a marker that identifies microglia.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Over 90% of hiPSCs in the derived lines expressed high levels of the pluripotency markers NANOG and OCT4 by flow cytometry.

    “Over 90% of hiPSCs in the derived lines expressed high levels of the pluripotency markers NANOG and OCT4 by flow cytometry.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    One cluster contained few cells (0.84% of total microglia, compared with an average of 6.20 ± 1.60% for other clusters).

    “One cluster contained few cells (0.84% of total microglia, for an average of 6.20 ± 1.60% for other clusters)”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: PU.1 is a marker that identifies microglia.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Except for cluster 13, which consisted of 97% cells from healthy control Control 11_AG, all donors and samples were represented in every cluster.

    “Except for cluster 13 consisting at 97% of cells from the healthy control Control 11_AG, all donors and samples were represented in every cluster.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: PU.1 is a marker that identifies microglia.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    The mean age of AD patients is 65 years old (55.5% female, 44.5% male).

    “The mean age of AD patients is 65 years old (55.5% female, 44.5% male).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The overall mean of the post-mortem delay interval was 9.8 hours.

    “The overall mean of the post-mortem delay interval was 9.8 hours.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    VAF obtained by ddPCR correlated with original VAF by sequencing (R2 0.93, p<0.0001).

    “VAF obtained by ddPCR correlated with original VAF by sequencing (R2 0.93, p<0.0001).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    The mean age of AD age-matched controls was 70 years old (60% female, 40% male).

    “the mean age of AD age-matched controls was 70 years old (60% female , 40% male).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Integrated_snn at resolution 0.2 outlined 16 microglia clusters.

    “Integrated_snn at resolution 0.2 outlined 16 microglia clusters.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: PU.1 is a marker that identifies microglia.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    40% (24/69) of the variants analyzed by ddPCR were called by both ShearwaterML and Mutect1.

    “40% (24/69) by both ShearwaterML+ Mutect1”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Ribosomal reads represented 0-1.6% of the total reads generated.

    “Ribosomal reads represented 0-1.6% of the total reads generated”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Microglia represented 94 ± 3% of total cells.

    “Microglia represented 94 ± 3% of total cells.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: PU.1 is a marker that identifies microglia.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Altogether 62/69 of variants tested (~90%) were confirmed.

    “Altogether we confirmed 62/69 of variants tested (~90%).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    The mean age of all controls is 54 years old (60% female, 40% male).

    “The mean age of all controls is 54 years old (60% female, 40% male)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Findingnone given

    Mutational load varies considerably across cell types and patients.

    “As detailed in the manuscript, the mutational load varies considerably across cell types and patients.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The median allelic frequency was ~1.34%.

    “The median allelic frequency was ~1.34% (Supplementary Table S3).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    ~44% (34/69) of the variants analyzed by ddPCR were called by Mutect1.

    “~44% (34/69) were called by Mutect1”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Wildtype CHEK2 showed robust activity, while the R346H mutant was inactive.

    “Wildtype CHEK2 showed robust activity, while the R346H mutant was inactive (RIGHT).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    A total of 826 variants were found.

    “Overall a total of 826 variants (Supplementary Table S3) were found”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The percent of mRNA bases averaged 79%.

    “the percent of mRNA bases averaged 79%.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause tumoral, developmental, and immune diseases.

    “Somatic genetic heterogeneity (mosaicism), resulting from post-zygotic DNA mutations, is widespread in human tissues, and a cause of tumoral, developmental, and immune diseases [23][24][25][26] .”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [23]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [24]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [25]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation

    Genetic variation in microglia may contribute to the pathogenesis of neurodegeneration and AD.

    “These data, together with transcriptional studies [20][21][22] support the hypothesis that genetic variation in microglia may contribute to the pathogenesis of neurodegeneration and AD.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [20]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [21]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [22]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    The majority of other germline AD-risk variants are located within or near genes expressed in microglia and in particular at microglia-specific enhancers.

    “the majority of the other germline AD-risk variants are located within or near genes expressed in microglia 15 and in particular at microglia-specific enhancers 19 .”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [15]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [19]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Human microglia develop in embryo and renew by local proliferation within the brain.

    “Human microglia are reported to develop in embryo and renew by local proliferation within the brain [38][39][40] .”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [38]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [39]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [40]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation

    Enrichment of putative pathogenic somatic mutations in the PI3K-AKT, MAPK, and AMPK pathway occurs in the brain of AD patients in comparison to controls.

    “showing that the enrichment of putative pathogenic somatic mutations in the PI3K-AKT, MAPK, and AMPK pathway do occur in the brain of patients in comparison to controls 30 .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [30]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Somatic variants that activate the PI3K-AKT-mTOR or MAPK pathways in neural progenitors are a cause of cortical dysplasia and epilepsy and developmental brain malformations.

    “Somatic variants that activate the PI3K-AKT-mTOR or MAPK pathways in neural progenitors are a cause of cortical dysplasia and epilepsy [31][32][33][34] and developmental brain malformations 35”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [31]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [32]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [33]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Bone marrow-derived myeloid cells can enter the brain, in particular during pathological processes, and may not be distinguishable from resident microglia by transcriptomics alone.

    “However, bone marrow-derived myeloid cells can enter the brain, in particular during pathological processes, and may not be distinguishable from resident microglia by transcriptomics alone 41 .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    APOE risk allele frequency for patients and controls was comparable to published series.

    “APOE risk allele frequency for patients and controls was comparable to published series [10][11][12] (Supplementary Fig. 1A)”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [10]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [11]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [12]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    More than 400 samples were used as background, leading to an average background coverage >400,000x.

    “More than 400 samples were used as background leading to an average background coverage >400.000x.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    CBL 404C/Y and isogenic CBL 404C/C microglia-like cells expressed similar amount of CBL total mRNA and protein, and CBL 404C/Y cells expressed wt and mutant mRNA in similar amounts, as expected assuming bi-allelic expression of CBL.

    “CBL 404C/Y and isogenic CBL 404C/C microglia-like cells expressed similar amount of CBL total mRNA and protein, and CBL 404C/Y cells expressed wt and mutant mRNA in similar amounts, as expected assuming bi-allelic expression of CBL”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: CBL is expressed bi-allelically in the microglia-like cells.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Germline variants of the DNA-repair transcription factor TP53, and DNA damage sensors ATR and CHEK2 were shown to promote accelerated neurodegeneration in human.

    “germline variants of the DNA-repair transcription factor TP53, and DNA damage sensors ATR and CHEK2 were shown to promote accelerated neurodegeneration in human 83,84 .”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [83]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [84]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CBL somatic and germ-line LOF variants such as R420Q have been previously associated with tumoral diseases including clonal myeloproliferative disorders and RASopathies.

    “CBL somatic and germ-line LOF variants such as R420Q have been previously associated with tumoral diseases including clonal myeloproliferative disorders [63][64][65][66][67][68][69][70][71][72][73] and RASopathies 92 respectively.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [63]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [64]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [65]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationcitation

    TET2, DNMT3 and KMT2C are frequently mutated in clonal hematopoiesis, suggesting that in contrast to other variants, the presence of TET2, DNMT3 and KMT2C/MLL3 in the brain of patients may reflect the entry of blood clones in the brain.

    “TET2, DNMT3 and KMT2C are frequently mutated in clonal hematopoiesis 58,59 , suggesting that in contrast to other variants, the presence of TET2, DNMT3 and KMT2C/MLL3 in the brain of patients may reflect the entry of blood clones in the brain.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [58]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [59]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation

    CBL is an E3 ubiquitin-protein ligase that negatively regulates RTK signaling via MAPK.

    “CBL is an E3 ubiquitin-protein ligase that negatively regulates RTK signaling via MAPK 91 .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [91]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    P-values were corrected for multiple testing using Benjamini & Hochberg's False Discovery Rate (FDR) with a q-value cutoff of 0.01.

    “P-values were corrected for multiple testing using Benjamini & Hochberg's False Discovery Rate (FDR) 19 and a q-value of cutoff of 0.01 was used to call somatic variants.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationown data

    The clonal diversity of microglia and blood both increase with age.

    “These data altogether indicate that the clonal diversity of microglia and blood both increase with age”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Heterozygous expression of pathogenic CBL variant in human microglia-like cells was sufficient to drive a transcriptional program that associates with increased metabolic activity and a neurotoxic inflammatory response, also observed in microglia from patients with MAPK-activating variants.

    “Heterozygous expression of pathogenic CBL variant in human microglia-like cells was sufficient to drive a transcriptional program that associates with increased metabolic activity and a neurotoxic inflammatory response, also observed in microglia from patients with MAPK-activating variants.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Clonal hematopoiesis is associated with a higher risk of several diseases related to ageing such as cardiovascular diseases, but inversely associated with the risk of AD.

    “This is consistent with recent studies showing that clonal hematopoiesis is associated with a higher risk of several diseases related to ageing such as cardiovascular diseases, but inversely associated with the risk of AD 60,61 .”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [60]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [61]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Neuroinflammation is an early event in AD pathogenesis, increasingly considered as critical in pathogenesis initiation and progression.

    “Neuroinflammation is an early event in AD pathogenesis, increasingly considered as critical in pathogenesis initiation and progression 16,125,126 .”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [16]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [125]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [126]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationown data

    A subset of AD patients (12/45, ~27% of this series) present with microglial clones carrying one or several oncogenic variants that activate the RTK/MAPK pathway, and are characterized by recurrent oncogenic variants in CBL and RIT1.

    “These data altogether indicate that a subset of AD patients (12/45, ~ 27% of this series) present with microglial clones carrying one or several oncogenic variants that activate the RTK/MAPK pathway, and are characterized by recurrent oncogenic variants in CBL and RIT1.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Our results are consistent with a recent study showing that clonal hematopoiesis was inversely associated with the risk of AD.

    “In addition, our results are consistent with a recent study showing that clonal hematopoiesis was inversely associated with the risk of AD 61 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [61]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationown data

    Heterozygosity for a CBL C404Y allele is sufficient to activate human microglia-like cells increasing their proliferation and ERK activation.

    “Altogether, these results showed that heterozygosity for a CBL C404Y allele is sufficient to activate human microglia-like cells increasing their proliferation and ERK activation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Microglia variants are frequently absent from blood, and the DNA sequencing barcoding approach does not support a model where blood cells massively infiltrate the brain or replace the microglia pool in patients from this series, but instead is consistent with the local maintenance and proliferation of microglia.

    “Microglia variants are frequently absent from blood, and our DNA sequencing barcoding approach does not support a model where blood cells massively infiltrate the brain or replace the microglia pool in patients from our series, but instead consistent with the local maintenance and proliferation of microglia 38,39 .”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [38]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [39]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Sorting purity was >95%.

    “Sorting purity was >95%.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Samples were sequenced to a mean depth of coverage of 1106x (control samples 1071x, AD samples 1100x).

    “Samples were sequenced to a mean depth of coverage of 1106x (Control samples: 1071x, AD samples 1100x).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    The RAS/MAPK signaling pathway is involved in microglia proliferation, activation and inflammatory response, neuronal death, neurodegeneration, and AD pathogenesis, and its activation has been proposed to be an early event in the pathophysiology of AD in human.

    “the RAS/MAPK signaling pathway is involved in microglia proliferation, activation and inflammatory response 120-122 , neuronal death, neurodegeneration, and AD pathogenesis 15,19,37,123 , and its activation has been proposed to be an early event in the pathophysiology of AD in human 124 .”
    Quote in the paperSubstantial claim0 of 8 sources checked
    • cites [120]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [121]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [122]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [15]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [19]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [37]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [123]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [124]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The main known genetic risk factor for sporadic AD is the APOE4 allele, responsible for an increased inflammatory response in the brain of APOE4 carriers.

    “the main known genetic risk factor for sporadic AD is the APOE4 allele, responsible for an increased inflammatory response in the brain of APOE4 carriers 16 .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    RIT1 is a RAS GTPase, and somatic or germ-line GOF variants such as RIT1 F82L and RIT1 M90I enhance MAPK signaling in malignancies and RASopathies.

    “RIT1 is a RAS GTPase, and somatic or germ-line GOF variants such as RIT1 F82L and RIT1 M90I, also enhance MAPK signaling in malignancies 74 and RASopathies 93,94 respectively.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [74]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [93]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [94]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationreasoning

    Blood clones have a detectable but minor contribution to microglia, consistent with local maintenance and proliferation.

    “and therefore that blood clones have a detectable but minor contribution to microglia, consistent with its local maintainance and proliferation 38,39 .”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [38]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [39]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Somatic variants that activate the MAPK pathway in tissue macrophages cause a clonal proliferative and inflammatory disease called Histiocytosis, strongly associated with neurodegeneration.

    “somatic variants that activate the MAPK pathway in tissue macrophages cause a clonal proliferative and inflammatory disease called Histiocytosis, strongly associated with neurodegeneration 37,[95][96][97]”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [37]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [95]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [96]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The CHEK2 R346H variant is located within the catalytic loop of the protein kinase domain.

    “The R346H variant is located within the catalytic loop of the protein kinase domain and shown in red on the 3D structure of CHEK2 kinase domain (pdb code: 2cn5) (LEFT).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites 2cn5

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Samples were neuropathologically evaluated and classified by the collaborating institutions as Alzheimer's disease (AD) or non-dementia controls.

    “Samples were neuropathologically evaluated and classified by the collaborating institutions as Alzheimer's disease (AD) [1][2][3][4][5] or non-dementia controls.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [1]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [2]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [3]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Among SNVs tested, 69/760 (9%) were tested and 90% were true.

    “SNV: 69/760 tested (9%): 90%true [P-SNV] : 38/96 (40%) tested, 95% true PU.1 P-SNV: 33/54(60%) tested 100% true”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Among P-SNVs, 38/96 (40%) were tested and 95% were true.

    “SNV: 69/760 tested (9%): 90%true [P-SNV] : 38/96 (40%) tested, 95% true PU.1 P-SNV: 33/54(60%) tested 100% true”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Removal of doublets using DoubletFinder and cells with high mitochondrial content (>10% mitochondrial RNA) yielded between 6,437 and 9,241 nuclei per patient and sample.

    “The removal of doublets using DoubletFinder and cells with high mitochondrial content (>10% mitochondrial RNA) yielded between 6,437 and 9,241 nuclei per patient and sample.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: PU.1 is a marker that identifies microglia.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The study was approved by the Institutional Review Board of St Thomas' Hospital; Guy's hospital; the King's College London University; the Memorial Sloan Kettering Cancer Center and by the Tri-institutional (MSKCC, Weill-Cornell, Rockefeller University) Embryonic Stem Cell Research Oversight (ESCRO) Committee.

    “The study was approved by the Institutional Review Board of St Thomas' Hospital; Guy's hospital; the King's College London University; the Memorial Sloan Kettering Cancer Center and by the Tri-institutional (MSKCC, Weill-Cornell, Rockefeller University) Embryonic Stem Cell Research Oversight (ESCRO) Committee.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Anatomical regions of the brain specimen and originating brain banks were not incorporated because models incorporating those parameters did not significantly improve the overall model fitting by likelihood ratio test.

    “Anatomical regions of the brain specimen and originating brain banks were not incorporated because the models incorporating those parameters did not significantly improve the overall model fitting by likelihood ratio test (see Methods).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The study was conducted according to the Declaration of Helsinki.

    “The study was conducted according to the Declaration of Helsinki.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Human tissues were obtained with patient-informed consent and used under approval by the Institutional Review Boards from Memorial Sloan Kettering Cancer Center (IRB protocols #X19-027).

    “Human tissues were obtained with patient-informed consent and used under approval by the Institutional Review Boards from Memorial Sloan Kettering Cancer Center (IRB protocols #X19-027).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Among PU.1 P-SNVs, 33/54 (60%) were tested and 100% were true.

    “SNV: 69/760 tested (9%): 90%true [P-SNV] : 38/96 (40%) tested, 95% true PU.1 P-SNV: 33/54(60%) tested 100% true”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Non-parametric tests were used when data did not follow a normal distribution.

    “Non-parametric tests were used when data did not follow a normal distribution (Normality test: D'Agostino-Pearson and Shapiro-Wilk test).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    To avoid possible contamination of sequencing data with mutations associated with donor's tumoral disease in the group of non-dementia controls, the authors refrained from selecting cases with blood malignancies or with brain tumors.

    “To avoid possible contamination of sequencing data with mutations associated with donor's tumoral disease in the group of non-dementia controls, we refrained from selecting cases with blood malignancies or with brain tumors.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Written informed consent was obtained according to the Helsinki convention.

    “Written informed consent was obtained according to the Helsinki convention.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Cells were maintained at 37°C and 5% CO2 and were routinely tested for mycoplasma and periodically assessed for genomic integrity by karyotyping.

    “Cells were maintained at 37°C and 5% CO2 and they were routinely tested for mycoplasma and periodically assessed for genomic integrity by karyotyping.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Variants were required to have at least one supporting read in each strand.

    “Variants were required to have a least one supporting read in each strand.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Somatic variants within 10bp of an indel were filtered out as they typically reflect mapping errors.

    “Somatic variants within 10bp of an indel were filtered out as they typically reflect mapping errors.”
    Quote in the paperNot a substantial claim
97 of 97

  • The authors state this as an assumption.

    MAPK activating variants detected in microglia from AD patients are heterozygous.

    “assuming heterozygosity”

  • The authors state this as an assumption.

    CBL is expressed bi-allelically in the microglia-like cells.

    “as expected assuming bi-allelic expression of CBL”

  • The paper relies on this without support.

    Pathogenic variants drive neuroinflammation.

    “Together with evidence that pathogenic variants drive neuroinflammation”

  • The paper relies on this without support.

    PU.1 is a marker that identifies microglia.

    “anti-Pu.1 antibody (microglia marker 1:50, Pu.1-AlexaFluor 647, 9G7 Cell Signaling)”

  • The paper relies on this without support.

    Variants classified as pathogenic by ClinVar and/or OncoKB are pathogenic.

    “mean number of pathogenic variants (P-SNV) as determined by ClinVar and/or OncoKB”

  • The paper relies on this without support.

    NeuN is a marker that identifies neurons.

    “NeuN + (neurons)”
6 of 6

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Patient 53 has conflicting CBL C416 substitutions

    Minor

    The patient listing gives CBL C416S alongside BRAF L505H for patient 53, whereas the supplementary sample description pairs BRAF L505H with CBL C416Y.

  • CBL C404Y cells are described as made by two different editing methods

    Minor

    The results say the heterozygous hiPSC clones were generated by prime editing, while the methods say the CBL C404Y variant was inserted by cytidine base editing.

  • CBL c.1211 reference and variant alleles are reversed

    Minor

    The methods identify CBL C404Y as a G>A change, but the supplementary figure legend labels A as the reference allele and G as the variant.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 5

  1. Socci N. 2022. BIC-variants_pipeline. Software Heritage. swh:1:rev:88967b8ddc1af03f95f09458d6c2d2073d5bed62. https://archive.softwareheritage.org/swh:1:dir:13c637223a69aa12214127c1c0acb2086fc87060;origin=https://github.com/soccin/BIC-variants_pipeline;visit=swh:1:snp:4023f60335e776fe63506fe5de3b52e21570d2c3;anchor=swh:1:rev:88967b8ddc1af03f95f09458d6c2d2073d5bed62.
  2. Socci N. 2025. Variant-postprocess. Software Heritage. swh:1:rev:8e72acb84500719afde95dcfcfd3b910c59127d2. https://archive.softwareheritage.org/swh:1:dir:f427243f5c4a23569c26502e8d02705422c092e4;origin=https://github.com/soccin/Variant-PostProcess;visit=swh:1:snp:a833ea5cad143d1c2005a12a599e407d46d50369;anchor=swh:1:rev:8e72acb84500719afde95dcfcfd3b910c59127d2.
  3. Vicario R. 2025. DNA sequencing of postmortem brain samples processed for selection of somatic variants. NCBI dbGaP. https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs002213.v1.p1. phs002213.v1.p1.
  4. Geissmann F, Vicario R. 2025. A microglia clonal inflammatory disorder in Alzheimer’s Disease. NCBI Gene Expression Omnibus. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE274037. GSE274037.
  5. Geissmann F, Vicario R. 2025. A microglia clonal inflammatory disorder in Alzheimer’s Disease II. NCBI Gene Expression Omnibus. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE286627. GSE286627.

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Authors
Vicario, R., Fragkogianni, S., Weber, L., Lazarov, T., Hu, Y., Hayashi, S. Y., Craddock, B., Socci, N. D., Alberdi, A., Baako, A., Ay, O., Ogishi, M., Lopez-Rodrigo, E., Kappagantula, R., Viale, A., Iacobuzio-Donahue, C. A., Zhou, T., Ransohoff, R. M., Chesworth, R., Bank, N. B., Abdel-Wahab, O., Boisson, B., Elemento, O., Casanova, J.-L., Miller, W. T., & Geissmann, F.
Affiliations
  • Immunology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York
  • Memorial Sloan Kettering Cancer Center (US)
  • Cornell University (US)
  • Stony Brook School (US)
  • Rockefeller University (US)
  • Human Oncology & Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York
  • Third Rock Ventures
Corresponding author
Geissmann, Frédéric
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2025-03-14
Volume
13
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.96519.3
PMC
PMC10849735
OpenAlex
W4408443692
Cited by
21
References
157
Citations by year
2026: 12 · 2025: 4 · 2024: 5
FWCI
5.71
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
Funders
  • National Institutes of Health (P30 CA008748)
  • NIH Office of the Director (1R01NS115715-01, 1 R01 HL138090 01, 1 R01 AI130345 01)
  • Ludwig Center for Cancer Immunotherapy (Basic and Translational Immunology Grants)
  • Cycle for Survival
  • American Association for Cancer Research (18-40-15-VICA)
  • New York State Stem Cell Science (C32559GG)
  • Charles H Revson Fellowship
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Inflammasome and immune disorders
Keywords
Alzheimer's disease, microglia, somatic mutations, MAPK signaling, ERK activation, neuroinflammation, neurodegeneration

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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

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