Variant-to-function mapping of late-onset Alzheimer’s disease GWAS signals in human microglial cell models implicates RTFDC1 at the CASS4 locus

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

In S. Pombe, removal of RTFDC1 (RTF2) from stalled replication forks during S phase DNA replication is essential to maintain replication stress and genome integrity.

“What is known to date is that in S. Pombe the removal of RTFDC1 (also known as RTF2) from stalled replication forks during S phase DNA replication is essential to maintain replication stress and genome integrity69.”

• cites [69] sourceDisputed

  The Abstract and Discussion identify removal of RTF2 from stalled forks as a protective process in mammalian or human cells, not S. pombe. The Results section on DDI1/2 instead says S. pombe Rtf2 is required to maintain fork stalling at a specific replication termination site. Page numbers were not provided. (LLM, conf=0.87)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

May combine two claims.

A limitation of GWAS is that it only reports the sentinel SNP, which serves as a tag for all other variants in LD; any one could be causal.

“A limitation of GWAS is that the approach only reports the sentinel single nucleotide polymorphism (SNP) at a given locus, which serves as a tag for all other variants in LD; as such, among this entire LD series of proxy SNPs, any one (or more) could be causal15.”

• cites [15] sourcePartly supported

  In “Cataloging SNPs and linkage disequilibrium” (page unavailable), the paper explains that tag SNPs can act as proxies for nearby variants. In “Genome-Wide Associations: Lessons” (page unavailable), it says association signals often do not identify the causal mutation, which may even be a non-SNP variant. It does not support the absolute claim that GWAS reports only one sentinel SNP per locus or that the sentinel tags all other variants in LD. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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

The most recent large-scale LOAD GWAS identified over 70 independent signals associated with LOAD, including variants near MAPT and APP.

“The most recent large-scale genome wide association study (GWAS), a method which which offers unbiased identification of susceptibility loci, identified over 70 independent signals associated with LOAD4, including variants near the MAPT (encoding tau) and APP (encoding the amyloid precursor protein) genes.”

• cites [4] sourcePartly supported

  The Discussion reports 75 independent loci associated with Alzheimer’s disease and related dementias (ADD), and the GWAS meta-analysis identifies loci at MAPT and APP. The claim describes these as signals specifically associated with late-onset Alzheimer’s disease (LOAD), whereas the reported association analysis is for ADD. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

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

The strongest associated obesity GWAS signal lies within an intron of FTO but principally influences expression of distal genes IRX3 and IRX5.

“One such example comes from the strongest associated obesity GWAS signal that lies within an intron of the FTO gene, but principally influences the expression of distal genes IRX3 and IRX517–19.”

• cites [17] sourcePartly supported

  The paper’s main text (indexed as Abstract; page unavailable) reports that obesity-associated variants in an FTO intron are associated with IRX3 expression in human brain, but not FTO expression. The passages do not establish an effect on IRX5 expression or that this is the strongest obesity GWAS signal. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [18] sourceSupported
• cites [19] sourceNot checked — no full text of the source

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

Both rare and common variants have been identified in core microglial genes, such as TREM2.

“Consistent with these enrichment analyses, both rare and common variants have been identified in core microglial genes, such as TREM213,14.”

• cites [13] sourceSupported
• cites [14] sourcePartly supported

  The Discussion (page unavailable) identifies TREM2 as a gene expressed in brain microglia and reports rare TREM2 coding variants associated with Alzheimer’s disease risk. It does not identify common variants in TREM2 or other microglial genes, so this source supports only the rare-variant part of the claim. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

The majority of sentinel SNPs and their proxies lie within non-coding regions of the genome, and may not necessarily influence the expression of the nearest gene; instead, they likely modulate distal regulatory elements such as enhancers, which regulate distal gene expression.

“Furthermore, the majority of these sentinel SNPs and their associated proxies lie within non-coding regions of the genome, and may not necessarily influence the expression of the nearest gene. Instead, these variants likely modulate the activity of distal regulatory elements, such as enhancers, which in turn regulate distal gene expression16, which is frequently not the nearest gene.”

• cites [16] sourceSupported

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

Genes near GWAS signals are enriched for key functional annotations and are under strong selective constraint, while genes near eQTLs lack most functional annotations and show relaxed constraint.

“it has been recently shown that genes near GWAS signals are enriched for key functional annotations and are under strong selective constraint, while genes near eQTLs lack most functional annotations and show relaxed constraint68”

• cites [68] sourceSupported

Classified as probably not a substantial claim.

Individuals homozygous for the APOEε2 allele have poorer cancer outcomes.

“as well as poorer cancer outcomes77”

• cites [77] sourcePartly supported

  The paper’s melanoma survival passage reports shorter median survival for APOE2 carriers than for APOE3 homozygotes or APOE4 carriers. It does not establish the result specifically for people homozygous for APOEε2, and its human outcome findings concern melanoma rather than cancer generally. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

May combine two claims.

H3K27ac was only enriched in microglia among four major brain cell types at the region harboring rs6024870, suggesting this region acts specifically as an enhancer in microglia.

“Of the four cell types, H3K27ac was only enriched in microglia, suggesting that this region acts specifically as an enhancer in microglia.”

• cites [22] sourcePartly supported

  The paper’s main text (page unavailable in our copy) reports H3K27ac profiling across four brain cell populations and identifies microglia-specific enhancers. It does not establish that H3K27ac was enriched only in microglia at the region harboring rs6024870; a search for that variant found no matching passage. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

The authors previously identified ING3 and EPDR1 as long-range effector genes regulated by osteoblast enhancers harboring bone mineral density GWAS SNPs at the WNT16-CPED1 and STARD3NL loci.

“Additionally, we identified ING3 and EPDR1 as the long-range effector genes regulated by osteoblast enhancers harboring bone mineral density GWAS SNPs at the WNT16-CPED1 and STARD3NL loci, respectively20.”

• cites [20] sourcePartly supported

  The Results (Functional studies; page unavailable) identify ING3 at the WNT16-CPED1 locus and EPDR1 at the STARD3NL locus, and report interactions between their promoters and regions containing BMD GWAS proxy SNPs in osteoblasts. The Discussion also describes both as implicated genes involved in osteoblast differentiation. The paper does not establish that the interacting regions are enhancers that regulate these genes, so that causal part of the claim is stronger than the evidence. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Two studies using proximity-ligation assays in human primary or iPSC-derived microglia have reported 3D genomic interactions between GWAS-associated variants and gene promoters in LOAD.

“Recently, two studies using proximity-ligation assays in either human primary or iPSC-derived microglia have reported 3D genomic interactions between GWAS-associated variants and gene promoters in LOAD22,65.”

• cites [22] sourceSupported
• cites [65] sourceSupported

May combine two claims.

In microglia from human brains, rs6024870 acts as an eQTL on RTFDC1 expression, with the minor A allele associated with reduced expression (GG average 93.40 TPM vs GA 85.30 TPM, P=0.0385).

“and found that GG individuals had on average 93.40 TPM of RTFDC1, significantly higher than GA individuals, which had on average 85.30 TPM (P=0.0385, Student’s 1-tailed T-test, Figure 6C).”

• Also stated in: § DISCUSSION · plain

  “our observation that rs6024870 acts as a modest eQTL on RTFDC1 expression in microglia from human brains, with the minor A allele (protective against LOAD) associated with reduced expression levels.”

• cites [58] sourceCould not verify

  The Materials and Methods section (2.9, page unavailable) describes analyses of amyloid-β uptake and cellular markers, not genotype-dependent gene expression. Searches for rs6024870, RTFDC1, eQTL, and the reported TPM values found no passage in our copy addressing the claimed association. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: The publicly available patient RNA-seq dataset of microglia from fresh human brain tissue is representative of microglial RTFDC1 expression in vivo.

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

A single enhancer may contact and regulate several promoters, a phenomenon previously seen in macrophages.

“This is consistent with the observation that a single enhancer may contact and regulate several promoters, a phenomenon previously seen in macrophages74.”

• cites [74] sourcePartly supported

  The Results section (‘Dynamic re-wiring of chromatin architecture’) describes a macrophage activation hub connecting distal enhancers to the promoters of both TPRG1 and BCL6, with increased expression of both genes. It does not establish that one individual enhancer contacts and regulates both promoters; elsewhere, the paper characterizes hubs chiefly as multiple enhancers connected to a promoter. No page number is available in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

ChIP-based proximity ligation assays, such as PLAC-seq, rely on antibodies to pull down pre-specified regions associated with histone marks that demarcate putative enhancer or promoter regions.

“Additionally, ChIP-based proximity ligation assays, such as PLAC-seq67, rely on the use of antibodies to pull down pre-specified regions associated with certain histone marks that can demarcate putative enhancer (H3K27ac) or promoter (H3K4me3) regions.”

• cites [67] sourceSupported

May combine two claims.

In the GWAS, both the minor alleles of the sentinel SNP (T) and rs6024870 (A) are protective against LOAD.

“In the GWAS, both the minor alleles of the sentinel SNP (T) and rs6024870 (A) are protective against LOAD.”

• cites [4] sourceCould not verify

  The GWAS meta-analysis tables report minor-allele odds ratios for many variants, but the passages in our copy do not identify the claimed sentinel SNP or report an association for rs6024870. Searches for “rs6024870” and “sentinel SNP (T) protective against LOAD” returned no passage establishing that either specified allele is protective. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [31] sourceCould not verify

  The Discussion reports a GWAS of late-onset Alzheimer’s disease, and Table 1 lists lead variants and tested alleles. Our copy does not identify rs6024870 or establish that its A allele and the unspecified sentinel SNP’s T allele are protective (searched: rs6024870, protective alleles, sentinel variant). (LLM, conf=0.74)

  Experimental — accuracy not yet measured

May combine two claims.

PIRs were significantly enriched for open chromatin regions detected in ATAC-seq and for active chromatin marks from ex-vivo microglia.

“PIRs were significantly enriched for open chromatin regions detected in our ATAC-seq experiments and for active chromatin marks from ex-vivo microglia22, suggesting a potential regulatory role (Suppl. Fig 1).”

• cites [22] sourcePartly supported

  The paper’s interactome passage (page unavailable) reports that a subset of chromatin interactions colocalized with increased ATAC-seq and active chromatin-mark signals. Its microglia profiling passage says those signals were highly correlated with profiles previously defined in ex-vivo microglia. It does not report that PIRs were significantly enriched for the citing authors’ ATAC-seq regions or for marks from ex-vivo microglia. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Individuals homozygous for the APOEε2 allele have an increased risk of reduced fertility.

“individuals homozygous for the APOEε2 allele also have an increased risk of reduced fertility76”

• cites [76] sourcePartly supported

  The Fertility section (page unavailable) says the ε2 allele appears to have the worst reproductive efficiency across the populations discussed, but cautions that offspring counts have confounding factors. It does not establish an increased risk specifically for people homozygous for APOEε2. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

In humans, RTFDC1 is required for appropriate localization of RNAseH2 to the replication fork to remove ribonucleotides from nascent DNA via ribonucleotide excision repair.

“However, more recent work has also shown that in humans, RTFDC1 is required for appropriate localization of RNAseH2 to the replication fork to remove ribonucleotides from nascent DNA via the ribonucleotide excision repair pathway70.”

• cites [70] sourcePartly supported

  The Results section (page not provided) shows that RTF2 loss reduces RNase H2 at replication forks and increases DNA-embedded ribonucleotides; the Introduction says this localization is necessary for proper ribonucleotide excision repair. However, the decisive RTF2-loss experiments used mouse embryonic fibroblasts. The passages do not establish the claim specifically for human RTFDC1. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

The strongest protective allele against LOAD, APOEε2, has the lowest frequency of the three alleles at the APOE locus.

“In fact, the strongest protective allele against LOAD, APOEε2, has the lowest frequency of the three alleles at the APOE locus75.”

• cites [75] sourcePartly supported

  The Introduction (page unavailable) identifies apoE3 as the most common of the three isoforms, but does not establish whether apoE2 is less frequent than apoE4. The Aβ-dependent effects section reports that apoE2 and apoE3 cleared more Aβ than apoE4 in transgenic mice; it does not establish that APOEε2 is the strongest protective allele against late-onset Alzheimer’s disease. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

May combine two claims.

CRISPR-Cas9-induced ~400bp deletion of the rs6024870-harboring enhancer provided variant-to-function evidence for its role in regulating DNA damage response, including downregulation of DNA repair pathways and loss of ability to resolve DNA DSBs as assayed by γH2AX accumulation.

“and DNA damage response (downregulation of DNA repair pathways, loss in ability to resolve DNA DSBs as assayed by fluorescent γH2AX accumulation).”

• AssumptionRests on an assumption: CRISPR-Cas9 deletion of the approximately 400bp region surrounding rs6024870 specifically ablates the rs6024870-harboring enhancer without affecting other regulatory elements.

May combine two claims.

RTFDC1 overexpression in enhancer-KO cells led to significantly more IL-8 secretion than in enhancer-KO counterparts (P<0.001), while IL-6 secretion did not significantly differ (P=0.198).

“Unexpectedly, RTFDC1 overexpression enhancer-KO cells secreted significantly more IL-8 than their enhancer-KO counterparts (P<0.001, one-way ANOVA with Tukey’s HSD test, Figure 4D), while the same cells did not significantly differ in secretion of IL-6 (P=0.198, Figure 4E).”

May combine two claims.

Of these contacts, 7 (19%) were to the nearest gene, 8 (22%) to both nearest and more distal genes, and 21 (58%) to distal genes only.

“Of these contacts, 7 (19%) were to the nearest gene, 8 (22%) to both nearest and more distal genes, and 21 (58%) to distal genes only.”

May combine two claims.

The enhancer region containing the minor (A) protective allele showed a nominal decrease in luciferase expression (2.0-fold compared to promoter-only controls) but did not reach significance.

“While the enhancer region containing the minor (A) protective allele did show a nominal decrease in luciferase expression, with a 2.0-fold expression change compared to promoter-only controls, it did not reach the threshold of significance (Figure 6B).”

May combine two claims.

Promoter-focused Capture-C and ATAC-seq identified 33 LOAD GWAS-associated variants contacting the promoters of 32 putative effector genes, 24 protein-coding, with 5 targets overlapping in both cellular models (AURKA, CSTF1, MIR4487, RTFDC1, and TCONS_00028450).

“Our combination of promoter-focused Capture-C and ATAC-seq data identified 33 LOAD GWAS-associated variants contacting the promoters of 32 putative effector genes, 24 of which code for a protein, with 5 targets overlapping in both cellular models (AURKA, CSTF1, MIR4487, RTFDC1, and TCONS_00028450).”

May combine two claims.

26 (79%) of the SNPs contacted one effector gene and 7 (21%) more than one.

“26 (79%) of the SNPs contacted one effector gene and 7 (21%) more than one.”

May combine two claims.

CRISPR-Cas9-induced ~400bp deletion of the rs6024870-harboring enhancer provided variant-to-function evidence for its role in regulating microglial inflammation, including upregulation of inflammatory pathways and increased secretion of IL-8 and IL-6.

“our CRISPR-Cas9 induced ~400bp deletion of this enhancer provided variant-to-function evidence for its role in regulating both microglial inflammation (upregulation of inflammatory pathways, increased secretion of IL-8 and IL-6)”

• AssumptionRests on an assumption: CRISPR-Cas9 deletion of the approximately 400bp region surrounding rs6024870 specifically ablates the rs6024870-harboring enhancer without affecting other regulatory elements.

May combine two claims.

RTFDC1 knockdown cells yielded significantly elevated levels of γH2AX at the 24 hour time point compared to mock transduced controls (P<2.2×10−16), and did not differ significantly from enhancer-deletion cells, resolving 18.5% of γH2AX fluorescence.

“Indeed, these RTFDC1 knockdown cells also yielded significantly elevated levels of γH2AX at the 24 hour time point when compared to mock transduced controls (P<2.2×10−16, two-way ANOVA (genotype by condition) with Tukey’s HSD), and did not differ significantly from enhancer-deletion cells, resolving 18.5% of γH2AX fluorescence (Figure 5D).”

• AssumptionRests on an assumption: The pool of siRNAs targeting RTFDC1 did not have off-target effects that confound the γH2AX results.

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

HMC3 clones heterozygous for the CRISPR deletion had both WT and deletion bands present on PCR.

“Clones heterozygous for the deletion had both WT and deletion bands present on the PCR.”

May combine two claims.

PCR products from mock transduced (WT) HMC3 cells were 889 bp and from CRISPR deletion cells were 495 bp, matching the predicted sizes.

“PCR products for both mock transduced and knockout cells were the predicted sizes: mock transduced (WT) PCR product was 889bp, and the CRISPR deletion product was 495bp.”

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

In HMC3 cells, PIRs were similarly enriched for active chromatin markers.

“PIRs in HMC3 were similarly enriched for active chromatin markers (Suppl. Fig. 1).”

May combine two claims.

RTFDC1 did not reach the threshold for significance but was downregulated by a fold change of 0.82 in enhancer-KO cells.

“While RTFDC1 did not reach the threshold for significance, it was downregulated by a fold change of 0.82.”

May combine two claims.

LOAD GWAS signals were significantly enriched in iMg Capture-C-prioritized regions (P=0.04, enrichment 47.2), while schizophrenia signals were not (P=0.81, enrichment 1.5).

“The LOAD GWAS signals were significantly enriched in iMg (P=0.04, enrichment 47.2), while schizophrenia signals were not (P=0.81, enrichment 1.5), which is in line with expectation given that schizophrenia loci are widely hypothesized to confer their effects principally via neuronal cell types.”

May combine two claims.

Mock transduced cells cleared 41.2% of γH2AX fluorescence from hour 0 to hour 24, whereas enhancer-KO cells only cleared 20.5%.

“Quantification of γH2AX clearance from hour 0 to hour 24, normalized to control fluorescence levels, showed that mock transduced cells cleared 41.2% of γH2AX fluorescence, whereas enhancer-KO cells only cleared 20.5%.”

May combine two claims. The quote only partly states this claim.

rs6024870 lies within open chromatin in both HMC3 and iMg, as well as in ex vivo human microglia.

“Notably, in both HMC3 cells and iMg, this variant lies within a region of open chromatin.”

May combine two claims.

Overexpression of RTFDC1 in enhancer-KO cells increased clearance of γH2AX fluorescence from hour 0 to hour 24 to 41.6%, and mean fluorescence at hour 24 was lower than even mock transduced controls (P<2.2X10−16).

“Indeed, overexpression of RTFDC1 increased the clearance of γH2AX fluorescence from hour 0 to hour 24 to 41.6%, and the mean fluorescence at hour 24 was in fact lower than even mock transduced controls (P<2.2X10−16, two-way ANOVA (genotype by condition) with Tukey’s HSD, Figure 5E).”

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

The observed rs6024870 genotype counts are consistent with Hardy-Weinberg proportions given a minor A allele frequency of 0.058 in European ancestry.

“This is consistent with expected Hardy Weinberg genotype proportions, as the minor A allele frequency (MAF) of rs6024870 is 0.058 in European ancestry.”

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

LOAD risk is substantially explained by heritable factors, with approximately 60-80% of risk attributed to genetic factors.

“Population studies have shown that approximately 60–80% of LOAD risk is explained by heritable factors3, suggesting a strong polygenic component.”

• cites [3] sourceNot checked — no full text of the source

May combine two claims.

Our Capture-C library assessed a comprehensive set of primary and alternative human gene promoters for both coding and non-coding genes, allowing probing of all known promoters' interactions without prior knowledge of histone acetylation status.

“Additionally, our Capture-C library assessed a comprehensive set of primary and alternative human gene promoters for both coding and non-coding genes, allowing us to probe all known promoters’ interactions without any prior knowledge of histone acetylation status.”

May combine two claims.

HMC3 cells do not secrete LOAD-relevant tumor necrosis factor α (TNF-α) or IL1-β.

“they do not secrete LOAD-relevant tumor necrosis factor α (TNF-α) or IL1-β78”

• cites [78] sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims. The quote only partly states this claim.

Final plasmid constructs were confirmed by Sanger sequencing and Oxford Nanopore sequencing.

“resulting plasmids were Sanger sequenced at both the 5’ and 3’ ends of the inserted sequences to confirm successful insertion.”

• Also stated in: § Luciferase Assays · plain

  “The final successful plasmid was sent for Oxford Nanopore sequencing by Plasmidsaurus to confirm fidelity of the entire sequence”

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

Digestion efficiencies of 3C libraries were assessed using undigested and digested controls by gel electrophoresis and quantitative PCR.

“Digestion efficiencies of 3C libraries were assessed using control 1 (undigested) and control 2 (digested) by gel electrophoresis on a 0.9% agarose gel and quantitative PCR (SYBR green, Thermo Fisher).”

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

A new standard curve was performed on each ELISA plate.

“A new standard curve was performed on each plate.”

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

PCA and heatmap clustering showed that technical replicates clustered together with no batch effects.

“PCA and heatmap clustering showed that technical replicates clustered together with no batch effects (Suppl. Fig. 2).”

May combine two claims.

The rs6024870-harboring regulatory element at the LOAD 'CASS4' GWAS locus influences both microglial inflammatory capacity and DNA damage resolution.

“Our variant-to-function approach therefore reveals that the rs6024870-harboring regulatory element at the LOAD ‘CASS4’ GWAS locus influences both microglial inflammatory capacity and DNA damage resolution, along with cumulative evidence implicating RTFDC1 as a novel candidate effector gene.”

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

Each γH2AX timepoint and genotype was plated in duplicate on two separate days, and each well was imaged in 3 separate locations, for a total of 6 images per genotype/timepoint combination.

“Each timepoint and genotype was plated in duplicate (2 wells) on two separate days, and each well imaged in 3 separate locations, for a total of 6 images per genotype/timepoint combination.”

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

HMC3 cells were originally immortalized by transfecting human embryonic microglial cells with a plasmid encoding the cDNA of the viral SV40 large T antigen.

“HMC3 cells were originally immortalized by transfecting human embryonic microglial cells with a plasmid encoding the cDNA of the viral SV40 large T antigen78.”

• cites [78] sourceNot checked — no full text of the source

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

ELISA for pro-inflammatory cytokines IL-6 and IL-8 were performed in technical triplicate.

“ELISA for pro-inflammatory cytokines IL-6 and IL-8 were performed in technical triplicate according to manufacturer’s protocol (Thermo Fisher KHC0061 and Thermo Fisher KHC0081, respectively), and resulting assay plate was read for optical density on the ID5 SpectraMax plate reader at a 450nm wavelength.”

May combine two claims.

These data suggest that overexpression of RTFDC1 did not rescue the pro-inflammatory phenotype, and may itself lead to increased IL-8 secretion, and that increased secretion of IL-6 in enhancer-deleted clones is independent of RTFDC1 expression.

“These data suggest that overexpression of RTFDC1 did not rescue the pro-inflammatory phenotype, and may itself lead to increased IL-8 secretion, and that increased secretion of IL-6 in enhancer-deleted clones is independent of RTFDC1 expression.”

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

Capture-C libraries from each cell type yielded high coverage (average ~2.6 billion reads per library) with >70% capture efficiency.

“Libraries from each cell type yielded high coverage (an average of ~2.6 billion reads per library) with >70% capture efficiency (% of unique valid reads captured; Suppl. Table 3).”

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

Regulation of additional genes by the enhancer may contribute to the pro-inflammatory and DNA-repair phenotypes observed in enhancer-deleted cells.

“where the regulation of these additional genes may contribute to the pro-inflammatory and the DNA-repair phenotypes we observe in our enhancer-deleted cells.”

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

Transductions were conducted in duplicate (2 wells at a time) to obtain 2 separate replicates of each transduction.

“Transductions were conducted in duplicate (2 wells at a time) to obtain 2 separate repliactes of each transduction.”

May combine two claims.

The region harboring rs6024870 acts as an enhancer on RTFDC1 expression in HMC3 cells, and the minor protective A allele trends towards decreased activity.

“These results revealed that the region harboring rs6024870 acts as an enhancer on RTFDC1 expression in HMC3 cells, and that the minor protective A allele trends towards decreased activity.”

May combine two claims.

The rs6024870-harboring enhancer is necessary to suppress secretion of cytokines in the HMC3 cell line, but does not solely function through RTFDC1 in this process.

“These results, together, indicate that the rs6024870-harboring enhancer is necessary to suppress secretion of cytokines in the HMC3 cell line, but does not solely function through RTFDC1 in this process.”

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

Cell culture supernatants were tested for mycoplasma contamination using the InvivoGen MycoStrip Detection kit.

“Supernatants were tested for the presence of mycoplasma using the InvivoGen MycoStrip™ Detection kit according to manufacturer’s protocol with no alterations.”

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

Many enhancers operate via binding with transcription factors to facilitate contact with promoters.

“Many enhancers operate via binding with transcription factors (TFs) to facilitate contact with promoters61.”

• cites [61] sourceNot checked — no full text of the source

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

RNA quality was assessed on an RNA 6000 Nano chip on the Agilent BioAnalyzer.

“Resulting RNA quality was assessed on an RNA 6000 Nano chip on the Agilent BioAnalyzer.”

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

Accurate insertion of sgRNAs into the lentiCRISPR v2-mCherry plasmid was checked via Sanger sequencing.

“Accurate insertion of sgRNAs into the plasmid was checked via Sanger sequencing.”

Classified as probably not a substantial claim.

Three individual enhancer-deleted clones were validated for the desired enhancer deletion, as well as three mock clones.

“We then isolated three individual enhancer-deleted clones that were validated for the desired enhancer deletion, as well as three mock clones transduced with lentivirus containing the tracrRNA scaffold and Cas9 gene, but no targeting gRNA sequence (Figure 2C).”

• AssumptionRests on an assumption: The CRISPR-Cas9-mediated deletion of the ~400bp region harboring rs6024870 did not have off-target effects that confound the observed phenotypes.

May combine two claims.

Inflammation and DNA damage repair are relevant microglial-specific pathways to the pathology of LOAD.

“we implicate both inflammation and DNA damage repair as relevant microglial-specific pathways to the pathology of LOAD”

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

In lieu of a loading control, whole-protein staining was performed for downstream quantification.

“In lieu of a loading control, we performed whole-protein staining for downstream quantification by incubating membranes for 25 minutes in No-Stain Protein Labeling Reagent (ThermoFisher Scientific), prepared according to manufacturer’s protocol.”

May combine two claims.

The genomic region harboring rs6024870 acts as a microglial enhancer and influences LOAD risk through regulation of both inflammatory and DNA damage related pathways.

“Altogether, we present variant-to-function evidence that the genomic region harboring rs6024870 acts as a microglial enhancer and influences LOAD risk through regulation of both inflammatory and DNA damage related pathways.”

The quote only partly states this claim.

The inability of HMC3 cells to secrete TNF-α or IL1-β limited the cytokines and chemokines that could be assayed in this study.

“which limited the cytokines and chemokines we could assay in our study.”