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PreprintCited by 1Genetic Associations and EpidemiologyGenetic Mapping and Diversity in Plants and AnimalsGenetic and phenotypic traits in livestock

Identification of Genetic Variants Regulating the Abundance of Clinically Relevant Plasma Proteins using the Diversity Outbred Mouse Model

Open access
Philtjens, Stéphanie, Acri, Dominic J., Kim, Byungwook, Kim, Hyewon, Kim, Jungsu·2021·bioRxiv (Cold Spring Harbor Laboratory)
DOI 10.21203/rs.3.rs-171899/v1
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Abstract

Background: Levels of plasma proteins are under control of environmental and genetic factors. To use plasma proteins in biomarker studies, we need to understand how genetic modifiers influence their abundance. Although there has been expression quantitative trait loci (eQTL) studies on a few limited numbers of proteins, the effect of genetic variants on the levels of multiple plasma proteins still warrants more systematic investigation.Results: To identify genetic modifiers that influence the levels of clinically relevant plasma proteins, we performed protein quantitative trait locus (pQTL) mapping on the 92 proteins present in the Olink Mouse Exploratory Panel using the Diversity Outbred (DO) mouse population. We identified 12 significant pQTL that were located in cis and 6 that were in trans. Among them, we discovered that the presence of coding variants in the gene encoding for the Aryl Hydrocarbon Receptor (Ahr) had a significant effect on its abundance in plasma. Most interestingly, we identified variants in the Regulatory Factor X1 (Rfx1) gene that influence the abundance of the IL-17A protein in plasma.Conclusion: Our study reports an innovative pipeline for the identification of genetic modifiers that may be targeted for drug development.

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  1. Registry recordVerified
  2. Full textRead the full text
  3. Reference list35 references found
  4. Reference check37 of 37 references checked
  5. StatisticsRecomputed
  6. Claims41 claims anchored to a quote
  7. Cited sourcesChecked
  8. Self-checkDidn’t finish — the rest of the report still applies.
  9. VerificationFindings verified

AI snapshot

Written from the paper’s full text.

TL;DR

Using 140 Diversity Outbred mice and the Olink Mouse Exploratory Panel, the authors mapped protein quantitative trait loci for 76 plasma proteins and identified 18 significant and 5 suggestive pQTL. They found coding/UTR variants in *Ahr* associated with AHR abundance and variants in *Rfx1* associated with IL-17A abundance.

Key findings

  • Among 76 reliably detected proteins from 92 measured, pQTL mapping identified 18 significant pQTL (12 cis, 6 trans) and 5 suggestive pQTL (1 cis, 4 trans).
  • A significant cis pQTL for AHR on chromosome 12 had a LOD score of 12.8; the *Ahr* missense variant p.K432R minor G allele increased AHR protein abundance (P = 3.41 × 10⁻⁷), while the 5′ UTR variant c.-100G>A minor A allele decreased AHR levels (P = 6.3 × 10⁻⁹).
  • A significant trans pQTL for IL-17A on chromosome 8 had a LOD score of 8.3; in *Rfx1*, two minor C alleles of p.F612S decreased IL-17A (P = 0.0036), heterozygous c.*935G>A decreased IL-17A (P = 0.0049), and p.A724 had no significant effect.
  • The authors propose *Rfx1* as a genetic modifier of plasma IL-17A, citing prior functional evidence from Zhao et al. linking RFX1 and IL-17A.

Methods

  • Male DO mice (n = 140, two batches, generation 28) were genotyped with GigaMUGA (143,259 markers); plasma was assayed with the Olink Mouse Exploratory Panel, measuring 92 proteins in 1 µL plasma, with 76 proteins detected in >10% of samples used for mapping.
  • pQTL mapping used R/qtl2: founder haplotype probabilities from a hidden Markov model, protein abundance regressed on haplotype probabilities, kinship as a random effect using leave-one-chromosome-out, genome scans, 1,000 permutations, LOD scores, and 95% Bayesian credible intervals; significance was P < 0.05 and suggestive was 0.05 < P < 0.1.
  • Follow-up included fine mapping, sequence alignment, MEME/Regma and PicTar/miRDB analyses, MetaCore network analysis, and one-way ANOVA with Tukey pairwise comparisons.

Limitations

  • The paper does not include a dedicated limitations section.
  • Evident from the text: the study used only male DO mice (n = 140) from two batches, and only 76 of 92 assayed proteins were reliably detected in >10% of samples and used for pQTL mapping.
  • Evident from the text: findings are in mice without independent human cohort validation, and functional support for the *Rfx1*–IL-17A link relies on cited prior literature rather than experiments performed here.

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

Integrity signals

Registry

Crossref · 10.21203/rs.3.rs-171899/v1

Cites

37 of 37 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

16 of 20 cited sources checked · 3 supported · 1 flagged · accuracy not yet measured

  • Interpretationown data

    Rfx1 is a genetic modifier for IL-17A abundance in plasma.

    “Therefore, our data suggest that Rfx1 is a genetic modi er for IL-17A plasma levels.”
    • Also stated in: another passage · boosted
      “We also report both coding and non-coding variants in the Rfx1 gene that alter IL-17A plasma protein abundance, replicating a known connection between RFX1 and IL-17A in mouse and human [28].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [28] source

      The Results (QTL analysis) and Discussion address genetic loci and candidate genes for testis weight, not plasma IL-17A levels. Searches for Rfx1, IL-17A, and their proposed relationship found no passage in our copy that addresses the claim; the indexed passages provide no page numbers. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
  • Findingcitation

    The c.*935G>A variant is not located in a known miRNA binding site according to PicTar and miRDB.

    “However, this variant is not located in a known miRNA binding site according to PicTar and miRDB database [22,23].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [22] source
    • cites [23] source

      The Results section on RFX1 regulation of the IL17A locus discusses predicted transcription-factor binding sites, not miRNA binding sites. Searches for c.*935G>A, PicTar, miRDB, and miRNA binding sites found no passage addressing the variant or the claimed database result. Page numbers were not available in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Findingcitation

    Fine mapping of the IL-17A pQTL region resulted in a locus of approximately 3 Mb.

    “Fine mapping of this region resulted in a locus of approximately 3 Mb in size (Fig. 3b) [20,21].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [20] source

      The Results and Discussion passages describe functional domains of the aryl hydrocarbon receptor, not an IL-17A pQTL. Searches for IL-17A, fine mapping, and a 3 Mb locus found no passage addressing the claimed locus size. Our copy therefore cannot verify the claim. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [21] source
  • Interpretationown data

    The paper's data suggest that coding variants in Ahr are responsible for the differences in AHR protein abundance.

    “Although p.K432R is not located in a known protein domain [26] nor is c.-100G > A located in a predicted 5' UTR motif, our data do suggest that coding variants in Ahr are responsible for the differences in AHR protein abundance.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [26] source

      In the citing sentence, citation [26] is attached to the statement about p.K432R and a known protein domain; the conclusion about Ahr coding variants and AHR protein abundance is presented as the citing authors’ own data. The indexed paper’s Results and discussion passages concern genetic associations with gut microbes and bile acids, and searches for Ahr, p.K432R, and AHR protein abundance found no passage addressing the quoted conclusion. (LLM, conf=0.77)

      Experimental — accuracy not yet measured
  • Premisecitation

    The results from Zhao et al. provide functional evidence directly linking RFX1 and IL-17A.

    “The results from Zhao et al. [27] provide functional evidence directly linking RFX1 and IL-17A.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [27] source

      The Results section (3.3, page unavailable) reports a link between Gpc5 expression and pancreatic lesion size in mice, not between RFX1 and IL-17A. Searches for “RFX1 IL-17A” and “RFX1 interleukin 17A” found no passage in our copy addressing the claimed functional link; its reference to a Zhao paper concerns GPC5 in lung adenocarcinoma. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 14
  • May combine two claims. 6
  • The quote only partly states this claim. 1

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

    Premisecitation

    QTL mapping in multiparent populations has identified genetic modifiers for viral response, kidney disease, atherosclerosis, and heart size.

    “For example, QTL mapping in these multiparent populations resulted in the identi cation of genetic modi ers for the viral response [13,14], kidney disease [15], atherosclerosis [16] and heart size [17].”
    Quote in the paperNot a substantial claim4 of 4 sources checked
    • cites [13] source
    • cites [14] source

      For this citation, the relevant claim is that QTL mapping identified a modifier of viral response, not the separately cited claims about kidney disease, atherosclerosis, or heart size. In “Genetic analysis suggests a polygenic control of susceptibility to ZIKV in CC mice” (page unavailable), the paper reports that its genome scan found no significant loci for the viral-load traits; the Discussion likewise says no genomic region was significantly associated with viral-load variation. (LLM, conf=0.89)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [15] source

      The Results (page not provided) report that QTL mapping in Diversity Outbred mice identified a locus affecting plasma cystatin C, a kidney biomarker. The Discussion (page not provided) says the study found no QTL for other kidney traits. This supports genetic mapping of a kidney-related biomarker, but not the stronger claim that it identified a modifier of kidney disease itself; this citation is attached only to the kidney-disease example. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [16] source

  • May combine two claims.

    Premisecitation

    The combination of eight founder strains results in greater genetic diversity than existing recombinant inbred lines and more uniform distribution of genetic variants across the genome than in other genetic reference populations.

    “The combination of eight founder strains results in a much greater level of genetic diversity than existing recombinant inbred lines and the genetic variants are more uniformly distributed across the genome than in other genetic reference populations [12].”
    Quote in the paperSubstantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    Zhao et al. showed that IL-17A mRNA and protein levels significantly increased after knock-down of RFX1 in CD4+ T cells, while a decrease was observed after overexpressing RFX1.

    “They showed that the levels of IL-17A mRNA and protein signi cantly increased after knock-down of RFX1 in CD4 + T cells, while a decrease in IL-17A level was observed after overexpressing RFX1.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [27] source

      Section 3.3 reports an association between a genetic variant and Gpc5 expression in mice, not an RFX1 experiment in CD4+ T cells. Searches for RFX1, IL-17A, knock-down, and overexpression found no passage supporting the claimed changes in IL-17A mRNA or protein. The References section names a Zhao paper, but describes it as a study of GPC5 in lung adenocarcinoma, not the claimed experiment. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The Olink technology allows the measurement of up to 92 proteins in 1 μL of plasma using the proximity extension assay (PEA).

    “The Olink technology allows the measurement of up to 92 proteins in 1 µL of plasma using the proximity extension assay (PEA).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [35] source

      The paper’s Sequence database search and Web server and user support sections describe tools for finding sequence motifs, not protein measurement in plasma. Searches for Olink, proximity extension assay, 92 proteins, and plasma found no passage addressing the claim; the page numbers are unavailable in our copy. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

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

    Premisecitation

    RFX1 plays an important role in methylation and acetylation of the promoter region of IL17A.

    “RFX1 also played an important role in methylation and acetylation of the promoter region of IL17A [27].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [27] source

      The Discussion and section 3.3 address pancreatic-cancer candidate genes and cis-regulation of Gpc5, not RFX1 or IL17A. Searches for RFX1, IL17A, and promoter methylation and acetylation found no passage in our copy addressing the claimed relationship. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Zhao et al. demonstrated that RFX1 directly affects IL-17A expression by binding one of its two binding sites upstream of the transcription start site of the IL17A gene.

    “Furthermore, they demonstrated that RFX1 directly affects IL-17A expression by binding one of its two binding sites upstream of the transcription start site of the IL17A gene.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [27] source

      Section 3.3 reports cis-regulation of Gpc5 in mice, not regulation of IL-17A by RFX1. Searches for RFX1, IL17A and the claimed upstream binding sites found no passage addressing that mechanism; the Zhao work listed in the References concerns GPC5 in lung adenocarcinoma. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The GigaMUGA contains 143,259 genetic markers specifically designed for genetic mapping in the DO mouse population.

    “The GigaMUGA contains 143,259 genetic markers that were speci cally designed for genetic mapping in the DO mouse population [19,33].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [19] source

      The Abstract identifies GigaMUGA as a 143,259-probe array and says the bulk of its content is optimized for genetic mapping in the Collaborative Cross and Diversity Outbred populations. It does not say all 143,259 markers were specifically designed for mapping in the DO; the Abstract and Results and Discussion describe probes serving other purposes, including copy-number detection and studies of wild mice. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [33] source

      The Materials and Methods section describes xenograft experiments in NMRI-nude mice, while the Results and Discussion describes a protein immunoassay. Searches for “GigaMUGA 143,259 genetic markers” and “Diversity Outbred mouse genotyping array” found no passage addressing the marker count or the array’s design for DO mouse mapping. The claim cannot be verified from our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Of the 5 suggestive pQTL, 1 was cis and 4 were trans.

    “while four of the suggestive pQTL were trans pQTL (Fig. 1c, blue lines) and one was a cis pQTL (Fig. 1c, red lines).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Two coding variants, p.F612S and p.A724, and one 3' UTR variant c.*935G>A were identified in Rfx1.

    “We identi ed two coding variants, p.F612S and p.A724, (Fig. 3d) in Rfx1, as well as one in the 3' UTR (c.*935G > A, Fig. 3d).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    A variant in the 5' UTR of Ahr, c.-100G>A, was identified.

    “In addition, we identi ed a variant located in the 5' untranslated region (UTR) of Ahr”
    Quote in the paperSubstantial claim

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

    Findingown data

    Protein sequence alignment showed conservation of the lysine at position 432 between mouse and human.

    “Protein sequence alignment showed conservation of the lysine at position 432 between mouse and human (Fig. S2a).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    76 of the 92 proteins in the Olink Mouse Exploratory Panel were reliably detected in more than 10% of the samples and were used for pQTL mapping.

    “Of the 92 proteins in the assay, 76 were reliably detected in more than 10% of the samples (Table S1) and were used for pQTL mapping.”
    • Also stated in: another passage · plain
      “83% (76/92) of the detected proteins were detected in more than 10% of the samples and were used for pQTL mapping (Table S1 and File S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The genetic diversity of the DO mouse model makes it possible to identify trans pQTL that can easily be translated to human, making this mouse model suitable for translational discovery studies.

    “Additionally, we show that the genetic diversity of the DO mouse model makes it possible to identify trans pQTL that can easily be translated to human, making this mouse model suitable for translational discovery studies.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    A random effect was included to account for kinship.

    “Genome scans were performed, and a random effect was included to account for kinship.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The kinship matrix was determined based on the leave-one-chromosome-out method to account for genetic similarity between mice.

    “To account for genetic similarity between mice, the kinship matrix was determined based on the leave-one-chromosome-out method.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All animal experiments were performed in accordance with the approved animal protocol and guidelines established by the Institutional Animal Care Committee at Mayo Clinic, Jacksonville, FL.

    “All animal experiments were performed in accordance with the approved animal protocol and guidelines established by the Institutional Animal Car Committee at Mayo Clinic, Jacksonville, FL (#A00003398-00).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    pQTL mapping was performed with genetic relatedness as a covariate.

    “To identify modi er genes that affect levels of plasma proteins, we performed pQTL mapping for the analytes measured using the Mouse Exploratory Panel from Olink Proteomics with genetic relatedness as a covariate.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Plasma samples of all 140 DO mice were randomly distributed across 96-well plates.

    “Plasma samples of all 140 DO mice were randomly distributed across 96-well plates and shipped to Olink Proteomics”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Significance thresholds were determined using 1,000 permutations.

    “Signi cance thresholds were determined using 1,000 permutations and the mapping statistic is the logarithm of the odds (LOD) score.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    This study was carried out in compliance with the ARRIVE guidelines.

    “This study was carried out in compliance with the ARRIVE guidelines.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Genotype quality was assessed using the R package argyle.

    “Genotype quality was assessed using the R package argyle.”
    Quote in the paperNot a substantial claim
21 of 21

  • The paper relies on this without support.

    The DO mouse population eliminates the effects of environment.

    “The DO mouse population was chosen because of the advantage of eliminating the effects of environment while pro ting from a genetic diversity similar to what is observed in humans.”

  • The paper relies on this without support.

    The DO mouse population has genetic diversity similar to what is observed in humans.

    “The DO mouse population was chosen because of the advantage of eliminating the effects of environment while pro ting from a genetic diversity similar to what is observed in humans.”

  • The paper relies on this without support.

    Each DO mouse is genetically unique with a high level of allelic heterozygosity, providing precision for mapping QTL with relatively small sample sizes compared to human mapping studies.

    “Each DO mouse is a genetically unique individual with a high level of allelic heterozygosity, providing precision for mapping QTL with relatively small sample sizes compared to human mapping studies.”
3 of 3

Self-check

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

This check didn’t finish.

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Génin E, Feingold J, Clerget-Darpoux F: Identifying modi er genes of monogenic disease: strategies and di culties. Hum Genet 2008, 124(4):357-368.
  2. Assarsson E, Lundberg M, Holmquist G, Bjorkesten J, Thorsen SB, Ekman D, Eriksson A, Rennel Dickens E, Ohlsson S, Edfeldt G et al: Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, speci city, and excellent scalability. PLoS One 2014, 9(4):e95192.

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Related

Authors
Philtjens, S., Acri, D. J., Kim, B., Kim, H., & Kim, J.
Affiliations
  • Indiana University Indianapolis (US)
  • Indiana University
  • Mayo Clinic (US)
Corresponding author
Kim, Jungsu
Venue
bioRxiv (Cold Spring Harbor Laboratory)
Publisher
Research Square Platform LLC
Published
2021-02-08
DOI
10.21203/rs.3.rs-171899/v1
OpenAlex
W4245995461
Cited by
1
References
35
Citations by year
2024: 1
Type
posted content
Language
en
Open access
Licence
Locations
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
Funders
  • Eli Lilly and Company
  • Indiana Clinical and Translational Sciences Institute (UL1TR002529)
  • National Institutes of Health (R01AG053500, UL1 TR002529, R01AG053242, R21AG050804)
  • National Institute on Aging (R01AG054102)
  • National Center for Advancing Translational Sciences (UL1TR002529)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Associations and Epidemiology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Mapping and Diversity in Plants and Animals
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic and phenotypic traits in livestock
Keywords
protein quantitative trait loci, plasma protein levels, Diversity Outbred mice, genetic modifiers, AHR, RFX1, IL-17A, cis and trans regulation, autoimmune disease biomarkers, Olink proteomics

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