Posadas-Mondragon2020 - TLR Polymorphisms and Clinical Forms of Dengue

Full citation: Posadas-Mondragón, A., Aguilar-Faisal, J. L., Zuñiga, G., Magaña, J. J., Santiago-Cruz, J. A., Guillén-Salomón, E., Alcántara-Farfán, V., Arellano-Flores, M. L., Salas-Benito, J. S., Neri-Bazán, R. M., Luna-Rojas, L., Avila-Trejo, A. M., & Chávez-Negrete, A. (2020). Association of genetic polymorphisms in TLR3, TLR4, TLR7, and TLR8 with the clinical forms of dengue in patients from Veracruz, Mexico. Viruses, 12(11), 1230. https://doi.org/10.3390/v12111230

Raw file: [[raw/Posadas-Mondragon2020.pdf]]

Scope note for this wiki. This is a host-genetics candidate-gene association study. It contains no B cell phenotyping, no flow cytometry, and no measurement of DN, DN2, atypical or plasmablast populations. It was ingested for the TLR7 axis — dengue virus is a positive-sense ssRNA flavivirus and TLR7 is the endosomal ssRNA sensor that the wiki’s DN2 mechanism runs on. What the paper can speak to is whether germline variation in TLR7 modifies dengue clinical form. It cannot speak to whether TLR7 signalling operates in dengue B cells. The two questions are separate and this page keeps them separate.

Summary

Posadas-Mondragón et al. genotyped 13 single-nucleotide polymorphisms across four Toll-like receptor genes — TLR3 (2 SNPs), TLR4 (6), TLR7 (2) and TLR8 (4) — in 165 adult dengue patients and 89 general-population controls from Veracruz, Mexico, sampled during the 2013 and 2015 epidemic seasons. Patients were split by WHO 1997 criteria into dengue fever (DF, n=100) and dengue haemorrhagic fever (DHF, n=65). All samples were drawn in the acute phase, 1–8 days after symptom onset. Alongside genotyping, the authors measured haematological values, serotyped by multiplex RT-qPCR, and quantified anti-dengue IgM, total IgG and IgG subclasses 1–4 by capture ELISA and an in-house EIA.

The primary genetic result is confined to TLR4. Two promoter variants were associated with protection from dengue relative to the general population: TLR4-rs2737190-G/G (codominant model, DEN vs GP, OR 0.34, 95% CI 0.14–0.8, p=0.038) and TLR4-rs11536865-G/C (OR 0.19, 95% CI 0.05–0.73, p=0.0092). A four-SNP TLR4 TGCG haplotype was likewise protective (OR 0.55, 95% CI 0.35–0.86, p=0.0084), while the TACG haplotype was over-represented in patients. For TLR3, TLR7 and TLR8 the authors state explicitly that “there was no statistical association in the crude analysis with clinical forms of the dengue disease.”

The paper’s second half is a large exploratory interaction analysis in which each SNP is cross-tabulated against haematological covariates (leukocytes, lymphocytes, monocytes) and antibody levels (IgM, total IgG, IgG1, IgG3) dichotomised by tertile, and against primary-versus-secondary infection status. This is where every TLR7-related observation in the paper lives. Because TLR7 and TLR8 are X-linked, these analyses were run separately for men and women. The interaction results are reported only as forest plots (Figures 1–4) with no tabulated odds ratios or confidence intervals, are unadjusted for multiplicity, and rest on single-digit strata. They are hypothesis-generating and are not treated as findings anywhere in this wiki.

Study Design

• Type: Cross-sectional genetic case-control association study, with a nested exploratory SNP × covariate interaction analysis
• Sample size: DF n=100; DHF n=65; combined dengue (DEN) n=165; general population (GP) n=89. Total n=254
• Setting: Family Medicine Unit No. 61 and General Hospital No. 71, Mexican Institute of Social Security (IMSS), Veracruz, Mexico. Blood drawn during the epidemic outbreaks of August–October 2013 and September–October 2015. Acute phase only, 1–8 days after onset of symptoms. No convalescent or follow-up sampling
• Population: Unrelated adult volunteers (>18 y), residents of Veracruz. Severity assigned by WHO 1997 criteria — DF = platelet count >100 × 10⁹/L; DHF = platelet count <100 × 10⁹/L plus any haemorrhagic manifestation. Diagnosis confirmed by serology and viral genome identification. Primary versus secondary assigned by IgM/IgG OD ratio (>1.2 primary, <1.2 secondary): DF 27% primary / 73% secondary; DHF 29.2% primary / 70.8% secondary
• Controls: GP subjects were healthy adults (>18 y) attending a routine medical check-up at the same units in the same period, with normal haematic biometry. Not screened for prior dengue exposure
• Serotype: Viral genome recovered in only 54/165 (33%) of acute samples. Of 46 typed DF samples: 3 (6.5%) DENV-1, 42 (91.3%) DENV-2, 1 (2.2%) DENV-4. All typed DHF samples were DENV-2. The cohort is effectively a DENV-2 cohort

Key Findings

TLR7 — the curator’s focus

• TLR7 SNPs studied: rs179008 (exonic, A/T, Gln11Leu) and rs3853839 (3′-UTR, C/G). Both lie on the X chromosome, so all TLR7 analyses were stratified by sex
• ★ The crude TLR7 analysis is null. Section 3.4, which covers TLR3, TLR7 and TLR8 together, closes with: “There was no statistical association in the crude analysis with clinical forms of the dengue disease.” No TLR7 genotype, allele or inheritance model distinguished DF from DHF, or dengue from the general population
• Descriptive frequencies: the ancestral allele rs179008-A and the minor allele rs3853839-G were the most frequent, as were the genotypes rs179008-A/A, rs179008-A/T and rs3853839-G/C. The actual TLR7 frequency table is supplementary (Table S2) and is not in the PDF — those numbers are therefore not available to this wiki
• ⚠ rs3853839 deviates from Hardy–Weinberg equilibrium in the dengue (DEN) group. This is the single TLR7 SNP the Discussion builds its argument on, and the HWE failure is stated in the same paragraph as the null result. HWE deviation in a case group can reflect genotyping error, population stratification, or a true association — the paper does not adjudicate. Weight every rs3853839 claim below accordingly
• Exploratory interaction findings (Figure 3, §3.8.3) — directional only, no numbers. Recorded for completeness; see the caveat block under Relevance & Notes before using any of them:
  • DHF women with low leukocytes → more likely rs179008-A/T and rs3853839-C/C
  • DHF women with high monocytes → more likely rs179008-A/A and rs3853839-C/C
  • DHF women with high total IgG → more likely rs179008-A/A, rs179008-A/T, rs3853839-C/G and rs3853839-C/C; those with high IgG3, however, had a lower probability of rs3853839-G/C and rs179008-A/T
  • DHF patients with high IgG1 → more likely rs3853839-C/C
  • DF and DEN patients with high monocytes → higher probability of rs179008-A/A than GP individuals
• ⚠ Results and Discussion disagree on whether the IgG1 finding is women-only. §3.8.3 reads “DHF patients with high levels of IgG1 were more likely to be TLR7-rs3853839-C/C”; the Discussion reads “DHF women patients with low levels of leukocytes, and high levels of total IgG and IgG1, were more likely to be TLR7-rs3853839-C/C.” Recorded as a discrepancy rather than resolved in one direction
• External context the authors supply for rs3853839 (transcribed from their reference list; not independently verified — see the [2026-08-16] PDF-only decision): the C/C genotype has been reported to associate with dengue infectivity in Indian patients (Mukherjee & Tripathi 2019, Immunobiology 224:774–785, their ref 30), and a G/C excess was reported in chikungunya-infected Indian patients (Kumar & Tripathi 2017, Virology 511:207–213, their ref 31)
• Background stated by the authors: TLR7 and TLR8 are endosomal receptors recognising non-methylated single-stranded viral RNA rich in guanosine or uridine (their refs 11–12, Diebold 2004 and Heil 2004, Science). Dengue virus is a positive-sense ssRNA virus, so the ligand is present by construction during viraemia

The only positive genetic result — TLR4

• TLR4-rs2737190 (promoter −2570, A/G): the G/G genotype was protective. DF vs GP codominant OR 0.29 (95% CI 0.10–0.84), p=0.035, AIC 260.6; recessive OR 0.29 (0.11–0.78), p=0.0095, AIC 258.6. DEN vs GP codominant OR 0.34 (0.14–0.8), p=0.038, AIC 328.5; recessive OR 0.36 (0.16–0.8), p=0.011, AIC 326.6
• TLR4-rs11536865 (promoter −728, C/G): DEN patients had a lower probability of the G/C genotype (OR 0.19, 95% CI 0.05–0.73, p=0.0092) and a higher probability of carrying allele G (DEN vs GP OR 5.12, 95% CI 1.34–19.5, p=0.0081). The C allele is rare — 8/178 chromosomes in GP, 3/330 in DEN — so these estimates rest on very few carriers
• Haplotype analysis (4 TLR4 SNPs): the TACG haplotype was over-represented in DF, DHF and DEN relative to GP; the TGCG haplotype was under-represented (GP vs DF OR 0.55, 0.34–0.91, p=0.021; GP vs DHF 0.59, 0.34–1.0, p=0.05; GP vs DEN 0.55, 0.35–0.86, p=0.0084; global haplotype association p=0.0019 for DEN). DF vs DHF showed no haplotype difference (OR 1.02, p=0.95; global p=0.98) — the TLR4 signal concerns acquiring symptomatic dengue, not severity
• In silico transcription-factor binding (PROMO): at rs2737190, allele G admits HNF-1B; allele A additionally admits HOX D9, HOX D10 and progesterone receptors PR-B and PR-A. At rs11536865, allele C admits only the glucocorticoid receptor (GR); allele G admits GR and GR-α, the isoform mediating steroid anti-inflammatory effects through transrepression of NF-κB and AP-1. This is prediction, not experiment. (Minor internal inconsistency: the Figure 5 caption says “five more transcription factors bind” but names four.)
• ★ Note for a B cell wiki: Pax-5 is among the transcription factors PROMO predicts at the rs2737190 site. Pax-5 is the master B-lineage commitment factor. The authors do not comment on this and no B cell data were collected, so it is an in-silico coincidence at present — but it is the only point in the paper at which a B-cell-intrinsic reading of a TLR4 promoter variant is even conceivable
• Why TLR4 features in a flavivirus study: the authors note that dengue NS1 in its hexameric form activates macrophages and PBMCs through TLR4 (their ref 13, Modhiran 2015, Sci Transl Med). TLR4 is the extracellular, NS1-facing receptor; TLR7 is the endosomal, RNA-facing one

Haematology — the most directly usable result

• DHF versus DF (Table 1, n=165):
  • Monocytes 14.44 ± 7.11% vs 10.33 ± 4.705%, OR 2.095 (95% CI 1.075–4.083), p=0.0001
  • Lymphocytes 35.23 ± 14.88% vs 24.77 ± 14.38%, OR 3.467 (95% CI 1.244–9.663), p<0.0001
  • Leukopenia OR 2.118 (1.096–4.09), p<0.001; neutropenia OR 2.34 (1.281–6.779), p<0.0001
  • Platelets 54.03 ± 30.48 vs 191.1 ± 65.07 × 10⁹/L (definitional, given the WHO 1997 split)
• Both dengue groups had higher monocyte percentages than the general population (GP 7.517 ± 2.646%)

Serology (detail canonical to dengue-wiki/)

• Anti-DENV IgM detected in 37% of DF and 63% of DHF sera; anti-DENV IgG in 93% of DF and 96.9% of DHF
• Tertile cut-offs: IgM 1.47, total IgG 1.678, IgG1 0.564, IgG2 0.018, IgG3 0.033, IgG4 0.193
• DHF sera were skewed high across isotypes: high IgM 58.46% (DF 14%), high IgG 55.38% (DF 16%), high IgG1 38.98% (DF 28.2%), high IgG4 35.59% (DF 30.77%). DF sera were predominantly IgM-negative (74%) and low-IgG (77%)
• IgG3 runs the other way — high IgG3 in 38.46% of DF versus 25.42% of DHF. IgG2 was negative in the great majority of both groups (DF 91.03%, DHF 84.75%)

Methods Used

• SNP Genotyping — 13 SNPs by real-time PCR using Applied Biosystems TaqMan genotyping assays; genomic DNA from leukocytes (Gentra Puregene Blood Kit). Five inheritance models (codominant, dominant, recessive, over-dominant, additive) compared by Akaike information criterion; haplotypes estimated by the expectation-maximisation algorithm; linkage disequilibrium (D, D′, r, χ²) computed for the TLR4 SNPs; X-linked TLR7/TLR8 analysed separately by sex
• In silico transcription-factor binding prediction — PROMO / ALGGEN virtual laboratory, applied to the TLR4-rs2737190 and rs11536865 promoter sites. Prediction only, with no experimental validation; no method page created
• Multiplex RT-qPCR serotyping — QIAamp viral RNA extraction from serum; flavivirus plus serotype-specific TaqMan probes to the NS5 region (Chien 2006); SuperScript III Platinum One-Step kit on a LightCycler 480. DENV-1 (Hawaii), DENV-2 (New Guinea), DENV-3 (H-87) and DENV-4 (H-241) as positive controls
• Anti-dengue IgM/IgG capture ELISA (DRG EIA-3470/EIA-3471) and an in-house anti-dengue IgG subclass EIA using DENV-2 (New Guinea) grown in Vero cells as capture antigen, with HRP-conjugated anti-human IgG1/2/3/4 monoclonals. Indices computed as (OD sample − OD blank)/(OD blank) and categorised by tertile. Serology methodology is canonical to dengue-wiki/; no method page is created here
• Automated haematology — COULTER LH 500 analyser for platelets, leukocytes, neutrophils and monocytes. This yields total monocyte percentage, not CD14/CD16 subset resolution

Entities Mentioned

TLR7, IgG, IgM, Inflammatory Monocyte

Named in the paper but deliberately given no entity page under the [2026-08-16] / [2026-08-18] evidence gate: TLR3 (dsRNA sensor — outside the DN/DN2/plasmablast spine), TLR4 (extracellular NS1-facing receptor, monocyte-side; carries this paper’s only positive result but has no B cell application in any ingested source — covered in prose above and tracked as a Watch Item), TLR8 (see the R848 note under Contradictions & Debates on TLR7), and the IgG subclasses IgG1–IgG4 (serology, canonical to dengue-wiki/).

Concepts Addressed

Toll-like Receptor Signaling in B Cells, Dengue Severity Classification, Antibody-Dependent Enhancement

Relevance & Notes

What this changes for the wiki’s TLR7 story: less than the title promises, but not nothing.

Every existing bullet on TLR7 asserts that TLR7 is required for DN2 generation and for DN2 → ASC differentiation. That evidence is in vitro, human, and overwhelmingly SLE-derived (Jenks2018 - DN2 B Cells and EF Pathway in SLE, Zumaquero2019 - IFN-gamma Programs T-bet-hi B Cells for ASC Differentiation). This is the first ingested source to ask a different question — whether germline variation in TLR7 changes who gets dengue or who gets DHF — and on n=254 the answer is no.

These are not in conflict, and the wiki should say why. A pathway can be functionally obligate and still show no genetic association: if the pathway is essential, common loss-of-function variation in it is selected against, so the variation that persists in the population is by construction the variation that does not break the pathway. A null association study of an essential gene is the expected result, not a refutation. The failure mode to guard against is a future session reading this null as evidence that TLR7 is unimportant in dengue — it is not evidence of that. The mirror-image failure mode is treating the §3.8.3 interaction results as support; they are not that either.

Where it does connect. Three threads:

1. X-linkage. Lamprinou2026 - ABCs and DN B Cells and Sanz2025 - Human Atypical B Cells Overview both build on TLR7 being X-encoded — gene dosage, XIST as an endogenous ligand, defective X-inactivation — as a partial explanation for the female bias in autoimmunity and ABC expansion. This paper is the wiki’s first infection cohort forced by that same X-linkage to analyse TLR7 separately in men and women. Its sex-stratified design is the methodological precedent; its underpowered strata are the cautionary half of the lesson.
2. Monocytes. The DHF monocyte elevation (14.44% vs 10.33%, p=0.0001, n=165) is an independent replication, in a Mexican adult cohort, of the monocyte expansion that Kwissa2014 - Monocytes Drive Plasmablast Differentiation in Dengue documented in a Thai cohort and then tied causally to plasmablast differentiation via BAFF/APRIL and IL-10. Kwissa also showed that R-848, a TLR7/8 agonist, reproduced DENV’s conversion of monocytes to the plasmablast-driving CD14⁺CD16⁺ phenotype whereas LPS did not. Posadas-Mondragón adds a genetic layer that is directionally consistent but formally uninterpretable: DF/DEN patients with high monocytes were more likely to carry TLR7-rs179008-A/A. Unadjusted, numberless, and one of dozens of comparisons — a coincidence worth remembering, not a result.
3. Antibody quantity is not antibody quality. DHF sera are high across IgM, IgG, IgG1 and IgG4 and low in IgG3. The wiki’s spine claim is that the atypical/EF route yields abundant, low-fidelity, cross-reactive antibody (GodoyLozano2016 - Lower IgG SHM Rates in Acute Dengue, Priyamvada2016 - Cross-Reactive Memory Plasmablasts in Secondary Dengue). A severity-linked isotype skew is consistent with that picture but does not test it — nothing here measures affinity, mutation load, neutralisation, or cell of origin.

Methodological weight — this is a weak study, and the weakness is concentrated in exactly the part the curator cares about.

• Multiplicity is uncontrolled. 13 SNPs × 5 inheritance models × 4 group comparisons × roughly 10 covariate strata, with no stated correction. Under those conditions the interaction analysis generates significant-looking cells by construction
• The interaction results are unreadable. Figures 1–4 are forest plots with no tabulated ORs or CIs. The TLR7 panel (Figure 3) has an abscissa running to about 1000 with intervals spanning orders of magnitude — the signature of sparse-data separation. No numeric OR from §3.8 can be quoted, and none is quoted on any wiki page
• Strata are tiny. DHF women number 28 before any covariate split; a “DHF women with high monocytes” cell is single-digit
• HWE failure at rs3853839 in the case group — see above
• The control group is not matched. GP subjects are substantially older (49.52 ± 22.99 y versus DF 37.86 ± 13.49) and sex-skewed opposite to the patients (GP 59 F / 30 M versus dengue 77 F / 88 M). (Read off Table 1 — the authors do not flag this.) No adjustment for age or sex is reported, and for X-linked TLR7/TLR8 the sex imbalance directly determines the size of every stratum
• Controls are not dengue-naive. They are healthy adults from an endemic region, with no serology reported for them. Since 93–97% of patients were anti-DENV IgG positive, an unknown and probably large fraction of “general population” controls have been exposed. “Protection from dengue” therefore means, at best, “protection from symptomatic, presenting dengue in this sample”
• DENV-2 cohort. Serotype was recovered in 33% of patients and was DENV-2 in 91.3% of typed DF and 100% of typed DHF samples. Findings do not generalise across serotypes
• WHO 1997, not WHO 2009. See Dengue Severity Classification for why this matters when comparing severity strata across dengue papers
• PROMO output is prediction. No EMSA, ChIP, reporter assay or expression measurement supports any transcription-factor claim, and no TLR mRNA or protein levels were measured at all — so even the positive TLR4 result has no mechanistic bridge to function

Questions Raised

• Does a null genetic association for TLR7 tell us anything about dengue B cell biology? The argument above says no — an essential pathway is expected to lack common functional variation. But the wiki has never articulated what evidence would count. Concretely: what would a positive result even look like, given that the informative comparison is not “carriers versus non-carriers among dengue cases” but “TLR7 responsiveness in sorted DN2 cells during acute dengue,” which nobody has measured?
• Is the female excess among general-population controls (66% F) versus patients (47% F) an artefact of recruitment, or does it interact with X-linked TLR7/TLR8 dosage? The paper does not address it. It is exactly the kind of imbalance that can manufacture an X-linked association or conceal one.
• Does the DHF monocyte elevation reflect the inflammatory (CD14⁺CD16⁺) monocyte expansion of Kwissa2014 - Monocytes Drive Plasmablast Differentiation in Dengue, or merely total monocyte count? A haematology analyser cannot distinguish them. If the curator’s cohort measures CD14/CD16, this is a cheap and directly comparable readout.
• Is the IgG3-low / IgG1-high / IgG4-high DHF isotype signature reproducible, and does it track the plasmablast burst? IgG3 moving opposite to IgG1 and IgG4 is the least expected part of the serology and is not discussed by the authors.
• Does TLR4-rs2737190 genotype affect anything measurable in B cells? The PROMO prediction places Pax-5 at that promoter site. No B cell data exist here, and the wiki has no source connecting TLR4 to B cell fate at all — but the NS1→TLR4 axis is the one innate arm of dengue with a documented receptor and no B cell page.