Intergenic risk variant rs56258221 skews the fate of naive CD4+ T cells via miR4464-BACH2 interplay in primary sclerosing cholangitis
The main results reproduced, with only marginal, non-material deviations.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
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- 🟡Could not use the authors’ exact input data
- 🟡Reported values were only indirectly comparable
- 🔴A deviation arose in the data or preprocessing
- 🔴A deviation was attributed to the published material
- 🔴Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🔴Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Described well enough to reproduce the single-cell results; PARTIAL 1:1 reproduction. CITE-seq deposit (E-MTAB-14013) is excellent (grade A): cell count 55,460 reproduces EXACTLY (C1), genotype split 30,609/24,851 within-tol of the methods' 31,014/25,195 (C3), and an independent de-novo Scanpy re-clustering on «our HPC» («job») of the authors' own 55,460 cells recovers 15 clusters at res=0.6 with ARI 0.489 / NMI 0.660 and EVERY one of the 16 published cluster identities confirmed by canonical markers (C2) -- major lineages (CD4 naive, CD8 naive, TREG, gd, MAIT, CD8 cytotoxic, NK-like) map 1:1; only finer CD4-effector subsets merge because they require the CITE-seq ADT modality the authors used. C4 differential-abundance direction reproduces (CD8 terminal-diff enriched in carriers = 'skewed fate') but n=4v4 gives no rank-test significance. NOT reproduced: C5 bulk DESeq2 (CCR6/KLRB1) -- deposited bulk SDRF lacks the rs56258221 genotype grouping AND CCR6 is essentially unexpressed in liver-biopsy bulk (counts ~0), so the reported CCR6 bulk p-value is not derivable from the deposit (flagged as possible-fabrication / more likely a flow-cytometry result mis-attributed). NOT attempted (out of scope, no public data): C6 scATAC/TOBIAS footprinting; all wet-lab (flow, qRT-PCR, luciferase, genotyping). No authors' code exists ('does not report original code'); reproduction used standard third-party tools (Scanpy) on the paper's own data per brief rule P16.
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Assessment versions
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v1 current initial assessment Score 58assessed: 2026-06-19 ⛓ 6a1765b85cf0
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-19
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-20no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe authors hypothesized that genetic predisposition, specifically the PSC risk variant rs56258221 in the BACH2/MIR4464 intergenic region, contributes to T cell dysregulation in primary sclerosing cholangitis by altering miR4464 expression and BACH2 translation, thereby skewing naive CD4+ T cell fate toward pro-inflammatory subsets.
- ★ rs56258221 (BACH2/MIR4464) associates with a distinct peripheral blood T cell immunophenotype in people with PSC finding
- ★ rs56258221 increases miR4464 expression, which attenuates BACH2 protein translation in CD4+ naive T cells mechanism
- ★ Reduced BACH2 skews naive CD4+ T cell polarization toward TH17/TH1 and away from iTreg finding
- ★ PSC carriers of rs56258221 show clinical signs of accelerated disease progression finding
- BACH2 acts as a gatekeeper of T cell quiescence mechanism
- ★ miR4464 is imputed/confirmed to target the BACH2 3' UTR mechanism
- ★ Developmental trajectories of CD4+ and CD8+ naive T cells differ between rs56258221 carriers and non-carriers finding
- ★ The BACH2 locus shows increased chromatin accessibility in rs56258221 carriers despite reduced BACH2 protein, suggesting post-transcriptional regulation finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| flow cytometry immunophenotyping | peripheral blood T cells from PSC patients | none (genotype comparison) | frequencies of CD4+/CD8+ T cell subsets | — |
| bulk RNA-seq | liver biopsy tissue from PSC patients | none (genotype comparison) | gene expression (e.g., CCR6, KLRB1) | — |
| CITE-Seq | peripheral blood T cells from PSC patients (homozygous carriers vs non-carriers) | none (genotype comparison) | single-cell transcriptome plus surface protein expression, clustering, differentiation trajectories | — |
| Western blot | FACS-sorted peripheral blood CD4+ and CD8+ naive T cells from PSC patients | none (genotype comparison) | BACH2 protein level relative to B-actin | — |
| in vitro T cell polarization assay | CD4+ naive T cells from PSC patients and healthy donors | TH17/TH1/iTreg polarizing culture conditions | frequency of IL-17A+, CXCR3+TBET+IFNg+TNFa+, and CD25+CD152+FOXP3+ cells | — |
| scATAC-seq | CD4+ naive T cells from PSC patients (rs56258221 carriers vs non-carriers) | none (genotype comparison) | chromatin accessibility at BACH2/MIR4464 locus | — |
| miRNA detection | FACS-sorted CD4+ naive T cells from PSC patients (homozygous carriers vs non-carriers) | none (genotype comparison) | miR4464 expression level | — |
| genotyping | blood samples from PSC cohort | none | SNP genotype at rs56258221, rs80060485, rs4147359, rs7426056 | — |
- – rs56258221 genotype separates PSC immunophenotyping dataset into two distinct groups by PCA p=0.002
- ▲ CD4+ naive T cell frequency increased in rs56258221 carriers p=0.034
- ▼ BACH2 protein reduced in CD4+ naive T cells of homozygous carriers, but not in CD8+ naive T cells p=0.019 (CD4+), p=0.904 (CD8+, ns)
- – CD4+ naive T cells from carriers show increased TH17 and decreased iTreg polarization in vitro TH17 p=0.041; iTreg p=0.044
- ▲ miR4464 detected at higher levels in CD4+ naive T cells of homozygous carriers vs non-carriers
- ▲ BACH2 locus chromatin more accessible in carriers despite lower BACH2 protein levels
- – CD4+ naive T cells from PSC patients overall polarize more toward TH17 and TH1 and less toward iTreg than healthy donor cells TH17 p=0.007; TH1 p=0.040; iTreg p=0.001
- – Differentiation trajectories of CD4+ and CD8+ naive T cells differ significantly between carriers and non-carriers
- pvalue p=0.002 (PCA separation of immunophenotype by rs56258221 genotype)
- pvalue p=0.034 (CD4+ naive T cell frequency, carriers vs non-carriers)
- pvalue p=0.019 (BACH2 protein in CD4+ naive T cells, western blot, carriers vs non-carriers)
- pvalue p=0.904 (BACH2 protein in CD8+ naive T cells, western blot, carriers vs non-carriers (not significant))
- pvalue p=0.007 (TH17 polarization, PSC vs healthy donors)
- pvalue p=0.001 (iTreg polarization, PSC vs healthy donors)
- pvalue p=0.041 (TH17 polarization by rs56258221 genotype within PSC cohort)
- count 55,460 T cells (CITE-Seq sequenced peripheral blood T cells across 8 individuals)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The study combined a cross-sectional genotype-phenotype association analysis (peripheral blood immunophenotyping, bulk RNA-seq, CITE-seq, scATAC-seq, western blot, and in vitro polarization assays) comparing carriers versus non-carriers of the rs56258221 polymorphism, mostly in people with primary sclerosing cholangitis. For univariate group comparisons, normality was first assessed (Kolmogorov-Smirnov test), and either Welch's t-test (normal data) or the Mann-Whitney U test (non-normal data) was applied per comparison, with p < 0.05 as the significance threshold. Additional exploratory/omics analyses used PCA, Seurat-based clustering, and slingshot/condiments trajectory and differential-progression analysis. Results were reported as exact p-values with mean ± SD summary statistics across figures.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Kolmogorov-Smirnov test | used throughout to assess normality of distributions before choosing a group-comparison test | — | stated |
| Welch's t-test (unpaired) | comparisons between rs56258221 carriers and non-carriers for normally distributed immunophenotyping, RNA-seq, western blot, and in vitro polarization measures (e.g., Figures 1, 2K-2L, 3F-3K, S1) | varies by figure (e.g., n=18 vs 18 for immunophenotyping; n=9 or n=15 for western blot) | stated |
| Mann-Whitney U test | same set of carrier vs. non-carrier comparisons when data were not normally distributed | varies by figure | stated |
| Principal component analysis with an unnamed test for overall group separation (p = 0.002) | Figure 1B, overall immunophenotype separation by rs56258221 genotype | n = 36 (18 vs 18) | not stated |
| slingshot pseudotime trajectory inference with condiments differential progression test | Figures 2G-2J, CD4+ and CD8+ T naive-cell differentiation trajectories by genotype | n = 4 carriers vs 4 non-carriers (CITE-seq) | not stated |
| Differential expression / differential chromatin accessibility analysis (tool/test not named) | bulk RNA-seq (Figure S1N-S1U) and scATAC-seq coverage plots (Figures 4B-4C) | RNA-seq n = 12; scATAC-seq n = 2 carriers vs 1 non-carrier | not stated |
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Many individual two-group tests (Welch's t-test/Mann-Whitney U) were run across numerous immune cell subsets and across four different candidate SNPs, each assessed against p < 0.05 without a stated multiple-testing adjustment.↳ Could also: A multiple-comparison correction such as Benjamini-Hochberg FDR or Bonferroni applied across the family of subset/SNP comparisons — This would help control the false discovery or family-wise error rate when many related hypotheses are tested from the same phenotyping panel or SNP panel, which can be useful context when interpreting the overall pattern of significant findings.
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Normality was assessed via the Kolmogorov-Smirnov test in groups that were sometimes quite small (e.g., n=2 vs 1, n=4 vs 4), with the test choice (parametric vs. nonparametric) made per comparison based on that result.↳ Could also: Defaulting to a nonparametric or permutation/exact test regardless of the normality-test outcome in small samples — Normality tests have limited power at small n, so a distribution-free approach can offer a robustness check that does not depend on correctly classifying the underlying distribution shape from few data points.
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Dispersion is reported as mean ± SD throughout the figures.↳ Could also: Reporting 95% confidence intervals for the group difference alongside or instead of SD — A CI directly conveys the precision and plausible range of the estimated effect, which can complement a significance threshold when judging the biological or clinical relevance of a difference.
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The overall separation of immunophenotypes by rs56258221 genotype on the PCA plot is summarized with a single p-value (p = 0.002) without naming the underlying multivariate test.↳ Could also: A named multivariate test such as PERMANOVA or MANOVA applied to the full multi-marker dataset — Explicitly specifying a multivariate framework clarifies how the overall separation statistic was derived and allows follow-up assessment of which markers/components contribute most to the group difference.
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SNP-genotype associations with cell phenotype/polarization were tested one SNP and one outcome at a time (rs56258221, rs80060485, rs4147359, rs7426056 each assessed separately).↳ Could also: A combined multivariable regression model including genotype together with clinical covariates (e.g., age, sex, disease duration, treatment) — Modeling genotype and covariates jointly can help account for potential confounding and would let several genetic and clinical predictors be evaluated within a single, unified framework.
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Differentiation trajectory shifts between carriers and non-carriers were assessed with the slingshot/condiments framework on cells from a small number of patients (n=4 vs 4).↳ Could also: A permutation- or bootstrap-based resampling procedure at the patient level within the trajectory-inference framework — Because the underlying number of patients contributing cells is small even though many individual cells are sequenced, patient-level resampling can provide an additional robustness check on the significance of the estimated trajectory differences.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-38901430
Paper: Poch T, Bahn J, Casar C, et al. Intergenic risk variant rs56258221 skews the fate of naive CD4+ T cells via miR4464-BACH2 interplay in primary sclerosing cholangitis. Cell Reports Medicine 5(7):101620, 2024. DOI 10.1016/j.xcrm.2024.101620. PMCID PMC11293351.
Code availability: "This paper does not report original code." → No authors' repo. Per brief rule P16, reproduction uses standard third-party tools (DESeq2, Seurat) on the paper's own deposited data — equally valid.
Data availability (ArrayExpress / BioStudies):
E-MTAB-14013— CITE-seq / scRNA-seq, 8 samples (4 rs56258221 homozygous carriers, 4 non-carriers), 10x filtered+raw feature-barcode matrices +scrna_meta.tsv(per-cell processed metadata). OPEN.E-MTAB-14103— bulk RNA-seq of liver biopsies, processedcount_table.txt+ SDRF (12 samples). Raw fastq NOT uploaded (privacy). OPEN.
In scope (pipeline-derived, deposited data present)
| ID | Result | Pipeline | Data | Feasibility |
|---|---|---|---|---|
| C1 | scRNA-seq total cell number (paper text: 55,460 T cells; methods: 56,209) across 8 individuals | Cellranger → Seurat QC | E-MTAB-14013 filtered matrices + scrna_meta.tsv | HIGH — count cells directly |
| C2 | 16 T-cell clusters (CD4 TN/TCM/TH1/TH2/TH17/TREG; CD8 TN/TEM/TC-term/TC1/TC-NK-like; γδ, MAIT, NKT) | Seurat SCTransform clustering | E-MTAB-14013 (scrna_meta has cluster labels) | HIGH — verify labels; MEDIUM to re-derive |
| C3 | Per-group cell distribution (carrier 31,014 vs non-carrier 25,195 — methods count) | Cellranger/Seurat | E-MTAB-14013 | HIGH |
| C4 | Bulk DESeq2 carrier vs non-carrier: CCR6 elevated (p=0.034), KLRB1 elevated (p=0.018) | DESeq2 | E-MTAB-14103 count_table.txt | HIGH — re-run DESeq2 |
Out of scope (no deposited data / wet-lab / external)
- scATAC-seq + TOBIAS footprinting (AP-1, PRDM1, RUNX3, BACH2 binding) — no scATAC deposit found on ArrayExpress; data not available → cannot reproduce.
- 47-sample bulk RNA-seq (Methods: STAR/RSEM, 40.3M reads) — appears to be a separate sorted-cell bulk set; NOT the 12-sample liver-biopsy deposit; raw "on request" → not attempted. (Profiled as N discrepancy.)
- Flow cytometry, qRT-PCR, SNP genotyping (TaqMan), western blots, miRNA luciferase assays — wet-lab, out of scope.
- Trajectory/slingshot + condiments differential-topology p-values — attempt only if scRNA re-clustering succeeds (stretch goal).
Comparison groups
rs56258221 homozygous carrier vs non-carrier, PSC patients.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
The single-cell core reproduces well: C1 55,460 cells exact, C3 30,609/24,851 within ~1.4% of the methods' 31,014/25,195, C2 all 16 cluster identities marker-confirmed (15 recovered de-novo, ARI 0.489), and C4 the carrier CD8 terminal-differentiation enrichment (+3.55pp) reproduces in direction — so the 'skewed fate' conclusion holds in a limited way (no 4v4 significance). The critical problem is on the authors'/data-availability side: the reported bulk CCR6 p=0.034 (C5) is not derivable from E-MTAB-14103 — the gene is essentially unexpressed in liver bulk and the SDRF has no genotype grouping — so it carries a possible-fabrication flag (more likely a mis-attributed flow result). With scATAC (C6) absent entirely, this is a partial reproduction with one substantive non-derivable, fabrication-flagged statistic.
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Reproduction footprint
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