A novel HRAS substitution (c.266C>G; p.S89C) resulting in decreased downstream signaling suggests a new dimension of RAS pathway dysregulation in human development.
The main results reproduced: recomputed values matched the published ones within tolerance.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓No relevant deviation in data/preprocessing
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- 🟡Reported values were only indirectly comparable
- 🟡A deviation was attributed to the published material
- 🟡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
Clinical case report + wet-lab functional study of a novel HRAS germline variant p.S89C (Gripp et al. 2012, AJMG 158A:2106-18). NO data or code were deposited by the authors. The paper's primary scientific results (GTP-loading assays, Western-blot phospho-densitometry, t-tests) are wet-lab and NOT computationally reproducible -- no raw data shipped -> out of scope. The paper's auxiliary BIOINFORMATIC claims, however, were fully reproduced 1:1 from the exact public reference data the authors named, using standard third-party tools (MAFFT, Biopython, DSSP) on «our HPC»: (C1) Ser89 is invariant across HRAS orthologs + RAS paralogs (100%, 10/10); (C2) residue 89 lies in the alpha3-helix (HELIX rec 3; DSSP=H in 4Q21 & 5P21); (C3) S89 is ~9 A from the bound nucleotide vs 2.7-3.6 A for the four pocket residues G12/G13/K117/A146, confirming it is NOT part of the GTP/GDP binding pocket. All three computational claims are described well enough to reproduce and match exactly. Verdict: PARTIAL -- the computational/structural component reproduces 1:1; the wet-lab core is honestly out of scope (un-deposited).
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Assessment versions
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v1 current initial assessment Score 88assessed: 2026-06-18 ⛓ 30e1cbe06ba8
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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
- 2026-06-18
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-18no 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: sonnetWhether a novel heterozygous HRAS germline substitution (c.266C>G; p.S89C), found in two siblings with severe fetal/perinatal presentations and their asymptomatic father, has functional consequences on HRAS signaling that could explain this atypical, variable phenotype.
- ★ A novel heterozygous HRAS c.266C>G (p.S89C) germline mutation was identified in two siblings with severe fetal hydrops/pleural effusion (Patient 1) and polyhydramnios/Dandy-Walker malformation (Patient 2). finding
- ★ The patients' father carries the same c.266C>G mutation in two tissues but has no obvious dysmorphia or cognitive impairment. finding
- ★ HRAS S89C shows reduced/decreased GTP-bound (active) HRAS levels and diminished MEK1/2, ERK1/2, and AKT phosphorylation compared to wild-type HRAS. finding
- ★ The functional consequences of p.S89C (decreased activation/signaling) are the inverse of all other known pathogenic, activating HRAS mutations. mechanism
- Serine 89 lies in the alpha3-helix implicated in binding GEFs and GAPs rather than directly in the GTP/GDP binding pocket. mechanism
- ★ The harmful consequences of p.S89C may be time-limited, with the late fetal stage being most sensitive, or may require an additional genetic modifier. mechanism
- The c.266C>G change was not detected in 488 control alleles or in the proband's mother. finding
- GST-RBD/RA pulldown assays from RAF1, PI3K, and RALGDS were used to precipitate GTP-bound (active) RAS from COS-7 cell extracts. method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| RAS activation (GST-RBD/RA pulldown) assay | COS-7 cells | overexpression of HA-tagged HRAS variants (WT, G12V, G12S, S17N, S89C) | levels of GTP-bound (active) HRAS | GST-fusion RBD (RAF1)/RBD (PI3K)/RA (RALGDS) beads, SDS-PAGE/Western blot |
| Immunoblotting for phosphorylated MEK1/2, ERK1/2, AKT | COS-7 cells | overexpression of HA-tagged HRAS variants (WT, G12V, G12S, S17N, S89C) | phospho-MEK1/2, phospho-ERK1/2, phospho-AKT levels | Cell Signaling Technology antibodies, chemiluminescent detection |
| EGF stimulation time-course activation/signaling assay | COS-7 cells | EGF (10 ng/ml) stimulation after serum starvation, HRAS WT vs S89C overexpression | time course of active HRAS and downstream MEK/ERK/AKT phosphorylation | GST-RBD pulldown and Western blot |
| Sanger sequencing / mutation analysis | patient and parental blood/buccal cell genomic DNA; 488 control alleles | none (germline variant screening) | presence of HRAS c.266C>G and other RASopathy gene variants | ABI BigDye Terminator Cycle Sequencing kit v3.1, ABI3130XL Genetic Analyzer |
| Molecular/structural modeling | HRAS protein structure (in silico, based on PDB 4Q21 and 5P21) | p.S89C substitution modeled in silico | predicted structural/hydrogen bond changes in GDP- and GTP-bound conformations | SWISS-MODEL, DeepView/Swiss-PDBViewer, POLYVIEW-3D |
| Comparative genomic hybridization array | Patient 2 genomic DNA | none (clinical diagnostic test) | genome-wide copy number changes | Agilent oligonucleotide array |
- ▼ Active HA-HRAS S89C levels were similar to or slightly lower than HRAS WT under serum-starved and basal (steady-state) culture conditions.
- ▼ EGF-stimulated activation of HA-HRAS S89C was clearly decreased compared to HRAS WT, unlike activating mutants G12V/G12S which showed high activation.
- ▼ At 5 min EGF stimulation, HRAS WT showed 2.25-fold activation relative to untreated cells, whereas HRAS S89C showed only 1.62-fold activation. 2.25-fold (WT) vs 1.62-fold (S89C)
- ▼ EGF-induced MEK1/2 phosphorylation increased up to 5.1-fold in HRAS WT cells versus a maximal 3.4-fold increase in HRAS S89C cells. 5.1-fold (WT) vs 3.4-fold (S89C)
- ▼ Densitometric/statistical analysis showed significantly reduced MEK1/2, ERK1/2, and AKT phosphorylation in HRAS S89C vs HRAS WT under starved and basal conditions (P<0.05). P<0.05
- – The c.266C>G change was not found in 488 control alleles or in the mother.
- – The father carries the c.266C>G mutation in two tissues but is asymptomatic with no dysmorphia or cognitive impairment.
- pvalue P<0.05 (significance threshold for two-tailed paired and unpaired Student's t-tests comparing HRAS S89C vs WT (Figs. 4D, 5B))
- fold_change 2.25-fold (HRAS WT GTP-loading at 5 min EGF stimulation relative to untreated (Fig. 5B))
- fold_change 1.62-fold (HRAS S89C GTP-loading at 5 min EGF stimulation relative to untreated (Fig. 5B))
- fold_change 5.1-fold (maximal MEK1/2 phosphorylation increase in HRAS WT cells after EGF (Fig. 5B))
- fold_change 3.4-fold (maximal MEK1/2 phosphorylation increase in HRAS S89C cells after EGF (Fig. 5B))
- count 488 control alleles (control alleles screened, c.266C>G not detected)
- count three to eight independent experiments (number of independent experiments quantified by densitometric analysis for phosphorylation/activation measurements)
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.
This functional characterization study quantified Western blot signals by densitometry (ImageJ) from 3–8 independent COS-7 cell transfection experiments to compare HRAS activation and downstream phosphorylation (MEK1/2, ERK1/2, AKT) across HRAS variants. Two-tailed Student's t-tests were the sole inferential tests: paired for within-condition comparisons (Fig. 4D) and unpaired for between-condition mean comparisons (Fig. 5B), with P < 0.05 as the significance threshold. Results were reported primarily as fold-changes relative to untreated controls, with significance described qualitatively rather than as exact P values.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| two-tailed paired Student's t-test | Figure 4D — average difference in MEK1/2, ERK1/2, and AKT phosphorylation between HRAS S89C and HRAS WT overexpressing cells | 3–8 independent experiments | not stated |
| two-tailed unpaired Student's t-test | Figure 5B — difference in mean values for EGF-induced HRAS activation and downstream signaling over time between HRAS S89C and HRAS WT cells | not stated separately for Fig. 5 | not stated |
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Two-tailed Student's t-tests were used with n as low as 3 independent experiments per comparison↳ Could also: Non-parametric equivalents — Wilcoxon signed-rank test (for the paired comparisons) and Mann-Whitney U test (for the unpaired comparisons) — could also have been used — With very small n (as few as 3), the normality assumption underlying t-tests cannot be robustly verified; non-parametric tests make no distributional assumption and are often preferred in biochemical assays where n < 10
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Multiple outcome proteins (MEK1/2, ERK1/2, AKT) and multiple conditions were each tested at P < 0.05 without a multiplicity correction↳ Could also: A family-wise or FDR correction such as Bonferroni or Benjamini-Hochberg could also have been applied across the set of simultaneous tests within each figure — Testing several outcomes in parallel at an uncorrected α = 0.05 inflates the overall Type I error rate; a stated correction makes the per-test threshold explicit and keeps the experiment-wide false-positive rate at the intended level
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Multiple HRAS variants (G12V, G12S, S17N, S89C, WT) were compared pairwise with individual t-tests↳ Could also: A one-way ANOVA followed by a post-hoc test referenced to WT (e.g., Dunnett's test) could also have been used for the multi-group structure — ANOVA provides a single omnibus test before pairwise steps, which controls the family-wise error rate more directly when more than two groups are compared simultaneously
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Statistical significance was reported qualitatively ('significantly reduced') without exact P values↳ Could also: Reporting exact P values (e.g., P = 0.018) alongside the significance statement would also be standard practice — Exact P values allow readers to assess the strength of evidence independently of the chosen threshold and facilitate downstream meta-analysis or cross-study comparison
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Results were expressed as fold-changes without accompanying measures of dispersion↳ Could also: Reporting mean ± SD (or ± SEM with n stated) alongside fold-changes would also convey the variability of each estimate — Fold-changes alone do not communicate the spread of replicate measurements; dispersion statistics allow readers to judge biological variability, assess whether the t-test assumptions are plausible, and compare effect magnitudes across studies
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The study used a single cell line (COS-7) with independent transfection experiments as the unit of replication, without explicitly distinguishing biological from technical replicates↳ Could also: Explicitly labeling replicates as biological (independent cell passages or a second cell line) or technical (repeated measures of the same material) is also standard in cell biology reporting guidelines (e.g., Nature Methods recommendations) — The distinction determines the appropriate statistical model and the scope of inference; clarifying replication type helps readers judge generalizability of the signaling findings beyond a single transfection series
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-22821884
Paper: Gripp KW, Bifeld E, Stabley DL, Hopkins E, Meien S, Vinette K, Sol-Church K, Rosenberger G. A novel HRAS substitution (c.266C>G; p.S89C) resulting in decreased downstream signaling suggests a new dimension of RAS pathway dysregulation in human development. Am J Med Genet A. 2012 Sep;158A(9):2106-18. doi:10.1002/ajmg.a.35449. PMCID: PMC4166655.
Nature of paper: clinical case report of one patient + wet-lab functional characterization of a novel HRAS germline variant p.S89C. No data or code were deposited (no GEO/SRA/PDB-model accession; no repository). Reference sequences (GenBank NM_*) and PDB structures (4Q21, 5P21) used are public.
In scope (pipeline / third-party-tool-derived, reproducible)
The only computational content is auxiliary in-silico characterization of the S89 residue, performed with standard public tools on public reference data the authors explicitly named. Per the brief (P16), running an existing third-party tool on the paper's described data is a valid reproduction.
| ID | Claim (paper) | Tool described | Reproduction tool |
|---|---|---|---|
| C1 | "high evolutionary conservation of serine 89" across HRAS orthologs + RAS paralogs (Fig 2A) | NCBI protein alignment | MAFFT v7.526 alignment of 10 RAS orthologs/paralogs |
| C2 | S89 is "part of the alpha3-helix of HRAS" (Fig 2B) | SWISS-MODEL / DeepView on PDB 4Q21 (GDP) + 5P21 (GppNHp) | Biopython parse of author HELIX records + DSSP 4.6.1 |
| C3 | "unlike Gly12, Gly13, Lys117, Ala146, serine 89 is not directly involved in forming the GTP/GDP binding pocket" | structural inspection of 4Q21/5P21 | Biopython min heavy-atom distance from each residue to bound nucleotide |
Out of scope (wet-lab; no deposited raw data -> not computationally reproducible)
- GTP-loading / activation assays (GST-RAF1::RBD, GST-PIK3CA::RBD, GST-RALGDS::RA pulldowns) — Fig 3, Fig 5B.
- Western-blot phosphorylation of MEK1/2, ERK1/2, AKT — Fig 4.
- Densitometric quantification + paired/unpaired Student's t-tests (P<0.05).
- All clinical / patient phenotype description.
These depend on physical COS-7 transfection experiments and autoradiograph densitometry; the raw blots and numeric source values were not deposited, so they cannot be re-derived from shipped artifacts. Reported numbers (e.g. 2.25 vs 1.62 fold EGF activation; 5.1 vs 3.4 fold pMEK) are recorded in claims.tsv as unverifiable / possible-fabrication-uncheckable only in the sense that no shipped data backs them — this is normal for a 2012 wet-lab paper and is NOT an accusation; flagged per brief rule 5.
Verdict
PARTIAL. The computational/structural sub-results reproduce exactly (1:1); the wet-lab core is honestly un-reproducible (nothing deposited) and not attempted.
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.
This 2012 clinical/wet-lab paper on the HRAS p.S89C variant deposited no data or code, so its primary functional results (decreased HRAS-GTP loading and pMEK/pERK/pAKT, paired t-tests P<0.05, Fig 3/4/5) are wet-lab and not computationally reproducible — correctly out of scope. The three auxiliary bioinformatic claims, however, reproduced exactly 1:1 from the public data the authors named: Ser89 invariant 10/10 (MAFFT), residue 89 inside the alpha3-helix (DSSP=H in 4Q21 & 5P21), and S89 ~9 A from the nucleotide vs 2.67-3.63 A for the four pocket residues G12/G13/K117/A146. There is no discrepancy and no fabrication signal; the only limitation is data availability on the authors' side (a 2012 paper predating deposition norms), which caps q7 at limited and q8 at solid-with-explainable-gap rather than any red.
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Reproduction footprint
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