A novel HRAS c.466C>T p.(Phe156Leu) variant in two patients with attenuated features of Costello syndrome.
The main result did not reproduce in this reproduction attempt. Where our recomputation produced values that differ from the published ones, those discrepancies are listed below. This is a single automated attempt — not peer review and not a finding of error or misconduct — and differences can also arise from data access, undocumented parameters or the computing environment. The verdict can be contested via “report an error”.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Any deviation was negligible
- 🔴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 central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
▸Reproduction agent’s raw note
DROP = non_pipeline. This is a clinical case report (European Journal of Human Genetics 2022; PMC9437031) of two unrelated boys carrying a novel germline HRAS c.466C>T p.(Phe156Leu) variant with attenuated Costello syndrome. Its substantive results are wet-lab functional assays (GST-effector co-immunoprecipitation; immunoblot EGF time-courses in HEK293T/MCF-7; Fig 2C n=3, two-tailed t-test P<0.05) plus clinical phenotyping - all out of scope (no bioinformatic pipeline). The variant was identified by trio exome sequencing, but no exome data is deposited (no SRA/ENA/EGA/dbGaP accession in the paper, PMC full text, or data-availability statement) and no pipeline is described (no aligner/caller/reference/parameters), so the variant call cannot be reproduced. This run independently RE-VERIFIED both pillars of the drop: (1) the PMC9437031 full text reports NO in-silico prediction scores (CADD/SIFT/PolyPhen/REVEL/MutationTaster/GERP), no structure modeling/MD, no named bioinformatics software - the data-availability statement names ONLY ClinVar; (2) the single ClinVar deposit VCV001327492 (Variation ID 1327492) resolves via NCBI E-utilities esummary as gene=HRAS, 'NM_005343.4(HRAS):c.466T>C (p.Phe156Leu)', Pathogenic - ClinVar's minus-strand c.466T>C equals the paper's sense-strand c.466C>T (same variant, expected notation difference). There is therefore no pipeline-derived computational result with obtainable data to reproduce; no «our HPC» compute was spent, by design. NOT attempted: rerunning the wet-lab assays (impossible / out of scope) and re-calling the variant (raw exome data absent, pipeline unspecified). This is a well-founded, healthy drop. Verdict is provisional and human-checkable.
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
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v1 current initial assessmentassessed: 2026-06-19 ⛓ dfba9d64e672
✎ I am an author of this paper
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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
- 2026-06-22
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-19no 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 paper investigates the clinical manifestations and functional/molecular consequences of the novel de novo HRAS c.466C>T p.(Phe156Leu) variant, testing whether it is pathogenic and causes an attenuated form of Costello syndrome distinct from the classic p.Gly12Ser phenotype.
- ★ HRAS c.466C>T p.(Phe156Leu) is a novel de novo pathogenic variant causing an attenuated Costello syndrome phenotype in two unrelated boys finding
- ★ HRAS Phe156Leu shows enhanced/stable binding to effectors RAF1, RALGDS, PIK3CA and PLCE1 compared to wild-type finding
- ★ HRAS Phe156Leu accumulates in the active (GTP-bound) form due to accelerated GDP/GTP nucleotide exchange rather than impaired GTPase activity as in p.Gly12Ser mechanism
- ★ HRAS Phe156Leu enhances downstream MEK1/2 and ERK1/2 phosphorylation and increases AKT phosphorylation in MCF-7 cells compared to wild-type finding
- ★ HRAS Phe156Leu impairs EGF growth-factor-induced signaling dynamics, showing elevated basal ERK1/2 phosphorylation with little further increase upon stimulation, unlike wild-type finding
- ★ Impaired/dysregulated signaling dynamics, rather than simple static hyperactivation, is the central pathomechanism underlying Costello syndrome and related RASopathies mechanism
- Phe156 lies in the α5-helix critical for HRAS structural stability, and its substitution to leucine weakens contacts with Mg2+ and guanine nucleotides mechanism
- ★ Clinical variability exists between the two patients with the identical HRAS Phe156Leu variant, particularly in musculoskeletal, cardiovascular and endocrinologic manifestations finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Trio exome sequencing | Patient blood/DNA (two boys with Costello syndrome features) | none | identification of de novo HRAS c.466C>T p.(Phe156Leu) variant (and incidental SCN1A variant in patient 1) | — |
| GST pull-down / effector-binding assay with immunoblotting | HEK293T cells | overexpression of HA-HRAS variants (WT, Gly12Val, Gly12Ser, Ser17Asn, Phe156Leu) | co-precipitation of HA-HRAS with GST-fused RAF1, RALGDS, PIK3CA, PLCE1 effector peptides under serum-starved, normal growth, or EGF-stimulated conditions | — |
| GAP-binding assay | HEK293T cells (HA-HRAS variants) | overexpression of HA-HRAS variants | binding efficiency to NF1 GTPase-activating protein | — |
| Western blot / immunoblotting for phosphorylated MEK1/2, ERK1/2, AKT | HEK293T and MCF-7 cells | overexpression of HA-HRAS variants (WT, Gly12Val, Gly12Ser, Ser17Asn, Phe156Leu) under serum-starved, basal, and stimulated conditions | downstream signaling pathway activation (phospho-MEK1/2, phospho-ERK1/2, phospho-AKT levels) | — |
| Time-course EGF stimulation with immunoblotting | HEK293T cells transiently expressing HRAS WT, Gly12Val, or Phe156Leu | EGF stimulation for 0, 5, 15, 30 min | phosphorylation levels of ERK1/2 and AKT relative to maximum, assessing signaling dynamics over time | — |
| Clinical/phenotypic examination and imaging (abdominal ultrasound, echocardiography, endoscopy) | Two human patients (boys) with HRAS Phe156Leu variant | none (natural disease course) | craniofacial, musculoskeletal, cardiovascular, dermatologic, neurologic, gastroenterologic/endocrinologic manifestations | — |
- ▲ HA-HRAS Phe156Leu amount elevated compared to HA-HRAS WT in effector co-precipitates (RAF1, RALGDS, PIK3CA, PLCE1) under all tested conditions
- ▲ HA-HRAS Phe156Leu shows moderately enhanced NF1 GAP binding compared to WT, but less pronounced than Gly12Ser/Gly12Val
- ▲ HA-HRAS Phe156Leu enhances MEK1/2 and ERK1/2 phosphorylation compared to WT in HEK293T cells
- ▲ HA-HRAS Phe156Leu induces stronger AKT phosphorylation than WT in MCF-7 cells
- – Cells expressing HA-HRAS Phe156Leu show enhanced basal (0 min) ERK1/2 phosphorylation with little further increase after EGF stimulation, unlike WT which peaks at 5 min then decreases
- – AKT phosphorylation is slightly stimulated by EGF in WT cells but marginally/not in Gly12Val and Phe156Leu expressing cells
- – Both patients had severe feeding difficulties, airway obstruction and developmental delay typical of CS, but differed significantly in musculoskeletal, cardiovascular and endocrinologic manifestations
- – Patient 2 required subtotal pancreatectomy at 8 months old for severe hyperinsulinism-associated hypoglycemia unresponsive to octreotide
- pvalue P < 0.05 (two-tailed t test) (significance of differences in untreated (0 min EGF) phosphorylation levels between HRAS variants across three experiments)
- count ~80% (proportion of CS-causing HRAS gene variants that are p.Gly12Ser)
- count N=30 (number of previously reported patients with HRAS p.Gly12Ser compiled for comparison table)
- count N=4 (number of previously reported patients with HRAS p.Ala146Pro/Val/Thr compiled for comparison table)
- count N=3 (number of previously reported patients with HRAS p.Thr58Ile compiled for comparison table)
- mean Birth weight >97th centile, length 75th centile, head circumference >98th centile (Patient 1 birth anthropometrics)
- mean At 2 2/3 years: weight and length <3rd centile, head circumference >95th centile (Patient 1 growth at follow-up)
- other HCM in 13/30 (43%), PS in 15-20%, ASD in 5-7% (cardiovascular abnormality frequencies in HRAS p.Gly12Ser cohort from Table 1)
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 is a clinical case report of two patients with a novel HRAS variant, combined with in vitro functional/biochemical experiments (GST-pulldown co-precipitation and immunoblotting in HEK293T and MCF-7 cells) comparing HRAS variants across signaling readouts. Clinical and phenotypic findings are presented descriptively in a comparison table against previously published cohorts, without inferential statistics. For one quantitative functional readout (EGF-induced phosphorylation kinetics), values are given as the mean of three experiments, with a single two-tailed t-test used to flag significance at untreated (0 min) time points.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| two-tailed Student's t-test | comparison of ERK1/2 (and AKT) phosphorylation levels between HRAS variants at the untreated (0 min) time point in the EGF stimulation time-course, Fig. 2C/S3 | mean of three experiments | not stated |
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A single two-tailed t-test was used to flag significance at individual (e.g., untreated) time points across several HRAS variants and conditions.↳ Could also: A two-way ANOVA (variant × time/condition) with post-hoc comparisons — Because the data involve two crossed factors (variant and time/condition), an ANOVA-based approach would model both factors jointly and provide a structured way to compare multiple groups across the full time course rather than at isolated points.
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Multiple comparisons are made across several HRAS variants, cell lines, and time points, with one t-test explicitly reported.↳ Could also: A multiplicity correction such as Bonferroni or Benjamini-Hochberg FDR — Applying a correction across the family of comparisons would help control the overall false-positive rate when many pairwise tests are considered together.
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Quantitative signaling data are summarized as a mean across three experiments without a stated measure of spread.↳ Could also: Reporting SD, SEM, or a 95% confidence interval alongside the mean — A stated dispersion measure would let readers gauge the variability of the underlying replicates, which is especially informative with a small number of experiments.
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Significance is reported only as a threshold (P < 0.05) for the t-test result.↳ Could also: Reporting the exact p-value — Exact p-values convey the precise strength of evidence and allow readers to make their own judgments about significance thresholds or apply their own corrections.
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With n=3 experiments, a parametric t-test was applied.↳ Could also: A non-parametric test such as the Mann-Whitney U test — For very small sample sizes where normality is difficult to verify, a non-parametric alternative can be used as it does not rely on distributional assumptions.
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The two patients' phenotypes were compared to prior published cohorts using a descriptive comparison table rather than formal statistical testing.↳ Could also: Formal genotype-phenotype statistical comparison (e.g., proportion tests) if pooled cohort data from multiple centers were available — With larger aggregated cohorts, statistical comparison of feature frequencies between variant groups could complement the descriptive summary, though for an n=2 case report a descriptive approach is the standard and expected presentation.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope analysis — pmid-35764878
Title: A novel HRAS c.466C>T p.(Phe156Leu) variant in two patients with attenuated features of Costello syndrome. Journal: European Journal of Human Genetics (2022) · DOI 10.1038/s41431-022-01139-1 · PMCID PMC9437031 Authors: Lindsey-Temple S, Edwards M, Rickassel V, Nauth T, Rosenberger G.
What kind of paper is this?
A clinical case report of two unrelated boys carrying a previously unreported germline HRAS missense variant (NM_005343.4 c.466C>T, p.(Phe156Leu)) presenting with attenuated Costello-syndrome features. The reported results are:
- Clinical phenotyping of the two patients (feeding difficulties, airway obstruction, developmental delay, musculoskeletal/cardiovascular/endocrine findings). — manual / clinical, OUT OF SCOPE.
- Trio exome sequencing that identified the variant. — sequencing, but see below.
- Wet-lab functional assays: GST-effector co-immunoprecipitation (RAF1, RALGDS, PLCE1, PIK3CA), immunoblotting under serum-starved/basal/EGF-stimulated conditions in HEK293T and MCF-7 cells; EGF time-course (0/5/15/30 min); quantification = "mean of three experiments, two-tailed t-test, P<0.05" (Fig 2C). — wet-lab molecular biology, OUT OF SCOPE.
- Structural interpretation of Phe156 (α5-helix, hydrophobic core) — cited from Quilliam et al. 1995 [ref 8], not computed in this paper. — OUT OF SCOPE.
- Database deposit: ClinVar VCV001327492 (Variation ID 1327492). — a single variant record, not a reproducible dataset.
In-scope (pipeline-derived computational) results
NONE. Searched the full text (PMC9437031), abstract, methods, and figure legends:
- No in-silico variant-prediction scores reported (no CADD / SIFT / PolyPhen-2 / REVEL / MutationTaster / GERP values) → nothing to reproduce or compare against.
- No exome bioinformatic pipeline described (no aligner, variant caller, reference build, or parameters given); raw exome data is patient/clinical data and is not deposited anywhere (no SRA/EGA/dbGaP accession) → not reproducible.
- No protein structure modeling / MD simulation performed in this paper (the structural claims are citations to prior literature).
- No proteomics/transcriptomics deposit (no PRIDE/GEO/SRA accession).
- Functional-assay quantification is wet-lab band densitometry from non-deposited blot images → not a bioinformatic pipeline and not reproducible from shipped data.
Verdict
DROP — non_pipeline. This publication contains no pipeline-derived
computational result with an identifiable reported value backed by obtainable data.
Its findings are clinical + wet-lab functional + a single ClinVar variant deposit.
A genuine, well-founded drop (correctly determined outcome), not an our-side failure.
No individual results have been recorded for this entry yet.
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 is a correctly-identified non_pipeline drop: an EJHG 2022 clinical case report (HRAS c.466C>T p.(Phe156Leu), attenuated Costello syndrome) whose results are wet-lab functional assays (GST co-IP; immunoblot EGF time-courses, Fig 2C n=3, two-tailed t-test P<0.05) and clinical phenotyping — no bioinformatic pipeline, no in-silico scores, and no deposited input data (only ClinVar VCV001327492). The reproducibility gap sits entirely on the data-availability/authors' side (trio WES not deposited, no pipeline params, raw blots absent), not on our methodology. Because nothing computational was reproduced there is no measured deviation (severity negligible), but the reported values are not derivable from the shared data, and the core claims can only be regarded as unconfirmed-but-not-refuted. No fabrication concern; the drop is healthy and human-checkable.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
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