An attenuated phenotype of Costello syndrome in three unrelated individuals with a HRAS c.179G>A (p.Gly60Asp) mutation correlates with uncommon functional consequences.
Part of the results reproduced; minor but material deviations remained.
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). PMID 25914166 (Gripp et al., Am J Med Genet A 2015, DOI 10.1002/ajmg.a.37128, PMC4830354) is a clinical case series of 3 unrelated individuals with a novel HRAS p.Gly60Asp Costello-syndrome variant, combined with wet-lab functional characterization (RAS effector pull-down, co-immunoprecipitation of RAF1/PIK3CA/PLCE1/RALGDS/NF1-GAP, and MEK/ERK/AKT phospho-immunoblots in COS-7 cells, quantified by gel densitometry). The paper is described well enough to understand fully, but there is NOTHING computationally reproducible: no bioinformatic pipeline, no code repository (own or third-party), and no public data accession (no GEO/SRA/ENA/dbGaP/PRIDE/figshare/zenodo; no Data Availability statement, confirmed in PMC full text). The HRAS variant was found by clinical RASopathy gene-panel testing of private patient samples. All 6 recorded claims (C1-C6) are wet-lab/clinical and recorded as uncheckable/not-attempted. No «our HPC» compute was warranted or submitted. This is an honest drop, not a failed reproduction.
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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: 2026-06-18 ⛓ 445978528766
✎ 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
- 2026-06-18
- 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: sonnetThe paper tests whether the novel HRAS c.179G>A (p.Gly60Asp) mutation causes Costello syndrome via the canonical gain-of-function/hyperactivation mechanism of RAS-MAPK signaling, or instead through an alternative mechanism such as disrupted HRAS reactivity/dominant-negative effects on effector binding.
- ★ HRAS c.179G>A (p.Gly60Asp) causes an attenuated Costello syndrome phenotype without severe failure-to-thrive, intellectual disability, or cancer predisposition finding
- ★ HRAS Gly60Asp shows strongly increased binding to effector RAF1 but not to other tested effectors (PI3K, RALGDS, PLCE1) finding
- ★ Hyperactivation of MAPK downstream signaling pathways is absent despite increased RAF1 binding finding
- ★ Hyperactivation of RAS downstream signaling does not entirely explain the molecular basis of Costello syndrome; disrupted HRAS reactivity is a critical molecular dysfunction mechanism
- ★ The HRAS p.Gly60Asp mutation was identified in three unrelated individuals, occurring de novo (paternal origin) in two and maternally transmitted in the third finding
- Glycine 60 is a highly conserved residue located in the HRAS switch II domain (amino acids 59-67) that mediates binding to regulator and effector proteins mechanism
- Expression constructs for wild-type and mutant HRAS (Gly12Val, Ser17Asn, Gly60Asp) were generated for functional characterization method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| RAS pull-down assay (GST-RBD/RA precipitation) | COS-7 cells transfected with HA-tagged HRAS constructs (WT, Gly12Val, Ser17Asn, Gly60Asp) | overexpression of mutant HRAS; serum starvation, normal growth, or EGF stimulation | GTP-bound (active) HRAS binding to RBD/RA domains of RAF1, PI3K (PIK3CA), RALGDS, PLCE1 | GST-fusion RBD/RA beads, SDS-PAGE, immunoblot (anti-HA) |
| Co-immunoprecipitation | transiently transfected COS-7 cells | overexpression of mutant HRAS | protein-protein interaction of HRAS with binding partners | EZview Red Anti-HA Affinity Gel, SDS-PAGE |
| Immunoblotting (phospho-signaling) | COS-7 and HEK293 cells | HRAS mutant overexpression | levels of MEK1/2, phospho-MEK1/2, ERK1/2, phospho-ERK1/2, Akt, phospho-Akt | PVDF membrane immunoblot with Cell Signaling Technology antibodies |
| Clinical mutation panel testing (Sanger sequencing/panel) | human patients, genomic DNA from buccal cells | none (germline variant detection) | presence of pathogenic variants in rasopathy genes (HRAS, BRAF, CBL, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, SHOC2, SOS1, RAF1) | — |
| Allele specific amplification (ASA) / microsatellite genotyping | patient and parental buccal-cell DNA | none | parental origin of the HRAS mutation using informative SNPs (rs12628, rs41258054) | — |
| Echocardiography | Individuals 1, 2, and 3 (human patients) | none | cardiac structure and function (valve morphology, ventricular wall thickness, arrhythmia) | — |
| Brain MRI / cranial CT | Individual 1 (MRI) and Individual 3 (CT) | none | cerebellar tonsillar ectopia, arachnoid cyst, foramen magnum crowding | MRI; CT |
| Dual energy x-ray absorptiometry (DEXA) | Individual 1 (human patient) | none | bone mineral density | DEXA scan |
- ▲ HA-HRAS Gly60Asp precipitated by RAF1[RBD] was clearly elevated compared to active HA-HRAS WT across serum starvation, normal growth, and EGF-stimulated conditions
- ▼ HA-HRAS Gly60Asp binding to RAF1[RBD] was decreased relative to constitutively active HA-HRAS Gly12Val
- – Constitutively active HA-HRAS Gly12Val was efficiently pulled down by RAF1[RBD] under all conditions, while dominant-negative HA-HRAS Ser17Asn was not pulled down
- – None of the three probands had severe failure-to-thrive, intellectual disability, or cancer, consistent with an attenuated phenotype
- – The HRAS c.179G>A mutation arose de novo with paternal origin in Individuals 1 and 2, and was maternally transmitted (from an affected mother) in Individual 3
- – Disease-associated mutations at the homologous glycine 60 position in KRAS (p.Gly60Arg) and NRAS (p.Gly60Glu) have been shown to exert strong impact on RAS function
- other >80% (proportion of Costello syndrome patients sharing the common HRAS c.34G>A (p.Gly12Ser) mutation)
- count 3 (unrelated individuals identified with the novel HRAS c.179G>A (p.Gly60Asp) mutation)
- other up to 257 beats/minute, maximum run length about 900 beats (Holter-monitored SVT episodes in Individual 1)
- other 1:5000 dilution (anti-HA), 1:1000 dilution (phospho-antibodies) (antibody dilutions used in immunoblotting protocol)
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 paper presents a descriptive case series of three unrelated individuals (plus a transmitting mother) carrying a novel HRAS c.179G>A (p.Gly60Asp) germline mutation, with clinical features compared narratively to a reference cohort of HRAS mutation-positive Costello syndrome patients enrolled in an ongoing IRB-approved study. Functional characterization employed cell-based biochemical assays (GST-RBD pull-down, co-immunoprecipitation, immunoblotting for MEK, ERK, and Akt phosphorylation) to assess effector binding and downstream MAPK/PI3K signaling across multiple HRAS constructs and cell-culture conditions. Results from biochemical experiments are presented qualitatively by visual description of Western blot band intensities, with no formal statistical inference tests described in the available text.
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Western blot band intensities for effector binding and downstream phosphorylation were compared qualitatively by visual inspection across HRAS variant constructs and culture conditions↳ Could also: Densitometric quantification of band intensities using software such as ImageJ/Fiji, normalised to a loading control (e.g., GAPDH), expressed as ratios with mean and SD across independently repeated experiments — Quantitative densitometry with a stated number of independent replicates and a measure of variability would allow readers to evaluate the consistency and magnitude of observed binding differences across experiments
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The number of independent biological (transfection) replicates for each cell-based assay is not stated in the available text↳ Could also: Pre-specifying at least three independent transfection replicates per condition, then applying a one-way ANOVA or Kruskal-Wallis test with post-hoc pairwise comparisons (e.g., Dunn's or Tukey HSD) on the quantified ratios — Stating replicate numbers and applying an inferential test separates systematic biological effects from run-to-run variability and makes the evidence for each comparison independently assessable
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Clinical features of the three p.Gly60Asp carriers were compared to the reference Costello syndrome cohort by narrative description only↳ Could also: Fisher's exact test for categorical phenotypic features (e.g., presence of failure-to-thrive, intellectual disability, cardiac arrhythmia) between the novel variant carriers and the reference cohort, with prevalences and 95% confidence intervals — Even with small case counts, exact tests and confidence intervals would quantify the degree of phenotypic attenuation relative to the typical Costello syndrome spectrum and convey uncertainty around prevalence estimates
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Anthropometric data for cases are reported as centile values from external growth references without a common continuous scale or summary across cases↳ Could also: Converting measurements to z-scores (SDS) from the same external references and reporting the distribution (median and range or IQR) alongside the reference Costello syndrome cohort — Z-scores place measurements on a continuous scale that is age-independent and directly comparable across individuals and cohorts, and support straightforward group-level summaries
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Parental origin of the de novo mutation was determined using a single informative SNP per family by allele-specific amplification↳ Could also: Haplotype phasing using multiple flanking microsatellite or SNP markers surrounding the mutation locus — Multiple flanking markers provide independent lines of evidence for the inferred parental chromosome of origin, reducing the possibility of a spurious result from a single locus
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The reference Costello syndrome cohort used for clinical comparison is described qualitatively without reporting its size, demographic composition, or how features were ascertained↳ Could also: Reporting cohort size, ascertainment method, and key demographic features in a summary table alongside the novel variant cases, with frequencies and denominators for each compared phenotypic feature — Transparent cohort description allows readers to evaluate comparability and generalisability, and is standard practice for case-series comparisons against historical or registry controls
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-25914166
Title: An attenuated phenotype of Costello syndrome in three unrelated individuals with a HRAS c.179G>A (p.Gly60Asp) mutation correlates with uncommon functional consequences. Authors: Gripp KW, Sol-Church K, Smpokou P, Graham GE, Stevenson DA, Hanson H, Viskochil DH, Baker LC, Russo B, Gardner N, Stabley DL, Kolbe V, Rosenberger G. Journal: American Journal of Medical Genetics Part A, 2015. DOI: 10.1002/ajmg.a.37128 · PMCID: PMC4830354 · PMID: 25914166
What kind of paper is this?
A clinical case series (3 unrelated patients) plus wet-lab functional characterization of a novel HRAS p.Gly60Asp variant in Costello syndrome. It is NOT a computational / bioinformatic-pipeline paper.
Reported results and their origin (in-scope vs out-of-scope)
| Reported result | Origin / method | In scope? |
|---|---|---|
| HRAS c.179G>A (p.Gly60Asp) variant identified in 3 individuals | Clinical RASopathy gene-panel sequencing of patients (private patient samples) | OUT — clinical genotyping, no public pipeline/data |
| Clinical phenotype description (attenuated Costello: no severe FTT, no intellectual disability, no cancer) | Clinical examination / chart review | OUT — wet/clinical |
| Gly60Asp binds RAF1 (elevated vs WT, reduced vs Gly12Val) | RAS effector pull-down + immunoblot + gel densitometry (COS-7, HA-tagged constructs) | OUT — wet-lab assay, no software-derived value |
| Gly60Asp binding to PIK3CA / PLCE1 / RALGDS ~ WT | Co-immunoprecipitation + immunoblot | OUT — wet-lab |
| NF1-GAP (FLAG-NF1₃₃₃) co-precipitation ~ WT | Co-IP + immunoblot | OUT — wet-lab |
| MEK1/2, ERK1/2, AKT phosphorylation similar/decreased vs WT | Transfection + phospho-immunoblot densitometry | OUT — wet-lab |
| Schematic of HRAS motifs / Gly60 position (figure) | Hand-drawn schematic, not structural modeling | OUT — illustration, not computed |
Pipeline-derived results in scope
None. No bioinformatic pipeline is used or described: no sequencing-analysis / variant-calling pipeline on public data, no RNA-seq, no molecular dynamics or computational structural modeling, no omics. All quantitative findings come from wet-lab immunoblot / pull-down densitometry on the authors' own (non-deposited) experimental gels and from clinical exam.
Data availability
No Data Availability statement; no public accession (no GEO/SRA/ENA/dbGaP/ PRIDE/figshare/zenodo deposit). Underlying data are patient clinical records (private) and wet-lab gel/blot images (not deposited). No code repository exists (authors' own or third-party) that operates on any deposited data of this paper.
Decision
DROP — non_pipeline. There is no computational-pipeline result to
reproduce and no public dataset to run a third-party tool against. Per the brief,
a drop with a controlled reason is a valid, recorded outcome. No «our HPC» compute
was warranted or used.
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.
Correctly identified non-pipeline drop. PMID 25914166 is a clinical case series of 3 individuals with a novel HRAS p.Gly60Asp Costello variant combined with wet-lab functional characterization (effector pull-down, co-IP, phospho-immunoblot densitometry in COS-7 cells). There is no bioinformatic pipeline, no code repository, and no public data accession (data_source/code_url null; no Data Availability statement in PMC), so nothing is computationally reproducible. The limitation is data availability / study scope, not authors' integrity — no fabrication concern and no observed deviation, so q5/q7 are yellow (cannot assess) rather than red.
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.