USH2A Molecular Research InitiativeProject 01 · USH2A G1526R Molecular Mechanism

Patient-initiated · computationally characterized · experimentally unresolved

Project 01 · USH2A G1526R Molecular Mechanism

From a Personal Variant to a Testable Molecular Hypothesis

An independent, patient-initiated investigation of USH2A p.Gly1526Arg.

The nucleotide and amino-acid changes are verified. Three splice models agree on a site-level effect, and one model returns a cryptic donor–exon 22 pair that can be reconstructed into a candidate transcript carrying a 50-nt deletion. Whether that RNA exists, how abundant it would be, and how much normally spliced G1526R protein still functions are unresolved. The project now seeks laboratory partners who can test the resulting questions.Supporting claims: C001sequence factC010predictionC016conditionalC019unresolvedC032unresolvedC042scope

Transcript
NM_206933.4:c.4576G>A
Protein
NP_996816.3:p.Gly1526Arg
Genome (GRCh38)
NC_000001.11:g.216175303C>T

The scientific question

USH2A encodes usherin and is associated in reference databases with Usher syndrome type 2A and nonsyndromic retinitis pigmentosa. Those gene-level associations motivate molecular investigation; they do not specify this variant’s mechanism or predict an individual clinical course.Supporting claims: C003annotation

The reference mapping is verified: USH2A lies on the minus strand, so the genomic and transcript substitutions are complementary, and at the normally spliced coding sequence GGG becomes AGG — glycine replaced by arginine at residue 1526.Supporting claims: C001sequence factC002sequence fact

What is still open is where the damage occurs. Does the change alter how USH2A RNA is spliced, does the amino-acid substitution alter the protein, or do both contribute? The dominant pathogenic mechanism is unknown, and the present evidence cannot quantify the relative contribution of each route.Supporting claims: C032unresolvedC020unresolved

Two candidate routes, one variant

Both routes below are working hypotheses. The RNA route is the clearest candidate to test; neither route has been experimentally established as dominant.

Route A — predicted RNA processing

Computationally supported; the reconstructed product has not been observed. C019unresolved

  1. USH2A c.4576G>A in exon 21 Sequence fact
  2. Cryptic donor position enhanced, annotated donor position reduced Computational prediction
  3. Predicted pair: donor 216175302 → exon-22 acceptor 216097213 Computational prediction
  4. Candidate transcript carrying a 50-nt deletion: c.4578_4627 deleted Conditional consequence
  5. Conditional frame: Arg1526–His1527–stop1528 Conditional consequence
  6. Stop 124 nt before the next junction: NMD-compatible geometry Conditional consequence

Route B — normally spliced protein

The alternative is an intrinsic effect of the amino-acid substitution. C031conditional

  1. Normally spliced G1526R transcript Sequence fact
  2. Full-length G1526R usherin retains the annotated membrane and cytoplasmic regions Conditional consequence
  3. Residual protein function unknown Unresolved question
  4. Local model differences do not establish native folding, maturation or interactions Unresolved question

Ranking matters here and is easy to over-read: the exon-22 acceptor ranks first among the cryptic donor’s 98 returned partners, yet the annotated exon 21–exon 22 junction still has the higher ALT prediction. Those are different comparisons, and neither is an RNA percentage.Supporting claims: C013predictionC032unresolved

A verified variant branches into a predicted cryptic-splicing route and a conditional missense-protein route. Both lead to unresolved RNA amount, protein fate and mechanism, with RNA assays and an intron-free protein comparison proposed.
FIG-01 · Variant-to-mechanism overview. Verified G1526R sequence identity is separated from the predicted RNA route and the conditional normally spliced missense route. The proposed deletion-containing RNA, potential NMD and downstream protein effects remain conditional or unresolved. Arrows organize research questions; they do not establish a causal sequence or dominant mechanism.

Limitations: No experimentally observed deletion-containing RNA, demonstrated NMD or native functional result is shown.

In words: A verified sequence change sits at the top. It branches into a predicted RNA route (left) and a conditional normally spliced protein route (right). Both branches end in unresolved quantities: RNA amount, protein fate and the dominant mechanism. The arrows organize research questions; they are not a demonstrated causal chain.

Claims shown in this figure: C001C010C011C016C018C019C029C032C036C039 Evidence: USH2A-E001, USH2A-E005, USH2A-E006, USH2A-E007, USH2A-E012, USH2A-E013 References: REF-001, REF-008
Text description of the figure

Verified variant identity branches into predicted RNA processing and conditional missense-protein routes. A 50-nt deletion, frameshift and possible NMD are conditional; abundance, function and predominant mechanism are unresolved.

A verified variant branches into a predicted cryptic-splicing route and a conditional missense-protein route. Both lead to unresolved RNA amount, protein fate and mechanism, with RNA assays and an intron-free protein comparison proposed.

The diagram is wider than the screen: scroll it horizontally (drag, swipe, or arrow keys once focused) to read every panel.

FIG-01 · Variant-to-mechanism overview. Verified G1526R sequence identity is separated from the predicted RNA route and the conditional normally spliced missense route. The proposed deletion-containing RNA, potential NMD and downstream protein effects remain conditional or unresolved. Arrows organize research questions; they do not establish a causal sequence or dominant mechanism.

Limitations: No experimentally observed deletion-containing RNA, demonstrated NMD or native functional result is shown.

What has been computed

Four analysis components support the questions above. Their scores use different scales and are never combined or averaged.

RNA site prediction

SpliceAI 1.3.1 · Pangolin v2 · AlphaGenome

All three agree on enhancement at the cryptic donor-associated position and reduction at the annotated donor-associated position. Only AlphaGenome supplies an explicit donor–acceptor pair in these runs.Supporting claims: C010prediction

Read the RNA evidence

Junction prediction

AlphaGenome 1-Mb context · two tissue tracks

Proposed junction outputs, unchanged from the model: eye REF 0.0006493872497230768 / ALT 0.005134492181241512; brain REF 0.00025533069856464863 / ALT 0.0007750487420707941. These are not read counts, RNA percentages or probabilities of complete transcripts.Supporting claims: C011predictionC014prediction

See the reconstructed consequence

Local protein structure

ColabFold 1.6.3 / AlphaFold2 pTM · six matched WT/G1526R pairs

Matched local Cα RMSDs 0.152–0.341 Å partly overlap the within-WT range 0.132–0.230 Å. Target pLDDT 88.69–90.56 (WT) versus 78.00–83.56 (G1526R) is model confidence, not an energy or function score.Supporting claims: C022predictionC023prediction

Read the protein analysis

Stability and dynamics

Rosetta 2025.51 · ThermoMPNN · OpenMM 8.6.1 (125 ns)

The primary Rosetta run failed geometry qualification in 26 of 30 mutant structures, so its large positive values were excluded. More-flexible runs gave +13.706 / +9.135 REU; ThermoMPNN forward means were +1.288 / +1.306 predicted kcal/mol with reverse means near +0.060 / +0.058. REU and predicted kcal/mol are not interchangeable. Paired 20-ns simulations showed local RMSD differences of −0.664 to +1.185 Å that reversed direction.Supporting claims: C024predictionC025predictionC026predictionC028prediction

Parameters and controls

What remains unknown

This list is the scientific centre of the project. None of it is a negative result; it is an evidence gap.

The experimental roadmap

Every stage below is proposed, not started, and no laboratory is represented as committed. The order reflects what would resolve the most uncertainty first.

  1. Establish whether the RNA junction is used — matched WT and variant contexts, sequence-confirmed products.
  2. Measure proportions and RNA fate — quantitative, NMD-aware comparison of native and alternative products.
  3. Resolve full transcript and isoform identity — junction-to-transcript-end linkage, long and short architectures analysed separately.
  4. Test the missense protein independently — matched intron-free constructs with transcript normalization.
  5. Test relevant cellular and isoform contexts — human-cell, iPSC or retinal-organoid systems where the relevant isoforms are expressed.
Two proposed experimental paths lead from sequence-confirmed RNA testing and intron-free protein comparison to quantitative, isoform-aware and relevant-cell studies. No stage is marked completed or scheduled.
FIG-06 · Proposed experimental validation roadmap. Parallel RNA and intron-free protein paths can distinguish altered processing from an intrinsic amino-acid effect. RNA work would establish the event, quantify products and link them to isoforms; protein work would test an independent phenotype. Relevant human-cell, iPSC and retinal-organoid contexts are sought. Every stage is proposed.

Limitations: No laboratory, material, schedule, approval or therapeutic outcome is implied. These are research questions, not detailed laboratory protocols.

In words: Two proposed experimental paths run in parallel. The RNA path would confirm the event, quantify products and link them to isoforms. The protein path would test an amino-acid effect independently of splicing. Both then feed relevant-cell studies. No stage is marked completed or scheduled.

Claims shown in this figure: C036C037C038C039C040C041C042C043 Evidence: USH2A-E013, USH2A-E014
Text description of the figure

Parallel RNA and intron-free protein paths distinguish processing effects from missense-protein effects. RNA work establishes, quantifies and links products; protein work tests an independent phenotype. Relevant human-cell and isoform contexts are proposed, not committed.

Two proposed experimental paths lead from sequence-confirmed RNA testing and intron-free protein comparison to quantitative, isoform-aware and relevant-cell studies. No stage is marked completed or scheduled.

The diagram is wider than the screen: scroll it horizontally (drag, swipe, or arrow keys once focused) to read every panel.

FIG-06 · Proposed experimental validation roadmap. Parallel RNA and intron-free protein paths can distinguish altered processing from an intrinsic amino-acid effect. RNA work would establish the event, quantify products and link them to isoforms; protein work would test an independent phenotype. Relevant human-cell, iPSC and retinal-organoid contexts are sought. Every stage is proposed.

Limitations: No laboratory, material, schedule, approval or therapeutic outcome is implied. These are research questions, not detailed laboratory protocols.

Where collaborators are needed

  • RNA splicing biology: cryptic and annotated donor use in matched backgrounds, junction confirmation.
  • Transcriptomics: linking alternative junctions to transcript ends with isoform-aware controls.
  • Human-cell functional validation: intron-containing versus intron-free designs with matched RNA and protein measurements.
  • iPSC and retinal organoids: relevant expression and developmental context.
  • Protein biochemistry: defined WT/G1526R constructs for maturation and folding-related behaviour.
  • Isoform-specific functional studies: long and short architectures tested separately.

Starting a conversation

Write to president@usher.org.tw with the hypothesis you could test, the expression context you work in and the readouts you could provide.

A first message does not create a collaboration: materials, data rights, authorship and responsibilities are agreed separately.

This public case concerns only USH2A c.4576G>A. It offers no private familial or association-member datasets, and no laboratory, funding source or institution is represented as committed.Supporting claims: C035scopeC042scope

Research status

  1. Reference identity and isoform mappingCompleted · computation

    MS01 · recorded 2026-10-11 · evidence USH2A-E001, USH2A-E002

  2. RNA predictor and reconstruction auditCompleted · computation

    MS02 · recorded 2026-10-11 · evidence USH2A-E005, USH2A-E006, USH2A-E007

  3. Local structure, stability controls and MD assessmentCompleted · computation

    MS03 · recorded 2026-10-12 · evidence USH2A-E008, USH2A-E009, USH2A-E010, USH2A-E011

  4. Independent-review junction packageCompleted · documentation

    MS04 · recorded 2026-10-12 · evidence USH2A-E006

  5. Scientific website handoffCompleted · documentation

    MS05 · recorded 2026-10-12 · evidence USH2A-E014, USH2A-E015

  6. Sequence-confirmed RNA event testingProposed · not started

    MS06 · requires relevant expression context and matched controls · evidence USH2A-E013

  7. RNA quantification, fate and isoform linkageProposed · not started

    MS07 · requires sequence-confirmed products and validated quantitative readouts · evidence USH2A-E013

  8. Independent protein and cellular comparisonsProposed · not started

    MS08 · requires matched intron-free constructs and interpretable expression context · evidence USH2A-E013

Completed entries record computation or documentation only. Proposed entries have no start date, no laboratory commitment and no result.

Read the proposed experiments · Download the evidence tables