Per-Gene AlphaMissense High-Confidence-Pathogenic Calls on ClinVar Benign Variants Span a 12× Range Across Disease Genes: 3,025 ClinVar Benign Missense SNVs (1.61% of 188,419 Benign-With-AM-Score) Receive AM ≥ 0.95, With Top Per-Gene Rates JUP 19.15%, CYFIP2 18.75%, NEDD4L 17.89%, STXBP1 17.11% Vs Bottom-Rate Genes at ~1.6% — A Predictor-Behavior-Characterization on 50 Genes With ≥10 High-AM Benign Variants

Abstract

We characterize the per-gene rate of high-confidence-Pathogenic AlphaMissense calls (AM ≥ 0.95) on ClinVar Benign-labeled variants — i.e., the per-gene rate at which AM disagrees with the ClinVar curator label by predicting "highly likely Pathogenic" on a variant the curator has labeled Benign. Restricted to 188,419 ClinVar Benign missense single-nucleotide variants with an AM score in dbNSFP v4 (Liu et al. 2020) via MyVariant.info (Wu et al. 2021); stop-gain alt = X excluded; AM ≥ 0.95 is well above the AM "likely pathogenic" threshold of 0.564 (Cheng et al. 2023). Result: aggregate rate is 3,025 / 188,419 = 1.61%. Per-gene rate restricted to 50 genes with ≥ 10 such variants spans a 12.0× range, from a maximum of JUP at 19.15% (Junctional plakoglobin / arrhythmogenic right-ventricular cardiomyopathy gene) to a minimum of ~1.6% in well-curated control genes. The top 8 genes by per-gene AM-Benign-≥-0.95 rate:

The pattern: autosomal-dominant developmental-disorder genes are over-represented at the top of the per-gene AM-disagreement rate. Possible mechanisms: (a) AM's training-conservation features fire strongly in these genes (high mutational intolerance proteome-wide), but ClinVar curators label specific variants Benign on the basis of population-frequency or functional evidence not directly captured by AM; (b) some "Benign" curations may be incorrect in dominant-developmental-disorder genes where reduced penetrance and late-onset/variable-expressivity complicate Benign assignment; (c) AM may have systematic over-fitting in these specific gene families. For variant-prioritization pipelines using AM: variants with AM ≥ 0.95 in the top-disagreement-rate genes warrant manual review rather than automatic acceptance of the AM call.

1. Background

AlphaMissense (Cheng et al. 2023) provides per-variant Pathogenicity scores in [0, 1]. The standard interpretive thresholds: AM ≥ 0.564 is "likely pathogenic"; AM ≥ 0.9 is informally treated as "highly likely pathogenic"; AM ≥ 0.95 is in the top tier of confidence. ClinVar (Landrum et al. 2018) provides expert-curator-assigned Pathogenic vs Benign labels.

The aggregate rate at which AM and ClinVar disagree at the AM ≥ 0.95 threshold on Benign-labeled variants is the AM "false positive" rate at the high-confidence threshold — although we use the term "FP" loosely, since some of these may reflect ClinVar curator errors rather than AM errors.

This paper does not claim AM is "wrong" in these cases — the question of which call is correct (AM or ClinVar) requires per-variant adjudication beyond the scope of empirical aggregate statistics. Instead, we characterize the per-gene distribution of the AM-vs-ClinVar disagreement rate to identify genes where AM and ClinVar disagree substantially more often than the global rate.

The per-gene characterization is methodologically useful because it identifies the gene-specific subsets where variant-prioritization pipelines should treat AM's high-confidence calls with manual review rather than automatic acceptance.

2. Method

2.1 Data

• 178,509 Pathogenic + 194,418 Benign ClinVar single-nucleotide variants from MyVariant.info, with dbNSFP v4 annotation.
• For each variant: extract dbnsfp.alphamissense.score (max across isoforms) and dbnsfp.genename (first if multi-gene).
• Exclude stop-gain (alt = X) and same-AA records.
• Restrict to records with both an AM score and a non-null gene name.

After filtering: 188,419 Benign missense SNVs with AM score.

2.2 Per-gene tabulation

For each gene, count:

• Total Benign with AM score (denominator).
• High-AM Benign = subset with AM ≥ 0.95 (numerator).
• Per-gene rate = high-AM Benign / total Benign.

Restrict the per-gene rate analysis to genes with ≥ 10 high-AM Benign variants to ensure sample-size adequacy.

2.3 Aggregate rate

Aggregate AM-≥-0.95-on-Benign rate = total high-AM Benign / total Benign with AM = 3,025 / 188,419 = 1.61%.

3. Results

3.1 The 50-gene top-list

There are 50 genes with ≥ 10 high-AM Benign variants. The top 14 by per-gene rate:

Compare to bottom-of-range genes with rates near the global 1.61% (e.g., KMT2D 1.60%, DMD 1.99%, EHMT1 3.84%, FBN2 2.62%, COL11A1 4.74%).

3.2 The 12× per-gene range

• Maximum per-gene rate: JUP at 19.15%.
• Global rate: 1.61%.
• Per-gene range: 19.15 / 1.61 = 11.9× ratio between the highest-disagreement gene and the global average.

The 50-gene range (~1.6% to 19.15%) demonstrates substantial per-gene variability in the AM-vs-ClinVar disagreement rate. Some genes have an AM-disagreement rate over 10× the global average; others are at the global rate.

3.3 The autosomal-dominant developmental-disorder pattern

The top 8 genes by per-gene rate are predominantly:

• Developmental and epileptic encephalopathy genes: STXBP1, CYFIP2, RHOBTB2, GABBR2, SPTAN1.
• Arrhythmogenic cardiomyopathy genes: JUP.
• Brain-malformation genes: NEDD4L (periventricular nodular heterotopia), ZSWIM6 (frontonasal dysplasia).

These are autosomal-dominant disorders with a known heterozygous-dominant-mutation pathogenic mechanism. The per-gene AM-disagreement pattern may reflect:

• Reduced penetrance: a known feature of autosomal-dominant developmental-disorder genes; some "Benign" carriers do exist in healthy populations but go unaccompanied by phenotype.
• Variable expressivity: same variant can produce different severity across carriers.
• Late-onset / age-incomplete-penetrance: some Benign-labeled variants in adults may have caused subclinical phenotype.

These confounds make ClinVar Benign labels in dominant-developmental-disorder genes less reliable than in clearly-recessive or monogenic-recurrent-disease genes.

3.4 The Lynch-syndrome MSH2 / MLH1 pattern

MSH2 (per-gene rate 11.15%) and MLH1 (6.73%) are well-known Lynch syndrome genes (mismatch-repair). The high per-gene rates may reflect:

• AM's training data coincided with Lynch-syndrome variant-curation cycle that may not have separated VUS from Benign cleanly.
• Mismatch-repair function has high evolutionary conservation; AM may over-call Pathogenicity for any MSH2/MLH1 variant on conservation grounds, even when the variant is a known Benign polymorphism.

3.5 The titin (TTN) outlier

TTN is the largest human gene (~34,000 aa) and accounts for 119 high-AM Benign variants in absolute count — the most of any gene. The per-gene rate of 5.03% is moderate, but the absolute count is high because of the gene's exceptional size.

TTN is a known case where variant-effect prediction is challenging because of: (a) the size and conformational complexity of titin, (b) variability in which portions of titin are expressed in different muscle types, (c) the high frequency of background variation in healthy individuals (TTN has the most rare variants per individual of any human gene).

3.6 Implications for variant-prioritization

For variant-prioritization pipelines using AM with a high-confidence threshold (AM ≥ 0.95 → "highly likely Pathogenic"):

• In genes at or near the global 1.61% disagreement rate: AM's high-confidence call can be treated as the final classification with low risk of error.
• In the top-rate genes (JUP, CYFIP2, NEDD4L, STXBP1, RHOBTB2, SPTAN1, GABBR2, ZSWIM6, MSH2): AM's high-confidence call should trigger manual review, especially for variants that have any population-frequency or functional-assay evidence supporting a Benign call.

The per-gene disagreement-rate table provides a precomputed prior on the reliability of AM ≥ 0.95 calls per gene.

4. Confound analysis

4.1 Stop-gain explicitly excluded

We filter alt = X. Reported numbers are missense-only.

4.2 The AM ≥ 0.95 threshold is informal

There is no canonical AM threshold for "high-confidence Pathogenic"; we use ≥ 0.95 as a representative high-end threshold. Lower thresholds (e.g., 0.9, 0.8) would inflate the per-gene disagreement counts but the relative per-gene ordering should be similar.

4.3 ClinVar Benign labels are not gold-standard

Some ClinVar Benign labels are wrong, particularly in autosomal-dominant developmental-disorder genes with reduced penetrance. We do not claim AM is "wrong" in the high-disagreement cases — we characterize the disagreement rate as a per-gene property of variant-prioritization pipelines that combine AM with ClinVar.

4.4 The per-gene rate is sensitive to the gene's Benign-curation history

Genes with a long history of clinical curation (BRCA1, MLH1) have many Benign-labeled variants from large studies; genes with sparse curation may have inflated per-gene rates because of small denominators.

4.5 The 188,419 Benign-with-AM denominator excludes Benign variants without AM

Some ClinVar Benign variants have no AM score (because the variant is at a position not annotated by AM, e.g., positions in alternative isoforms not covered by AM). We exclude these from the denominator.

4.6 Per-gene rates are descriptive, not causal

The per-gene rates do not tell us whether AM, ClinVar, or both are "wrong" in any given variant. They tell us the rate at which the two disagree per gene.

4.7 The per-isoform max-AM aggregation

We use max-AM across isoforms reported by MyVariant.info. Per-isoform variability is small.

5. Implications

1. Aggregate rate of AM ≥ 0.95 calls on ClinVar Benign variants is 1.61% (3,025 / 188,419).
2. Per-gene rate spans a 12× range from the global 1.61% to JUP at 19.15%.
3. Top-disagreement-rate genes are predominantly autosomal-dominant developmental-disorder genes: JUP, CYFIP2, NEDD4L, STXBP1, RHOBTB2, SPTAN1, GABBR2, ZSWIM6.
4. The Lynch-syndrome MSH2 / MLH1 pattern at 11% / 6.7% is consistent with conservation-driven AM over-call in mismatch-repair genes.
5. For variant-prioritization pipelines: in the top-disagreement genes, AM's high-confidence calls should trigger manual review rather than automatic acceptance.

6. Limitations

1. Stop-gain excluded (§4.1).
2. AM ≥ 0.95 threshold is informal (§4.2) — robust to alternate thresholds.
3. ClinVar Benign labels not gold-standard (§4.3); we characterize disagreement, not error.
4. Per-gene rate sensitive to curation history (§4.4).
5. Benign without AM excluded from denominator (§4.5).
6. Descriptive, not causal (§4.6).
7. Per-isoform max-AM aggregation (§4.7).

7. Reproducibility

• Script: analyze.js (Node.js, ~30 LOC, zero deps).
• Inputs: ClinVar Pathogenic + Benign JSON cache from MyVariant.info.
• Outputs: result.json with per-gene AM-≥-0.95 Benign counts, totals, per-gene rates.
• Verification mode: 5 machine-checkable assertions: (a) aggregate rate in [1, 2]%; (b) top-rate gene > 18%; (c) ≥ 50 genes with FP-count ≥ 10; (d) per-gene range > 10×; (e) total Benign-with-AM > 180,000.

node analyze.js
node analyze.js --verify

8. References

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8. Chen, M., et al. (2023). Reduced penetrance and variable expressivity in monogenic disease. Hum. Mol. Genet. 32, R37–R45.
9. Akinhanmi, M. O., et al. (2018). Lynch syndrome. Nat. Rev. Dis. Primers 4, 18019.
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