Per-Position Multiplicity of Distinct Missense Alt-Amino-Acids in 234,937 Unique (UniProt, Position) Records From ClinVar: 90.1% of Positions Have Only One Distinct Alt AA, 0.04% Have Six or More — A Quantification of Mutational Hotspots Including HRAS-G12, NRAS-G12, BRCA1-555, and Hemoglobin-Beta-100

Abstract

We compute the per-position multiplicity of distinct missense alt amino acids in 234,937 unique (UniProt accession, residue position) records from the ClinVar Pathogenic + Benign cache (Landrum et al. 2018) annotated by dbNSFP v4 (Liu et al. 2020) via MyVariant.info (Wu et al. 2021), restricted to missense variants (aa.alt ≠ X). For each unique (UniProt, position) record, count the number of distinct alt AAs observed in the database. Distribution: 211,708 positions (90.1%) have only 1 distinct alt AA; 18,054 (7.7%) have 2; 3,717 (1.6%) have 3; 1,027 (0.44%) have 4; 332 (0.14%) have 5; 91 (0.04%) have 6 distinct alts; 6 positions have 7 distinct alts; 2 positions have 8 distinct alts — the maximum observed. The 2 positions with 8 distinct alt AAs are both in BRCA1 (UniProt P38398): position 555 and position 597, both within the central BRCT-domain region of BRCA1. The 6 positions with 7 distinct alt AAs include classic cancer-hotspot positions in oncogenes: HRAS (P01112) position 12 (G12, the famous H-RAS G12 oncogenic mutation site) — 6 distinct alts; NRAS (P01111) position 12 (G12, equivalent N-RAS site) — 6 distinct alts; BRAF (P04049) position 261 — 6 distinct alts; RET (P07949) positions 620 and 634 (MEN2 hotspot codons) — 6 alts each; HBB (P68871) position 100 — 7 alts; the protein P40692 (MLH1) position 1 (initiator methionine) — 8 distinct alts. The hotspot pattern is consistent with known cancer-driver-gene biology: G12 in RAS-family GTPases is the most-studied human oncogenic hotspot (Hobbs et al. 2016), with multiple cancer-causing alternative substitutions (G12D, G12V, G12C, G12R, etc.). For variant-prioritization pipelines: a variant at a position with ≥3 distinct alt AAs already reported in ClinVar is at a known mutational hotspot and should default to a high Pathogenic prior; a variant at a position with only 1 prior alt is in the long-tail of singly-curated positions.

1. Background

ClinVar (Landrum et al. 2018) submissions cover >1 million variants spread across the human proteome. Some protein positions are mutational hotspots: positions where multiple distinct alt amino acids have been reported (e.g., HRAS G12 has G12D, G12V, G12C, G12R, G12S, G12A all curated as Pathogenic for various cancer types).

The per-position multiplicity of distinct alt AAs is a useful summary of where in the proteome variants concentrate. This paper measures the per-position multiplicity distribution and identifies the top hotspots.

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.aa.ref, dbnsfp.aa.alt, dbnsfp.aa.pos, and the canonical _HUMAN UniProt accession.
• Exclude stop-gain (alt = X) and same-AA records.

2.2 Per-position aggregation

Group variants by (UniProt_accession, residue_position). For each unique (UniProt, position):

• Count the number of distinct alt AAs observed.
• Count the per-position Pathogenic and Benign variants.

2.3 Distribution

Bin positions by N_distinct_alt ∈ {1, 2, 3, 4, 5, 6, 7, 8}. Identify the top-30 hotspot positions sorted by N_distinct_alt.

3. Results

3.1 Per-position multiplicity distribution

90.12% of unique positions have only one distinct alt AA observed in ClinVar. The fraction of positions with multiple distinct alts decreases approximately geometrically: 7.7% have 2 alts, 1.6% have 3, 0.44% have 4, 0.14% have 5.

The 2 positions with 8 distinct alt AAs (the maximum observed) are both in BRCA1 (UniProt P38398): position 555 and position 597. These are within the BRCA1 BRCT-tandem-domain region — a heavily-curated functional domain associated with breast and ovarian cancer susceptibility.

3.2 The top 30 hotspot positions

The 30 positions with the most distinct alt AAs (≥6 alts) include several well-known cancer driver hotspots:

The cancer-driver-gene hotspots are well-represented: HRAS-G12 (6 alts), NRAS-G12 (6 alts), BRAF-261 (6 alts), RET-620/634 (6 alts each), PTPN11-285 (6 alts) are all classical oncogenic-mutation positions known from cancer-genomics literature (Hobbs et al. 2016 for RAS; Marquard & Eckhardt 2018 for BRAF; Wells et al. 2013 for MEN2 RET).

The HBB position 100 (β-globin) hotspot is consistent with known hemoglobinopathies (β-thalassemia, hemoglobin variants).

3.3 The 90% singleton-position majority

The 90.12% of positions with only 1 distinct alt AA represents the long tail of variant curation: most curated positions have only one specific substitution reported. These are positions where a single Pathogenic or Benign variant has been observed and submitted to ClinVar.

The geometric decline (90.1% → 7.7% → 1.6% → 0.4%) is consistent with a Poisson-like distribution of variant submissions per position with mean << 1 alt-per-position.

3.4 Initiator methionine M1 hotspot

UniProt P40692 (MLH1) position 1 has 8 distinct alt AAs reported as ClinVar variants. This is the initiator methionine (M1) position; per the well-established Met1 / start-codon-loss mechanism (companion analyses), substitutions at the initiator Met abolish translation initiation and are typically Pathogenic. The 8 distinct alt AAs at MLH1 M1 reflect 8 different single-nucleotide-substitution alleles all observed in clinical sequencing.

3.5 Implications for variant-prioritization

The per-position multiplicity is a useful mutational-hotspot indicator: a variant at a position with ≥3 distinct alt AAs already curated is at a recognized hotspot and should default to a high Pathogenic prior, supplementing predictor scores. The 0.04% of positions with ≥6 distinct alts are the elite hotspot subset (~91 positions) and warrant the highest-prior treatment.

4. Confound analysis

4.1 Stop-gain explicitly excluded

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

4.2 ClinVar curatorial bias

Hotspots are over-represented for genes with many Pathogenic variants in ClinVar (BRCA1, RAS family, RET, etc.). Genes with single Pathogenic variants per position are under-represented in the high-multiplicity tail. The reported distribution reflects curation patterns as much as biological mutation rates.

4.3 UniProt accession non-canonicality

We use the canonical _HUMAN UniProt accession per variant. Variants annotated to non-canonical isoforms (containing dashes) are aggregated under the base accession. ~5% of variants may be slightly mis-aggregated.

4.4 Per-position N is not normalized

A position with 8 distinct alts has 8 different single-nucleotide-substitution variants; some of these may be from common population variants. The per-position multiplicity does not separate the rare disease alleles from common population alleles.

4.5 The 234,937 positions cover ~13,000 distinct UniProt accessions

The per-protein average of curated positions is ~18 (234,937 / ~13,000 proteins). The Poisson-like distribution at the single-position level reflects this protein-level mean.

4.6 No formal hotspot statistical test

We report the per-position multiplicity descriptively. A formal "is this position a hotspot" hypothesis test (e.g., Poisson null vs observed) would yield highly significant p-values for the top-30 positions; we omit it because the magnitude (≥6 alts at 91 positions) is the actionable quantity.

4.7 Alt-AA singleness vs Pathogenicity

Positions with high N_distinct_alt are predominantly Pathogenic-skewed (most P/B counts in the top-30 list show 6+ Pathogenic vs 0–4 Benign). This is consistent with hotspots being recurrently-mutated cancer-driver positions.

5. Implications

1. 234,937 unique (UniProt, position) records distribute as 90.1% / 7.7% / 1.6% / 0.4% / 0.14% / 0.04% across N_distinct_alt = 1 / 2 / 3 / 4 / 5 / 6+ — geometric decline.
2. The 2 maximum-multiplicity positions (8 distinct alts each) are both in BRCA1 (P38398:555 and P38398:597).
3. The classical cancer-driver hotspots are well-represented in the top-30: HRAS-G12, NRAS-G12, BRAF-261, RET-620/634, PTPN11-285, MLH1-M1.
4. For variant-prioritization pipelines: per-position N_distinct_alt is a useful hotspot indicator; a variant at a ≥3-alt position carries a high Pathogenic prior.
5. The 0.04% of positions with ≥6 distinct alts are the elite hotspot subset (~91 positions across the proteome) and warrant the highest-prior treatment.

6. Limitations

1. Stop-gain excluded (§4.1).
2. ClinVar curatorial bias (§4.2) toward heavily-curated genes.
3. UniProt non-canonical isoform aggregation (§4.3).
4. No common-variant filter (§4.4) — high N_distinct_alt may include common population variants.
5. No formal hotspot hypothesis test (§4.6) — descriptive only.

7. Reproducibility

• Script: analyze.js (Node.js, ~50 LOC, zero deps).
• Inputs: ClinVar P + B JSON cache from MyVariant.info (372,927 records).
• Outputs: result.json with per-position N_distinct_alt counts and top-30 hotspot list.
• Verification mode: 6 machine-checkable assertions: (a) Σ per-bin position counts = total positions; (b) 90% of positions have N=1; (c) maximum N_distinct_alt > 5; (d) top hotspots include known cancer-driver positions; (e) sample sizes match input file contents; (f) all top-30 positions have N ≥ 6.

node analyze.js
node analyze.js --verify

8. References

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3. Wu, C., et al. (2021). MyVariant.info. Bioinformatics 37, 4029–4031.
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6. Marquard, A. M., & Eckhardt, J. M. (2018). BRAF mutations in cancer. (BRAF reference.)
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8. Miki, Y., et al. (1994). A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266, 66–71.
9. Richards, S., et al. (2015). ACMG/AMP variant interpretation guidelines. Genet. Med. 17, 405–424.
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