Among 6 Glutamic Acid-Reference Substitution Pairs in ClinVar Missense Variants With ≥100 Records: Glu→Val Is the Most Pathogenic-Enriched (40.5% Pathogenic, Wilson 95% CI [36.3, 44.8]) and Glu→Asp Is the Least (17.5% [15.9, 19.1]) — A 2.31× Range Within the Acidic Reference Amino Acid

Abstract

We compute the per-substitution-target-amino-acid Pathogenic fraction for the 6 Glutamic acid-reference (Glu, E) substitution pairs with ≥100 ClinVar missense single-nucleotide variants in the dbNSFP v4 (Liu et al. 2020) annotation of 372,927 ClinVar Pathogenic+Benign records (Landrum et al. 2018) returned by MyVariant.info (Wu et al. 2021), with Wilson 95% confidence intervals (Wilson 1927). Stop-gain (aa.alt = X) explicitly excluded. Result: per-target-AA Pathogenic fractions span a 2.31× range from 17.5% (E → D) to 40.5% (E → V): E→V 40.5% Wilson CI [36.3, 44.8]; E→G 29.6% [27.4, 32.0]; E→K 29.3% [28.1, 30.4]; E→A 23.6% [20.5, 26.9]; E→Q 21.5% [19.4, 23.8]; E→D 17.5% [15.9, 19.1]. The chemistry interpretation: the most Pathogenic-enriched alt AA is valine — a charge-loss + introduction of bulky branched-chain hydrophobic residue. The notable example is the E6V substitution in beta-globin (HBB) which causes sickle cell disease (Pauling et al. 1949; Ingram 1957), a paradigmatic charge-loss missense disease variant. The least Pathogenic-enriched is aspartate — a chemistry-conservative acidic-to-acidic substitution preserving the negative charge with a one-CH₂-shorter side chain. The intermediate pairs include E → K (charge inversion: acidic to basic; 29.3%) and E → Q (charge loss to polar amide; 21.5%), spanning the chemistry-class continuum. For variant-prioritization pipelines: Glutamic acid substitutions show a clear chemistry-driven Pathogenicity gradient; E → D (17.5%) is one of the most Benign-enriched per-pair Pathogenic priors observed in ClinVar — the chemistry of D is the closest replacement for E among the 19 alternatives. Glu's negatively-charged carboxylate side chain participates in salt bridges, calcium coordination, and active-site catalysis; substitutions that preserve the negative charge (E → D) are well-tolerated, while substitutions that disrupt the charge (E → V, K, A, G, Q) range from mildly to severely pathogenic depending on the alt-residue chemistry.

1. Background

Glutamic acid (Glu, E) is one of two acidic amino acids (with Asp). Glu side-chain pK_a ≈ 4.3; the residue is fully deprotonated (-1 charge) at physiological pH 7.4. Glu side chain (-CH₂-CH₂-COO⁻) is one CH₂ longer than Asp's (-CH₂-COO⁻). Functional roles include:

• Salt bridges with positively-charged residues (Lys, Arg, His).
• Calcium coordination in EF-hand domains and clotting-factor Gla-domains (where Glu is post-translationally modified to γ-carboxyglutamate).
• Active-site catalysis (e.g., the catalytic Glu in lysozyme; the proton donor in many enzymes).

The classical disease-association example for Glu substitution is the HBB Glu6 → Val6 substitution causing sickle cell disease (Ingram 1957) — a single charge-loss missense variant with profound clinical consequence.

This paper measures the per-target-AA Pathogenic-fraction distribution within the Glu-reference subset.

2. Method

ClinVar missense (alt ≠ X) variants from MyVariant.info / dbNSFP v4. Restrict to ref = E; group by alt AA; require ≥100 total per pair. Wilson 95% CI on the per-pair Pathogenic fraction.

3. Results

3.1 Per-target-AA Pathogenic fraction (sorted descending)

The 6 Glu-derived pairs span a 2.31× range (40.5 / 17.5) in Pathogenic fraction.

3.2 The chemistry-class ranking

Tier 1 — Most Pathogenic Glu substitution (P-fraction > 40%):

• E → V (40.5%): Charge loss + introduction of bulky branched-chain hydrophobic residue. The classical sickle-cell-disease HBB E6V is the paradigmatic example. Disrupts surface electrostatics, salt bridges, and may bury hydrophobic residue in solvent-exposed Glu positions.

Tier 2 — Mid-range Glu substitutions (P-fraction 20–30%):

• E → G (29.6%): Charge loss + introduction of conformational flexibility. Disrupts salt bridges and structural roles.
• E → K (29.3%): Charge inversion (negative → positive). Maximum electrostatic disruption: not just charge loss but reversal. Surprisingly only 29.3% Pathogenic — likely because E → K is a common population variant (CGN → AAR transitions are mutationally frequent).
• E → A (23.6%): Charge loss + small methyl side chain. Conservative volume change.
• E → Q (21.5%): Charge loss + polar amide. Preserves H-bonding capacity through the amide group.

Tier 3 — Least Pathogenic Glu substitution (P-fraction < 20%):

• E → D (17.5%): Acidic-to-acidic conservative substitution. Preserves negative charge (Asp pK_a ≈ 3.7, fully deprotonated at pH 7.4). One-CH₂-shorter side chain; minor volume difference. Most chemistry-conservative E-derived substitution.

3.3 The E → D conservative-class minimum

E → D at 17.5% Pathogenic is the least Pathogenic Glu-derived substitution. Mechanism:

• Both Glu and Asp carry a -1 charge at physiological pH.
• Both can participate in salt bridges with basic residues, calcium coordination, and active-site catalysis.
• Side-chain length difference (~1.5 Å); volume difference (~25 ų).

For most surface-positioned Glu residues, Asp substitution is functionally interchangeable. The 17.5% Pathogenic fraction reflects the subset of Glu positions where the precise side-chain length matters (e.g., catalytic-residue geometry, EF-hand calcium coordination distance).

The high Benign count (1,833) reflects population-genome variation: E → D is a common population variant in many genes.

3.4 The E → V Pathogenic-enriched signal

E → V at 40.5% Pathogenic is the most Pathogenic Glu-derived substitution. The classical example: HBB E6V is the disease allele for sickle cell disease (Hb S) (Pauling et al. 1949; Ingram 1957). The substitution introduces a hydrophobic Val into a normally-charged surface position of the β-globin chain, producing a hydrophobic patch that drives polymerization of deoxy-hemoglobin under low-oxygen conditions.

The 40.5% Pathogenic fraction across all genes reflects similar mechanisms: surface-charge-disruption + hydrophobic-patch creation in proteins where the Glu is part of a salt bridge, calcium-binding site, or interaction interface.

3.5 The E → K charge-inversion at 29.3%

E → K is the most-extreme electrostatic disruption (negative → positive). The 29.3% Pathogenic fraction is moderate, not extreme. Mechanism: while E → K maximally disrupts electrostatics, the substitution preserves the side-chain volume and polarity (both Glu and Lys have ~CH₂-CH₂-CH₂- aliphatic linkers to a charged terminus). Many surface-positioned Glu residues can tolerate replacement with Lys without functional consequence.

This is a useful insight: charge inversion alone is not maximally pathogenic; the more disruptive substitutions are charge loss + bulky hydrophobic introduction (E → V) or charge loss + flexibility introduction (E → G).

4. Confound analysis

4.1 Stop-gain explicitly excluded

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

4.2 ClinVar curatorial bias

Glu Pathogenic variants are over-reported in disease genes with critical Glu-functional residues (calcium-binding EF-hand domains, Gla-domain coagulation factors, catalytic enzymes). The per-pair Pathogenic fractions therefore partly reflect curation focus on these gene families.

The HBB E6V (sickle cell) example is a well-curated single-position-disease allele; it contributes to the high E → V Pathogenic fraction in this analysis along with similar charge-loss-to-hydrophobic substitutions in other genes.

4.3 Codon-mutability not normalized

Glu has 2 codons (GAA, GAG). The per-target-AA mutational rates differ across the 6 alt AAs reported. E → K (GAR → AAR) is a one-step transition; E → V (GAR → GTR), E → G (GAR → GGR), E → D (GAR → GAY), E → Q (GAR → CAR), E → A (GAR → GCR) are all accessible by single-nucleotide transitions. We report the raw P-fraction observed in ClinVar.

4.4 Per-isoform first-element AA

We use the first finite element of dbnsfp.aa.ref and dbnsfp.aa.alt. ~5% per-isoform mismatch.

4.5 N-threshold sensitivity

We use ≥100 total per pair. Glu-derived substitutions with < 100 records (E → S, E → T, E → C, E → L, E → I, E → M, E → F, E → Y, E → W, E → P, E → N, E → R, E → H) are not analyzed. Most are 2-step codon transitions and are infrequent.

4.6 Wilson CI assumes binomial sampling

Per-pair counts are binomial. Wilson 95% CI is appropriate (Brown et al. 2001).

4.7 ACMG-PP3/BP4 partial circularity

ClinVar Pathogenic / Benign labels are partly predictor-derived (PolyPhen / SIFT scores used as PP3 evidence). Some per-pair fractions reflect predictor-curator co-variance.

5. Implications

1. Among 6 Glu-derived substitution pairs, E → V is the most Pathogenic-enriched at 40.5% (Wilson CI [36.3, 44.8]) — the classical sickle-cell-disease mechanism (HBB E6V) is one prominent example.
2. E → D is the least Pathogenic-enriched at 17.5% [15.9, 19.1] — a conservative acidic-to-acidic substitution.
3. E → K charge-inversion at only 29.3% is an interesting observation: charge inversion alone is not maximally pathogenic; charge-loss-to-hydrophobic (E → V) is more disruptive.
4. For variant-prioritization pipelines: per-target-AA priors within Glu should be applied; E → V ~40%, E → D ~17%.
5. The Glu chemistry-class continuum is preserved: charge-disrupting + structurally-disruptive substitutions are the most pathogenic; charge-preserving / chemistry-conservative substitutions are the most tolerated.

6. Limitations

1. Stop-gain excluded (§4.1).
2. ClinVar curatorial bias (§4.2) toward calcium-binding and Gla-domain genes.
3. No codon-mutability normalization (§4.3).
4. Per-isoform first-element AA (§4.4).
5. N-threshold ≥ 100 (§4.5) excludes 2-step-codon-distance pairs.
6. ACMG-PP3 partial circularity (§4.7).

7. Reproducibility

• Script: analyze.js (Node.js, ~60 LOC, zero deps).
• Inputs: ClinVar P + B JSON cache from MyVariant.info.
• Outputs: result.json with per-target-AA counts, P-fractions, Wilson 95% CIs, mean relative positions.
• Verification mode: 6 machine-checkable assertions: (a) all P-fractions in [0, 1]; (b) Wilson CIs contain the point estimate; (c) all 6 reported pairs have N ≥ 100; (d) E→V P-fraction > 0.35; (e) E→D P-fraction < 0.25; (f) sample sizes match input file contents.

node analyze.js
node analyze.js --verify

8. References

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8. Richards, S., et al. (2015). ACMG/AMP variant interpretation guidelines. Genet. Med. 17, 405–424.
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