Among 7 Aspartic Acid-Reference Substitution Pairs in ClinVar Missense Variants With ≥100 Records: Asp→Tyr Is the Most Pathogenic-Enriched (54.7% Pathogenic, Wilson 95% CI [51.6, 57.7]) and Asp→Glu Is the Least (16.3% [14.8, 17.9]) — A 3.4× Range Within the Acidic Reference Amino Acid

Abstract

We compute the per-substitution-target-amino-acid Pathogenic fraction for the 7 Aspartic acid-reference (Asp, D) 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 3.4× range from 16.3% (D → E) to 54.7% (D → Y): D→Y 54.7% Wilson CI [51.6, 57.7]; D→V 51.3% [48.1, 54.6]; D→H 46.5% [43.5, 49.5]; D→A 44.0% [39.4, 48.7]; D→G 39.2% [37.0, 41.4]; D→N 23.3% [22.1, 24.6]; D→E 16.3% [14.8, 17.9]. The chemistry interpretation: the most Pathogenic-enriched alt AAs are tyrosine (charge loss + bulky aromatic introduction) and valine (charge loss + branched-chain hydrophobic). The least Pathogenic-enriched is glutamate — the chemistry-conservative acidic-to-acidic substitution preserving the negative charge with one CH₂ longer side chain. The next-least is asparagine (charge loss + amide preserving similar geometry to Asp, 23.3%). Aspartate substitutions show a clear chemistry-driven Pathogenicity gradient: charge-preserving substitutions (D → E at 16%, D → N at 23%) are well-tolerated; charge-disrupting substitutions with bulky residue introduction (D → Y at 55%, D → V at 51%) are most pathogenic. For variant-prioritization pipelines: the per-target-AA chemistry within Aspartic acid spans a 3.4× range; a D → Y substitution should default to ~55% Pathogenic prior, while D → E should default to ~16%.

1. Background

Aspartic acid (Asp, D) is one of two acidic amino acids (with Glu). Asp side-chain pK_a ≈ 3.7; the residue is fully deprotonated (-1 charge) at physiological pH 7.4. Asp side chain (-CH₂-COO⁻) is one CH₂ shorter than Glu's (-CH₂-CH₂-COO⁻). Functional roles include:

• Salt bridges with positively-charged residues (Lys, Arg, His).
• Calcium coordination in EF-hand domains (alongside Glu); also in coagulation factor Gla-domains where Asp/Glu carboxylates coordinate Ca²⁺.
• Active-site catalysis (e.g., the catalytic Asp in HIV protease, aspartyl proteases, and many enzyme catalytic triads).
• Phosphorylation acceptor in two-component signaling systems (less common in eukaryotes).

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

2. Method

ClinVar missense (alt ≠ X) variants from MyVariant.info / dbNSFP v4. Restrict to ref = D; 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 7 Asp-derived pairs span a 3.4× range (54.7 / 16.3) in Pathogenic fraction.

3.2 The chemistry-class ranking

Tier 1 — Most Pathogenic Asp substitutions (P-fraction > 50%):

• D → Y (54.7%): Charge loss + bulky aromatic ring introduction (Tyr is one of the largest amino acids). Maximum volume increase among the D-derived pairs.
• D → V (51.3%): Charge loss + branched-chain hydrophobic introduction. Disrupts surface electrostatics and may bury hydrophobic residue at solvent-exposed positions.

Tier 2 — Mid-range Asp substitutions (P-fraction 35–50%):

• D → H (46.5%): Charge inversion (negative → partial-positive imidazole). Disrupts salt bridges and may alter active-site catalytic chemistry.
• D → A (44.0%): Charge loss + small methyl side chain. Conservative volume change.
• D → G (39.2%): Charge loss + introduction of conformational flexibility (Gly is the smallest AA). Disrupts both electrostatic and structural roles.

Tier 3 — Least Pathogenic Asp substitutions (P-fraction < 25%):

• D → N (23.3%): Charge loss + amide group preserving similar geometry. Asn's amide can H-bond with similar partners as Asp's carboxylate; the chemistry change is the loss of -1 charge.
• D → E (16.3%): Acidic-to-acidic conservative substitution. Preserves -1 charge (Glu pK_a ≈ 4.3, fully deprotonated at pH 7.4). One-CH₂-longer side chain; minor volume difference. Most chemistry-conservative D-derived substitution.

3.3 The D → E conservative-class minimum

D → E at 16.3% Pathogenic is the least Pathogenic Asp-derived substitution. Mechanism:

• Both Asp and Glu carry -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 Asp residues, Glu substitution is functionally interchangeable.

The high Benign count (1,808) reflects population-genome variation: D → E is a common population variant in many genes.

The 16.3% Pathogenic fraction reflects the subset of Asp positions where the precise side-chain length matters (e.g., catalytic-Asp geometry in aspartyl proteases; EF-hand calcium coordination distance).

3.4 The D → N near-conservative substitution

D → N at 23.3% Pathogenic is the second-least-Pathogenic D-derived substitution. The chemistry change is the loss of the -1 charge while preserving the side-chain geometry (Asn's amide is isoelectronic with Asp's carboxylate, both terminal-CH₂-CN-OH or -CH₂-CO-NH₂). For Asp positions where the H-bonding capacity matters more than the charge, Asn substitution is well-tolerated.

The much higher Benign count (3,370) reflects D → N being a common population variant.

3.5 The D → Y maximum: charge loss + maximum volume increase

D → Y at 54.7% Pathogenic is the most Pathogenic Asp-derived substitution. Mechanism: Tyr is one of the largest amino acids (~30% larger than Asp by side-chain volume), with an aromatic ring + hydroxyl. The substitution introduces:

• Charge loss (essential for any structural role of the Asp -1 charge).
• Steric clash from the bulky aromatic ring in positions that fit a small Asp side chain.
• Hydrophobic-patch creation on what was a hydrophilic surface.

Combined, these effects make D → Y a highly Pathogenic substitution.

4. Confound analysis

4.1 Stop-gain explicitly excluded

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

4.2 ClinVar curatorial bias

Asp Pathogenic variants are over-reported in disease genes with critical Asp-functional residues (calcium-binding EF-hand, Gla-domain coagulation factors, catalytic Asp in aspartyl proteases, kinase catalytic loop Asp residues). The per-pair Pathogenic fractions partly reflect curation focus on these gene families rather than a generic Asp-pathogenicity rule.

4.3 Codon-mutability not normalized

Asp has 2 codons (GAT, GAC). The per-target-AA mutational rates differ across the 7 alt AAs reported. D → N (GAY → AAY) is a one-step transition; D → E (GAY → GAR) is a one-step transition; D → Y (GAY → TAY), D → H (GAY → CAY), D → A (GAY → GCY), D → G (GAY → GGY), D → V (GAY → GTY) are also 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. Asp-derived substitutions with < 100 records (D → S, D → T, D → C, D → L, D → I, D → M, D → F, D → W, D → P, D → Q, D → R, D → K) are not analyzed.

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 7 Asp-derived substitution pairs, D → Y is the most Pathogenic-enriched at 54.7% (Wilson CI [51.6, 57.7]) — driven by charge loss + maximum volume increase.
2. D → E is the least Pathogenic-enriched at 16.3% [14.8, 17.9] — a conservative acidic-to-acidic substitution.
3. D → N at 23.3% is the next-least, preserving Asp's side-chain geometry but losing the charge.
4. For variant-prioritization pipelines: per-target-AA priors within Asp should be applied; D → Y ~55%, D → E ~16%.
5. The Asp chemistry-class continuum is preserved: charge-disrupting + volume-increasing substitutions are most pathogenic; charge-preserving (E) or geometry-preserving (N) substitutions are least pathogenic.

6. Limitations

1. Stop-gain excluded (§4.1).
2. ClinVar curatorial bias (§4.2) toward EF-hand calcium-binding and catalytic-Asp gene families.
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 7 reported pairs have N ≥ 100; (d) D→Y P-fraction > 0.5; (e) D→E P-fraction < 0.2; (f) sample sizes match input file contents.

node analyze.js
node analyze.js --verify

8. References

1. Landrum, M. J., et al. (2018). ClinVar. Nucleic Acids Res. 46, D1062–D1067.
2. Liu, X., Li, C., Mou, C., Dong, Y., & Tu, Y. (2020). dbNSFP v4. Genome Med. 12, 103.
3. Wu, C., et al. (2021). MyVariant.info. Bioinformatics 37, 4029–4031.
4. Wilson, E. B. (1927). Probable inference, the law of succession, and statistical inference. J. Am. Stat. Assoc. 22, 209–212.
5. Brown, L. D., Cai, T. T., & DasGupta, A. (2001). Interval estimation for a binomial proportion. Stat. Sci. 16, 101–133.
6. Davies, D. R. (1990). The structure and function of the aspartic proteinases. Annu. Rev. Biophys. Biophys. Chem. 19, 189–215. (Aspartyl protease catalytic-Asp reference.)
7. Strynadka, N. C., & James, M. N. (1989). Crystal structures of the helix-loop-helix calcium-binding proteins. Annu. Rev. Biochem. 58, 951–998. (EF-hand reference.)
8. Richards, S., et al. (2015). ACMG/AMP variant interpretation guidelines. Genet. Med. 17, 405–424.
9. Cooper, D. N., & Krawczak, M. (1990). The mutational spectrum of single base-pair substitutions causing human genetic disease. Hum. Genet. 85, 55–74.
10. Grantham, R. (1974). Amino acid difference formula to help explain protein evolution. Science 185, 862–864.