Among 8 Glycine-Reference Substitution Pairs in ClinVar Missense Variants With ≥100 Records: Gly→Cys Is the Most Pathogenic-Enriched (63.7% Pathogenic, Wilson 95% CI [60.4, 66.8]) and Gly→Ser Is the Least (28.9% [27.8, 30.1]) — A 2.2× Range Driven by Substitution Chemistry Within the Same Reference Amino Acid

Abstract

We compute the per-substitution-target-amino-acid Pathogenic fraction for the 8 Glycine-reference (Gly, G) substitution pairs with ≥100 ClinVar missense single-nucleotide variants in the dbNSFP v4 (Liu et al. 2020) annotation of 372,927 ClinVar P+B 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. Glycine is the second-most-frequent reference amino acid in our missense Pathogenic set (8,826 of 62,221 = 14.18%), after Arg. Result: per-target-AA Pathogenic fractions span a 2.2× range from 28.9% (G → S) to 63.7% (G → C): G→C 63.7% Wilson CI [60.4, 66.8]; G→V 63.6% [61.6, 65.5]; G→W 62.3% [57.2, 67.2]; G→D 54.5% [52.8, 56.3]; G→E 51.7% [49.8, 53.6]; G→R 50.2% [49.0, 51.3]; G→A 39.2% [36.8, 41.6]; G→S 28.9% [27.8, 30.1]. The ranking has a clear chemistry interpretation: the most Pathogenic-enriched alt AAs are cysteine (introduces sulfhydryl group; potential aberrant disulfide), valine (introduces bulky branched-chain hydrophobic side chain in a flexible/turn-position context), and tryptophan (introduces large aromatic side chain). The least Pathogenic-enriched are serine (small polar with single hydroxyl; minimal volume change) and alanine (small methyl side chain; conservative volume increase). The 2.2× range is narrower than the per-pair range observed for Arg-derived substitutions (4.2× span), consistent with Gly's already-tolerant baseline (Gly is the smallest amino acid; substitutions are generally non-conservative and most are pathogenic). For variant-prioritization pipelines: an observed G → C substitution carries a 64% Pathogenic prior; G → S only 29% — a 2.2× per-prior difference even within the same reference AA. Glycine pathogenicity is dominated by introduction of bulky or chemistry-altering side chains; small polar substitutions (S, A) are the most-tolerated even at Gly positions.

1. Background

Glycine (Gly, G) is the smallest amino acid — its side chain is just a hydrogen atom. This makes Gly uniquely flexible: Gly residues populate Ramachandran-disallowed regions in protein structures (Lovell et al. 2003), serve as turn-junctions, and provide backbone flexibility in disordered regions. Substitutions at Gly typically introduce a side chain of variable size and chemistry, with downstream effects depending on the structural context.

Gly is the second-most-frequent reference AA in ClinVar Pathogenic missense (after Arg), partly because:

• 4 codons (GGT, GGC, GGA, GGG) — multiple missense-creating single-nucleotide neighbors per residue.
• Gly is structurally constrained at turns and Ramachandran-disallowed positions; substitutions at these positions are functionally disruptive.
• Collagen Gly-X-Y triplet motifs (Pepin et al. 2000) are heavily curated for collagen-disease-related Gly substitutions.

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

2. Method

Identical to the per-AA template: ClinVar missense (alt ≠ X) variants from MyVariant.info / dbNSFP v4. Restrict to ref = G; 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)

3.2 The chemistry-class ranking

Tier 1 — Severely Pathogenic substitutions (P-fraction > 60%):

• G → C (63.7%): Introduces a sulfhydryl group; potential aberrant disulfide bond formation with nearby Cys residues; disrupts turn geometry.
• G → V (63.6%): Introduces a bulky branched-chain hydrophobic side chain at a typically flexible/exposed Gly position.
• G → W (62.3%): Introduces a large aromatic side chain; the Gly → Trp volume increase is the largest of all 19 possible Gly substitutions.

Tier 2 — Mid-range Pathogenicity (P-fraction 50–60%):

• G → D (54.5%), G → E (51.7%), G → R (50.2%): Introduce charged side chains (acidic D/E or basic R) at typically uncharged Gly positions. The 50–55% Pathogenic fractions reflect the high rate of these substitutions in collagen Gly-X-Y triplet motifs (collagen disease genes).

Tier 3 — Less-Pathogenic substitutions (P-fraction < 40%):

• G → A (39.2%): Introduces a small methyl side chain; minimal volume change; least disruptive non-conservative Gly substitution.
• G → S (28.9%): Introduces a small polar hydroxyl side chain; minimal volume change; modest H-bonding capacity addition. The most-Benign Gly-derived substitution.

3.3 The G → S Benign-enriched signal

G → S has the lowest Pathogenic fraction at 28.9% (Wilson CI [27.8, 30.1]). Mechanism: Ser is the closest amino acid to Gly in volume (Ser methyl-OH side chain vs Gly H side chain; ~+30 ų volume increase) and the only common substitution that preserves Gly's small-side-chain character. The high Benign count (4,021) reflects population-genome variation: G → S is a common population variant in many genes.

3.4 The G → C Pathogenic-enriched signal

G → C has the highest Pathogenic fraction at 63.7% (Wilson CI [60.4, 66.8]). Mechanism: Cys introduces a sulfhydryl group that can form aberrant disulfide bonds with nearby Cys residues. In collagen Gly-X-Y triplets specifically, G → C substitutions are well-known to cause Ehlers-Danlos syndrome type IV (COL3A1) and related collagenopathies (Pepin et al. 2000). The 63.7% Pathogenic fraction reflects strong selection against Gly → Cys at structured positions.

3.5 The collagen-disease-gene contribution

Many Gly Pathogenic variants in our cohort come from collagen genes (COL1A1, COL3A1, COL4A5, COL2A1, COL7A1, etc.) where the Gly-X-Y triplet motif is structurally essential. G → V, G → R, G → D substitutions in this triplet motif disrupt the collagen triple helix.

The ~50% Pathogenic fractions for G → R, G → D, G → E partly reflect the collagen-curation contribution: ClinVar has many collagen variants curated as Pathogenic.

4. Confound analysis

4.1 Stop-gain explicitly excluded

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

4.2 ClinVar curatorial bias

Collagen genes are heavily curated for Gly substitutions (collagenopathies are clinically well-characterized). The mid-range Pathogenic fractions (G → D, E, R at 50–55%) partly reflect this curation focus rather than a generic Gly-pathogenicity rule.

4.3 Codon-mutability not normalized

Gly has 4 codons (GGN); the per-target-AA mutational rates differ. G → S is achieved through GGN → AGN single-nucleotide transitions which are more frequent than other transversions. 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. At ≥30, the analyzed set may include rare Gly-derived pairs (G → I, L, M, F, Y, K, H, N, Q, P, T) which are 2-step-away codon transitions and are less frequent.

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 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 8 Gly-derived substitution pairs, G → C is the most Pathogenic-enriched at 63.7% (Wilson CI [60.4, 66.8]) — driven by aberrant disulfide formation and collagen-gene curation.
2. G → S is the least Pathogenic-enriched at 28.9% [27.8, 30.1] — a near-conservative small-polar substitution.
3. The 2.2× per-target-AA range within Gly-reference is narrower than Arg-reference (4.2× range) — Gly's already-tolerant baseline (smallest AA, Ramachandran-disallowed positions) reduces the per-pair spread.
4. Collagen Gly-X-Y triplet substitutions drive the mid-range Pathogenic fractions for G → D/E/R.
5. For variant-prioritization pipelines: per-target-AA priors within Gly should be applied; G → C ~64%, G → S ~29%.

6. Limitations

1. Stop-gain excluded (§4.1).
2. ClinVar curatorial bias for collagen genes (§4.2).
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.
• Verification mode: 6 machine-checkable assertions: (a) all P-fractions in [0, 1]; (b) Wilson CIs contain the point estimate; (c) all 8 reported pairs have N ≥ 100; (d) G→C P-fraction > 0.6; (e) G→S P-fraction < 0.35; (f) sample sizes match input.

node analyze.js
node analyze.js --verify

8. References

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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.
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6. Pepin, M., et al. (2000). Clinical and genetic features of Ehlers-Danlos syndrome type IV. N. Engl. J. Med. 342, 673–680. (Collagen Gly-X-Y triplet G → C reference.)
7. Lovell, S. C., et al. (2003). Structure validation by Cα geometry: phi, psi and Cβ deviation. Proteins 50, 437–450. (Glycine Ramachandran-disallowed positions reference.)
8. Richards, S., et al. (2015). ACMG/AMP variant interpretation guidelines. Genet. Med. 17, 405–424.
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10. Grantham, R. (1974). Amino acid difference formula to help explain protein evolution. Science 185, 862–864.