Acetylcholine-induced vascular smooth muscle contraction and cholinergic synapse pathway variants in variant angina: an exome sequencing study
S Shibutani, T Soma, T Iwasaki, H TomitaAbstract
Background
Variant angina (VA) is characterized by rest angina with transient ST-segment elevation and angiographically normal or non-obstructive coronary arteries. Coronary spasm can be provoked by intracoronary acetylcholine (ACh), yet the genetic basis of ACh-induced coronary hyper-reactivity remains unclear. We hypothesized that rare predicted deleterious variants in genes involved in ACh signaling and calcium-dependent vascular smooth muscle contraction contribute to susceptibility to VA.
Methods
We performed whole-exome sequencing in 73 patients with VA and 30 elderly controls without a history of coronary artery disease. Standard sample- and variant-level quality control was applied, and principal component analysis showed no evidence of population stratification between groups. From KEGG hsa04270 (“Vascular smooth muscle contraction”), we prespecified a mechanistic core set of 40 genes implicated in ACh-induced and calcium-dependent vascular smooth muscle contraction. Rare variants were defined as minor allele frequency <1% in public databases and predicted deleterious by in silico tools (e.g., CADD/REVEL/SpliceAI). The primary analysis compared the proportion of carriers of ≥1 rare predicted deleterious variant in this 40-gene subset between cases and controls using logistic regression (Firth penalization when appropriate). As a secondary, exploratory analysis, we applied the same framework to the KEGG hsa04725 (“Cholinergic synapse”) gene set.
Results
In the ACh-related vascular smooth muscle contraction 40-gene subset, carriers of ≥1 rare predicted deleterious variant were more frequent among VA cases than controls (28/73 [38%] vs 5/30 [17%]; OR 3.11, 95% CI 1.14–10.05; p=0.038). No single gene reached statistical significance; variants were distributed across multiple pathway components, including L-type calcium channels, IP₃ receptors, and myosin light chain phosphorylation–related genes, consistent with a polygenic architecture. As supportive evidence, an exploratory analysis of the Reactome pathway ‘PLC beta mediated events’ (R-HSA-112043) showed enrichment of rare damaging variants in VA (28/73 vs 4/30; OR 3.69, 95% CI 1.32–12.56; p=0.011), providing convergent support for proximal PLCβ–IP₃–Ca²+ signaling. In the cholinergic synapse gene set, carrier frequency was also higher in cases than controls (38/73 [52%] vs 9/30 [30%]; OR 2.53, 95% CI 1.05–6.52; p=0.044).
Conclusions
VA was associated with an increased burden of rare damaging variants in genes related to ACh-induced vascular smooth muscle contraction, supporting a polygenic contribution of ACh-related contraction pathways to coronary spasm susceptibility.