GWAS Identifies CACNA2D3 Associated With Asthma and Atopic Dermatitis Multimorbidity in Children

Abstract

Allergic diseases significantly impact children's health—especially asthma and atopic dermatitis (AD) multimorbidity [1]. Genome-wide association studies (GWASs) have revealed genetic variants linked to these conditions, elucidating disease mechanisms and potential therapeutic targets. However, most GWASs focus on European populations [2, 3]. We conducted a GWAS with 955 Korean children (mean age 8.4 years) to identify genetic variants associated with asthma and AD multimorbidity, and thereby improve our understanding of these conditions in diverse populations (Table S1).

We analyzed 6.6 million SNPs for asthma and AD multimorbidity. The genomic inflation factor (λ) was 1.03, indicating no systemic bias (Figure S1). Three loci reached genome-wide suggestive significance—CACNA2D3 on chromosome 3p14.3 (p = 5.03 × 10⁻⁷), SMYD2 on chromosome 1q32.3 (p = 2.52 × 10⁻⁷), and SULF2 on chromosome 20q13.12 (p = 6.50 × 10⁻⁷) (Figure 1A and Table 1)—neither of which is reported in previous GWASs on asthma and AD multimorbidity. In our replication cohort of 274 individuals (mean age 8.5 years), we attempted to replicate 20 variants of these three novel loci (Figure S2). 16 SNPs in CACNA2D3 were significantly associated with asthma and AD multimorbidity (p = 0.003), while the SMYD2 and SULF2 variants were not (p = 0.418 and p = 0.109, respectively; Table 1).

The 16 significant SNPs in CACNA2D3 were in the same LD block. (Figure S3) All 16 SNPs located in the CACNA2D3 locus lost statistical significance after conditioning on the lead SNP, rs78970585, indicating a single association signal in this region (Figure S4). The CG and GG genotypes of rs78970585 were less common in the multimorbidity group (p < 0.001; Figure S5). We investigated 12 previously reported SNPs, finding significant associations at the 17q12 locus in IKZF3 (p = 4.76 × 10⁻⁵), and 5q31.1 in KIF3A (p = 0.01; Table S2). We performed in silico analysis using HaploReg v4.2 to examine the function of the lead SNP in CACNA2D3. This revealed that rs78970585 is purportedly related with key regulatory motifs for NF-κB, PPARγ, and RREB1, which relate to inflammation, metabolic regulation, and cell growth, implying a possible functional influence on asthma and AD multimorbidity (Table S3).

To examine the function of CACNA2D3, we conducted functional studies by manipulating CACNA2D3 expressions in both HaCaT and RAW 264.7 cell lines. Specifically, CACNA2D3 overexpression in HaCaT cells (Figure S6A,B) and RAW 264.7 cells (Figure S6C,D) resulted in a marked upregulation of IL-6 and IL-1β mRNA levels compared to vehicle controls. These data support a pro-inflammatory role for CACNA2D3, potentially linking it to the pathophysiology of asthma and AD multimorbidity.

We further analyzed data from the Genotype-Tissue Expression (GTEx) portal, which revealed CACNA2D3 expression in lungs, skin, and whole blood (Figure S7A). Single-cell analysis demonstrated CACNA2D3 expressions were identified including in lung immune cells (Figure S7B). We investigated CACNA2D3 expression in skin at the single-cell level, using a publicly available scRNA-seq dataset (GSE230575) including samples from 4 healthy controls and 10 AD patients (Figure S8A–C). Cluster analysis identified nine distinct cell types, with epithelial cells most abundant in AD patients, and fibroblasts predominant in healthy controls (Figure S8D–G). CACNA2D3 expression in macrophages was significantly lower in AD patients versus healthy controls (Figure S8H,I). Similarly, CACNA2D3 expression was dynamically elevated in the lung's mononuclear phagocytic system in response to allergen-induced inflammation, reinforcing its potential involvement in the asthma and AD multimorbidity (Figure S9A–J). This study elucidates the genetic underpinnings of asthma and AD multimorbidity in a Korean pediatric population, identifying a novel locus, CACNA2D3, associated with decreased risk. In the replication cohort, 16 genome-wide suggestive variants in CACNA2D3 exhibited significant associations (p = 0.003) and retained the same effects in the phenotype homogeneity test (Table S4). The consistent association across various control groups highlights its specific influence on the multimorbid condition, distinct from asthma or AD alone. CACNA2D3 encodes a subunit of voltage-dependent calcium channels, and is crucial in calcium signaling, impacting immune cell functions and inflammatory responses. Calcium channels play pivotal roles in immune cell activation and function [4, 5]. Calcium influx modulation can influence cytokine production, cell proliferation, and apoptosis, which are critical in allergic response development [6]. Variants in CACNA2D3 are associated with lung function metrics [7] (e.g., FEV1, FVC, and FEV1/FVC ratio) and airway inflammation markers (e.g., exhaled nitric oxide [8]), suggesting a possible involvement in asthma severity, progression, and exacerbation risk, even in patients receiving ICS therapy [9].

These findings suggest that CACNA2D3 genetic variants that reduce calcium channel function and/or expression can promote a tempered immune response, thereby mitigating the risk and severity of asthma and AD multimorbidity. The G allele of rs78970585 in CACNA2D3, the identified signal for asthma and AD multimorbidity, is more prevalent in East Asians (0.08) compared to Finnish (0.03) or non-Finnish European (0.01) populations (gnomAD), potentially explaining why it has been underrecognized in European-based studies. The limitations of this study encompass the use of the Infinium HumanCoreExome-24 BeadChip, which has incomplete genomic coverage focused on exonic variants; the relatively small sample size for multimorbidity GWAS; and single-cell validation data derived from an Austrian cohort, which may restrict generalizability to the Korean population; and the absence of an assessment of environmental interactions. While our in silico research indicates a possible regulatory function for rs78970585, we recognize that the lack of direct evidence, such as expression quantitative trait loci (eQTL) data, constitutes a drawback. Nevertheless, our findings underscore a significant genetic marker for asthma and AD multimorbidity in East-Asian populations and support the potential role of CACNA2D3 in immune regulation.

All authors made substantial contributions to the conception or design and data acquisition of the work. D.Y.K., K.W.K., and H.Y.G. designed the study, enrolled the patients, and wrote the paper. Y.S., S.L. contributed to the analysis of single-cell RNA-sequencing. J.H.J. analyzed the clinical data. S.L., E.G.K., M.N.K., S.Y.K., and M.H.S. made substantial contributions to the literature review and data collection. D.Y.K., K.W.K., and H.Y.G. performed the analysis and interpretation of data. D.Y.K., K.W.K., and H.Y.G. drafted the work, and reviewed and edited the manuscript. All authors revised the manuscript for relevant content and gave final approval of the version submitted for publication.

The authors declare no conflicts of interest.