Atrial Fibrillation associated chromosome 4q25 variants are not associated with PITX2c expression in human adult left atrial appendages

Abstract

Atrial Fibrillation (AF), the most common sustained arrhythmia, has a strong genetic component, but the mechanism by which common genetic variants lead to increased AF susceptibility is unknown. Genome-wide association studies (GWAS) have identified that the single nucleotide polymorphisms (SNPs) most strongly associated with AF are located on chromosome 4q25 in an intergenic region distal to the PITX2 gene. Our objective was to determine whether the AF-associated SNPs on chromosome 4q25 were associated with PITX2c expression in adult human left atrial appendages. Analysis of a lone AF GWAS identified four independent AF risk SNPs at chromosome 4q25. Human adult left atrial appendage tissue was obtained from 239 subjects of European Ancestry and used for SNP analysis of genomic DNA and determination of PITX2c RNA expression levels by quantitative PCR. Subjects were divided into three groups based on their history of AF and pre-operative rhythm. AF rhythm subjects had higher PITX2c expression than those with history of AF but in sinus rhythm. PITX2c expression was not associated with the AF risk SNPs in human adult left atrial appendages in all subjects combined or in each of the three subgroups. However, we identified seven SNPs modestly associated with PITX2c expression located in the introns of the ENPEP gene, ∼54 kb proximal to PITX2. PITX2c expression in human adult left atrial appendages is not associated with the chromosome 4q25 AF risk SNPs; thus, the mechanism by which these SNPs are associated with AF remains enigmatic.

Figure 1. Adjusted and unadjusted expression of PITX2c in human left atrial appendages in AF controls.

Log2 PITX2c expression, normalized to ACTC1, in the 16 donor and 24 surgical No AF samples uncorrected (A), or after correction for age and sex (B). There was no significant difference in PITX2c expression between these groups by non-parametric Mann-Whitney t-test. Individual values are shown along with median and interquartile range.

Figure 2. Identification of four SNPs independently associated with AF at the 4q25 locus in the Cleveland Clinic Lone AF GWAS.

A. AF associations of all genotyped SNPs in the 4q25 locus using the marginal model described in Table 3. The locations of the PITX2c and ENPEP genes are shown above. The dashed line is the Bonferroni corrected level of significance for the 150 SNPs tested at p = 0.01. B. Since many of the SNPs in this region are in LD with each other, the AF associations were recalculated after adjustment for the remaining significant SNPs using the full model described in Table 3. The four independently associated SNPs are in the center of the circles in both panels.

Figure 3. PITX2 expression was associated with AF history and rhythm status.

A. Unadjusted levels of PITX2c in the different AF history/rhythm groups revealed a U-shaped relationship with increased AF-status. PITX2c median levels were 17% higher (antilog2 transformed) in the AF/AF vs. AF/SR groups (p<0.001). B. PITX2c levels adjusted for sex and age. The U-shaped relationship was maintained with PITX2c median levels 24% higher (antilog2 transformed) in the AF/AF vs. AF/SR groups (p<0.001). Individual subject data is shown along with the median and interquartile range.

Figure 4. QQ plot of PITX2c eQTLs in the 4q25 region.

Seven SNPs were far above the expected p-value range for association with adjusted PITX2c levels.

Figure 5. PITX2c eQTLs for SNPs in the 4q25 region.

All genotyped SNPs +/−500 kb from PITX2c were evaluated for PITX2c eQTLs. The four AF associated SNPs are shown with arrows. Seven SNPs within introns of the ENPEP gene were associated with expression of PITX2c (p<0.01).

Figure 6. Correlation analysis of PITX2c with SHOX2 expression in human left atrial appendages.

A. SHOX2 expression was inversely correlated with PITX2c expression in all 239 subjects (r = −0.20 p = 0.0021) B. Among the three AF status/rhythm groups, the inverse correlation between SHOX2 and PITX2c was only found in the No AF subgroup (r = −0.47, p = 0.0023).