A genomic region on chromosome 1q33-q32.31 met genome-wide significance for asthma in the Isle of Wight cohort. Subsequent detailed examination using a combination of targeted genotyping and haplotype analysis in the primary population, along with genotyping plus imputation in consortia controls, confirmed the association with an LD block containing ATPAF1, C1ORF223, and KIAA0494 genes. Furthermore, asthma severity was associated with SNPs and haplotypes in this LD block. Replication studies pursued in eight independent populations revealed instances of strict replication with specific SNPs and haplotypes in a Caucasian European population. Further gene-level association was found with additional SNPs and haplotypes (genotyped using other platforms) in the other three non-Hispanic replication populations. Thus, while not all significant findings in the primary population were replicated, major trends in association were identified across the LD block in all but the populations of Hispanic descent. Finally, data demonstrating differential upregulation of ATPAF1 expression in asthmatics as compared to control subjects lend support for a role for this gene, which is novel to asthma.
While no previous asthma linkage or association has been as finely mapped on 1p3 as the current study, our work supports previous studies that implicated the 1p31 to 1p36 region.29–38
Indeed, the chromosome region 1p has been consistently identified in genome screens for asthma and related phenotypes in populations of different races and ethnicities.29–31, 33
Thus, our finding of association of asthma with a 115 kb LD block at 1p33-p32.31 both support and extend these earlier studies.
are adjacent genes on chromosome 1p33-p32.31 and occupy much of a 115 kb LD block ().38
Little is known about C1ORF223
(open reading frame for protein LOC37497, which is expressed predominately in testes)39
(widely expressed inferred calcium binding ion protein)39
genes and due to the overlap of coding and regulatory sequence among the three genes, additional studies on each gene are warranted. While we cannot exclude C1ORF223
from having a role in asthma, we chose to prioritize further study of ATPAF1
because 1) there is a well-established relationship between puringeric (ATP and adenosine) signalling and bronchoconstriction,40, 41
and 2) epithelial cell expression of the Th2 promoting cytokine IL-33 is regulated by purinergic signalling.42
is a nuclear gene encoding ATPAF1 (ATP11), which is a soluble mitochondria protein that binds to unassembled β subunits of the F1
and prevents the F1
alpha and beta subunits from aggregating in the matrix.44
The mechanism of correct assembly of the ATP synthase F1
complex requires ATPAF145
and is preserved in all eukaryotic lineages capable of ATP synthesis via oxidative phosphorylation.46 ATPAF1
is widely expressed, including in whole lung tissue.47
Functional significance is predicted for several of the SNPs associated with asthma in this study.38, 48
Specifically, sequence encompassing rs1258000 is typical of regulatory elements.38, 49
Similarly, rs620431 has high regulatory potential and also lies 60 bp downstream of the exon 6/intron 6 boundary making it a potential splicing modulating element for the alternatively spliced exon 7.38
The most direct evidence of functional relevance of the ATPAF1
gene in asthma comes from its differential expression in bronchial tissue between asthmatics and controls (). ATPAF1
was highly expressed in bronchial biopsies from those with severe asthma. Not only does this suggest a mechanism by which the gene may modify asthma risk, but it is also consistent with the findings of Chen et al50
in which they report that genes that are differentially expressed have a greater likelihood of containing variants that cause disease. Furthermore, the elevated ATPAF1
expression in bronchial tissue from severe asthmatics is consistent with and builds upon our findings of SNP and haplotype associations with asthma severity among the Isle of Wight children. Indeed, the importance of ATP-signalling in bronchoconstriction makes the link that we have identified between asthma severity and ATPAF1
expression and genetic variants all the more compelling.
The risk of reporting statistical significance merely by chance is a major concern in association studies in which a high number of tests are conducted. However, this is unlikely in the present study as several SNPs retained significance at the genome-wide level after correction for multiple testing, the outcomes were consistent across individual SNP and haplotype analyses, and SNPs and haplotypes showed association with asthma in the replication populations of the same race. In addition, data showing functional relevance of ATPAF1 further reinforce the validity of our findings.
For replication, we chose populations that had previously been studied for asthma genetics. We did not limit our selection to populations of the same ancestry as the primary cohort because inclusion of diverse populations broadens the relevance of the information generated. Indeed, variability in replication between cohorts has been a feature of studies of asthma genetics.7, 51, 52
We found significant associations in all the Caucasian populations, along with the one African American population, and one population of other ethnicity (not Caucasian, African American, or Hispanic). Interestingly, none of the populations of Hispanic descent (Hispanic, Puerto Rican and Mexican) showed association, indicating a race-specific trend. As expected, there were allele frequency differences between the populations of different races (Table E6
). The allele frequencies across the four populations of Hispanic origin were fairly consistent with one another, with their minor allele frequencies in most cases ≥10% higher than in the three Caucasian populations. However, the minor allele frequency differences tended to be greatest between the Caucasian and African populations, which would not explain the observed trend. Other possible explanations for lack of replication in the Hispanic ancestry populations include type II error due to lack of power and differences in environmental exposures between cohorts.
In conclusion, our sequential strategy, as well as the use of well-phenotyped populations, led to identification of an association between ATPAF1 region variants and asthma. Studies to further understand the mechanistic role of this gene in asthma are being pursued.