Genetic studies of asthma are few in Hispanic populations, and to our knowledge, this work presents the first asthma GWAS in Mexicans and the most extensive coverage of genetic variation for an asthma GWAS in any Hispanic population. The GWAS included 492 Mexican case-parent trios. Given the moderate GWAS sample size, no SNP met genome-wide significance. However, the ranking of GWAS SNPs highlighted a potentially important candidate region for childhood asthma susceptibility, chromosome 9q21.31. Several chromosome 9q21.31 SNPs with small GWAS p-values were located in TLE4 and its upstream region, and two of these SNPs (rs2378383 and rs2378377) were tested for replication in an independent study of 177 case-parent trios of Mexican ethnicity. Despite the small sample size for replication, both SNPs gave p-values close to 0.05 and the same direction and magnitude of association as the GWAS. Neither rs2378383 nor rs2378377 have a known impact on TLE4 expression, but given their location upstream of TLE4, it is possible that these SNPs reside in a TLE4 regulatory region.
Ancestry analysis in this chromosomal region provided supportive evidence that rs2378383 (G) is associated with a decreased risk of childhood asthma in Mexicans. Ancestry and transmission-based association analyses provide complementary but not completely independent lines of evidence. At each SNP, the log-linear method only used parents who were heterozygous in genotype, while ancestry analysis used all parents who are heterozygous in ancestry, including parents who are homozygous in genotype. We did not a priori expect that ancestry analysis results would corroborate log-linear association results.
Our ancestry analysis uses the same principles that underlie admixture mapping and relies on the key assumption of different risk allele frequencies between the ancestral populations, primarily Native American and European in this study. Under this assumption, individuals with disease in the admixed population would be expected to share an excess of ancestry from the population with the highest risk allele frequency at the disease locus 
. In contrast, individuals with disease in the admixed population would be expected to share a shortage of ancestry from the population with the highest protective allele frequency at the disease locus. At chromosome 9q21.31, there was less Native American ancestry than expected, suggesting that the Native American ancestral population had a higher frequency of the protective rs2378383 allele (G).
Ancestry analysis implicated chromosome 9q21.31 as a chromosomal region that may underlie ethnic differences in childhood asthma. Complex diseases with differing disease prevalence rates in the ancestral populations are most suitable for ancestry analysis 
. Prevalence rates of childhood asthma in the true ancestral Native American, European, and African populations are unknowable, but it is interesting to note that Mexicans have the highest Native American ancestry and the lowest asthma prevalence rate among Hispanic populations 
. Differing frequencies of genetic risk factors in the ancestral populations presumably contribute to the differing prevalence rates of childhood asthma in modern populations. Our study found an association between Native American ancestry and a lower disease risk. Similarly, Native American ancestry was associated with milder asthma in a previous study of subjects of Mexican ethnicity from the GALA study 
. These findings collectively suggest that the Native American ancestral population had higher frequencies of alleles that decrease prevalence and severity of asthma in the modern Mexican population. A comparison of asthma prevalence and severity among modern Native Americans, Europeans, and Africans would further support this interpretation, but such data are scarce 
The evidence for locus-specific ancestry around rs2378383 has implications for replication. Because rs2378383 (G) occurs at relatively low frequency in European, African and East Asian populations, genetic association studies in these populations are likely to suffer from lack of power at this locus. In contrast, the G allele occurs at moderate frequency in the Native American populations surveyed in HGDP 
. Such disparate allele frequencies facilitate ancestry analysis in the region and improve the statistical power of transmission-based tests, as there are many more heterozygous parents in the Mexican population than a European, African or East Asian population. In fact, we obtained association results for SNPs in the chromosome 9q21.31 region from previous GWASs and found that SNPs in this region had only nominal evidence for association with asthma in the GWASs in white populations 
. It is not surprising that substantial evidence for replication was not found given the ethnicity differences (whites for Moffatt et al. and Himes et al. 
and Puerto Ricans for Choudhry et al. 
). Future replication and fine-mapping of the region would be most effective if performed in Native American populations, or admixed populations with high Native American ancestral contributions.
The chromosome 9q21.31 SNPs associated with childhood asthma in the GWAS map to TLE4
and its upstream region. The TLE family of proteins in humans is homologous to the Drosophila Groucho protein, which participates in cell fate determination for neurogenesis and segmentation. The highly conserved structure among the Drosophila Groucho and human TLE gene products suggest similar functions as transcriptional regulators in cell fate determination and differentiation 
. Six genes encode the TLE family of proteins in humans (TLE1
), as deposited in the NCBI database. The distinct expression patterns among the TLE genes suggest a complex mechanism in humans involving non-redundant roles for the TLE genes 
. The TLE4
gene, in particular, shows ubiquitous expression across many tissues 
, and TLE4 functions as a transcriptional co-repressor in several key developmental pathways 
. More specifically, TLE4 has been implicated in early B-cell differentiation. TLE4 interacts with the transcription factor Paired box 5 (PAX5; OMIM 167414), which activates B-cell specific genes and represses alternative lineage fates 
. A spliced version of TLE4 acts as a negative regulator for the PAX5/TLE4 function 
. An alteration of B-cell differentiation involving TLE4 could be relevant to immune development and thus asthma.
TLE interacts with Runt-related transcription factor 3 (RUNX3; OMIM 600210) in a manner that may be directly relevant to asthma. In mice, loss of RUNX3 function results in an allergic asthma phenotype due to accelerated dendritic cell maturation and resulting increased efficacy to stimulate T cells 
. Interaction with TLE is required for RUNX3 to inhibit dendritic cell maturation 
. A recent paper provides support for the interaction of RUNX3 specifically with TLE4 
. Interestingly, the chromosome 9q21.31 SNPs rs2378383 and rs2378377 near TLE4
are associated with asthma as well as degree of atopy in our data, and their associations with asthma became more pronounced when considering only the asthmatic children with atopy and their parents. These findings suggest that the influence of TLE4
on asthma may be related to its influence on immune system development.
Childhood asthma is a complex disease, and there are likely many susceptibility genes influencing immune system development and asthma in the Mexican population. The examination of GWAS in the context of genome-wide expression illustrated the biological plausibility of our GWAS findings and showed consistency with the involvement of multiple genes. Genes expressed in the lung show association signals that differ most significantly from the association signals from genes not expressed in the lung when compared to 50 other human tissues. The lung represents a major pathogenic site for asthma, and this finding implies that multiple genes expressed in the lung are collectively associated with an increased risk of childhood asthma. Some of the other implicated tissues may represent false positives, but several of the highlighted tissues are biologically plausible for childhood asthma, including trachea, bronchial epithelium, smooth muscle, and immune tissues such as thymus, tonsil, and lymph node.
Other GWASs have implicated different susceptibility loci. Several SNPs implicated in the first asthma GWAS by Moffatt et al. in the ORMDL3
were associated with childhood asthma in our GWAS [including rs9303277 (p
0.036), rs11557467 (p
0.014), rs8067378 (p
0.020), rs2290400 (p
0.037), and rs7216389 (p
0.042)] but were not ranked among our top 5,000 SNPs. More recent GWASs have implicated loci other than ORMDL3
. The PDE4D
SNPs implicated by Himes et al. 
were not associated with childhood asthma in our GWAS at p<0.05. Two nearby SNPs, not in LD with the implicated SNPs, were associated [rs13158277 (p
0.030) and rs7717864 (p
0.015)] but were also not ranked among our top 5,000 SNPs. Chromosome 5q23 SNPs implicated by Choudhry et al. 
were not associated with childhood asthma in our GWAS at p<0.05. Initial GWAS findings regarded as replicated may not be ranked among the front runners in a genome-wide scan in the replication populations for statistical 
as well as other biological reasons (such as ethnic differences, phenotypic heterogeneity, genetic heterogeneity, differing patterns of interacting environmental exposures, or multigenic etiology). This trend in discordant GWAS findings is quite common for various complex diseases 
, and follow-up studies are crucial in separating true genetic associations from false positives.
The major limitation of this study is the sample size for the GWAS and replication study. The Mexican population is largely under-studied given its size, and only moderate sample sizes are currently available for the study of asthma genetics. In our study, no SNPs met genome-wide significance, and no replication SNPs met the significance threshold when using a conservative Bonferroni correction for multiple testing. Despite this limitation, top GWAS findings, replication in an independent population, and ancestry analysis taken together implicate a novel region for association with asthma in Mexican children.
This study has several strengths. The case-parent trio design and the log-linear analysis protects against bias due to population stratification 
, so our GWAS results are not confounded by population stratification in this admixed population. Also, disease misclassification is minimal. Although bronchial hyper-reactivity was not tested, children with asthma were given reliable diagnoses based on clinical grounds by pediatric allergists at a pediatric allergy specialty clinic. The allergy clinic is a tertiary referral clinic, so the children with asthma were previously seen by a generalist and a pediatrician over time for recurrent asthma symptoms. Physician diagnosis of asthma has been shown to have a high level of validity in children after the first few years of life 
. Further, the asthmatic children were predominantly atopic to aeroallergens based on skin prick testing limiting heterogeneity of the disease phenotype.
The GWAS and replication association results and the supporting ancestry analysis implicate chromosome 9q21.31 as a novel susceptibility locus for childhood asthma in the Mexican population. This region contains a biologically plausible novel susceptibility gene for childhood asthma, TLE4, but further work is needed to decipher whether TLE4 or a nearby gene explain the signals from the chromosome 9q21.31 region. Further, childhood asthma is a complex disease with a proposed multigenic etiology, but most single studies will not have sufficient power to examine such complex relationships. Identification of important interacting risk factors in childhood asthma and other complex diseases will require very large sample sizes. This work identifies chromosome 9q21.31 (including TLE4) as a novel candidate susceptibility locus for childhood asthma, suggests that this region may underlie ethnic differences in childhood asthma, and emphasizes the presence of multiple genetic risk factors in the complex mechanism leading to childhood asthma.