This article has been updated to reflect clarifications and additonal information by Kathleen Barnes.
NEW YORK (GenomeWeb) – While people typically expect asthma inhalers to work on children and adults of all ages and ethnic backgrounds, recent genetic research suggests that common asthma inhalers fail minority children groups.
Two different groups — one at the University of California, San Francisco and another at the University of Colorado-Denver — are independently making progress in their quests to identify the genetic factors contributing to the inefficacy of bronchodilators in groups with African ancestry.
One of the most common chronic childhood diseases, asthma causes children to struggle breathing due to airway inflammation. Inhaling drugs called bronchodilators can alleviate the condition by soothing the muscles lining the airway and allowing them to reopen.
While researchers have detected clusters of genes on chromosome 17 associated with childhood asthma using multiple genome-wide association studies, many have found that linkage disequilibrium on the chromosome makes it difficult to identify causal variants for asthma. Therefore, researchers believe that the associations seen on chromosome 17 could act as decoys or signals for the actual causal variants outside the cluster.
In a study published last month in the American Journal of Respiratory and Critical Care Medicine, Esteban Burchard, director of the UCSF Asthma Collaboratory, and his team performed the first large-scale whole-genome sequencing study of asthma drug response in African American and Latino children in order to identify genetic factors that cause reduced albuterol response.
According to Burchard, his team at UCSF has been studying the genetics of asthma in minority populations for over ten years. The group has previously identified genetic risk factors for higher rates of asthma and poor bronchodilator response (BDR) in minority populations. Burchard explained that his team wanted to find out the pharmacogenetic basis behind the ethnic and racial differences.
In order to test for albuterol response, the researchers measured patients' ability to blow into a breathing machine. Giving each patient a standard dosage of albuterol, the researchers waited 15 minutes to allow the airways relax and open up, then repeated the breathing measurement to determine the the drug's impact and then collected the patient's blood sample.
In total, Burchard and his team amassed 1,441 whole blood samples from three different racial groups, representing individuals with asthma who had either very high or very low response to albuterol. Plotting out their responses, Burchard's group performed WGS on the blood samples in order to detect potential genetic variants.
Burchard explained that the investigators initially analyzed each group's data separately, and then performed a meta analysis of all three groups. While they did not find any significant associations from the population-specific analyses, they identified 27 unique variants in 10 different genetic regions linked to reduced albuterol response, implicating genes involved in lung capacity, immune response, and responses to blockers and related medicine in patients.
"The 27 SNPs explain 23 percent, 16 percent, and 18 percent of the variation in BDR status in Puerto Ricans, Mexicans, and African American children respectively," Burchard noted. "Many of the loci were racially or population-specific rare variants."
In order to prioritize associated variants for further evaluation, they performed chromatin immunoprecipitation sequencing (ChIP-seq) analysis to identify variants overlapping with regulatory regions in primary bronchial smooth muscle cells (BSMC). Using an algorithm called Diverse Convergent Evidence (DiCE) to estimate the strength of information supporting each mutation, they then focused on variants with the highest score for downstream functional analysis.
After integrating information from its WGS analysis, publicly available bioinformatics data, and the ChiP-sequencing experiments, the team found that the NFKB1 locus had the highest DiCE score and strongest evidence of functional relevance to BDR variation.
"We settled on NFKB1, which was interesting because in addition to being the variant associated with low drug response, it also tracks African ancestry," Burchard explained. "It's higher in Caribbean, African American, and Puerto Rican populations."
In order to demonstrate the NFKB1 variant's importance, the researchers tested two regions that overlapped with the BDR-associated NFKB1 locus using luciferase enhancer assays in BSMCs. They saw that one enhancer, NFKB1 Region 2, showed significantly increased activity over a empty vector control.
The team also performed RNA-seq experiments in African American children with asthma to see if the NFKB1 SNPs managed expression of neighboring genes. Burchard and colleagues found that the low-BDR associated T-allele of NFKB1 was linked to decreased expression of SLC39AB in the blood, suggesting that the allele has a higher frequency in populations of African descent.
Burchard believes that NFKB1 could act as a regulatory element for other genes besides SLC39AB. In addition, the researchers hope that additional new risk biomarkers in the genome could be used to predict which children do not respond well to albuterol and other anti-asthma drugs.
Burchard emphasized certain limitations in the study, including the fact that it is impossible to identify age-matched and population matched cohorts to replicate the study's data, primarily because there is a dearth of research data that has been published on children of ethnic minorities with asthma..
In the future, Burchard's team plans to perform further functional studies to establish the role of NFKB1 and other potential gene variants on BDR, eventually focusing on more genes that his team believes will be strong targets for biomarkers that cover a wider population. Burchard has long called for diversity in clinical and biomedical research in respect to asthma.
"The NIH has answered the call, and have invested a lot of money in doing WGS in minority populations, and the data is forthcoming," Burchard said. However, he also noted that continued funding may be an issue in the future. "Although the [National Institutes of Health] has a commitment to improve diversity, it doesn't translate well to the study section," Burchard explained. "Despite our great success, it's been challenging to earn additional NIH funding."
Multi-omic analyses
On the other side of the Rocky Mountains, researchers at the University of Colorado-Denver aim to identify certain genetic biomarkers in airway epithelial cells that they believe could contribute to asthma risk in individuals with African ancestry. They recently completed a WGS study on 1,100 African Caribbean asthmatics and non-asthmatics, phenotyping and following participants for more than 20 years who lived in Barbados.
Now, under a larger five-year NIH grant, the team is recruiting a subset of the Barbados subjects to build a new genome catalog that could provide information on genetic variations in patients of African descent that cause resistance to first-line drugs for asthma like albuterol.
Kathleen Barnes, professor of medicine at CU-Denver and principal investigator, and her team will perform an expression quantitative trait locus (eQTL) study combining WGS and transcriptome data. She explained that her team has partnered with the University of West Indies and Barbados since 1991 to perform different types of clinical studies on the population.
The team seeks to find genetic determinants that may indicate increased risk of asthma, and if variation in the transcriptome of the nasal epithelium methylome might mediate the relationship between genotype, phenotype, and environments.
Barnes' group will first attempt to identify cis- and trans- effects of variants identified in the transcriptome for isolated nasal epithelial cells from atopic asthmatics. They will examine local and distal signals that might indicate genetic resistance against drugs like albuterol.
The team will then use eQTL mapping to identify patterns from nasal epithelial cells specific to atopic asthma. While Barnes has genomic data from the previous study, her team will now focus on asthmatics who are allergic and then compare them to controls who are allergic but do not have asthma.
Barnes explained that the study will include 400 samples in the discovery phase, in addition to 200 samples to replicate signals identified in the first phase. The team will use data from the 600 samples to test whether polymorphisms control asthma by regulating gene transcription and methylation patterns in nasal airway epithelial cells.
Because bronchoalveolar tissue collection can be invasive and relatively expensive, Barnes' team instead performs a process called "nasal brushing," which involves sticking a soft brush into a subject's upper nasal cavity to capture epithelial cells. By collecting samples that act as a proxy for the lower airway environment, the team can reduce sample heterogeneity and focus on the tissue they believe is most relevant to cases of atopic asthma.
After tissue collection, Barnes' team will isolate DNA from samples to identify methylation changes associated with atopic asthma. In addition, they will isolate RNA from samples to perform RNA-seq.
"By using the multi-omic approach [and] combining gene expression data generated by the upper airway epithelium, where we're looking at epigenetic effects, we are optimistic that it will help us home in on the actual causal gene," Barnes explained. "We may certainly identify new pathways we'd not think about before the genomic revolution."
Barnes noted that the study's major initial limitations will stem from logistical and public relation issues while collecting samples in the field. She explained that many participants in Barbados can be weary of being subjects of repeated research collection. Sensitive to this fatigue, Barnes and her team often provide updates to the population about their cooperation in studies and provide results for previous genetic-based tests on their visits to Barbados.
In addition, Barnes acknowledges some of the logistical issues of delivering samples back to the lab in Colorado. Simple elements, such as having enough dry ice to preserve tissue samples long enough to arrive at the lab, can be logistical challenges that she said people often do not think about when performing research in the field.
"One of the challenges in global health research [is] that the place you're conducting your research does not always have the same infrastructure you can count on, especially when you have to transport [samples] half a world away," Barnes explained.
While the CU-Denver and UCSF-led research teams are examining minority population groups in different geographical locations, both are aiming to identify the genetic cause behind reduced first-line asthma drug response for these populations. In addition, they both believe that more than one gene may cause resistance to asthma drugs in populations with African descent.
While the CU-Denver study is in its early stages, Barnes wants to leverage different types of omics datasets to produce predictive genetic risk scores for multiple diseases including cancer and cardiovascular disease.
"That's really what we want to garner from this study, to have data points to develop predictive risk scores for these kids and save lives," Barnes noted. "Our goal is to move into a space where no one dies of asthma, because with the right therapeutic, it can be managed."
When asked about the UCSF study, Barnes said the results are potentially meaningful because the team has identified new loci associated with resposne to asthma therapy that may be unique to underrepresented minority populations.
"The study is novel because it's the first of its kind leveraging whoe genome sequence data in a relatively large dataset of minorities, but it falls short of what has been shown previoulsy in asthma pharmacogenetics in terms of differnece according to ancestry," Barnes explained.
Burchard acknowleges that the UCSF study's findings could be potentially helpful in the Barbados study as her team begins to rerecruit. She also noted that the UCSF study could be utilized further by comparing findings in a European population.
"At the end of the day, we want to identify biomarkers for asthmatic individuals," Barnes explained. "If a patient walks into the clinic, and you suspect or want to detect if they will develop asthma, and wish there was something you could collect on the spot, then you [might eventually] use this test as a predictor."