**7. Pharmacogenetics of asthma**

The US Food and Drug Administration definition for pharmacogenomics is "the study of variations in DNA and RNA characteristics as related to drug response" [138]. "*Pharmacoge‐ nomics*" differs from "*Pharmacogenetics*" in that the former is concerned with the whole genome, its components, and regulators, while the latter is focused only on the DNA sequences of individual gene. Thus, in sense, pharmacogenetics is thought to be a subset of pharmacoge‐ nomics [139].

Because it is a complex trait, the drug response to asthma is diversely heterogeneous even among patients with apparently similar clinical profiles [7]. It is estimated that up to 50% difference in therapeutic response has been attributed to genetic variations between individ‐ uals [140]. Although several possible mechanisms have been postulated, genetic variants affect the pharmacogenetic response to drugs in two different ways:


Single nucleotide polymorphism, SNP, denoted by a reference sequence (rs) number, represents a class of polymorphism that is derived from a one-base point mutation in which a single nucleotide is substituted with another one. SNPs may be located in the gene regulatory or coding regions, and so it may affect the gene expression in more than one way; however, in majority of cases, most discovered SNPs do not change the gene function in a significant manner [141]. Consequently, it is essential to investigate whether the DNA sequence variances would actually cause significant functional impacts (i.e., resulting in an altered observed biology), or is a linkage disequilibrium marker of another DNA variant, which is the real cause of the response variability, or is generally nonsignificant. Because of its strong importance, since 15 years, catalogs of SNPs have started to outline the most common genetic polymorphisms among different population groups [141, 142], and this process has attracted more attention during the last couple of years [143].

its cellular receptors [132]. Several humanized IL-13-neutralizing antibodies have entered asthma phase I/II clinical trials—anrukinzumab [133], QAX576 [134], and CAT354 [135].

TNF-α, a cytokine produced by Th1 cells and macrophages, has diverse biological functions. TNF-α shows crucial, and previously extensively documented, role in Crohn's disease, rheumatoid arthritis, and psoriasis pathogenesis. The association between TNF-α increase and these disease progressions had inspired studies aiming to extend anti-TNF-α therapies also for the treatment of severe asthma and COPD [136]. Infliximab and Golimumab, two anti-TNFα mAbs, and Etanercept, a decoy soluble TNF-α receptor, are both able to biologically neutralize TNF-α cytokine, and blunt the immune response, thereby abolishing TNF-α effects

The US Food and Drug Administration definition for pharmacogenomics is "the study of variations in DNA and RNA characteristics as related to drug response" [138]. "*Pharmacoge‐ nomics*" differs from "*Pharmacogenetics*" in that the former is concerned with the whole genome, its components, and regulators, while the latter is focused only on the DNA sequences of individual gene. Thus, in sense, pharmacogenetics is thought to be a subset of pharmacoge‐

Because it is a complex trait, the drug response to asthma is diversely heterogeneous even among patients with apparently similar clinical profiles [7]. It is estimated that up to 50% difference in therapeutic response has been attributed to genetic variations between individ‐ uals [140]. Although several possible mechanisms have been postulated, genetic variants affect

**1.** *Pharmacodynamic genetic variations* are variations in which the receptor binding the drug ligand or another member of the drug target pathway is altered resulting in different drug effect. Most of the current pharmacogenetic research fall into this mechanistic category. Populations are stratified into responders and nonresponders, and then analyzed for DNA

**2.** *Pharmacokinetic genetic variations* are related to altered uptake, distribution, and/or metabolism of the administered drug. Fewer examples fall in this category; however, the most common research subfield here is the area of investigating drug-catabolizing or excreting enzymes. An important example here is the cytochrome *P450* (CYP450) family, a widely recognized metabolizing enzyme with several variable pharmacogenetic

Single nucleotide polymorphism, SNP, denoted by a reference sequence (rs) number, represents a class of polymorphism that is derived from a one-base point mutation in which a single nucleotide is substituted with another one. SNPs may be located in the gene regulatory

the pharmacogenetic response to drugs in two different ways:

polymorphisms, which distinguish these two groups apart.

*6.4.4. Anti-TNF-α*

in asthma [137].

nomics [139].

patterns.

**7. Pharmacogenetics of asthma**

160 Asthma - From Childhood Asthma to ACOS Phenotypes


**Figure 4.** Pharmacogenetically significant genes with relevance to corticosteroids, β-adrenergic, and leukotriene bio‐ logical pathways. Left side: Candidate gene approach studies, Right side: GWAS (Genome Wide association studies).

All genes contain huge number of SNPs and copy-number variations (CNVs). CNVs are another form of structural variations, which account for 13% of the human genome bulkiness, and manifest as kilo-to-mega bases of deletions or duplications [144]. Conjointly, it is challenging to outline which polymorphism is influencing the treatment response and which are not relevant. Two major approaches declaiming this challenge have been practiced so far: candidate gene approach and GWAS. As it combines transcriptomic, proteomic and metabolomic profiling traits, a third approach, the integrative system biology approach, had led to a more comprehensive pharmacogenetic view [3]. To differentiate, candidate gene approach is based on a prior evidence according to the knowledge of the drug pharmacody‐ namics or/and pharmacokinetics, by contrast, GWAS methodology identifies new associations with the null hypothesis being that no associations exist. GWAS picks the variations which are associated with observable phenotypes by scanning SNP markers that tag, via linkage disequilibrium, the complete human genome. GWAS and integrative system biology approaches are modern tools contributing to the recent advancements of genotyping and statistical technologies.

Current pharmacogenetic studies of the corticosteroids, β-adrenergic, and leukotriene pathways are mostly candidate gene studies, with some GWAS, however, altogether have identified several genetic loci in strong association with therapeutic responsiveness to asthma. **Figure 4** summarizes the pharmacogenetically significant genes with relevance to the corticosteroids, β-adrenergic, and leukotriene biological pathways.

#### **7.1. Corticosteroid pathway pharmacogenetics**

In cytosol, the glucocorticoids bind to their corresponding glucocorticoid receptor, forming a hetero-complex that is activated by ligand binding, and translocate into the nucleus. In the nucleus, this complex binds to the glucocorticoid response elements in some target genes' promoter region resulting in their expression regulation. The core role of glucocorticoids is mediated via activating the transcription of anti-inflammatory genes, and suppressing the transcription of pro-inflammatory genes [145, 146]. The glucocorticoid pharmacogenetic studies formerly focused on candidate gene approach. Those candidate genes covered functions related to the corticosteroid biosynthetic pathway, the hetero-complex receptor formation, and the related chaperone proteins.

Corticotrophin-releasing hormone *(CRHR1)*, stress-inducible protein 1 *(STIP1), TBX21, CYP3A4, GLCCI1*, T gene, and *FBXL7* are the most up-to-date potential pharmacogenetic biomarker targets for predicting patients' response to ICS [147]. Studies of the corticotrophinreleasing hormone gene are considered to be as one of the oldest and remarkable footsteps in asthma pharmacogenetics. CRHR1 protein, also known as CRF1, is the primary receptor controlling the adrenocorticotropic hormone release; hence, it plays a pleiotropic and vital role in steroid actions. A candidate gene study of *CRHR1* in 1117 asthmatics administrating ICS therapy, from three clinical cohorts, revealed two SNPs (rs242941 and rs1876828) associated with different response in lung functions [148]. Tantisira et al. [148] found that *CRHR1* gene variation was frequently related to augmented therapy response in each of the three studied cohorts. Since 2004, *CRHR1* gene studies opened the doors for all other corticosteroid phar‐ macogenetics and the possible future therapeutic outcomes.

*STIP1* or *HOP* (abbreviated for Hsp70-Hsp90-Organizing Protein) gene mainly functions to reversibly link Hsp70 and Hsp90 together as a co-chaperone [149]. *STIP1* pharmacogenetic studies in one adult cohort revealed three SNPs (rs2236647, rs6591838, and rs1011219) within this heat shock-organizing protein and related to improved lung response during ICS therapy [150]. *STIP1* rs2236647 variant analysis in healthy and asthmatic children showed that this SNP could serve as an asthma marker for choosing the population who receives corticosteroid therapy[151]; however, further replication studies should be held to confirm those results.

One significant aspect of pharmacogenomics is that it investigates the interactions with genes of other pathways. *TBX21* gene is one good example for observing the ICS response outside the glucocorticoid pathway. *TBX21* is one of the conserved genes of a family sharing a common DNA-binding domain; the T-box encodes T-box transcription factor TBx21 protein. Tbx21 protein is a Th1 (T-helper1) transcription factor, which regulates one of the Th1 cytokine expression, interferon-gamma (IFNG). In 2004, a nonsynonymous SNP rs2240017 (His33Glu) in the *TBX21* gene was linked to improvements in bronchial hyperresponsiveness or "bronchoprotection" in response to ICS in individuals participating in the Child Asthma Management Program (CAMP) cohort [152]. This finding was also observed in an independent Korean cohort in 2009 [153]. Thus, *TBx21* may be an important determinant pharmacogenetic candi‐ date gene for predicting asthmatics' response to inhaled corticosteroid therapies.

namics or/and pharmacokinetics, by contrast, GWAS methodology identifies new associations with the null hypothesis being that no associations exist. GWAS picks the variations which are associated with observable phenotypes by scanning SNP markers that tag, via linkage disequilibrium, the complete human genome. GWAS and integrative system biology approaches are modern tools contributing to the recent advancements of genotyping and

Current pharmacogenetic studies of the corticosteroids, β-adrenergic, and leukotriene pathways are mostly candidate gene studies, with some GWAS, however, altogether have identified several genetic loci in strong association with therapeutic responsiveness to asthma. **Figure 4** summarizes the pharmacogenetically significant genes with relevance to the

In cytosol, the glucocorticoids bind to their corresponding glucocorticoid receptor, forming a hetero-complex that is activated by ligand binding, and translocate into the nucleus. In the nucleus, this complex binds to the glucocorticoid response elements in some target genes' promoter region resulting in their expression regulation. The core role of glucocorticoids is mediated via activating the transcription of anti-inflammatory genes, and suppressing the transcription of pro-inflammatory genes [145, 146]. The glucocorticoid pharmacogenetic studies formerly focused on candidate gene approach. Those candidate genes covered functions related to the corticosteroid biosynthetic pathway, the hetero-complex receptor

Corticotrophin-releasing hormone *(CRHR1)*, stress-inducible protein 1 *(STIP1), TBX21, CYP3A4, GLCCI1*, T gene, and *FBXL7* are the most up-to-date potential pharmacogenetic biomarker targets for predicting patients' response to ICS [147]. Studies of the corticotrophinreleasing hormone gene are considered to be as one of the oldest and remarkable footsteps in asthma pharmacogenetics. CRHR1 protein, also known as CRF1, is the primary receptor controlling the adrenocorticotropic hormone release; hence, it plays a pleiotropic and vital role in steroid actions. A candidate gene study of *CRHR1* in 1117 asthmatics administrating ICS therapy, from three clinical cohorts, revealed two SNPs (rs242941 and rs1876828) associated with different response in lung functions [148]. Tantisira et al. [148] found that *CRHR1* gene variation was frequently related to augmented therapy response in each of the three studied cohorts. Since 2004, *CRHR1* gene studies opened the doors for all other corticosteroid phar‐

*STIP1* or *HOP* (abbreviated for Hsp70-Hsp90-Organizing Protein) gene mainly functions to reversibly link Hsp70 and Hsp90 together as a co-chaperone [149]. *STIP1* pharmacogenetic studies in one adult cohort revealed three SNPs (rs2236647, rs6591838, and rs1011219) within this heat shock-organizing protein and related to improved lung response during ICS therapy [150]. *STIP1* rs2236647 variant analysis in healthy and asthmatic children showed that this SNP could serve as an asthma marker for choosing the population who receives corticosteroid therapy[151]; however, further replication studies should be held to confirm those results.

corticosteroids, β-adrenergic, and leukotriene biological pathways.

**7.1. Corticosteroid pathway pharmacogenetics**

formation, and the related chaperone proteins.

macogenetics and the possible future therapeutic outcomes.

statistical technologies.

162 Asthma - From Childhood Asthma to ACOS Phenotypes

In 2005, another example demonstrated the glucocorticoid pathway interactions with one other pathway. *ADCY9*, adenylyl cyclase type 9, gene encodes a membrane-bound enzyme in the β2-adrenergic receptor pathway, which catalyzes the production of cyclic adenosine mono‐ phosphate (AMP) from adenosine triphosphate (ATP). This candidate gene contains a pharmacogenetic nonsynonymous SNP, Met772Ile, which was correlated to enhanced Salbu‐ tamol (SABA) bronchodilator effects only in patients treated with ICS [154]. An independent Korean cohort replicated the trial, using Formoterol (LABA) treatment in combination with ICS, and confirmed those results [155].

Cytochromes P450s belong to a heme cofactor-containing superfamily of metabolizing enzyme proteins that potentially control the metabolism of drug (i.e., pharmacokinetics), and conse‐ quently treatment response in many diseases. For asthma, *CYP3A4*, *CYP3A5*, and *CYP3A7* candidate genes have been studied among a retrospective analysis of 413 asthmatic children treated with the ICS Fluticasone propionate [156]. The three candidate CYPs of all subjects were genotyped for nine SNPs. Results showed that asthmatics with the *CYP3A4\*22* allele demonstrated a significant symptom control compared with those lacking that allele. This study included a small number of participants (*n* = 20), so further large-scale replication is required.

Tantisira et al. [157] conducted the first pharmacogenetic GWAS for ICS treatment in asthma and identified an SNP (rs37972) in the promoter of the glucocorticoid-induced transcript-1 gene (*GLCCI1*), which significantly associates with lung functions. Replicated in four inde‐ pendent populations (935 persons in total), this candidate SNP was linked to substantial decrements in the response to the ICS in asthmatics. The wild-type allele homozygotes (CC) showed greater forced expiratory volume in 1 s (FEV1) in response to the ICS compared with those identified with the homozygote variant allele (TT). Another functionally correlated SNP (rs37973) in the promoter of the same gene was further validated within *in vitro* studies [157]. Results showed declined luciferase reporter activity in cells with the minor allele. *GLCCI1* GWAS outlines that drug response to asthma treatment is subjected to wide inter-individual variation, and GWAS would uncover more novel pharmacogenetic associations in the future. Tantisira et al. conducted a second GWAS among 418 asthmatics randomized to ICS treatment from the Childhood Asthma Research and Education (CARE), Asthma Clinical Research Network (ACRN), and CAMP trial cohorts. The *T-gene* (encoding the Brachyury transcription factor protein) compromised two SNPs (rs3127412 and rs6456042) that were associated, out of the successfully genotyped 47 SNPs, with altered lung function response to ICS [158].

#### **7.2. β-adrenergic receptor pathway pharmacogenetics**

β2-adrenergic receptor gene remains to be the most studied pharmacogenetic loci among the beta-agonist pathways. *ADRB2* gene has several polymorphic variants that were discovered in multi-ethnic genetic asthma cohorts [159, 160]. ADRB2 protein is a cell membrane-spanning receptor that binds epinephrine, but not norepinephrine, unlike the other adrenergic receptors, and consequently mediates both smooth muscle relaxation and bronchodilation [161, 162]. Early ADRB2 studies showed that Gly16Arg, a prevalent coding variant of the amino acid at position 16 of ADRB2, is associated with altered bronchodilator response to SABAs [163].

The BARGE (Beta-Agonist Response by Genotype) study [164], held by the National Heart, Lung and Blood Institute Asthma Clinical Network, was one of the first genotype "stratified" pharmacogenetic studies for asthma. In this study, only Gly16Arg homozygotes for ADRB2 were included (i.e., Arg/Arg and Gly/Gly). Participants were randomly receiving either intermittent or regular albuterol, and then crossed over to receive the alternative treatment dose. For statistical stratification, this study ensured that the Arg16 homozygotes, who are less frequent, were appropriately randomly distributed to both SABA intermittent and regular protocols. Compared to Gly16 homozygotes, the BARGE study showed that the Arg16 homo‐ zygotes were good responders only to acute intermittent SABAs rather than to long-term regular treatments, a finding that does not coincide with the current clinical asthma treatment guidelines [165] which recommend SABA as for on-demand intermittent usage. Since the 16th amino acid of ADRB2 controls regular response to albuterol, bronchodilator medications other than SABAs would be more appropriate for Arg/Arg asthmatics.

Collectively, the BARGE study [164], along with some other pharmacogenetic studies [163, 166–168] of Gly16Arg and SABAs' exposure, provided insights for further studies [169–171] on LABAs. In contrast to SABAs, a large cohort [169] of 2250 asthmatics, randomly assigned to formoterol plus budesonide, demonstrated no pharmacogenetic action due to *ADRB2* variation on therapeutic response. Furthermore, a multicenter trial [170] showed that asth‐ matics with both Arg/Arg and Gly/Gly genetic signatures had improved airway functions, if they received combination treatment with Salmeterol and ICS, when compared with ICS therapy alone. Similarly, the results of another prospective trial cohort [171] of 544 subjects, also randomized by genotyping, demonstrated no evidence of any pharmacogenetic action due to *ADRB2* variation in response to Salmeterol. Together, these findings, confirmed among several asthma populations, suggest that in contrast to SABAs, asthmatics can still be treated with LABAs plus ICS irrespective of their genotyping status.

Genetic variants' occurrences among different ethnic groups are quantified by their percentage of allele frequencies. Usually, frequent and common variants have only little or modest impacts on disease susceptibility and, subsequently, therapeutic response. On the other hand, as the variant is characterized to be rare or more "private," its effect size on disease progression and therapeutic response dramatically increases [172]. Early *in vitro* studies had investigated a rare polymorphism of *ADRB2* within the fourth transmembrane domain, the Thr164Ile variant. For the Ile164 genotype, results showed significant lowering in Gs-protein signaling and different SABA- and LABA-binding affinities [173, 174]. While the Thr164Ile polymorphism is pointed out to be a rare coding variant (i.e., <5%), population studies showed that this variant is more common in non-Hispanic white populations [159, 160], a finding that requires further phar‐ macogenetic investigation in different and larger populations. To replicate results, a study of two large Copenhagen population cohorts [175], with more than 55,000 participants, was held to investigate the relation of Thr164Ile variation and lung responses. Among the general population, the Copenhagen study reported that the Thr164 genotype was associated with decreased FEV1, diminished lung function, and increased the overall COPD risk.

factor protein) compromised two SNPs (rs3127412 and rs6456042) that were associated, out of

β2-adrenergic receptor gene remains to be the most studied pharmacogenetic loci among the beta-agonist pathways. *ADRB2* gene has several polymorphic variants that were discovered in multi-ethnic genetic asthma cohorts [159, 160]. ADRB2 protein is a cell membrane-spanning receptor that binds epinephrine, but not norepinephrine, unlike the other adrenergic receptors, and consequently mediates both smooth muscle relaxation and bronchodilation [161, 162]. Early ADRB2 studies showed that Gly16Arg, a prevalent coding variant of the amino acid at position 16 of ADRB2, is associated with altered bronchodilator response to SABAs [163]. The BARGE (Beta-Agonist Response by Genotype) study [164], held by the National Heart, Lung and Blood Institute Asthma Clinical Network, was one of the first genotype "stratified" pharmacogenetic studies for asthma. In this study, only Gly16Arg homozygotes for ADRB2 were included (i.e., Arg/Arg and Gly/Gly). Participants were randomly receiving either intermittent or regular albuterol, and then crossed over to receive the alternative treatment dose. For statistical stratification, this study ensured that the Arg16 homozygotes, who are less frequent, were appropriately randomly distributed to both SABA intermittent and regular protocols. Compared to Gly16 homozygotes, the BARGE study showed that the Arg16 homo‐ zygotes were good responders only to acute intermittent SABAs rather than to long-term regular treatments, a finding that does not coincide with the current clinical asthma treatment guidelines [165] which recommend SABA as for on-demand intermittent usage. Since the 16th amino acid of ADRB2 controls regular response to albuterol, bronchodilator medications other

Collectively, the BARGE study [164], along with some other pharmacogenetic studies [163, 166–168] of Gly16Arg and SABAs' exposure, provided insights for further studies [169–171] on LABAs. In contrast to SABAs, a large cohort [169] of 2250 asthmatics, randomly assigned to formoterol plus budesonide, demonstrated no pharmacogenetic action due to *ADRB2* variation on therapeutic response. Furthermore, a multicenter trial [170] showed that asth‐ matics with both Arg/Arg and Gly/Gly genetic signatures had improved airway functions, if they received combination treatment with Salmeterol and ICS, when compared with ICS therapy alone. Similarly, the results of another prospective trial cohort [171] of 544 subjects, also randomized by genotyping, demonstrated no evidence of any pharmacogenetic action due to *ADRB2* variation in response to Salmeterol. Together, these findings, confirmed among several asthma populations, suggest that in contrast to SABAs, asthmatics can still be treated

Genetic variants' occurrences among different ethnic groups are quantified by their percentage of allele frequencies. Usually, frequent and common variants have only little or modest impacts on disease susceptibility and, subsequently, therapeutic response. On the other hand, as the variant is characterized to be rare or more "private," its effect size on disease progression and therapeutic response dramatically increases [172]. Early *in vitro* studies had investigated a rare polymorphism of *ADRB2* within the fourth transmembrane domain, the Thr164Ile variant. For

the successfully genotyped 47 SNPs, with altered lung function response to ICS [158].

**7.2. β-adrenergic receptor pathway pharmacogenetics**

164 Asthma - From Childhood Asthma to ACOS Phenotypes

than SABAs would be more appropriate for Arg/Arg asthmatics.

with LABAs plus ICS irrespective of their genotyping status.

In addition to ADRB2 Gly16Arg and Thr164Ile variants, the (-376 In-Del) polymorphism was extensively studied as another significant pharmacogenetic *ADRB2* variant. Presented primarily among African Americans and Puerto Ricans [159, 160], the 24-bp promoter insertion at −376, related to the start codon, is associated with asthma-related hospitalization in asth‐ matics treated with LABA [160]. Altogether, these variants, being unique to different popula‐ tions, highlight the increasing need of personalized-based treatments.

Adenylyl cyclase type 9, encoded in humans by *ADCY9* gene, is a membrane-bound enzyme that catalyzes the formation of cyclic AMP from ATP. ADCY9 is a widely abundant adenylyl cyclase, and it is stimulated via beta-adrenergic receptor activation [176]. Ile772Met is a coding variant of *ADCY9* gene that has been associated with both acute FEV1 bronchodilation in response to SABAs [154] and long-term FEV1 response for LABAs [155]. CRHR2 (which is more commonly known as CRF2) is a type-2 G protein-coupled protein receptor for the corticotropin-releasing hormone [177]. Out of the 28 studied SNPs in *CRHR2*, five SNPs were significantly correlated with acute bronchodilator response in one, or frequently more than one, cohort. Among those, variant rs7793837 was associated with altered SABA response in all three cohorts of the *CRHR2* study containing 607, 427, and 152 participants, respectively [178].

Different variants of *ARG1* (Arginase 1) and *ARG2* (Arginase 2) show altered acute response to SABAs, while the endothelial nitric oxide synthase (*NOS3*) shows altered acute response to LABAs. NO (nitric oxide), an endogenous vasorelaxing bronchodilator, is generated by the action of NOS3 on L-arginine. Since ARG1 and ARG2 are metabolizing L-arginine, so it is expected that the entire three genes, *ARG1*, *ARG2*, and *NOS3*, might be implicated in asthma pharmacogenetics. Combined association evidence, surviving Bonferroni correction for multiple testing from the CAMP four asthma cohorts [179], points to SNP rs2781659 in *ARG1*. C-allele homozygotes for SNP rs2781667 in arginase 1 showed significantly less response to the inhaled corticosteroid treatments [180]. Arginase-2 variants rs17249437 and rs3742879 correlated with increased airway obstruction and airway hyperresponsiveness, and lower reversibility of airway constriction following treatment with beta-2 agonists [180]. A small candidate gene study [181] of *NOS3* had revealed one possible variant (Asp298Glu) correlated with lung function response to ICS/LABA combined treatment; however, this result still needs to be replicated in larger cohorts. *THRB* [182], *SLC24A4* [183], *SLC22A15* [183], *SPATS2L* [184], and SNPs (rs892940, rs77441273, rs1281748/rs1281743, and rs295137, respec‐ tively) show promising loci for further pharmacogenetic investigations.

#### **7.3. Leukotriene pathway pharmacogenetics**

Relative to the corticosteroid and β-adrenergic pathways, the cysteinyl leukotriene pathway pharmacogenetic studies are generally fewer and have smaller sample sizes. The oldest of these studies [185], held in 1999, had investigated the tandem repeat polymorphism in *ALOX5* promoter. Among 114 asthmatics, it has been shown that the *ALOX5* promoter repeat is associated with altered lung functions in response to a 5-LO inhibitor [185]. It has been shown in children that those who had more or less than five repeats (3, 4, and 6) of the *ALOX5* promoter-binding motif experienced increased urinary leukotriene E4 (the terminal cysteinyl leukotriene metabolite) concentrations and reduced FEV1 baseline than the wild-type geno‐ type with five repeats [186]. Further pharmacogenetic studies revealed that the *ALOX5* promoter polymorphism, along with the *ALOX5* SNPs rs892690, rs2029253, and rs2115819, influences leukotriene pathway antagonist therapy [187–190]. Moreover, variants of *LTC4S*, encoding Leukotriene C4 synthase, and *MRP1* (or *ABCC1*), encoding multidrug resistanceassociated protein 1, have been linked to lung function response while treatment with Zileuton and Montelukast [189, 190].

Arg312Gln, rs12422149, which is a coding variant in *SLCO2B1* (solute carrier organic anion transporter family member 2B1 gene), has been related to symptom control during Montelu‐ kast therapy. This fact was due to the interindividual variability of carrier-mediated Monte‐ lukast transport in the intestines, and consequently its plasma levels [191]. By contrast, two other studies, probably due to their small sample sizes, were unable to replicate similar *SLCO2B1* pharmacokinetic effects [192, 193]. Overall, larger replicate cohorts, for the leuko‐ triene pathway identified loci, are still needed.
