Cysteinyl Leukotrienes Pathway Genes, Atopic Asthma and Drug Response: From Population Isolates to Large Genome-Wide Association Studies.

Genetic variants associated with asthma pathogenesis and altered response to drug therapy are discussed. Many studies implicate polymorphisms in genes encoding the enzymes responsible for leukotriene synthesis and intracellular signaling through activation of seven transmembrane domain receptors, such as the cysteinyl leukotriene 1 (CYSLTR1) and 2 (CYSLTR2) receptors. The leukotrienes are polyunsaturated lipoxygenated eicosatetraenoic acids that exhibit a wide range of pharmacological and physiological actions. Of the three enzymes involved in the formation of the leukotrienes, arachidonate 5 lipoxygenase 5 (ALOX5), leukotriene C4 synthase (LTC4S), and leukotriene hydrolase (LTA4H) are all polymorphic. These polymorphisms often result in variable production of the CysLTs (LTC4, LTD4, and LTE4) and LTB4. Variable number tandem repeat sequences located in the Sp1-binding motif within the promotor region of the ALOX5 gene are associated with leukotriene burden and bronchoconstriction independent of asthma risk. A 444A > C SNP polymorphism in the LTC4S gene, encoding an enzyme required for the formation of a glutathione adduct at the C-6 position of the arachidonic acid backbone, is associated with severe asthma and altered response to the CYSLTR1 receptor antagonist zafirlukast. Genetic variability in the CysLT pathway may contribute additively or synergistically to altered drug responses. The 601 A > G variant of the CYSLTR2 gene, encoding the Met201Val CYSLTR2 receptor variant, is associated with atopic asthma in the general European population, where it is present at a frequency of ∼2.6%. The variant was originally found in the founder population of Tristan da Cunha, a remote island in the South Atlantic, in which the prevalence of atopy is approximately 45% and the prevalence of asthma is 36%. In vitro work showed that the atopy-associated Met201Val variant was inactivating with respect to ligand binding, Ca2+ flux and inositol phosphate generation. In addition, the CYSLTR1 gene, located at Xq13-21.1, has been associated with atopic asthma. The activating Gly300Ser CYSLTR1 variant is discussed. In addition to genetic loci, risk for asthma may be influenced by environmental factors such as smoking. The contribution of CysLT pathway gene sequence variants to atopic asthma is discussed in the context of other genes and environmental influences known to influence asthma.

Cellular signalling of cysteinyl leukotriene type 1 receptor variants CysLT1-G300S and CysLT1-I206S.

Cysteinyl-leukotrienes are pro-inflammatory lipid mediators, involved in allergic asthma, that bind the G-protein-coupled receptors CysLT1, CysLT2 and GPR99. A polymorphism in one of these receptors, CysLT1-G300S was strongly associated with atopy, whereas the CysLT1-I206S polymorphism was not. In the present work, our aim was to characterize these two variants by studying their cellular signalling. Cell surface expression of mutant receptors in transfected HEK-293 cells was comparable to that of the wild-type receptor. Compared to CysLT1-WT, production of inositol phosphates as well as IL-8 and IL-13 promoter transactivation in response to either LTD4 or LTC4 was significantly increased in CysLT1-G300S-transfected cells. Moreover, LTD4-induced phosphorylation of the signalling effector Erk, but not p38, p65 or c-Jun was higher in CysLT1-G300S-transfected cells. On the other hand, the variant CysLT1-I206S did not show a significant difference in its signal transduction compared to the wild-type receptor. Taken together, our results indicate that the variant CysLT1-G300S can induce a greater signal than the CysLT1-WT receptor, a feature that may be relevant to its association with atopy.

Pharmacogenetics of the G protein-coupled receptors.

Pharmacogenetics investigates the influence of genetic variants on physiological phenotypes related to drug response and disease, while pharmacogenomics takes a genome-wide approach to advancing this knowledge. Both play an important role in identifying responders and nonresponders to medication, avoiding adverse drug reactions, and optimizing drug dose for the individual. G protein-coupled receptors (GPCRs) are the primary target of therapeutic drugs and have been the focus of these studies. With the advance of genomic technologies, there has been a substantial increase in the inventory of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms and insertion or deletions that have potential to alter GPCR expression of function. In vivo and in vitro studies have determined functional roles for many GPCR variants, but genetic association studies that define the physiological impact of the majority of these common variants are still limited. Despite the breadth of pharmacogenetic data available, GPCR variants have not been included in drug labeling and are only occasionally considered in optimizing clinical use of GPCR-targeted agents. In this chapter, pharmacogenetic and genomic studies on GPCR variants are reviewed with respect to a subset of GPCR systems, including the adrenergic, calcium sensing, cysteinyl leukotriene, cannabinoid CB1 and CB2 receptors, and the de-orphanized receptors such as GPR55. The nature of the disruption to receptor function is discussed with respect to regulation of gene expression, expression on the cell surface (affected by receptor trafficking, dimerization, desensitization/downregulation), or perturbation of receptor function (altered ligand binding, G protein coupling, constitutive activity). The large body of experimental data generated on structure and function relationships and receptor-ligand interactions are being harnessed for the in silico functional prediction of naturally occurring GPCR variants. We provide information on online resources dedicated to GPCRs and present applications of publically available computational tools for pharmacogenetic studies of GPCRs. As the breadth of GPCR pharmacogenomic data becomes clearer, the opportunity for routine assessment of GPCR variants to predict disease risk, drug response, and potential adverse drug effects will become possible.

Hyperphosphatasia with seizures, neurologic deficit, and characteristic facial features: Five new patients with Mabry syndrome.

Persistent hyperphosphatasia associated with developmental delay and seizures was described in a single family by Mabry et al. 1970 (OMIM 239300), but the nosology of this condition has remained uncertain ever since. We report on five new patients (two siblings, one offspring of consanguineous parents, and two sporadic patients) that help delineate this distinctive disorder and provide evidence in favor of autosomal recessive inheritance. Common to all five new patients is facial dysmorphism, namely hypertelorism, a broad nasal bridge and a tented mouth. All patients have some degree of brachytelephalangy but the phalangeal shortening varies in position and degree. In all, there is a persistent elevation of alkaline phosphatase activity without any evidence for active bone or liver disease. The degree of hyperphosphatasia varies considerably ( approximately 1.3-20 times the upper age-adjusted reference limit) between patients, but is relatively constant over time. In the first family described by Mabry et al. 1970, at least one member was found to have intracellular inclusions on biopsy of some but not all tissues. This was confirmed in three of our patients, but the inclusions are not always observed and the intracellular storage material has not been identified.

Vitamin B-12 and neural tube defects: the Canadian experience.

Although early epidemiologic studies showed a protective effect of adequate maternal folic acid (FA) status against neural tube defects (NTDs), the role of adequate vitamin B-12 nutrition in the putative reduction of NTD frequency has remained uncertain. Evaluating vitamin B-12 status was complicated by the need to control for altered FA status after fortification in Canada. More recent studies have made use of better biomarkers of vitamin B-12 status, including methylmalonic acid and holotranscobalamin (holoTC). HoloTC provides a useful measure of vitamin B-12 status because it represents the bioavailable fraction of circulating vitamin B-12. By assessing bioavailable vitamin B-12 status in a large Canadian cohort accrued before and after FA fortification, we found a 3-fold increase in the risk of NTDs in mothers who had vitamin B-12 status in the lower quartile, regardless of FA fortification. Our work suggests that vitamin B-12 fortification, analogous to the FA fortification program, may reduce NTDs more than FA fortification alone. A multicenter randomized controlled trial comparing periconceptional vitamin B-12 in combination with FA against FA alone is warranted.

G protein-coupled receptor pharmacogenetics.

Common G protein-coupled receptor (GPCR) gene variants that encode receptor proteins with a distinct sequence may alter drug efficacy without always resulting in a disease phenotype. GPCR genetic loci harbor numerous variants, such as DNA insertions or deletions and single-nucleotide polymorphisms that alter GPCR expression and function, thereby contributing to interindividual differences in disease susceptibility/progression and drug responses. In this chapter, these pharmacogenetic phenomena are reviewed with respect to a limited sampling of GPCR systems, including the beta(2)-adrenergic receptors, the cysteinyl leukotriene receptors, and the calcium-sensing receptor. In each example, the nature of the disruption to receptor function that results from each variant is discussed with respect to the regulation of gene expression, expression on cell surface (affected by receptor trafficking, dimerization, desensitization/downregulation), or perturbation of receptor function (by altering ligand binding, G protein coupling, and receptor constitutive activity). Despite the breadth of pharmacogenetic knowledge available, assessment for genetic variants is only occasionally applied to drug development projects involving pharmacogenomics or to optimizing the clinical use of GPCR drugs. The continued effort by the basic science of pharmacogenetics may draw the attention of drug discovery projects and clinicians alike to the utility of personalized pharmacogenomics as a means to optimize novel GPCR drug targets.

Metabolic syndrome features and risk of neural tube defects.

BACKGROUND: Maternal obesity and pre-pregnancy diabetes mellitus, features of the metabolic syndrome (MetSyn), are individual risk factors for neural tube defects (NTD). Whether they, in combination with additional features of MetSyn, alter this risk is not known. We evaluated the risk of NTD in association with maternal features of the MetSyn. METHODS: We used a population-based case-control study design in the province of Ontario, Canada. Cases and controls were derived from women who underwent antenatal maternal screening (MSS) at 15 to 20 weeks' gestation. There were 89 maternal cases with, and 434 controls without, an NTD-affected singleton pregnancy. Maternal features of MetSyn were defined by the presence of pre-pregnancy diabetes mellitus, body weight > or = 90th centile among controls, non-white ethnicity and/or serum highly sensitive C-reactive protein (hsCRP) > or = 75th centile of controls. Since hsCRP naturally increases in pregnancy, analyses were performed with, and without, the inclusion of hsCRP in the model. RESULTS: Mean hsCRP concentrations were exceptionally high among study cases and controls (6.1 and 6.4 mg/L, respectively). When hsCRP was excluded from the model, the adjusted odds ratios for NTD were 1.9 (95% confidence interval 1.1-3.4) in the presence 1 feature of MetSyn, and 6.1 (1.1-32.9) in the presence of 2 or more features. When hsCRP was included, the respective risk estimates were attenuated to 1.6 (0.88-2.8) and 3.1 (1.2-8.3). CONCLUSION: We found about 2-fold and 6-fold higher risk for NTD in the presence 1, and 2 or more features, of the metabolic syndrome, respectively. It is not clear whether this risk is altered by the presence of a high serum hsCRP concentration.

A functional G300S variant of the cysteinyl leukotriene 1 receptor is associated with atopy in a Tristan da Cunha isolate.

Atopy is a well-defined immune phenotype that is reported to be a risk factor for asthma. Among the many loci that contribute to a genetic predisposition to asthma, the cysteinyl leukotriene receptor genes and their variants have been important subjects of study because they are functionally and pharmacologically implicated in the atopy phenotype affecting many asthma subjects. Moreover, the product of cysteinyl-leukotriene 1 receptor gene (CysLT1), located at Xq13.2, is targeted by LT receptor antagonists. In our earlier association study, the M201V variant of the cysteinyl-leukotriene 2 receptor gene (CysLT2), located at 13q14, was implicated in atopic asthma. Here we report the screening of the coding region of the CysLT1, gene in the highly asthmatic Tristan da Cunha population. In this population, we discovered a CysLT1 G300S variant that is carried with a significantly higher frequency in atopics and asthmatics from the Tristan da Cunha population. Furthermore, we report the asthma independent association of the CysLT1 G300S variant with atopy. Subsequently, we compared the changes conferred by each SNP on CysLT function. The CysLT1 300S receptor interacts with LTD4 with significantly greater potency. For the 300S variant, a statistically significant decrease in the effector concentration for half-maximum response (EC50) for intracellular Ca flux and total InsP generation is observed. Other aspects of the receptor function and activity, such as desensitization, pharmacologic profile in response to montelukast, and cellular localization, are unchanged. These in vitro analyses provide evidence that the 300S CysLT1 variant, found more commonly in atopics in the Tristan da Cunha population, encodes a functionally more sensitive variant.

Vitamin B12 and the risk of neural tube defects in a folic-acid-fortified population.

BACKGROUND: Low maternal vitamin B(12) status may be a risk factor for neural tube defects (NTDs). Prior studies used relatively insensitive measures of B(12), did not adjust for folate levels, and were conducted in countries without folic acid food fortification. In Canada, flour has been fortified with folic acid since mid-1997. METHODS: We completed a population-based case-control study in Ontario. We measured serum holotranscobalamin (holoTC), a sensitive indicator of B(12) status, at 15 to 20 weeks' gestation. There were 89 women with an NTD and 422 unaffected pregnant controls. A low serum holoTC was defined as less than 55.3 pmol/L, the bottom quartile value in the controls. RESULTS: The geometric mean serum holoTC levels were 67.8 pmol/L in cases and 81.2 pmol/L in controls. There was a trend of increasing risk with lower levels of holoTC, reaching an adjusted odds ratio of 2.9 (95% confidence interval = 1.2-6.9) when comparing the lowest versus highest quartile. CONCLUSIONS: There was almost a tripling in the risk for NTD in the presence of low maternal B(12) status, measured by holoTC. The benefits of adding synthetic B(12) to current recommendations for periconceptional folic acid tablet supplements or folic-acid-fortified foods need to be considered. It remains to be determined what fraction of NTD cases in a universally folate-fortified environment might be prevented by higher periconceptional intake of B(12).

Cysteinyl-leukotrienes and their receptors in asthma and other inflammatory diseases: critical update and emerging trends.

Cysteinyl-leukotrienes (cysteinyl-LTs), that is, LTC4, LTD4, and LTE4, trigger contractile and inflammatory responses through the specific interaction with G protein-coupled receptors (GPCRs) belonging to the purine receptor cluster of the rhodopsin family, and identified as CysLT receptors (CysLTRs). Cysteinyl-LTs have a clear role in pathophysiological conditions such as asthma and allergic rhinitis (AR), and have been implicated in other inflammatory conditions including cardiovascular diseases, cancer, atopic dermatitis, and urticaria. Molecular cloning of human CysLT1R and CysLT2R subtypes has confirmed most of the previous pharmacological characterization and identified distinct expression patterns only partially overlapping. Interestingly, recent data provide evidence for the immunomodulation of CysLTR expression, the existence of additional receptor subtypes, and of an intracellular pool of CysLTRs that may have roles different from those of plasma membrane receptors. Furthermore, genetic variants have been identified for the CysLTRs that may interact to confer risk for atopy. Finally, a crosstalk between the cysteinyl-LT and the purine systems is being delineated. This review will summarize and attempt to integrate recent data derived from studies on the molecular pharmacology and pharmacogenetics of CysLTRs, and will consider the therapeutic opportunities arising from the new roles suggested for cysteinyl-LTs and their receptors.

G-protein-coupled receptors and asthma endophenotypes: the cysteinyl leukotriene system in perspective.

Genetic variation in specific G-protein coupled receptors (GPCRs) is associated with a spectrum of respiratory disease predispositions and drug response phenotypes. Although certain GPCR gene variants can be disease-causing through the expression of inactive, overactive, or constitutively active receptor proteins, many more GPCR gene variants confer risk for potentially deleterious endophenotypes. Endophenotypes are traits, such as bronchiole hyperactivity, atopy, and aspirin intolerant asthma, which have a strong genetic component and are risk factors for a variety of more complex outcomes that may include disease states. GPCR genes implicated in asthma endophenotypes include variants of the cysteinyl leukotriene receptors (CYSLTR1 and CYSLTR2), and prostaglandin D2 receptors (PTGDR and CRTH2), thromboxane A2 receptor (TBXA2R), beta2-adrenergic receptor (ADRB2), chemokine receptor 5 (CCR5), and the G protein-coupled receptor associated with asthma (GPRA). This review of the contribution of variability in these genes places the contribution of the cysteinyl leukotriene system to respiratory endophenotypes in perspective. The genetic variant(s) of receptors that are associated with endophenotypes are discussed in the context of the extent to which they contribute to a disease phenotype or altered drug efficacy.

The G protein-coupled receptors: pharmacogenetics and disease.

Genetic variation in G-protein coupled receptors (GPCRs) is associated with a wide spectrum of disease phenotypes and predispositions that are of special significance because they are the targets of therapeutic agents. Each variant provides an opportunity to understand receptor function that complements a plethora of available in vitro data elucidating the pharmacology of the GPCRs. For example, discrete portions of the proximal tail of the dopamine D1 receptor have been discovered, in vitro, that may be involved in desensitization, recycling and trafficking. Similar in vitro strategies have been used to elucidate naturally occurring GPCR mutations. Inactive, over-active or constitutively active receptors have been identified by changes in ligand binding, G-protein coupling, receptor desensitization and receptor recycling. Selected examples reviewed include those disorders resulting from mutations in rhodopsin, thyrotropin, luteinizing hormone, vasopressin and angiotensin receptors. By comparison, the recurrent pharmacogenetic variants are more likely to result in an altered predisposition to complex disease in the population. These common variants may affect receptor sequence without intrinsic phenotype change or spontaneous induction of disease and yet result in significant alteration in drug efficacy. These pharmacogenetic phenomena will be reviewed with respect to a limited sampling of GPCR systems including the orexin/hypocretin system, the beta2 adrenergic receptors, the cysteinyl leukotriene receptors and the calcium-sensing receptor. These developments will be discussed with respect to strategies for drug discovery that take into account the potential for the development of drugs targeted at mutated and wild-type proteins.

A cysteinyl leukotriene 2 receptor variant is associated with atopy in the population of Tristan da Cunha.

The clinical heterogeneity of asthma suggests that the contribution of genetic variability in candidate gene loci to well-defined phenotypes, such as atopy, may be examined to identify appropriate genetic risk factors for asthma. The gene encoding the cysteinyl leukotriene 2 (CysLT2) receptor has been implicated in atopy since it is localized to a region of chromosome 13q14 that has been linked to atopy in several populations and the cysteinyl leukotrienes are known to activate eosinophils and mast cells in atopy. Accordingly, we analysed the contribution of CysLT2 receptor gene variation to atopy in the inhabitants of Tristan da Cunha, a population characterized by both a founder effect and a 47% prevalence of atopy. Single-stranded conformational polymorphism analysis revealed four variants. Among these, the M201V [corrected] variant was activated with four-fold less potency by leukotriene D4 (LTD4) in a calcium flux assay. The CysLT2 receptor partial agonist, BAY u9773, also showed four-fold lower potency on the M201V [corrected] variant. The M201V [corrected] mutation is located within the extracellular region of the fifth transmembrane spanning domain of CysLT2 receptor, a position that may alter ligand binding and effector signalling. The novel M201V [corrected] CysLT2 receptor variant was associated with atopy (21%) on Tristan da Cunha compared with those who were non-atopic (7%) (Fisher's exact test, P=0.0016) in a manner that was independent of asthma (two-way ANOVA, P=0.0015). This represents the first association of a coding mutation in the CysLT2 receptor gene, located on chromosome 13q14, with the atopic phenotype found in the Tristan da Cunha population.