Genome-Wide Association Study Identifies Novel Pharmacogenomic Loci For Therapeutic Response to Montelukast in Asthma

Background

Genome-wide association study (GWAS) is a powerful tool to identify novel pharmacogenetic single nucleotide polymorphisms (SNPs). Leukotriene receptor antagonists (LTRAs) are a major class of asthma medications, and genetic factors contribute to variable responses to these drugs. We used GWAS to identify novel SNPs associated with the response to the LTRA, montelukast, in asthmatics.

Methods

Using genome-wide genotype and phenotypic data available from American Lung Association - Asthma Clinical Research Center (ALA-ACRC) cohorts, we evaluated 8-week change in FEV₁ related to montelukast administration in a discovery population of 133 asthmatics. The top 200 SNPs from the discovery GWAS were then tested in 184 additional samples from two independent cohorts.

Results

Twenty-eight SNP associations from the discovery GWAS were replicated. Of these, rs6475448 achieved genome-wide significance (combined P = 1.97 x 10^-09), and subjects from all four studies who were homozygous for rs6475448 showed increased ΔFEV₁ from baseline in response to montelukast.

Conclusions

Through GWAS, we identified a novel pharmacogenomic locus related to improved montelukast response in asthmatics.     

Association between Hemoglobin Concentration and the Progression or Development of Albuminuria in Diabetic Kidney Disease

Aims

Anemia, which might contribute to pathogenesis of kidney dysfunction, is a common finding in patients with type 2 diabetes. The aim of this study was to investigate if hemoglobin concentration is associated with the degree of change in urinary albumin-creatinine ratio or the development of albuminuria in patients with type 2 diabetes.

Methods

We measured hemoglobin concentration in 470 (296 men and 174 women) consecutive type 2 diabetic patients without albuminuria. We performed a follow-up study to assess the progression or development of albuminuria, the interval of which was 3.0 years. Then we evaluated relationships between hemoglobin concentration and albuminuria, using multivariate linear regression analyses and logistic regression analyses.

Results

Eighty four patients developed albuminuria during follow-up duration. In multivariate analyses, hemoglobin concentration was negatively associated with a change in urinary albumin-creatinine ratio in men (ß = -0.259, P = 0.0002) and women (ß = -0.194, P = 0.030). Moreover, multivariate adjusted odds ratio associated with 1 g/L in hemoglobin for the development of albuminuria was 0.93 (95% confidence interval; 0.89-0.96) in men and 0.94 (95% confidence interval; 0.88-0.99) in women, respectively. And, multivariate analyses revealed that adjusted odds ratios for the development of albuminuria were 4.78 (95% confidence interval; 1.65-13.91) in men and 4.62 (95% confidence interval; 1.34-16.68) in women with anemia (hemoglobin < 130 g/L for men and < 120 g/L for women), which were higher than those without anemia.

Conclusions

Low hemoglobin concentration could be a predictor for the progression and development of albuminuria in patients with type 2 diabetes.     

Clinical Features of Autoimmune Autonomic Ganglionopathy and the Detection of Subunit-Specific Autoantibodies to the Ganglionic Acetylcholine Receptor in Japanese Patients

Autoimmune autonomic ganglionopathy (AAG) is a rare acquired channelopathy that is characterized by pandysautonomia, in which autoantibodies to ganglionic nicotinic acetylcholine receptors (gAChR) may play a central role. Radioimmunoprecipitation (RIP) assays have been used for the sensitive detection of autoantibodies to gAChR in the serum of patients with AAG. Here, we developed luciferase immunoprecipitation systems (LIPS) to diagnose AAG based on IgGs to both the α3 and β4 gAChR subunits in patient serum. We reviewed the serological and clinical data of 50 Japanese patients who were diagnosed with AAG. With the LIPS testing, we detected anti-α3 and -β4 gAChR antibodies in 48% (24/50) of the patients. A gradual mode of onset was more common in the seropositive group than in the seronegative group. Patients with AAG frequently have orthostatic hypotension and upper and lower gastrointestinal tract symptoms, with or without anti-gAChR. The occurrence of autonomic symptoms was not significantly different between the seropositive and seronegative group, with the exception of achalasia in three patients from the seropositive group. In addition, we found a significant overrepresentation of autoimmune diseases in the seropositive group and endocrinological abnormalities as an occasional complication of AAG. Our results demonstrated that the LIPS assay was a useful novel tool for detecting autoantibodies against gAChR in patients with AAG.     

Mechanical Stimulation by Postnasal Drip Evokes Cough

Cough affects all individuals at different times, and its economic burden is substantial. Despite these widespread adverse effects, cough research relies on animal models, which hampers our understanding of the fundamental cause of cough. Postnasal drip is speculated to be one of the most frequent causes of chronic cough; however, this is a matter of debate. Here we show that mechanical stimuli by postnasal drip cause chronic cough. We distinguished human cough from sneezes and expiration reflexes by airflow patterns. Cough and sneeze exhibited one-peak and two-peak patterns, respectively, in expiratory airflow, which were also confirmed by animal models of cough and sneeze. Transgenic mice with ciliary dyskinesia coughed substantially and showed postnasal drip in the pharynx; furthermore, their cough was completely inhibited by nasal airway blockade of postnasal drip. We successfully reproduced cough observed in these mice by injecting artificial postnasal drip in wild-type mice. These results demonstrated that mechanical stimulation by postnasal drip evoked cough. The findings of our study can therefore be used to develop new antitussive drugs that prevent the root cause of cough.     

Stopped-flow Measurement of Reaction Rate of Triose Reductone with 2,6-Dichlorophenolindophenol and Its Application to the Determination of Triose Reductone

The reduction of 2, 6-dichlorophenolindophenol by triose reductone was studied in a wide pH range (pH 2~11), using a stopped-flow apparatus. The apparent first-order rate constant, k_app, and the second-order rate constant, k, were obtained. The k-pH profile resembles that of l-ascorbic acid, though the rate of l-ascorbic acid is considerably larger than that of triose reductone. Determination of triose reductone by stopped-flow method is possible, even in the presence of various organic substances.     

Effects of synthetic antifreeze glycoprotein analogue on islet cell survival and function during cryopreservation

The antifreeze glycoprotein (AFGP), found in the blood of polar fish, is known to prevent ice crystal growth and to depress the freezing temperature, which may in turn protect tissues from freezing injury. The chemical synthesis of AFGP is an attractive alternative to its difficult isolation from natural sources, and this would permit quality control and mass production. In spite of recent success in islet transplantation for the treatment of type 1 diabetes mellitus, existing methods for the long-term preservation of islets are considered to be suboptimal and inadequate, which indicates the need for the development of improved methods. Rat islets were isolated from male Wistar rats, using intraductal collagenase distention, mechanical dissociation, and Ficoll-Conray gradient purification. Islets were cultured overnight and then cryopreserved in RPMI1640 in the presence of dimethyl sulfoxide (Me2SO) and 10% FCS with various concentrations of syAFGP, followed by slow cooling (0.3 degrees C/min) and rapid thawing (200 degrees C/min) as described by Rajotte. The freezing process was observed by cryomicroscopy. Islet recovery post-cryopreservation was 85.0 +/- 6.2% with syAFGP and 63.3 +/- 14.2% without syAFGP, both compared with the pre-cryopreservation counts (P < 0.05). The in vitro islet function measured by insulin release was equivalent to a static stimulation index of 3.86+/-0.43 for the islets that were frozen-and-thawed with syAFGP, compared to 2.98 +/- 0.22 without syAFGP (P < 0.05). At a concentration of around 500 microg/ml syAFGP, a strong attenuation of ice crystal growth and formation was observed by cryomicroscopy and these ice crystals did not cause cryoinjury. In conclusion, the attenuation of ice crystallization by syAFGP improves islet survival and function following cryopreservation and thawing.     

DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells

Although gemcitabine is the most effective chemotherapeutic agent against pancreatic cancer, a growing concern is that a substantial number of patients acquire gemcitabine chemoresistance. To elucidate the mechanisms of acquisition of gemcitabine resistance, we developed gemcitabine-resistant cell lines from six human cancer cell lines; three pancreatic, one gastric, one colon, and one bile duct cancer. We first analyzed gemcitabine uptake using three paired parental and gemcitabine resistant pancreatic cancer cell lines (PK-1 and RPK-1, PK-9 and RPK-9, PK-59 and RPK-59) and found that uptake of gemcitabine was rapid. However, no DNA damage was induced in resistant cells. We further examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance and found a remarkable reduction in the expression of deoxycytidine kinase (DCK). DCK is a key enzyme that activates gemcitabine by phosphorylation. Genetic alterations and expression of DCK were studied in these paired parental and derived gemcitabine-resistant cell lines, and inactivating mutations were found only in gemcitabine-resistant cell lines. Furthermore, siRNA-mediated knockdown of DCK in the parental cell lines yielded gemcitabine resistance, and introduction of DCK into gemcitabine-resistant cell lines invariably restored gemcitabine sensitivities. Mutation analyses were expanded to three other different paired cell lines, DLD-1 and RDLD-1 (colon cancer cell line), MKN-28 and RMKN-28 (gastric cancer cell line), and TFK-1 and RTFK -1 (cholangiocarcinoma cell line). We found inactivating mutations in RDLD-1 and RTFK-1 and decreased expression of DCK in RMKN-28. These results indicate that the inactivation of DCK is one of the crucial mechanisms in acquisition of gemcitabine resistance.     

Functional Variants in NFKBIE and RTKN2 Involved in Activation of the NF-κB Pathway Are Associated with Rheumatoid Arthritis in Japanese

Rheumatoid arthritis is an autoimmune disease with a complex etiology, leading to inflammation of synovial tissue and joint destruction. Through a genome-wide association study (GWAS) and two replication studies in the Japanese population (7,907 cases and 35,362 controls), we identified two gene loci associated with rheumatoid arthritis susceptibility (NFKBIE at 6p21.1, rs2233434, odds ratio (OR) = 1.20, P = 1.3×10⁻¹⁵; RTKN2 at 10q21.2, rs3125734, OR = 1.20, P = 4.6×10⁻⁹). In addition to two functional non-synonymous SNPs in NFKBIE, we identified candidate causal SNPs with regulatory potential in NFKBIE and RTKN2 gene regions by integrating in silico analysis using public genome databases and subsequent in vitro analysis. Both of these genes are known to regulate the NF-κB pathway, and the risk alleles of the genes were implicated in the enhancement of NF-κB activity in our analyses. These results suggest that the NF-κB pathway plays a role in pathogenesis and would be a rational target for treatment of rheumatoid arthritis.     

Genome-wide association analysis in asthma subjects identifies SPATS2L as a novel bronchodilator response gene

Bronchodilator response (BDR) is an important asthma phenotype that measures reversibility of airway obstruction by comparing lung function (i.e. FEV(1)) before and after the administration of a short-acting β(2)-agonist, the most common rescue medications used for the treatment of asthma. BDR also serves as a test of β(2)-agonist efficacy. BDR is a complex trait that is partly under genetic control. A genome-wide association study (GWAS) of BDR, quantified as percent change in baseline FEV(1) after administration of a β(2)-agonist, was performed with 1,644 non-Hispanic white asthmatic subjects from six drug clinical trials: CAMP, LOCCS, LODO, a medication trial conducted by Sepracor, CARE, and ACRN. Data for 469,884 single-nucleotide polymorphisms (SNPs) were used to measure the association of SNPs with BDR using a linear regression model, while adjusting for age, sex, and height. Replication of primary P-values was attempted in 501 white subjects from SARP and 550 white subjects from DAG. Experimental evidence supporting the top gene was obtained via siRNA knockdown and Western blotting analyses. The lowest overall combined P-value was 9.7E-07 for SNP rs295137, near the SPATS2L gene. Among subjects in the primary analysis, those with rs295137 TT genotype had a median BDR of 16.0 (IQR = [6.2, 32.4]), while those with CC or TC genotypes had a median BDR of 10.9 (IQR = [5.0, 22.2]). SPATS2L mRNA knockdown resulted in increased β(2)-adrenergic receptor levels. Our results suggest that SPATS2L may be an important regulator of β(2)-adrenergic receptor down-regulation and that there is promise in gaining a better understanding of the biological mechanisms of differential response to β(2)-agonists through GWAS.