GSNO Reductase and 2 Adrenergic Receptor Gene-gene Interaction: Bronchodilator
Responsiveness to Albuterol
†*
Shweta Choudhry, Ph.D., M.Sc.1, *Loretta G. Que, M.D.2 Zhonghui Yang, Ph.D.2, Limin Liu, Ph.D.3,
Celeste Eng, B.S.1, Sung O. Kim, Ph.D.4, Gunjan Kumar, B.S.1, Shannon Thyne, M.D.1, Rocio Chapela,
M.D.5, Jose R. Rodriguez-Santana, M.D.6, William Rodriguez-Cintron, M.D.7, Pedro C. Avila, M.D.8,
Jonathan S. Stamler, M.D.2, and Esteban G. Burchard, M.D., M.P.H.1,9
*
These authors contributed equally to this manuscript
1
Lung Biology Center and Institute for Human Genetics, University of California, San Francisco, CA
2
Duke University Medical Center, Durham, NC
3
Microbiology and Immunology, University of California, San Francisco, CA
4
Bipion Co. LTD, Seoul, Republic of Korea
5
Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico
6
Centro de Neumologia Pediatrica, CSP, San Juan, PR
7
Veterans Caribbean Health Care System, San Juan, PR
8
Division of Allergy-Immunology, Northwestern University, Chicago, IL
9
Department of Biopharmaceutical Sciences, University of California, San Francisco, CA
†Correspondence
and reprint requests: Shweta Choudhry, Ph.D. UCSF/Lung Biology Center,
University of California, San Francisco, San Francisco, California 94143-2911; telephone: 415-5149927, fax: 415-514-4365, e-mail: shweta.choudhry@ucsf.edu
Running Title: GSNOR-2AR interaction in bronchodilator response
1
Supported By: This work was supported by National Institutes of Health (HL078885, HL088133, U19
AI077439, ES015794), Flight Attendant Medical Research Institute (FAMRI), and the RWJ Amos
Medical Faculty Development Award to EGB), HL086887 to LGQ, National Institute of Environmental
Health Sciences 5U19-ES012496 and NHLBI R01HL96673-01 to JSS, American Thoracic Society
“Breakthrough Opportunities in Lung Disease” (BOLD) Award and Tobacco-Related Disease Research
Program New Investigator Award (15KT-0008) to SC, the Ernest S. Bazley Grant to PCA, and the
Sandler Center for Basic Research in Asthma and the Sandler Family Supporting Foundation.
2
Genotyping
Five GSNOR SNPs, +3207 in the potential promoter region and +16701, +16777, +17059 and +17250
in the 3’UTR region were genotyped in the entire GALA cohort. We selected these five SNPs for
genotyping based on their allele frequency (minor allele frequency > 5%), location (exonic or in
potential promoter region) and the haplotype structure of the GSNOR gene.
All five SNPs were
genotyped using the AcycloPrime-FPTM (PerkinElmer) method.(1) The PCR cocktail included: 2.4-4.0
ng genomic DNA, 0.1-0.2 µM primers, 2.5 mM MgCl2, 50 µM dNTPs, 6 µl volume with Platinum Taq
PCR buffer and 0.1-0.2 units Platinum Taq (Invitrogen) plus 1 µl extra water to counteract evaporation.
PCR cycling conditions were as follows: 95°C for 2 minutes, 35 cycles of 92°C for 10 seconds, 58°C for
20 seconds, 68°C for 30 seconds, and final extension at 68°C for 10 minutes. We used AcycloPrime-FP
kits for enzymatic cleanup and single base extension genotyping reactions. Plates were read on an
EnVision fluorescence polarization plate reader (PerkinElmer, Waltham, MA).
Constructs and Cell Transfections
Luciferase reporter gene constructs
The full length GSNOR 3’UTR was amplified from human cDNA. The resulting PCR products were
digested using proper restriction enzymes and cloned into both forward (F) and reverse (R) orientations
in the pGL3 plasmid containing an SV40 promoter upstream of the firefly luciferase gene (Promega
Corporation, Madison, WI).
Site directed mutagenesis
Site directed mutagenesis of the 3’UTR was performed using QuikChange® II XL Site Directed
Mutagenesis Kit (Stratagene, CA) according to the manufacturer’s instructions. Individual mutants
3
mimicking the SNPs associated with asthma in our patient population were generated by substituting
C+16701→T, A+17059→C and T+17250→C (data not shown). Another mutant was created containing
the three individual mutants to mimic the GSNOR haplotype associated with asthma. The mutated
fragments were amplified using the forward primer: TAATTCAAAAGAGAAAAATAATGTCCATCC
and the reverse primer: ATGTGACTACATTAAAACTTTATTCAAC, then ligated into the pGL3
promoter vector. All constructs used in this study were restriction-mapped and sequenced to confirm
authenticity and correct orientation in the vector.
Transfection experiments and Luciferase assay
The luciferase reporter gene constructs were transiently transfected into HEK293 cells with
Lipofectamine 2000 (Invitrogen; Carlsbad CA) according to the manufacturer’s protocol.
Approximately 1x106 HEK293 cells were seeded on each well of a 6-well tissue culture plate and
transfected with each plasmid construct (0.2 g). Co-transfection with the pRL vector (9 ng) containing
the Renilla luciferase gene allowed monitoring of transfection efficiencies. HEK293 cells were chosen
because they are of human origin, have high transfection efficiency, and also express the 2AR. Cells
were incubated for 48 hours and harvested by adding 500 l of reporter lysis buffer. Specific substrates
were used to measure the activities of the firefly and Renilla luciferase with a TD-20/20 luminometer
(Turner Designs, Sunnyvale, CA). Firefly luminescence was normalized to Renilla luminescence and
reported as relative luciferase units (RLU). All experiments were done in duplicate and independently
performed at least three times. Differences among cell transfection groups were evaluated using a
student’s t-test. A p value of < 0.05 was considered statistically significant.
4
References for online supplement
1.
Chen X, Levine L, Kwok PY. Fluorescence polarization in homogeneous nucleic acid analysis.
Genome Research 1999;9:492-498.
5
Table 1S. SNPs identified in resequencing of the GSNOR exons, exon-intron boundaries and potential promoter regions in Mexican
and Puerto Rican subjects with asthma.
Frequency
SNP
bp
position*
rs number
-749
SNP location
Alleles
Reference
Allele
Mexicans
(n = 24)
Puerto
Ricans
(n = 24)
Promoter 1
CAAA/-
-
0.02
0.02
Amino Acid
change
-745
rs3974877
Promoter 1
-/GAAA
GAAA
0.17
0.27
-421
rs17216887
Promoter 1
G/C
C
0.02
0.02
Promoter 2
C/T
T
0.02
0.02
Promoter 2
C/T
T
0.12
0.24
+6494
Intron 2
T/C
C
0.0
0.02
+6519
Intron 2
T/C
C
0.08
0.08
+6551
Intron 2
T/A
A
0.02
0.02
+6564
Intron 2
G/A
A
0.0
0.02
+7205
Intron 3
T/-
-
0.06
0.08
+11651
Intron 4
A/G
G
0.0
0.02
+12207
Exon 6
T/C
C
I --> T
0.0
0.02
+12216
Exon 6
G/A
A
C --> Y
0.02
0.0
+12443
Intron 6
G/T
T
0.0
0.04
+13597
Intron 6
TTG/-
-
0.08
0.08
+3003
+3207
rs1154410
6
Frequency
SNP
bp
position*
rs number
SNP location
Alleles
Reference
Allele
Mexicans
(n = 24)
Puerto
Ricans
(n = 24)
Intron 7
T/C
C
0.03
0.0
Intron 7
C/A
A
0.09
0.06
+16019
Exon 8
T/C
C
0.02
0.02
+16166
Intron 8
G/A
A
0.02
0.02
+16354
Exon 9
G/A
A
0.02
0.02
+13816
+15983
rs17595424
Amino Acid
change
Syn (L)
+16476
rs12697
3' UTR
T/C
C
0.11
0.11
+16502
rs12106
3' UTR
T/C
C
0.11
0.11
+16701
rs1803037
3' UTR
G/A
A
0.08
0.08
3' UTR
T/C
C
0.02
0.0
+16770
+16777
rs13832
3' UTR
T/G
G
0.21
0.29
+16858
rs6827292
3' UTR
A/G
G
0.02
0.02
+16999
rs1061187
3’ UTR
C/T
T
0.08
0.08
+17059
rs11547772
3’ UTR
T/G
G
0.08
0.08
+17200
3’ UTR
C/T
T
0.02
0.02
+17201
3’ UTR
G/T
T
0.02
0.02
3’ UTR
A/G
G
0.10
0.10
+17250
rs7669660
*The bp position of the SNP is calculated with reference to ATG with A as bp
7
African Americans
Figure 1S. Pair wise linkage disequilibrium (LD) as estimated by r2 statistic for the SNPs in the GSNOR (ADH5) gene with > 5%
frequency in Mexican and Puerto Rican subjects with asthma. r2 statistic of 0 means no LD and 1 means complete LD.
Puerto Ricans
Mexican
8