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Association of polymorphism in glutamate-cysteine ligase catalytic subunit gene with coronary vasomotor dysfunction and myocardial infarction

Shun-ichi Koide, MD^*, Kiyotaka Kugiyama, MD, PhD^*,*, Seigo Sugiyama, MD, PhD^*, Shin-ichi Nakamura, MD^*, Hironobu Fukushima, MD^*, Osamu Honda, MD^*, Michihiro Yoshimura, MD, PhD^* and Hisao Ogawa, MD, PhD^*

^* Department of Cardiovascular Medicine, Kumamoto University School of Medicine, Kumamoto, Japan
Second Department of Internal Medicine, Yamanashi University School of Medicine, Yamanashi, Japan

View larger version (53K): [in a new window]   Figure 1 Ethidium bromide gel showing three genotypes from a single nucleotide polymorphism (C or T) in the promoter region 129 bases upstream of the GCLC gene. The 613-bp polymerase chain reaction amplification fragment contains an invariant Tsp45I restriction site, yielding a constant 113-bp fragment seen in all lanes. Individuals homozygous for the C allele have no additional Tsp45I restriction sites and consequently show only two bands: at 500-bp and the invariant band at 113-bp. The –129T allele creates an additional Tsp45I site such that homozygous T-allele individuals have three bands at 302-, 198-, and 113-bp. Heterozygous individuals show all four bands.

View larger version (16K): [in a new window]   Figure 2 Percent changes (mean ± SEM) in lumen diameter from baseline in response to acetylcholine in the proximal and distal segments of the left anterior descending coronary arteries in subjects with the –129C/T or T/T genotype (solid bars) (n = 31) and in age-matched subjects with the –129C/C genotype (open bars) (n = 31).

View larger version (15K): [in a new window]   Figure 3 Effects of the –129C/T polymorphism on GCLC promoter activity in human umbilical vein endothelial cells. The transfected cells were incubated for 18 h with H2O2 (100 µmol/l) or phosphate-buffered saline (PBS) (as a time control). Promoter activity is expressed as relative luciferase activity normalized to Renilla activity. Data are presented as the mean value ± SEM from eight independent experiments. *p < 0.05, p < 0.01. Open bars = –129C; solid bars = –129T.

View larger version (69K): [in a new window]   Figure 4 Electrophoretic mobility shift assay: allele-specific binding of nuclear protein to the –129C/T polymorphic site. Nuclear extracts were obtained from human umbilical vein endothelial cells after treatment for the indicated time with H2O2 (100 µmol/l) or phosphate-buffered saline (PBS) (as a time control). Left panel = interaction with the –129C probe; right panel = interaction with the –129T probe.