Sequencing of the putative promoter region of the cocaine- and amphetamine-regulated-transcript gene and identification of polymorphic sites associated with obesity.
OBJECTIVE
We tested the hypothesis that polymorphisms in the cocaine- and amphetamine-regulated-transcript (CART) gene is associated with the development of obesity.


METHODS
Five-hundred and twenty-eight subjects (325 men and 203 women) aged 49.6+/-11.0 y with body mass index (BMI) of 26.9+/-5.1.


METHODS
The 5(')-flanking region of the CART gene was cloned using adaptor-ligated genomic DNA fragments. The CART gene including the 5(')-flanking region was screened for mutation by PCR-single strand conformation polymorphism and direct sequencing. Associations between polymorphisms and obesity were investigated by PCR-restriction fragment length polymorphism analysis and direct sequencing.


RESULTS
The 5(')-flanking region of the CART gene up to -1072 bp from the transcription initiation site was sequenced. The region contained a putative cyclic AMP-responsive element and four E-box motifs upstream of a TATA box. Six polymorphic sites were identified in the upstream region; A-->G at -156, T-->C at -390, T-->G at -484, G-->T at -915, G-->C at -929 and C-->T at -962. The nucleotide substitution at -156 was significantly associated with greater BMI (P=0.036). The allele frequency of the -156 variant was significantly higher in obese subjects with BMI > or = 30 than in non-obese subject (0.41 vs 0.32, P=0.0076). The -929 variant allele in linkage disequilibrium with the -156 variant was also more common in obese subjects. No mutation was found in the coding regions. A single nucleotide insertion/deletion polymorphism at +1355 in the 3(') untranslated region was not associated with obesity.


CONCLUSIONS
The 5(')-flanking region of the CART gene was highly polymorphic. The -156 polymorphism or polymorphisms in linkage disequilibrium with the site may be associated with genetic predisposition to obesity.
Yamada
K
K
Department of Endocrinology and Metabolism, Kurume University School of Medicine, Kurume, Japan. yamada@med.kurume-u.ac.jp
Yuan
X
X
Otabe
S
S
Koyanagi
A
A
Koyama
W
W
Makita
Z
Z
D001483:Base Sequence; D017931:DNA Primers; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009765:Obesity / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein
D017931:DNA Primers; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein