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. SUBJECTS: 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. MEASUREMENTS: 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 greater than or equal to 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. CONCLUSION: 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.