Functional characterization of a -100_-102delAAG deletion-insertion polymorphism in the promoter region of the HTR3B gene.
OBJECTIVE
The HTR3B gene encodes the B-subunit of the type 3 serotonin receptor (5-HT3). A -100_-102delAAG deletion in the promoter region has been associated with poor response to antiemetic medication and susceptibility to bipolar affective disorders. The molecular mechanisms underlying these associations, however, remained unclear.


METHODS
We performed electrophoretic mobility shift and luciferase reporter gene assays to elucidate the effect of this polymorphism on the HTR3B promoter activity in PC-12 and HEK293 cells. The reporter constructs carried a 2171 bp fragment of the native HTR3B promoter or 30 bp of the polymorphic locus in tandem triplication upstream of the thymidine kinase minimal promoter.


RESULTS
Deletion mapping indicated that the sequence around the -100_-102delAAG polymorphism had significant promoter activity. Electrophoretic mobility shift assays indicated differential binding of nuclear proteins to the polymorphic DNA region with stronger binding to the insertion than to the deletion allele. The activity of the native promoter carrying the deletion allele was 25% higher in PC-12 (P=0.016) and 40% higher in HEK cells (P=0.016) compared with the respective insertion construct. Constructs carrying the deletion allele in tandem triplicates showed 43% (PC-12 cells, P=0.002) and 28% (HEK293 cells, P=0.015) higher activity than those carrying the insertion allele. The polymorphism was not linked with known amino acid substitutions in HTR3A and HTR3B.


CONCLUSIONS
The -100_-102delAAG 3 bp deletion increases the HTR3B promoter activity in vitro. The consequences of this for the structure and the function of the resulting 5-HT3 receptors remain to be elucidated.
Meineke
Cornelia
C
Department of Clinical Pharmacology, Georg-August-University Göttingen, Göttingen, Germany.
Tzvetkov
Mladen Vassilev
MV
Bokelmann
Kristin
K
Oetjen
Elke
E
Hirsch-Ernst
Karen
K
Kaiser
Rolf
R
Brockmöller
Jürgen
J
D000818:Animals; D001483:Base Sequence; D002477:Cells; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D006239:Haplotypes; D006801:Humans; D015810:Linkage Disequilibrium; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D016716:PC12 Cells; D010597:Pharmacogenetics; D011401:Promoter Regions, Genetic; D051381:Rats; D011985:Receptors, Serotonin / Q000235:genetics* / Q000378:metabolism; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D017931:DNA Primers; C509410:HTR3B protein, human; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins; D004247:DNA; D008156:Luciferases