A common polymorphic allele of the human luteinizing hormone beta-subunit gene: additional mutations and differential function of the promoter sequence.
A common genetic variant (V) of the human luteinizing hormone (LH) beta-subunit gene was recently discovered. The V-LH molecules have higher bioactivity in vitro, but shorter half-life in circulation, which apparently is related to the alterations of LH function observed in individuals homo- and heterozygous for the V-LHbeta allele. We have now studied whether additional mutations in the V-LHbeta promoter sequence could contribute to the altered physiology of the LH variant molecules. The 661 bp 5'-flanking region of the V-LHbeta gene, retrieved from human genomic DNA by PCR, contained eight single-nucleotide changes, as compared with the wild-type (wt) LHbeta promoter. The finding was consistent in DNA samples of different ethnic groups. Reporter constructs with various lengths of the wt- and V-LH promoter sequences, driving the firefly luciferase reporter gene, were transfected into an immortalized mouse pituitary cell line, LbetaT(2), known to express the endogenous LHbeta gene, and into a non-endocrine human embryonic kidney cell line, HEK 293. Basal expression levels of the V-LHbeta promoter constructs were on average 36% higher in LbetaT(2)cells ( P < 0.001; n = 29), and 40% higher in HEK 293 cells ( P < 0.001; n = 16), as compared with the respective wt sequences. Numerous qualitative and quantitative differences were found between the two cell lines in responses of the two promoter sequences to stimulation with 12- O -tetradecanoylphorbol-13-acetate, forskolin, 8-bromo-cAMP, progesterone and gonado- tropin-releasing hormone. In conclusion, the V-LHbeta promoter has higher basal activity, and differs in response to hormonal stimulation, as compared with the wt-LHbeta promoter. The altered promoter function of the V-LHbeta gene provides evidence for differences in regulation of the wt- and V-LHbeta genes, which may contribute to the differences observed in pituitary-gonadal function between carriers of the two LHbeta alleles. The findings also suggest a novel evolutionary mechanism whereby polymorphic changes resulting in altered bioactivity of a gene product may be compensated for by additional mutations in the cognate promoter sequence, changing transcription of the same gene.
Jiang
M
M
Department of Physiology, University of Turku, Kiinamyllynkatu, Finland.
Pakarinen
P
P
Zhang
F P
FP
El-Hefnawy
T
T
Koskimies
P
P
Pettersson
K
K
Huhtaniemi
I
I
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D005576:Colforsin / Q000494:pharmacology; D004252:DNA Mutational Analysis; D005006:Ethnicity / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D005787:Gene Frequency; D017930:Genes, Reporter; D005838:Genotype; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D007986:Luteinizing Hormone / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000166:cytology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011374:Progesterone / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D011993:Recombinant Fusion Proteins; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D007987:Gonadotropin-Releasing Hormone; D011374:Progesterone; D007986:Luteinizing Hormone; D008156:Luciferases; D001616:beta-Galactosidase; D013755:Tetradecanoylphorbol Acetate