A Common Polymorphic Allele of the Human Luteinizing Hormone β-Subunit Gene: Additional Mutations and Differential Function of the Promoter Sequence

Abstract

A common genetic variant (V) of the human luteinizing hormone (LH) β-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-LHβ allele. We have now studied whether additional mutations in the V-LHβ promoter sequence could contribute to the altered physiology of the LH variant molecules. The 661 bp 5′-flanking region of the V-LHβ gene, retrieved from human genomic DNA by PCR, contained eight single-nucleotide changes, as compared with the wild-type (wt) LHβ 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, LβT₂, known to express the endogenous LHβ gene, and into a non-endocrine human embryonic kidney cell line, HEK 293. Basal expression levels of the V-LHβ promoter constructs were on average 36% higher in LβT₂ 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 gonadotropin-releasing hormone. In conclusion, the V-LHβ promoter has higher basal activity, and differs in response to hormonal stimulation, as compared with the wt-LHβ promoter. The altered promoter function of the V-LHβ gene provides evidence for differences in regulation of the wt- and V-LHβ genes, which may contribute to the differences observed in pituitary-gonadal function between carriers of the two LHβ 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.