CONSEQUENCES OF HUMAN PIT-1 AND GH PROMOTER MUTATIONS

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Pituitary growth hormone production is essential for normal postnatal growth. Recent studies in the applicant's laboratory have linked mutations of the Pit-1 gene to a human growth disorder involving deficiencies of the anterior pituitary hormones growth hormone, prolactin and thyrotropin. Humans with a substitution of proline for alanine at amino acid 158 differ from Snell dwarf mice with a mutation at amino acid 261 in that they have normal anterior pituitary size and only mild thyrotropin deficiency. The observation that an alteration in the POU-specific domain of Pit-1 eliminates transcriptional activation of its known target genes growth hormone and prolactin, with minimal impairment of binding to these genes and no impairment of anterior pituitary growth was not predicted by studies of mutant Pit-1 proteins produced in the laboratory. It suggests that sequence variation in the Pit-1 gene and its target gene promoters may account for a broader spectrum of human growth disorders. The proposed studies address this issue through examination of Pit-1 gene sequence in a large number of families with combined GH, Prl and TSH deficiencies, autosomal recessive isolated growth hormone deficiency, or autosomal dominant isolated growth hormone deficiency. The alternative possibility of an abnormality of regulatory elements in the growth hormone gene promoter is addressed through sequencing of the growth hormone promoter in families with autosomal recessive isolated growth hormone deficiency. Sequence variation is detected through polymerase chain reaction amplification of genomic DNA or cDNA and localized through direct DNA sequencing. The functional consequences of Pit-1 gene and growth hormone promoter sequence abnormalities are assessed by mobility shift DNA-binding assays, co-transfection of activator and reporter gene constructs in HeLa cells, and assays for activity of wild type and mutant Pit-1 in Adenovirus DNA replication assays. Studies of DNA sequence variation in children with severe disorders of growth are accompanied by studies of polymorphic variation in the same sequences among subjects at the extremes for normal growth. This project will define the importance of variations in Pit-1 and its target gene sequences in the pathogenesis of anterior pituitary hormone deficiency and in the generation of differences in patterns of growth among normal individuals.