The understanding of the molecular mechanisms whereby prohormones are processed to mature hormones has been greatly enhanced by the identification and characterisation of two subtilisin-like prohormone convertases, PC I and PC2. both of which cleave prohormones at the carboxyl end of dibasic sequences. In the proposed host laboratory, two families have recently been identified in which primary disorders of prohormone processing appear to lead to complex endocrine pathologies. In Family 1, the pattern of defective prohormone processing strongly suggests a defect in the activity of the prohormone convertase PC 1, whereas in Family 2 the biochemical feature are more suggestive of a defect in PC2-mediated prohormone cleavage. The objectives of the project are to examine the genes encoding PC I and PC2 for mutations in these families and subsequently to examine the relevant genes in other families with similar phenotypes. To achieve these aims the following studies will be performed. I . As the orgamsation of the human PC I gene has not been established, the first step will involve the characterisation of the intron-exon boundaries of the normal human PC1 gene by PCR from an available YAC clone containing the entire PC 1 gene. Using the information derived from these initial studies, the nucleotide sequence of the PC I coding region in the proband of Family 1 will be determined by direct sequencing of each exon amplified by PCR.
2. The human PC2 gene has been characterised and a highly polymorphic CA repeat identified. The cosegregation of this polymorphic marker in the PC2 gene with the disease in Family 2 will be studied. If there is evidence that the PC2 gene could be linked to the phenotype in the Family 2, then the nucleotide sequence of the coding region of PC2 will be determined. 3. Subjects with clinical phenotypes related to those seen in the original families will be screened biochemically for evidence of defective prohormone processing. Where the biochemical findings are consistent with defective prohormone processing the appropriate prohormone convertase gene will be examined for mutations.
4. The functional properties of any mutant prohormone convertase molecules that are found will be examined.