Molecular Pathophysiology of Human Growth Disorders

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DESCRIPTION (adapted from the application) We have undertaken a patient-based study of the role of molecular defects in families and individuals with growth disorders that are likely to be due to genetic aberrations in constituents of the Growth Hormone axis. Families and individuals with growth disorders-isolated Growth Hormone (GH) deficiency or GH excess, as well as multiple pituitary hormone deficiencies-are evaluated for mutations in candidate genes chosen according to the specific endocrine phenotype of that family/individual. The objective of this study is to understand the mechanism by which mutations in genes result in abnormal growth. The elucidation of the causes o abnormal growth-and the specific derangements wrought by them-will allow a greater understanding of the physiology of 'normal' growth and shed light on the larger question of what proportion of human attributes (e.g., height) are determined by genetics. The Principal Investigator currently holds an NIDDK-funded Mentored Clinical Scientist Development Award (K-08 DK02569) through June 30, 2002, and is requesting additional funds to improve the ability to complete the goals as stated in this and the original application. We hypothesize that a subset of individuals with growth disorders have a mutation in a gene within the Growth Hormone axis. After a detailed endocrine evaluation, we evaluate families for mutations in PROP-1, PIT-1, LHX3, LHX4, Growth Hormone Releasing Hormone (GHRH), the GHRH Receptor (GHRHR), the Growth Hormone Secretagogue (GHS, or GHRELIN), the GHS receptor (GHSR) and Growth Hormone (GH1). We analyze for genetic linkage/haplotype disequilibrium to identify a candidate gene, then screen exon-by-exon, by either Denaturing High-Performance Liquid Chromatography (DHPLC) or Single Strand Conformation Polymorphism Analysis (SSCA) then directly sequence the conformationally unique exon in affected and unaffected members of a family. We then confirm mutation status with an automated Single Nucleotide Polymorphism (SNP) analyzer, or alternatively, through the use of restriction fragment length polymorphism (RFLP) analysis (including induced-mutagenesis, when necessary). The mutated gene product is then transfected into an appropriate cell line (e.g., a mammalian somatotrope or somatomammotrope) and expressed to determine the resulting specific cellular derangement. As our previous work has shown, the specific cell line used for expression is critical to obtaining physiologically relevant results. Both genomic DNA and tumor cDNA are used as templates for mutation analysis in tumor tissue, allowing for differentiation between germ-line and somatic mutations. A novel aspect of this grant is the full spectrum analysis of these subjects-proceeding from patient to hormones to DNA to cell system. The transfection studies meant to complete the initial work has been started and will be the major focus of the remaining two years of the K-08 grant period. The enhanced funding will increase throughput in all phases by increasing personnel and equipment, improving the Principal Investigator's prospects for obtaining an Individual Investigator Research Grant (R-01), and his ultimate goal of becoming an independent investigator.