The proximal promoter region of the human pituitary expressed growth hormone (GH1) gene is highly polymorphic, containing at least 15 single nucleotide polymorphisms (SNPs). This variation is manifest in 40 different haplotypes, the high diversity being explicable in terms of gene conversion, recurrent mutation, and selection. Functional analysis showed that 12 haplotypes were associated with a significantly reduced level of reporter gene expression whereas 10 haplotypes were associated with a significantly increased level. The former tend to be more prevalent in the general population than the latter (p<0.01), possibly as a consequence of selection. Although individual SNPs contributed to promoter strength in a highly interactive and non-additive fashion, haplotype partitioning was successful in identifying six SNPs as major determinants of GH1 gene expression. The prediction and functional testing of hitherto unobserved super-maximal and sub-minimal promoter haplotypes was then used to test the efficacy of the haplotype partitioning approach. Electrophoretic mobility shift assays demonstrated that five SNP sites exhibit allele-specific protein binding. An association was noted between adult height and the mean in vitro expression value corresponding to an individual's GH1 promoter haplotype combination (p=0.028) although only 3.3% of the variance of adult height was found to be explicable by reference to this parameter. Three additional SNPs, identified within sites I and II of the upstream locus control region (LCR), were ascribed to three distinct LCR haplotypes. A series of LCR-GH1 proximal promoter constructs were used to demonstrate that 1) the LCR enhanced proximal promoter activity by up to 2.8-fold depending upon proximal promoter haplotype, and that 2) the activity of a given proximal promoter haplotype was also differentially enhanced by different LCR haplotypes. The genetic basis of inter-individual differences in GH1 gene expression thus appears to be extremely complex.

In short non-GH-deficient SGA children, both spontaneous growth rate and responsiveness to 66 microg/k.d GH therapy were similar for each d3/fl-GHR genotype carried.

Communicated by Mark H. PaalmanSubtle mutations in the growth hormone 1 (GH1) gene have been regarded as a comparatively rare cause of short stature. Such lesions were sought in a group of 41 individuals selected for short stature, reduced height velocity, and bone age delay; a group of 11 individuals with short stature and idiopathic growth hormone deficiency (IGHD); and a group of 154 controls. Heterozygous mutations were identified in all three groups but disproportionately in the individuals with short stature, both with (odds ratio 25.2; 95% CI, 5.1-132.2) and without (odds ratio 3.6; 95% CI, 1.0-12.9) IGHD. Twenty-four novel GH1 gene lesions were found. Thirteen novel missense mutations were characterized by assaying the signal transduction activity of in vitro expressed variants; six (T27I, K41R, N47D, S71F, S108R, and T175A) exhibited a reduced ability to activate the JAK/STAT pathway. Molecular modeling suggested that both K41R and T175A might compromise GH receptor binding. Seven GH variants (R16C, K41R, S71F, E74K, Q91L, S108C, and a functional polymorphism, V110I) manifested reduced secretion in rat pituitary cells after allowance had been made for the level of expression attributable to the associated GH1 proximal promoter haplotype. A further leader peptide variant (L-11P) was not secreted. Eleven novel mutations in the GH1 gene promoter were assessed by reporter gene assay but only two, including a GH2 gene-templated gene conversion, were found to be associated with a significantly reduced level of expression. Finally, a novel intron 2 acceptor splice-site mutation, detected in a family with autosomal dominant type II IGHD, was shown to lead to the skipping of exon 3 from the GH1 transcript. A total of 15 novel GH1 gene mutations were thus considered to be of probable phenotypic significance. Such lesions are more prevalent than previously recognized and although most may be insufficient on their own to account for the observed clinical phenotype, they are nevertheless likely to play a contributory role in the etiology of short stature. Hum Mutat 21:424-440,

A B S T R A C T This study examines the ontogenesis of somatomedin and insulin receptors in man. Particulate plasma membranes were prepared by ultracentrifugation from various tissues removed from fetuses after abortion and classified as <17, 17-25, and >25 cm in length. The binding of iodinated insulinlike growth factors 1 (IGF-1) and 2 (IGF-2), somatomedin A (SMA), multiplication-stimulating activity (MSA), and insulin was examined at the different ages.In the liver, cross-reaction studies revealed separate insulin and IGF-2 receptors. The Scatchard plots of insulin binding to liver membranes were curvilinear and showed an increase in the concentration of insulin receptors with advancing age. A single IGF-2 receptor was found on liver and no alteration was observed during development. The brain contained a lower concentration of insulin receptors. A change in the brain receptors for somatomedins occurred during development. Early in gestation, a high concentration of a low-affinity IGF-1 receptor was found. After approximately the 17th wk of gestation a higher affinity IGF-1 receptor appeared, which then increased in concentration. Cross-reaction studies also revealed changes in the specificity of these receptors during development. In the youngest fetal group IGF-2 was preferentially bound. Around midgestation a separate IGF-1 receptor, indicated by the preferential displacement of iodinated IGF-1 by IGF-1, appeared. In contrast, iodinated IGF-2 bound to a receptor where IGF-1 and IGF-2 were equipotent. Dr. M. Misaki's present address is

Our data showed significant differences in the frequency distribution of the d3/fl-GHR genotypes between a normally distributed adult height population and short SGA children, with the biologically less active fl/fl genotype being almost twice as frequent in SGA patients. These data suggest that the d3/fl-GHR polymorphism might be considered among the factors that contribute to the phenotypic expression of growth.