Reduced pericellular sensitivity to IGF-I in fibroblasts from girls with Turner syndrome: a mechanism to impair clinical responses to GH.

Girls with Turner syndrome (TS) are treated with supraphysiological doses of growth hormone (GH) to improve final height; however in some girls, the growth response can be poor. This may reflect aberrations in GH and/or IGF-I actions at the cellular level, and thus this study compared the response of skin fibroblasts from normal children (n = 5) and girls with TS (n = 8) to GH, IGF-I, or a combination, by assessing the IGF binding protein (IGFBP) profile of conditioned medium harvested over 7 d. The two cell types had a comparable IGFBP profile; IGFBP-3 and IGFBP-4 were the most abundant species. TS fibroblasts produced more IGFBP-3 (d 7, 51.4 ± 45 ng/mL versus 20 ± 22 ng/mL; p < 0.05) than control cells; levels of IGFBP-4 were similar (21 ± 12 ng/mL versus 30 ± 21 ng/mL). GH did not influence IGFBP production. IGF-I treatment did not affect IGFBP-4 levels but enhanced the production of IGFBP-3 by both cell types (p < 0.05). However, the response of TS fibroblasts to IGF-I was approximately half that observed in normal cells (p < 0.05). Altered IGF-I activity, because of reduced bioavailability and/or reduced sensitivity, could contribute to the need for high GH doses in TS and for the poor response to GH in some girls with TS.

The effect of different patterns of growth hormone administration on the IGF axis and somatic and skeletal growth of the dwarf rat.

Normal childhood growth is determined by ultradian and infradian variations in GH secretion, yet GH treatment of children with short stature is restricted to daily fixed doses. We have used GH-deficient dwarf rats to determine whether variable GH dose regimens promote growth more effectively than fixed doses. Animals were treated with saline or 4.2 mg of recombinant bovine GH given as 1) 700 microg/wk in 100 microg/day doses, 2) alternating weekly doses of 966 (138 microg/day) or 434 microg (62 microg/day), or 3) 700 microg/wk in randomized daily doses (5-250 microg/day). Body weight and length were measured weekly. Femur and tibia lengths and internal organ, fat pad, and muscle weights were recorded at the end of the study (6 wk); blood was collected for IGF axis measurements. GH promoted femur [F(3,60) = 14.67, P < 0.05], tibia [F(3,60) = 14.90, P < 0.05], muscle [F(3,60) = 10.37, P < 0.05], and organ growth [liver: F(3,60) = 9.30, P < 0.05; kidney: F(3,60) = 2.82, P < 0.05] and an increase in serum IGF-I [F(3,60) = 9.18, P < 0.05] and IGFBP-3 [F(3,60) = 6.70, P < 0.05] levels. IGF-I levels correlated with final weight (r = 0.45, P < 0.05) and length (r = 0.284, P < 0.05) in the whole cohort, but within each group, growth parameters correlated with serum IGF-I only in animals treated with random GH doses. The variable regimens promoted femur length (P < 0.05) and muscle (P < 0.05) and kidney (P < 0.05) weight more effectively than treatment with the fixed regimen. This study demonstrates that aspects of growth are improved following introduction of infradian variation to GH treatment in a GH-deficient model. The data suggest that varying the pattern of GH doses administered to children may enhance growth performance without increasing the overall GH dose.

A pilot study to evaluate gene expression profiles in peripheral blood mononuclear cells (PBMCs) from children with GH deficiency and Turner syndrome in response to GH treatment.

Response to GH treatment is variable and dependent on diagnosis and dose. We used a pharmacogenomic approach to assess whether this variability is reflected in patterns of GH-induced gene expression in peripheral blood mononuclear cells (PBMCs) taken from three children with GH deficiency (GHD) and three girls with Turner syndrome (TS). Analysis of the response to GH treatment revealed that in GHD, 15 probe sets (11 genes) showed a fold change > +/- 1.4 at a P-value < 0.0005 (and a false detection rate

The relationship between nocturnal urinary leptin and gonadotrophins as children progress towards puberty.

BACKGROUND/AIMS: Leptin is necessary for normal human pubertal development but its exact role in the period leading up to the onset of puberty has not been defined. This study has assessed the relationship between leptin and gonadotrophin secretion over time as children progress into puberty. SUBJECTS AND METHODS: Twenty children (13 boys and 7 girls) judged to be close to the initiation of puberty were recruited. Three consecutive first morning urine samples were collected from each subject each month over 6 months. At the end of the study, the children were classified into those who remained physically prepubertal (n = 7) and those that had advanced in puberty (n = 13). Leptin and gonadotrophins were measured by immunoradiometric and immunofluorometric assay, respectively. RESULTS: Total urinary leptin excreted over 6 months was higher in girls than in boys, both prepubertally and in early puberty, and in both sexes, was higher in those advancing into puberty than in those remaining prepubertal (girls 8.0 vs. 3.4 ng/l and boys 3.6 vs. 1.7 ng/l; both p < 0.05). In the whole group, when controlling for gender, there was a significant correlation between both leptin and luteinizing hormone (LH; r = 0.43, p < 0.001) and leptin and follicle-stimulating hormone (FSH; r = 0.32, p = 0.001). The possibility of a lead relationship was explored by pairing leptin values with the gonadotrophin values in the following month. Leptin was significantly correlated with FSH but not LH in both pre- and peripubertal children (prepubertal r = 0.45, p = 0.01; peripubertal r = 0.32, p = 0.01). CONCLUSIONS: This study has shown that in children approaching and progressing into puberty, leptin is associated with LH and FSH over the same time frame, and with FSH when leptin is acting as the lead hormone. These data imply that leptin is an important facilitator of the early phases of human puberty.

The contributions of plasma IGF-I, IGFBP-3 and leptin to growth in extremely premature infants during the first two years.

We determined the contributions of IGF-I, IGFBP-3 and leptin to growth in extremely premature infants over the first two years. Weight (Wt), crown-to heel length (CHL), plasma IGF-I, IGFBP-3 and leptin were measured in infants (gestation 24-33 wk) at birth (n = 54), expected date of delivery (EDD) and 6, 12 and 24 mo post-EDD (n = 29). Area under the curve (AUC) for hormone levels was calculated over 4 periods: birth-EDD, EDD-200 d, EDD-350 d and EDD-700 d. IGFBP-3, but not IGF-I or leptin, on day 1 correlated with birth Wt SD scores (SDS) (r = 0.46, p = 0.002) and CHL SDS (r = 0.41, p = 0.01). Wt SDS at EDD correlated with AUC IGF-I, IGFBP-3 and leptin (birth-EDD), but leptin was the best predictor in multiple regression (r = 0.65, p < 0.0001). Wt at EDD + 700 d correlated with AUC leptin (EDD-700 d) (r = 0.62, p = 0.002). CHL SDS at EDD correlated with AUC IGFBP-3 and leptin (birth-EDD), but IGFBP-3 was the best predictor (r = 0.55, p < 0.0001). CHL at EDD + 700 d correlated with AUC IGF-I and IGFBP-3 (EDD-700 d), but IGFBP-3 was the best predictor (r = 0.47, p = 0.01). Wt and CHL at birth were associated with IGFBP-3 levels in these infants. Wt at EDD and EDD + 700 d was predicted by concurrent leptin output while linear growth at EDD and EDD + 700 d was predicted by IGFBP-3 output.

Phenotypic variation in constitutional delay of growth and puberty: relationship to specific leptin and leptin receptor gene polymorphisms.

OBJECTIVES: Constitutional delay of growth and puberty (CDGP) is a variant of normal pubertal timing and progress, often with dominant inheritance. It is likely that one or more genes will be associated with CDGP. Possible candidates are the leptin (L) and the leptin receptor (LR) genes, as the leptin axis links nutritional status to pubertal development. This study has assessed whether a) L or LR gene polymorphisms were associated with CDGP and b) the CDGP phenotype was influenced by these polymorphisms. DESIGN: Case-control and transmission disequilibrium tests were used to test genetic association of L and LR polymorphisms with CDGP. METHODS: We genotyped L (3'CTTT repeat) and LR polymorphisms (Gln > Arg substitution, exon 6) in 81 CDGP children and 94 controls in the UK and 88 CDGP children from the US and assessed the effect of genotype on their anthropometric characteristics. RESULTS: There was no association of these L or LR gene polymorphisms with CDGP. There was no difference in height or bone age delay within L or LR genotypes. However, UK CDGP children homozygous for the L short allele were heavier than heterozygotes and long allele homozygotes, with a similar trend in the US cohort. UK CDGP children with severe pubertal delay, who were thin, had significantly greater bone age delay and an increased frequency of parental pubertal delay than other groups and were less likely to be L short allele homozygotes. CONCLUSIONS: There was no association of specific L or LR polymorphisms with CDGP, but L short allele carriage influenced the phenotype within CDGP.

Variability in anterior pituitary size within members of a family with GH deficiency due to a new splice mutation in the GHRH receptor gene.

OBJECTIVE: Mutations in the GHRH receptor (GHRHR) gene (GHRHR) cause autosomal recessive isolated GH deficiency (IGHD), and are usually associated with anterior pituitary hypoplasia (APH) (defined as pituitary height more than 2 SDS below normal). We searched for GHRHR mutations and studied pituitary morphology in three prepubertal sibs with severe IGHD, who were born from consanguineous parents. DESIGN: We sequenced the 13 exons and the intron-exon boundaries of the GHRHR of the index patient. After identifying a novel mutation, we sequenced the same area in the other family members. In addition, we performed magnetic resonance imaging (MRI) study of the pituitary (at age 8, 4 and 3 years) in the three affected subjects. RESULTS: The three children were homozygous for a new GHRHR mutation that alters the second base of the invariant 5' splice site (GT) of intron 12 [IVS12 + 2T-->A]. The parents and an unaffected sibling were heterozygous for the same change. MRI did not show frank APH (by height criteria) in any of the subjects: pituitary height was normal (5.6 mm, +1.8 SDS) in the oldest sibling, and it was low but not below 2 SDS by age-adjusted criteria in the second (3 mm, -1.4 SDS), and third sibling (2.8 mm, -1.7 SDS). Calculated pituitary volume was below -2 SDS in the youngest patient. CONCLUSIONS: These data demonstrate that pituitary height may fall within 2 SDS from the norm in patients with severe IGHD due to a homozygous GHRHR mutation, and that pituitary size may vary within patients with identical mutations who belong to the same family.

A new missense mutation in the growth hormone-releasing hormone receptor gene in familial isolated GH deficiency.

OBJECTIVE: Mutations in the GH-releasing hormone (GHRH) receptor (GHRHR) gene (GHRHR) cause autosomal recessive familial isolated GH deficiency (IGHD). We searched for GHRHR mutations in two siblings with IGHD type IB and a history of parental consanguinity. DESIGN: We analyzed peripheral genomic DNA of an index patient. After identifying a novel mutation in the GHRHR, we performed functional studies in order to confirm that the mutation causes receptor malfunction. METHODS: The entire GHRHR was analyzed in the index case by denaturing gradient gel electrophoresis. Abnormally migrating bands were isolated and sequenced. The mutated area was then sequenced in all family members whose DNA was available. The newly found mutation was inserted into a GHRHR cDNA. Wild-type and mutant cDNAs were expressed into CHO cells and the cyclic AMP (cAMP) response to GHRH was measured. In order to determine whether the mutant receptor was properly expressed on the cell membrane surface, CHO cells were transfected with wild-type or mutant GHRHR cDNA containing a FLAG epitope tag in the extracellular N-terminus. RESULTS: Both patients were homozygous for a new missense mutation in codon 176, corresponding to the second transmembrane domain of the receptor protein that replaces alanine with valine (A176V). The mother and three unaffected siblings were heterozygous for the mutation; DNA from the father was not available. Cells expressing the A176V receptor had a significantly reduced cAMP response to GHRH, despite appropriate expression on the cell surface. CONCLUSIONS: We describe two siblings with IGHD due to a new mutation in the GHRHR that disrupts GHRH signaling and leads to GHRH resistance.

Leptin measurement in urine in children and its relationship to other growth peptides in serum and urine.

OBJECTIVE: Leptin has been implicated in the interaction between nutrition, energy balance and sexual maturation in humans. A non-invasive method of measuring leptin would greatly facilitate longitudinal studies of changes in leptin in normal children. The aim of this study was to evaluate the use of urinary leptin as a surrogate for serum leptin measurements. DESIGN: We have modified and validated a serum immunoradiometric assay (IRMA) kit for the measurement of leptin in urine, and subsequently investigated the relationship between urinary leptin and other growth-related proteins. METHODS: Cross-sectional study: urinary leptin, measured in the first morning urine voided and expressed as ng excreted overnight, and serum concentrations of leptin, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 were determined in a cohort of 188 healthy schoolchildren aged 5-19 years (88 males, 100 females). Height, weight and pubertal status were assessed in all children. Longitudinal study: urinary levels of leptin, IGF-I and GH were measured daily in two adults (one male, one female) over a period of 6 weeks. RESULTS: The detection limit of this modified assay was 0.59 ng/L. The intra- and interassay coefficients of variation range was 4-8% and 4-9%, respectively. The recovery of recombinant leptin added to urine was 98-108%, and the assay had a recovery rate for serial dilution in the range of 106-112%. Urinary leptin correlated significantly with serum leptin (r = +0.65, P < 0.01). Urinary leptin showed similar changes through puberty to those of serum leptin, with levels rising in females throughout puberty, whereas in males levels peaked at G2/G3 then decreased. BMI SDS was the main determinant of urinary leptin, as it was for serum leptin. In the cross-sectional study urinary leptin correlated significantly with serum IGF-I (r = +0.41, P = 0.001), IGF-II (r = +0.19, P = 0.05), IGFBP-3 (r = +0.29, P = 0.001) and IGFBP-1 (r = -0.25, P = 0.001). In the adult study, leptin was also detected in urine with similar night-to-night variability to that found for IGF-I and GH. CONCLUSION: Urinary leptin is a valid marker of serum leptin concentrations, and therefore this non-invasive assay would be a useful tool for longitudinal assessment of changes in leptin in children.

GH and epidermal growth factor signaling in normal and Laron syndrome fibroblasts.

We have investigated and compared GH and epidermal growth factor (EGF) signaling in primary human skin fibroblasts from normal subjects and subjects with GH-binding protein-positive Laron syndrome (LS). In normal human fibroblasts, GH and EGF activate the tyrosine phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT5b; in LS fibroblasts, EGF does, but GH does not. GH also activates the tyrosine phosphorylation of Janus kinase (JAK)2 in normal, but not LS, fibroblasts. Similarly, both GH and EGF activate MAPK in normal fibroblasts, but only EGF does in the LS fibroblasts. As in the 3T3-F442A mouse preadipocyte cell line, GH signaling to mitogen-activated protein kinase is partially inhibited by wortmannin treatment, indicating a role for phosphatidylinositol 3-kinase (PI3K) in this signaling pathway. The exogenous expression of the GH receptor in one family of LS fibroblasts (H1) but not the other (M) restores signaling to a STAT5 reporter element. Together, these results indicate that the mechanism of defective GH signaling in two families of LS fibroblasts are different but that both occur at a level close to, and specific for, the GH receptor.