A proteomic approach identified growth hormone-dependent nutrition markers in children with idiopathic short stature

Background  

The broad range in growth observed in short prepubertal children receiving the same growth hormone (GH) dose is due to individual variation in GH responsiveness. This study used a pharmaco-proteomic approach in order to identify novel biomarkers that discriminate between short non-GH-deficient (GHD) children who show a good or poor growth response to GH treatment.     

Growth response to recombinant human growth hormone (GH) in children with idiopathic growth retardation by level of maximum GH peak during GH stimulation tests

Due to their lack of reproducibility, it is unlikely that GH stimulation tests can provide reliable diagnostic information to distinguish partial isolated GH deficiency (GHD) from idiopathic short stature (ISS). We hypothesized that the classical distinction between these groups, essentially based on stimulatory GH peaks, is artificial and that, as a consequence, the average response to GH treatment will not be different between them. The hypothesized lack of prognostic validity of stimulatory GH peaks was studied in 435 prepubertal children with nonorganic growth retardation. Children were categorized as 'severe GHD', 'partial GHD' or 'ISS', if the maximum rise in their serum GH during two GH stimulation tests was 0--10 mU/l, 10--20 mU/l, or >20 mU/l, respectively. Children with 'partial GHD' had short-term (1- and 2-year) and long-term (till final adult height) growth responses similar to those of children with ISS, significantly lower than the response seen in children with 'severe GHD'. In children with stimulatory GH peaks >10 mU/l, including those currently considered partially GH deficient, the maximum GH peak was not a significant determinant of growth response in the short or the long term. In conclusion, 'partial GHD' is ill defined and cannot be distinguished from ISS based on the currently applied auxological or GH stimulation test criteria alone. More research is required for better identification of (all) children who will respond to GH treatment, whether or not GH deficient.     

Plasma levels of insulin-like binding protein-2 in prepubertal short children and its diagnostic value in the evaluation of growth hormone deficiency

AIM: This study was designed to investigate whether determination of plasma insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) levels could be of benefit in the evaluation of childhood growth hormone (GH) deficiency (GHD). METHOD: A retrospective analysis was performed on 91 prepubertal children referred for investigation of short stature. Maximal GH levels in plasma after provocative stimuli were between 1.0 and 93.0 mU/l, 6 subjects exhibiting peak values of <5 mU/l. Initially a GH peak of 20 mU/l was used as a cutoff limit to define GHD and idiopathic short stature (ISS) patients. The results of GH provocative tests were compared to age- and gender-based standard deviation scores (SDS) of plasma IGFBP-2, IGF-I, IGFBP-3 and the molar ratios of the latter two to IGFBP-2. The respective normative range values for these parameters were determined in plasma samples from 353 healthy children (i.e. 171 girls, 182 boys). RESULTS: Circulating IGFBP-2 levels did not correlate with height SDS, height velocity SDS or the peak GH levels after provocative stimuli. A weak negative relationship was found between IGFBP-2 and IGF-I. Plasma levels of IGFBP-2 in GHD patients were higher than those of ISS children, who had normal levels. Although at the optimal cutoff point of -0.71 SDS 91.5% of the GHD patients were identified correctly, a substantial proportion (71.9%) of the ISS subjects also had IGFBP-2 levels above this limit. The use of various combinations of IGFBP-2, IGF-I, IGFBP-3 and the derived ratios only slightly improved the diagnostic efficiency as compared to the results of the individual tests. Neither IGFBP-2 nor the IGFBP-3/IGFBP-2 and IGF-I/IGFBP-2 ratios were found to be related to the short- (1 year) or long-term (3 years) growth response to GH therapy. CONCLUSION: It is concluded that none of the tests investigated, either alone or in various combinations, are reliable in either predicting the peak GH level after provocative stimuli in prepubertal short children or in predicting their growth response to GH.     

Growth prediction models,concept and use

There is a principal and qualitative difference between using regression results on data from groups of children, and using validated prediction models for individual children. Using accurate models, it is now possible to predict the growth response to growth hormone (GH) treatment in a slowly growing child with GH deficiency (GHD) or in a child with idiopathic short stature (ISS). The growth response to the standard dose of GH can be regarded as a bioassay for GH (i.e. the tissue GH responsiveness) and the information on this growth response can be used for different purposes: to decide about treatment or not, for monitoring, and for adjusting the GH dose in order to reach a defined goal for height. This last concept is now used in an ongoing prospective randomized GH dose-finding trial.     

Should we treat children with idiopathic short stature?

The use of growth hormone (GH) to treat short children who are clearly GH-deficient is now well accepted. However, GH treatment of short children who have no currently recognizable abnormalities in their GH-insulin-like growth factor I axis remains controversial. Whether such children with so-called idiopathic short stature (ISS) should be treated with GH was the subject of an international workshop held in St.-Paul-de-Vence, France, in April 1999. This article summarizes the issues discussed at the workshop, including the definition of ISS, ethical and health-economic aspects of treatment, results from clinical trials and surveillance studies, and the use of prediction models in aiding treatment decisions.     

Normal progression of testicular size in boys with idiopathic short stature and isolated growth hormone deficiency treated with growth hormone: experience from the KIGS

BACKGROUND: The aim of this retrospective analysis was to evaluate the effects of growth hormone (GH) treatment on testicular development in boys with idiopathic short stature (ISS) and isolated GH deficiency (IGHD) followed in the KIGS (Pharmacia International Growth Database). METHODS: For inclusion in the study, the patients had to have received more than 1 year of prepubertal GH treatment, at least 4 consecutive years of GH treatment in total, and to have attained their final height, defined as a height velocity of less than 2 cm/year. Data on 107 boys in the KIGS database have been analyzed. RESULTS: No significant differences in duration of GH treatment and testicular volume at the start of treatment or at final height were found between the boys with ISS and those with IGHD. The progression of testicular volume in boys with ISS or IGHD during GH treatment did not differ from the reference population. CONCLUSIONS: This analysis shows that GH treatment does not alter testicular growth in boys with ISS or IGHD. However, prospective controlled studies are needed to rule out moderate attenuating or stimulating effects.     

Skeletal dysplasia, growth hormone treatment and body proportion: comparison with other syndromic and non-syndromic short children

Skeletal dysplasias comprise a diverse group of conditions that usually compromise both linear growth and body proportions. It is of theoretical interest to evaluate the effect of GH treatment on linear growth, body proportion and final height in the different skeletal dysplasias. Reported experience of GH treatment in short children with skeletal dysplasia is sparse and often limited to short treatment periods and knowledge of its effects on final height and body proportion is generally lacking. Formal studies are almost all confined to achondroplasia as the most common entity. First-year response is typically a 2-3 cm increase in growth velocity in prepubertal children, or a gain of about 0.5 SDS or less in relative height from a baseline level of -4 to -5 SDS. GH treatment for up to 5 years in achondroplasia can produce a total height gain of about 1 SDS. Apart from achondroplasia, treatment of hypochondroplasia and dyschondrosteosis with GH has been reported in a small number of patients. Long-term data are, however, lacking. Of theoretical interest is that in many syndromic or non-syndromic short-statured children body proportion, i.e. trunk to leg length ratio, does not seem to be dependent on the degree of GH sufficiency and does not seem to be changed by GH treatment. GH treatment, at least in the prepubertal period, does seem to influence degree of disproportion.     

Comparative proteomic analysis in children with idiopathic short stature (ISS) before and after short‐term recombinant human growth hormone (rhGH) therapy

This study was undertaken to identify growth hormone (GH) responsive proteins and protein expression patterns by short‐term recombinant human growth hormone (rhGH) therapy in patients with idiopathic short stature (ISS) using proteomic analysis. Seventeen children (14 males and three females) with ISS were included. They were treated with rhGH at a dose of 0.31 ± 0.078 mg/kg/week for 3 months. Immunodepletion of six highly‐abundant serum proteins followed by 2D DIGE analysis, and subsequent MALDI TOF MS, were employed to generate a panel of proteins differentially expressed after short‐term rhGH therapy and verify the differences in serum levels of specific proteins by rhGH therapy. Fourteen spots were differentially expressed after rhGH treatment. Among them, apo E and apo L‐1 expression were consistently enhanced, whereas serum amyloid A was reduced after rhGH therapy. The differential expressions of these proteins were subsequently verified by Western blot analysis using sera of the before and after rhGH treatment. This study suggests that rhGH therapy influences lipoprotein metabolism and enhances apo L‐1 protein expression in ISS patients.     

Challenges of Securing Growth Hormone Coverage for Idiopathic Short Stature: Review of the 7-Year Experience at one Institution

Objective: Despite U.S. Food & Drug Administration (FDA) approval of growth hormone (GH) for idiopathic short stature (ISS), many providers face challenges obtaining insurance coverage. We reviewed the insurance coverage experience for ISS at our hospital to identify factors predictive of approval or denial.Methods: We reviewed charts of patients who underwent GH stimulation testing from July 1, 2009, to April 30, 2017, to identify ISS patients (height <-2.25 SD, subnormal predicted adult height (PAH) and peak GH >10 ng/mL).Results: Eighty-seven patients met ISS criteria, of whom 47 (29 male/18 female) had a GH request submitted to insurance. Mean age, height, and growth velocity were 8.6 ± 2.7 years, 2.83 ± 0.4 SD, and 4.4 ± 1.7 cm/year, respectively. Mean PAH based on bone age was -2.50 ± 0.9 SD, equaling 62 inches for males and 58 inches for females. Most had private managed care insurance (74%). Overall, 17/47 (36%) received treatment approval, 7 immediately and 10 more on appeal. There were no differences in age, height SD, growth rate, insurance type, or PAH between the 17 who were approved and the 30 denied. For 21 patients who were treated, a mean increase in 0.6 SD in height was seen after 1 year.Conclusion: At our institution, GH coverage requests for ISS included very short children mostly ages 6 to 11, with heights well below -2.25 SD and poor PAH. Only 36% were approved even after appeal. This highlights the challenge in our area to secure GH treatment for a FDA-approved indication. Collaboration between pediatric endocrinologists and insurers focusing on height SD and PAH, may improve cost-effective coverage to deserving short children who meet FDA guidelines for ISS treatment.Abbreviations: FDA = Food and Drug Administration; GH = growth hormone; IGF-1 = insulin-like growth factor 1; ISS = idiopathic short stature; PAH = predicted adult height     

Metabolic consequences of growth hormone treatment in paediatric practice

No metabolic side-effects of clinical significance have been reported during a 5-year study of growth hormone (GH) therapy in children with GH deficiency, Turner syndrome, idiopathic short stature or chronic renal insufficiency. In particular, insulin levels increase but remain within the normal range, as do glucose and haemoglobin A(1c). A recent study showed that the effects of growth on insulin sensitivity in prepubertal children with idiopathic short stature represent the changes in carbohydrate tolerance observed during normal adolescence. Thus, GH treatment may lead to prolongation of the physiological state of insulin resistance observed in normal puberty. Insulin levels during the fasting state and 2 h after a standard glucose load showed no further rise after the first 3 years of continuous GH therapy. The hyperinsulinaemia observed during GH therapy may, therefore, amplify the anabolic effects of insulin on protein metabolism during puberty.     

Oral clonidine provocative test in the diagnosis of growth hormone deficiency in childhood: should we make the timing uniform?

INTRODUCTION: Oral clonidine is one of the most frequent drugs used for the diagnosis of growth hormone deficiency (GHD), but the duration of the test, depending on which European centres use it, is not uniform and can vary from 120 to 150 min or even 180 min. SUBJECTS AND METHODS: To standardize this test, evaluating the possibility to shorten it to 90 min, we investigated the response of GH to the oral clonidine test in 291 children evaluated for short stature (height <-2 SD). Of these, 164 were diagnosed as idiopathic short stature (ISS) and 127 as GHD. In these patients, we calculated: (1) the frequency distribution of the GH peaks to clonidine in GHD and in ISS at various times; (2) the percentage of GH peaks to clonidine before and after 90 min in all and in ISS children; (3) the percentage of the first GH value >or=10 ng/ml before 90 min and after 90 min in ISS. RESULTS: GH peak distribution varied between 30 and 180 min, even though the vast majority of peaks occurred between 30 and 60 min. There was no significant difference (p > 0.05) in the peak distribution between ISS and GHD children. The percentages of GH peaks within 90 min were 92.1% in all children and 95.7% in ISS. If considering the first value of GH >or=10 ng/ml this last percentage reaches 96.3%. CONCLUSION: Our study suggests that the oral clonidine test can be administered for only 90 min without significantly changing its validity. This test should be standardized at 90 min in European protocols just as in those currently used in the USA in order to reduce the discomfort of patients and the cost of this diagnostic procedure.     

Spontaneous prolactin secretion in growth hormone-deficient children.

OBJECTIVE: To establish the spontaneous nocturnal prolactin (PRL) release in relation to growth hormone (GH)-deficient children and idiopathic short-stature children (ISS). METHODS: A total of 32 prepubertal children (11 girls, 21 boys) aged between 3 and 12 years were studied retrospectively and sorted according to diagnosis: idiopathic GH deficiency (GHD, n = 9), neurosecretory deficiency of GH secretion (NSD, n = 10) and ISS (n = 13). Nocturnal spontaneous hormone secretion was studied by intermittent venous sampling. Secretion profiles and copulsatility were analyzed using Pulsar and AnCoPuls software. RESULTS: (median, range in mug/l): Children with GHD and NSD had significantly lower GH and area-under-the-curve (AUC) levels than normal children (p < 0.001), whereas ISS children showed normal values. In contrast, prolactin levels were significantly higher (p < 0. 05) in children with GHD and NSD (11.1, 4.9 - 13.0 and 10.3, 8. 8 - 19. 6, respectively) compared to the ISS children (8.0, 4.9 - 13.0). In addition, prolactin AUC and peak height were higher (p < 0.05) in GH-deficient patients, whereas all other secretion parameters were the same. Correlation and copulsatility analysis revealed no evidence for a direct relation between PRL and GH secretion. CONCLUSIONS: PRL secretion is significantly higher in children with GHD and NSD compared to ISS children but PRL and GH show no copulsatile secretion pattern.     

Resolution of apparent growth hormone-dependent growth failure during puberty: a case report

A prepubertal boy with apparent growth hormone (GH)-dependent growth failure displayed a marked increase in growth velocity, normal GH responses to arginine/insulin infusion and a fourfold increase in spontaneous 24-hour GH secretion following the onset of normal puberty. The case supports earlier observations of a transient form of GH insufficiency in some short prepubertal children, but represents the first evidence that puberty restores spontaneous as well as stimulated GH secretion in such patients.     

Bone age progression during the first year of growth hormone therapy in pre-pubertal children with idiopathic growth hormone deficiency, Turner syndrome or idiopathic short stature, and in short children born small for gestational age: analysis of data from KIGS (Pfizer International Growth Database)

BACKGROUND/AIMS: The beneficial effects of growth hormone (GH) therapy on statural growth in children are well established, but the effects on skeletal maturation are less clear. The progression of bone age (BA) was therefore studied during the first year of GH treatment in pre-pubertal children with idiopathic GH deficiency (GHD), Turner syndrome (TS) or idiopathic short stature (ISS), and in short pre-pubertal children born small for gestational age (SGA). METHODS: Cross-sectional data on 2,209 short children with idiopathic GHD, 694 with TS, 569 with ISS and 153 with SGA were analysed. Longitudinal data were also analysed from 308 children with idiopathic GHD, 99 with TS, 57 with ISS and 29 with SGA. All patients included in the study were enrolled in KIGS (Pfizer International Growth Database) and were being treated with recombinant human GH (Genotropin). BA was assessed using the Greulich and Pyle method at baseline and after 1 year of GH therapy. RESULTS: In all groups of patients the mean progression of BA was 1 year during the year of GH therapy, although there was considerable individual variation. Progression of BA was not correlated with chronological age, BA, height SD score (SDS) or body mass index SDS at the onset of GH therapy. There was also no consistent effect of the GH dose on BA progression. CONCLUSION: Progression of BA appears to be normal in patients receiving GH in these diagnostic groups, at least over the first year of treatment in pre-puberty.     

Non-compliance with growth hormone treatment in children is common and impairs linear growth

Background

GH therapy requires daily injections over many years and compliance can be difficult to sustain. As growth hormone (GH) is expensive, non-compliance is likely to lead to suboptimal growth, at considerable cost. Thus, we aimed to assess the compliance rate of children and adolescents with GH treatment in New Zealand.

Methods

This was a national survey of GH compliance, in which all children receiving government-funded GH for a four-month interval were included. Compliance was defined as ≥85% adherence (no more than one missed dose a week on average) to prescribed treatment. Compliance was determined based on two parameters: either the number of GH vials requested (GHreq) by the family or the number of empty GH vials returned (GHret). Data are presented as mean ± SEM.

Findings

177 patients were receiving GH in the study period, aged 12.1±0.6 years. The rate of returned vials, but not number of vials requested, was positively associated with HVSDS (p<0.05), such that patients with good compliance had significantly greater linear growth over the study period (p<0.05). GHret was therefore used for subsequent analyses. 66% of patients were non-compliant, and this outcome was not affected by sex, age or clinical diagnosis. However, Maori ethnicity was associated with a lower rate of compliance.

Interpretation

An objective assessment of compliance such as returned vials is much more reliable than compliance based on parental or patient based information. Non-compliance with GH treatment is common, and associated with reduced linear growth. Non-compliance should be considered in all patients with apparently suboptimal response to GH treatment.     

Growth hormone response to a bolus injection of 1-44 growth-hormone-releasing hormone in very short children with intrauterine onset of growth failure

The release of growth hormone (GH) during the 120 min following a bolus venous injection of 1-44 GH-releasing hormone (GHRH) 2 micrograms/kg was studied in 52 prepubertal children aged 8.4 +/- 2.1 years, having a nonfamilial growth deficiency of prenatal onset (-3.26 +/- 1.13 SDS at birth, -3.22 +/- 0.88 SDS at the time of study) and a normal response to conventional GH stimulation tests. GH release reached a peak level of 96.1 +/- 60.2 microU/ml, being significantly higher than that found in 68 non-GH-deficient very short children whose growth failure had a postnatal onset, and not significantly correlated with the response to conventional tests. 26 of the 52 intrauterine growth retardation (IUGR) patients were re-tested with GHRH in similar conditions after 6-12 months of daily subcutaneous injections of GH and 2 days without. They reached at the second test a peak plasma GH level of 91.7 +/- 56.1 microU/ml, not different from their response to the first test. These data could be taken into consideration for long-term studies of the clinical effects of GH in IUGR children with persisting severe growth deficiency.     

Change in the growth hormone (GH) response to GH-releasing factor (GRF) caused by exogenous GH in short children

To determine whether exogenous GH induces feedback of GH release in children, growth hormone-releasing factor (GRP) tests were performed before and after 10-day GH administration. Sixteen non-obese short boys, aged 5-14 yr, with normal GH response to pharmacological tests were studied. Mean basal and peak serum GH levels in GRF tests before and after exogenous GH were not significantly different. The subjects were divided into two groups, A and B, according to the percent change in integrated areas under the GH curves in GRF tests (GH AUC) before and after 10-day GH administration. Group A consisted of 6 boys with decreased GH AUC and group B consisted of 10 boys with increased GH AUC. Mean peak GH in GRF tests and mean GH AUC were significantly higher before exogenous GH in group A than in group B. The boys in group A were all prepubertal, while 4 boys in group B had begun their early pubertal change. The mean age in group A (7.8 +/- 1.8 yr) was significantly lower than that of group B (11.9 +/- 2.4 yr). GH AUC before exogenous GH showed a significant correlation with the percent change in AUC (= -0.742, p less than 0.01). These data demonstrated that the exogenous GH suppressed the GH response to GRF in prepubertal children with good response to GRF before exogenous GH, while it exaggerated the GH response to GRF in older children with relatively poor response before GH.     

Genetic variations at the human growth hormone receptor (GHR) gene locus are associated with idiopathic short stature

GH plays an essential role in the growing child by binding to the growth hormone receptor (GHR) on target cells and regulating multiple growth promoting and metabolic effects. Mutations in the GHR gene coding regions result in GH insensitivity (dwarfism) due to a dysfunctional receptor protein. However, children with idiopathic short stature (ISS) show growth impairment without GH or GHR defects. We hypothesized that decreased expression of the GHR gene may be involved. To test this, we investigated whether common genetic variants (microsatellites, SNPs) in regulatory regions of the GHR gene region were associated with the ISS phenotype. Genotyping of a GT‐repeat microsatellite in the GHR 5′UTR in a Montreal ISS cohort (n = 37 ISS, n = 105 controls) revealed that the incidence of the long/short (L/S) genotype was 3.3× higher in ISS children than controls (P = 0.04, OR = 3.85). In an Italian replication cohort (n = 143 ISS, n = 282 controls), the medium/short (M/S) genotype was 1.9× more frequent in the male ISS than controls (P = 0.017, OR = 2.26). In both ISS cohorts, logistic regression analysis of 27 SNPs showed an association of ISS with rs4292454, while haplotype analysis revealed specific risk haplotypes in the 3′ haploblocks. In contrast, there were no differences in GT genotype frequencies in a cohort of short stature (SS) adults versus controls (CARTaGENE: n = 168 SS, n = 207 controls) and the risk haplotype in the SS cohort was located in the most 5′ haploblock. These data suggest that the variants identified are potentially genetic markers specifically associated with the ISS phenotype.     

Physiological growth hormone secretion in children with short stature and intra-uterine growth retardation

31 prepubertal children with short stature [mean height standard deviation score (SDS) -2.84] and low birth weight (mean -2.82 SDS) were studied. Mean age was 6.0 years and mean height velocity SDS was -0.76. Patients were classified as having either the clinical characteristics of Russell-Silver syndrome (RSS) (4 F, 13 M) or not (4 F, 10 M). All children had an overnight profile of spontaneous growth hormone (GH) secretion. 4 children achieved a maximum GH concentration of less than 20 mU/l. 9 children with RSS secreted only one large GH peak during the night. Most of the non-RSS group had normal GH pulse frequency but 3 boys had a fast-frequency pattern. Abnormal GH secretion may contribute towards growth failure in children with low birth weight/RSS.     

Plasma growth hormone response to synthetic GH-RH1-44 in 52 children and adults with growth hormone deficiency of various etiologies

52 patients (42 children and 10 adults) with growth hormone deficiency (GHD), grouped into four diagnostic categories, and 6 children with constitutional short stature who served as controls were tested for plasma GH response to synthetic GH-RH1-44 given in an intravenous bolus. The response was classified into three degrees according to the magnitude of the maximal rise: Good, greater than 9 ng/ml; Partial, 3.1-9.0 ng/ml; None, less than or equal to 3 ng/ml. Among the GHD patients the highest response was observed in patients with partial growth hormone deficiency (PGHD), and 60% of the children with isolated GH deficiency (IGHD) showed an increase in plasma GH levels. Nevertheless, the response of the GHD patients was lower than that in the control group. In the children and adolescents with PGHD and IGHD the response was not age related. Among those with multiple pituitary hormone deficiencies-idiopathic (MPHD-ID) there was no response in the adolescents although a hypothalamic disorder had been documented by other tests. Among those with MPHD-organic (MPHD-ORG) the GH-RH stimulated GH secretion in the patients with glioma, who had received only irradiation treatment, and in the youngest of the patients with craniopharyngioma. Of the 10 young adults tested none showed a good response. It is concluded that GH-RH is useful in differentiating between GH deficiency of hypothalamic origin and that of pituitary origin, and in selecting those patients who might benefit from long-term treatment with GH-RH in the future.