Near-final height outcomes in children with idiopathic short stature responsive to the IGF-1 generation test: a multicenter retrospective study of rhGH therapy

Abstract

Background

Children with idiopathic short stature (ISS) have reduced height potential without identifiable endocrine or systemic causes. Selecting appropriate candidates for recombinant human growth hormone (rhGH) therapy remains challenging. The aim of this study was to describe near-final height (NFH) outcomes and to identify clinical predictors of growth response in children with ISS who were selected for rhGH therapy based on a positive IGF-1 generation test (IGFGT).

Methods

In this multicenter retrospective study (2009–2018) across four centers in Türkiye, patients diagnosed with ISS, baseline IGF-1 < 0 SDS, and ≥ 20% IGF-1 increment after IGFGT were included. GH1 gene sequencing was performed to exclude biologically inactive GH, a condition marked by normal or elevated GH secretion but impaired biological activity. NFH was defined as bone age ≥ 14 years in girls and ≥ 16 years in boys. Height SDS gain from baseline to NFH was evaluated.

Results

Sixty-three patients (28 females, 19 pubertal) were enrolled, of whom 55 had NFH data and were included in the final analysis. Mean age at treatment initiation was 10.8 ± 3.0 years. No GH1 mutations were identified. Among 55 patients with NFH data, mean height SDS increased from − 2.90 ± 0.77 at baseline to -1.92 ± 0.90 at NFH (p < 0.001), with a mean height SDS gain of 0.93 ± 0.68. The median difference between NFH SDS and baseline predicted adult height (PAH) SDS was − 0.06 (− 0.32–0.07). Predictors of NFH gain included female gender, target height (TH) SDS, baseline height-TH difference, baseline height-PAH difference, and first-year growth velocity SDS.

Conclusion

rhGH therapy was associated with improvements in height SDS and NFH in this selected cohort of children with ISS. However, the absence of an untreated control group and the minimal difference between NFH and baseline PAH indicate that these findings should be interpreted with caution. Although no GH1 mutations were identified, the relatively high rate of parental consanguinity suggests that other genetic factors within the GH–IGF-1 axis or epiphyseal growth plate pathways may underlie the etiology of short stature.

Clinical trial number

Not applicable.

What is already known on this topic

• Idiopathic short stature (ISS) is a diagnosis of exclusion in children with growth failure, characterized by normal GH levels and no identifiable cause.

• There is limited data on the long-term growth outcomes of patients diagnosed with ISS, particularly regarding height SDS gains after rhGH therapy.

What this paper adds

• This is the largest multicenter cohort from Türkiye evaluating near-final height outcomes in patients with ISS.

• The study demonstrates that height SDS gains can be achieved with appropriate rhGH treatment in ISS.

• Children with ISS who were selected for rhGH therapy according to national treatment criteria, including a positive IGF-1 generation test, demonstrated gains in near-final height.

Introduction

Idiopathic short stature (ISS) is defined as a diagnosis of exclusion in children whose height is below − 2 SDS, with normal birth parameters and body proportions, normal growth hormone (GH) secretion on stimulation testing, and no evidence of systemic, endocrine, nutritional, or chromosomal disorders [1]. Within this heterogeneous group, a subset of patients has been proposed to exhibit “biologically inactive GH,” characterized by normal or elevated GH levels but impaired biological activity, leading to reduced insulin-like growth factor-1 (IGF-1) production and growth failure [2]. Biologically inactive GH syndrome was first described by Kowarski et al. [3] in 1978. Subsequent studies have identified pathogenic mutations in the GH1 gene as a rare cause of biologically inactive GH [4–6]. These GH1 variants have been implicated in growth failure through various mechanisms, including reduced binding affinity to the GH receptor (GHR), disruption of disulfide bond formation, impaired interaction with GH binding proteins, defective GHR dimerization, and antagonistic effects on wild-type GH [2].

As diagnostic tools and genetic technologies advance, the ability to exclude underlying genetic causes of short stature continues to evolve. Moreover, whether biologically inactive GH represents a distinct clinical entity or substantially overlaps with ISS remains uncertain. Another important consideration is that, historically, ISS encompassed several genetic diagnoses that were not recognized at the time. With contemporary genetic techniques, approximately 25–40% of children previously diagnosed with ISS can now receive a molecular diagnosis. Monogenic defects—such as mutations in short stature homeobox (SHOX), aggrecan (ACAN), fibroblast growth factor receptor 3 (FGFR3), natriuretic peptide receptor 2 (NPR2), or other components of the GH/IGF-1 axis—should now be excluded from the ISS category through advanced genetic testing. This extreme heterogeneity within the ISS population significantly impacts responses to recombinant human growth hormone (rhGH) therapy. Furthermore, establishing a molecular diagnosis is crucial, as it can guide treatment decisions, support individualized management strategies, and enable appropriate genetic counseling [1].

rhGH has been approved for the treatment of ISS in many countries, and several studies have demonstrated significant improvements in growth velocity and final adult height, particularly when treatment is initiated at a younger age and with higher doses [1]. In cases of suspected biologically inactive GH, exogenous rhGH may bypass the impaired bioactivity of endogenous GH and stimulate growth [2]. Previous reports support the efficacy of rhGH in promoting linear growth in short-statured children with reduced GH bioactivity [7, 8], and similar benefits have been documented in patients with ISS receiving long-term treatment [9–12]. However, despite these findings, evidence regarding the long-term efficacy of rhGH therapy, particularly its impact on near-final height (NFH), remains limited in children with ISS.

This multicenter retrospective study aimed to evaluate the long-term efficacy of rhGH therapy on linear growth and NFH in children clinically diagnosed with ISS who were selected for rhGH therapy according to national treatment criteria that include a positive IGF-1 generation test (IGFGT), and to investigate potential GH1 gene variants in these patients.

Methods

Study design and patients

This multicenter retrospective study included 63 consecutively enrolled patients diagnosed with ISS and followed in four centers in Türkiye between 2009 and 2018.

Inclusion criteria were: (1) height standard deviation score (SDS) < − 3, or height SDS between − 2 and − 3 with an annual growth velocity SDS < − 1, or a decrease in height SDS > 0.5 over two years; in children without overt short stature, a one-year growth velocity SDS < − 2 or two-year growth velocity SDS < − 1.5; (2) basal serum IGF-1 level < 0 SDS; (3) sufficient response (peak GH > 10 ng/mL) to GH stimulation tests; and (4) an IGF-1 increase ≥ 20% from baseline following the IGFGT.

In Türkiye, demonstration of an IGF-1 increment ≥ 20% in the IGFGT is required for reimbursement of rhGH therapy in patients without confirmed GHD. Therefore, all patients underwent IGFGT to meet reimbursement criteria and assess GH responsiveness.

Exclusion criteria included any chronic systemic disease, malnutrition, skeletal dysplasia, or other conditions potentially affecting growth. All patients were born appropriate for gestational age with normal birth weight and length according to gestational age. At baseline evaluation, body proportions were assessed clinically and no disproportionate short stature suggestive of skeletal dysplasia was observed. Systemic, endocrine, nutritional, or syndromic disorders affecting growth were excluded during the diagnostic work-up. In female cases, karyotype analysis confirmed a normal 46,XX karyotype, thereby excluding Turner syndrome.

Anthropometric data, pubertal stages, peak GH responses to stimulation tests, basal and stimulated IGF-1 levels, and other relevant clinical parameters were recorded. Height and weight SDS values were calculated using national reference standards and an online pediatric endocrinology calculator [13]. Pubertal status was determined according to Tanner staging, with breast stage ≥ 2 in girls and testicular volume ≥ 4 mL in boys considered indicative of puberty. Bone age was assessed using the Greulich and Pyle atlas, predicted adult height (PAH) was estimated using the Bayley-Pinneau method, and target height (TH) was calculated as:

• (mother’s height + father’s height) / 2 + 6.5 cm for boys.

• (mother’s height + father’s height) / 2–6.5 cm for girls.

Definition of near-final height

Near-final height was defined as a bone age of ≥ 14 years in girls and ≥ 16 years in boys, indicating near completion of linear growth.

Analyses related to NFH were restricted to patients with available follow-up data (n = 55).

Outcome measures

The primary outcome of the study was height SDS gain, expressed as the change in height SDS.

Height SDS gain (Δheight SDS) was defined as the difference between NFH SDS and baseline height SDS.

Variables such as height SDS – TH SDS and height SDS – PAH SDS represent the difference between the patient’s height SDS and the corresponding TH SDS or PAH SDS.

Duration of rhGH treatment was defined as the interval between treatment initiation and treatment discontinuation, and did not necessarily correspond to the entire interval between treatment initiation and NFH assessment.

Growth hormone stimulation tests

All patients underwent GH stimulation testing during the diagnostic evaluation of short stature. During the study period, a GH peak response threshold of > 10 ng/mL was used to exclude classical GH deficiency.

Although lower thresholds have been proposed in more recent clinical guidelines, the cut-off value used in this study reflects the diagnostic criteria applied at the time when the patients were evaluated.

Pharmacological stimulation tests included L-dopa, clonidine, or insulin tolerance tests.

IGF-1 generation test

Patients received subcutaneous rhGH injections at a dose of 100 µg/kg/day for four days. This dosing regimen differs from the weekly dosing used for long-term rhGH therapy. Serum IGF-1 levels were measured at baseline and 8–16 h after the final dose. The percentage increment in IGF-1 was then calculated.

A ≥ 20% increase in serum IGF-1 concentration was considered a positive response. This criterion reflects national treatment reimbursement for rhGH therapy rather than a validated predictive test for GH treatment response.

IGF-1 concentrations were expressed as IGF-1 SDS calculated according to age- and sex-specific reference ranges based on chronological age [14].

GH1 gene molecular working methods

The GH1 gene variations were analyzed using a standardized protocol and equipment. 5 mL of peripheral venous blood from 63 patients was taken into tubes with EDTA. If the blood can be delivered to the laboratory within 1 week, it was stored at + 4 °C. In case of delivery to the laboratory in a longer time, the blood was stored at -20 °C. Genomic DNA isolation was performed automatically from peripheral blood lymphocytes from patients using the MagNA Pure LC 2.0 device (Roche Applied Science, Pleasanton, CA, USA). Exons 2, 3, 4, and 5 of the GH1 gene were amplified using appropriate primers by PCR to cover the intron-exon junction regions [15]. The PCR primers and amplification protocol were based on the methodology described in this reference and represent the protocol routinely used in our laboratory at the time the genetic analyses were performed. It was not included in the study because there was no mutation information about the first exon. BigDye Terminator v3.1 Cycle Sequencing Kit on an ABI PRISM 3130 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) was used for Sanger sequencing and subsequently analyzed by SeqScape Software version 2.7 and Sequencing Analysis Software version 5.1.

Ethics

This study was conducted in accordance with the ethical standards of the institutional and national research committees and the 1964 Helsinki Declaration and its later amendments. Ethical approval was obtained from the Clinical Research Ethics Committee of Kırıkkale University (Project No: 2009/134). Written informed consent was provided by parents or legal guardians for all participants.

Statistical analysis

Data were analyzed using Jamovi statistical software (version 2.6.44) and IBM SPSS Statistics version 21.0 (IBM Corp., Armonk, NY, USA). Categorical variables are presented as frequencies (n) and percentages (%), and continuous variables as means ± standard deviation (SD) or median (interquartile range). Normality was assessed using the Shapiro–Wilk test. Group comparisons were made using the chi-square or Fisher’s exact test for categorical variables, the Mann–Whitney U test for non-normally distributed continuous variables, and the Student’s t-test or Welch’s t-test for normally distributed continuous variables. Paired comparisons of baseline and NFH height SDS were performed using the Wilcoxon signed-rank test. Pearson correlation and multiple linear regression analyses were used to identify predictors of NFH outcomes. A post-hoc power analysis was performed using the G*Power module in Jamovi to evaluate the statistical power of group comparisons. Power was calculated for comparisons with non-significant results, including patients with and without parental consanguinity (n = 12 vs. n = 43) and pubertal status groups (n = 18 vs. n = 37), assuming a moderate effect size (Cohen’s d = 0.5), a two-tailed test, and α = 0.05. A p-value < 0.05 was considered statistically significant.

Results

Demographic and auxological characteristics at the diagnosis

A total of 63 patients (28 females, 19 pubertal) clinically diagnosed with ISS and fulfilling the inclusion criteria were enrolled from four centers. However, analyses related to near-final height (NFH) were restricted to the 55 patients with available follow-up data, and all baseline characteristics presented in Table 1 refer to this subgroup.

Table 1.

Demographic characteristics of patients at diagnosis

Variable	Total (n = 55)	Female (n = 24)	Male (n = 31)	p-value	Prepubertal (n = 37)	Puberty (n = 18)	p-value
Chronologic age (years) &	10.8 ± 3.0	10.5 ± 2.5	10.9 ± 3.3	0.238	9.8 ± 2.5	13.4 ± 2.2	< 0.001
Female/male, n (%)	24 (43.6)/31 (56.4)	-	-	-	18 (48.6)/19 (51.4)	6 (33.3)/12 (66.7)	0.283
Prepubertal/pubertal, n (%)	37 (67.3)/18 (32.7)	18 (75)/6 (25)	19 (61.3)/12 (38.7)	0.283	-	-	-
Consanguineous parents, n (%)	12 (21.8)	4 (16.6)	8 (25.8)	0.416	10 (27)	2 (11.1)	0.298
Bone age (years) &	8.4 ± 3.4	8.4 ± 2.8	8.3 ± 3.9	0.952	7.2 ± 3.2	10.9 ± 2.5	< 0.001
Chronologic age-Bone age (years) &&	2.00 (1.50–2.70)	1.80 (1.50–2.20)	2.40 (1.67–3.92)	0.030	1.95 (1.50–2.50)	2.20 (1.60–2.70)	0.749
Weight SDS&&	-2.31 (-2.86–-1.86)	-2.33 (-3.12–-2.06)	-2.31 (-2.75–-1.70)	0.252	-2.31 (-2.80–-1.66)	-2.33 (-3.08–-2.12)	0.206
Height SDS&&	-2.90 ± 0.77	-2.89 ± 0.73	-2.91 ± 0.81	0.940	-2.93 ± 0.78	-2.83 ± 0.78	0.649
TH SDS&	-1.36 ± 0.97	-1.57 ± 1.04	-1.21 ± 0.90	0.209	-1.58 ± 0.92	-0.89 ± 0.92	0.016
Height SDS-TH SDS&	-1.41 ± 1.12	-1.16 ± 1.18	-1.61 ± 1.06	0.156	-1.25 ± 1.16	-1.77 ± 0.98	0.104
PAH SDS&	-1.95 ± 1.37	-2.39 ± 1.08	-1.53 ± 1.51	0.044	-2.29 ± 1.32	-1.36 ± 1.29	0.036
Height SDS-PAH SDS&	-0.93 ± 1.11	-0.50 ± 0.80	-1.33 ± 1.22	0.014	-0.63 ± 0.99	-1.44 ± 1.13	0.022
PAH SDS-TH SDS&	-0.66 ± 1.67	-0.79 ± 1.64	-0.54 ± 1.73	0.633	-0.68 ± 1.83	-0.64 ± 1.4	0.939
Peak GH after 1st stimulus (ng/mL)&	9.1 (5.8–11.8)	7.9 (6.3–12.1)	9.7 (5.4–11.6)	0.617	9.3 (5.0–10.8)	10.9 (7.5–19.9)	0.041
Peak GH after 2nd stimulus (ng/mL)&	14.1 (11.7–16.7)	14.5 (12.9–17.1)	12.3 (11.5–15.9)	0.257	13.7 (11.5–16.7)	14.1 (12.9–16.3)	0.617
GH peak (ng/mL) &	13.9 (11.5–17.4)	14.3 (12.0–17.8)	12.3 (11.4–17.2)	0.455	13.2 (11.4–16.6)	14.9 (12.0–20.4)	0.149
Baseline IGF-1 (ng/mL) &	139 (98–199)	149 (110–187)	132 (77–216)	0.549	118 (81–159)	216 (185–286)	< 0.001
Baseline IGF-1 SDS&	-0.67 ± 0.93	-0.55 ± 0.85	-0.76 ± 0.99	0.427	-0.66 ± 0.96	-0.71 ± 0.89	0.850
Peak IGF-1 (ng/mL) &	249 (159–387)	244 (176–343)	251 (77–397)	0.595	208 (139–280)	374 (261–445)	< 0.001
IGF-1 increment compared to baseline (%)&	53.2 (36.9–89.0)	53.0 (38.0–79.0)	57.0 (34.4–94.3)	0.560	60.0 (37.0–92.0)	51.5 (33.4–66.0)	0.237
Duration of active rhGH treatment (months) &	36 (24–48)	36 (24–48)	36 (22–45)	0.324	36 (24–48)	30 (24–36)	0.162
Total dose (mg/kg/week) &	0.20 ± 0.01	0.20 ± 0.01	0.20 ± 0.02	0.680	0.20 ± 0.01	0.20 ± 0.03	0.100

^&: Means ± SD, ^&&: Median (interquartile range)

Normally distributed variables are presented as mean ± standard deviation and compared using the independent samples t-test or Welch’s t-test, as appropriate. Non-normally distributed variables are expressed as median (interquartile range) and compared using the Mann–Whitney U test. Categorical variables were compared using the chi-square or Fisher’s exact test

Abbreviations: SDS, standard deviation score; TH, target height; PAH, predicted adult height; GH, growth hormone; IGF-1, insulin-like growth factor-1; rhGH, recombinant human growth hormone

At treatment initiation, the mean chronological age was 10.8 ± 3.0 years, and the mean bone age was 8.4 ± 3.4 years. Mean baseline height SDS was − 2.89 ± 0.73 in females and − 2.91 ± 0.81 in males. Parental consanguinity was present in 21.8% of the patients.

Among the 63 patients, four (6.3%) had baseline height SDS values slightly above − 2.0 (range, − 1.90 to − 1.60). All four patients fulfilled national treatment criteria because of documented growth deceleration and were included in the NFH analysis cohort of 55 patients.

GH stimulation tests using L-dopa, clonidine, and insulin tolerance tests showed sufficient peak GH responses (> 10 ng/mL) in 52.3% (33/63), 100% (25/25), and 100% (5/5) of patients, respectively. Median baseline IGF-1 level was 139 (98–199) ng/mL and mean IGF-1 SDS was − 0.67 ± 0.93.

Following the IGFGT, the median peak IGF-1 level increased to 249 (159–387) ng/mL, with a median percentage increment of 53.2 (36.9–89.0)% relative to baseline. Prior to rhGH treatment, the mean growth velocity SDS was − 1.43 ± 1.33. No GH1 gene variants were identified in any patient within the sequenced regions of the GH1 gene (exons 2–5). Table 1 summarizes the demographic and auxological characteristics at diagnosis.

Growth outcomes of rhGH treatment

Among the 55 patients who reached NFH, pubertal staging at treatment initiation showed that 37 (67.3%) were prepubertal (Tanner stage 1), while 9 (16.4%), 6 (10.9%), and 3 (5.5%) were at Tanner stages 2, 3, and 4, respectively.

The distribution of sex across pubertal groups was as follows: among prepubertal patients, 18 (48.6%) were female and 19 (51.4%) were male, whereas among pubertal patients, 6 (33.3%) were female and 12 (66.7%) were male. There was no statistically significant difference in sex distribution between prepubertal and pubertal groups (p = 0.283).

Growth velocity during the first year of therapy was available for 47 patients who completed at least one year of treatment. The median duration of rhGH therapy was 36 (24–48) months, representing the period of active rhGH treatment rather than the total interval from treatment initiation to NFH assessment. The mean rhGH dose was 0.20 ± 0.01 mg/kg/week. Growth velocity and growth velocity SDS peaked during the first year of treatment (8.83 ± 1.02 cm/year and 2.0 ± 0.71, respectively). Although growth velocity declined in subsequent years, it remained higher than baseline levels (Table 2).

Table 2.

Growth velocity and growth velocity standard deviation score of patients during the treatment

	n	Growth velocity (cm/year) &	Growth velocity SDS&
T0 (baseline/before rhGH therapy)	55	4.01 ± 1.45	-1.43 ± 1.33
T1 (1st year of rhGH therapy)	47	8.83 ± 1.02	2.0 ± 0.71
T2 (2nd year of rhGH therapy)	41	6.66 ± 1.79	0.98 ± 0.94
T3 (3rd year of rhGH therapy)	27	6.32 ± 2.59	1.06 ± 1.13
T4 (4th year of rhGH therapy)	15	5.72 ± 1.92	0.74 ± 0.98
T5 (5th year of rhGH therapy)	7	5.22 ± 2.32	0.04 ± 1.09
T6 (6th year of rhGH therapy)	4	4.29 ± 1.49	0.00 ± 1.30
T7 (7th year of rhGH therapy)	2	5.20 ± 0.99	0.30 ± 1.18
T8 (8th year of rhGH therapy)	1	6.26	1.11

^&: Means ± SD

Abbreviations: SDS, standard deviation score

Auxological characteristics at near-final height

Near-final height data were available for 55 patients (87.3% of the original cohort), and all analyses evaluating NFH outcomes were performed in this subgroup together with their corresponding baseline characteristics. At NFH, the mean chronological age and median bone age of the patients were 16.1 ± 1.2 and 16 (15–16) years, respectively.

The mean height SDS increased from − 2.90 ± 0.77 at baseline to − 1.92 ± 0.90 at NFH (p < 0.001), with a mean Δheight SDS of 0.93 ± 0.68, indicating considerable inter-individual variability. The difference between mean height SDS and TH SDS also decreased significantly, from − 1.41 ± 1.12 at baseline to − 0.45 ± 1.16 at NFH (p < 0.001). Trends of height SDS and height SDS-TH SDS at the baseline and the NFH are shown in Fig. 1.

Fig. 1.

Changes in height SDS and the difference between height SDS and target height SDS from baseline to near-final height (NFH). * Wilcoxon signed rank test

There were no significant differences in Δheight SDS between female and male patients (p = 0.925) or between prepubertal and pubertal patients at treatment initiation (p = 0.960). Although pubertal females and prepubertal males demonstrated higher mean Δheight SDS, these differences were not statistically significant. In females, mean Δheight SDS was 1.25 ± 0.76 in pubertal patients and 0.85 ± 0.58 in prepubertal patients (p = 0.445), whereas in males the corresponding values were 1.02 ± 0.88 in prepubertal patients and 0.77 ± 0.44 in pubertal patients (p = 0.227).

Among patients with available NFH data, 20 (42.6%) achieved a height above − 2 SDS, whereas 24 (51.1%) reached NFH within their TH range, defined as TH SDS ± 1.5 SDS.

No significant differences were observed in baseline auxological characteristics or growth outcomes between patients with and without parental consanguinity (all p > 0.05). A post-hoc power analysis for the comparison between patients with and without parental consanguinity (n = 12 vs. n = 43) yielded a statistical power of 0.32 to detect a moderate effect size (Cohen’s d = 0.5), indicating limited ability to detect small-to-moderate differences.

No serious adverse events related to rhGH therapy were observed during follow-up.

When NFH was compared with baseline PAH, the median difference in the overall cohort was − 0.06 SDS (− 0.32– 0.07). This difference did not differ significantly between females and males (− 0.05 [− 0.39–0.01] vs. − 0.07 [− 0.24–0.39] SDS, p = 0.236). Similarly, no significant difference was observed between patients who were prepubertal and those who were pubertal at treatment initiation (p = 0.230). For the comparison between pubertal and prepubertal patients (n = 18 vs. n = 37), the post-hoc power was 0.46 for detecting a moderate effect size (Cohen’s d = 0.5), indicating limited statistical power to detect small or moderate differences.

Table 3 summarises the auxological characteristics at the NFH.

Table 3.

Auxological characteristics at the NFH

At the NFH	Total (n = 55)	Female (n = 24)	Male (n = 31)	p-value	Prepubertal at the start of treatment (n = 37)	Pubertal at the start of treatment (n = 18)	p-value
Current age NFH (years) &	16.1 ± 1.2	15.7 ± 1.3	16.4 ± 1.1	0.043	15.7 ± 1.1	16.3 ± 1.1	0.002
Bone age NFH (years) &&	16 (15–16)	15 (14–16)	16 (16–16)	0.007	16 (14–16)	16 (16–17)	0.030
HeightNFH SDS&	-1.92 ± 0.90	-1.96 ± 0.86	-1.88 ± 0.95	0.762	-2.02 ± 0.98	-1.70 ± 0.67	0.275
Δheight SDS &	0.93 ± 0.68	0.95 ± 0.63	0.94 ± 0.74	0.925	0.94 ± 0.74	0.95 ± 0.59	0.960
HeightNFH SDS - PAHbaseline SDS&	-0.06 (-0.32–0.07)	-0.05 (-0.39–0.01)	-0.07 (-0.24–0.39)	0.236	-0.04 (-0.34–0.10)	-0.09 (-0.25 – -0.03)	0.606
Height NFH SDS - TH SDS&	-0.45 ± 1.16	-0.16 ± 1.14	-0.68 ± 1.15	0.152	-0.24 ± 1.23	-0.90 ± 0.88	0.092
Δ height SDS - TH SDS&	1.00 ± 0.80	0.95 ± 0.65	1.04 ± 0.91	0.731	0.94 ± 0.75	1.13 ± 0.91	0.453

^&: Means ± SD, ^&&: Median (interquartile range)

Normally distributed variables are presented as mean ± standard deviation and compared using the independent samples t-test. Non-normally distributed variables are expressed as median (interquartile range) and compared using the Mann–Whitney U test

Abbreviations: NFH, near-final height; SDS, standard deviation score; Δheight SDS Height _NFH SDS - Height_baseline SDS; PAH, predicted adult height; TH, target height

Correlation and predictive factors for near-final height

Height SDS at NFH correlated positively with height_baseline SDS, TH SDS, PAH_baseline SDS, and first-year growth velocity SDS (Fig. 2). We also evaluated correlations between NFH outcomes and baseline bone age delay, duration of rhGH therapy, rhGH dose, and stimulated IGF-1 levels during the IGFGT. None of these variables showed a statistically significant association with NFH SDS or Δheight SDS in the regression analyses.

Fig. 2.

Correlation of height SDS at the NFH with heightbaseline SDS, TH SDS, PAHbaseline SDS, and first-year growth velocity SDS

Multiple regression analysis identified female gender (β = −0.624, p = 0.009), TH SDS (β = 0.400, p = 0.003), baseline height SDS relative to TH (height SDS–TH SDS) (β = −0.494, p = 0.032), baseline height SDS relative to PAH (Δheight SDS–PAH SDS) (β = −0.286, p = 0.012), and first-year growth velocity SDS (β = 0.204, p = 0.018) as significant predictors of height SDS at NFH (Table 4).

Table 4.

Multiple linear regression analysis for NFH and height SDS gain

Variable	Height SDS at the NFH				Δheight SDS
	Adjusted R2 = 0.735 F = 9.31 p = < 0.001				Adjusted R2 = 0.617 F = 5.84 p = < 0.001
	β	SE	t	p-value	β	SE	t	p-value
Reference	-1.388	0.683	-2.032	0.056	-1.388	0.683	-2.032	0.056
Agebaseline	0.095	0.057	-1.658	0.113	0.095	0.057	-1.658	0.113
Gender (1 = male, 0 = female)	-0.624	0.217	-2.870	0.009	-0.624	0.217	-2.870	0.009
Puberty (1 = pubertal, 0 = prepubertal)	-0.115	0.219	-0.525	0.606	-0.115	0.219	-0.525	0.606
Heightbaseline SDS	0.269	0.177	1.517	0.145	-0.730	0.177	-4.116	< 0.001
TH SDS	0.400	0.117	3.403	0.003	0,400	0.117	3.403	0.003
Δ Heightbaseline SDS-TH SDS	-0.494	0.214	-2.303	0.032	-0.494	0.214	-2.303	0.032
Δ Heightbaseline SDS-PAHbaseline SDS	-0.286	0.104	-2.749	0.012	-0.286	0.104	-2.749	0.012
IGF-1 SDS at the baseline	-0.126	0.101	-1.248	0.227	-0.126	0.101	-1.248	0.227
Duration of rhGH treatment	0.006	0.006	1.059	0.302	0.006	0.006	1.059	0.302
First-year growth velocity SDS	0.204	0.079	2.582	0.018	0.204	0.079	2.582	0.018

Abbreviations: SDS, standard deviation score; NFH, near-final height; TH, target height; PAH, predicted adult height; IGF-1, insulin-like growth factor-1; rhGH, recombinant human growth hormone

When height gain was analyzed as Δheight SDS, baseline height SDS was a significant predictor (β = −0.730, p < 0.001), indicating that children with more severe baseline short stature tended to achieve greater catch-up growth during rhGH therapy (Table 4).

Discussion

This study assessed long-term growth outcomes in children clinically diagnosed with ISS and treated with rhGH after fulfilling national treatment criteria, including a positive IGFGT response. Among 63 patients treated with rhGH, 55 reached NFH, achieving a mean Δheight SDS of approximately + 1 SDS. Although the mean NFH SDS remained close to − 2, nearly half of the patients achieved a height above − 2 SDS and more than half reached a height within their target height range, suggesting clinically meaningful improvement even when complete normalization of height SDS is not achieved. These findings indicate that clinically selected ISS patients may show meaningful height gains during rhGH therapy. However, because this study lacked an untreated control group, the observed changes cannot be attributed solely to rhGH treatment. Parental consanguinity was relatively frequent in our cohort (12/55, 21.8%); however no significant differences in growth outcomes were observed between children with and without parental consanguinity. The limited number of such patients may have reduced the statistical power to detect potential differences. Consistent with this, post-hoc power analyses demonstrated low statistical power for comparisons based on parental consanguinity (power = 0.32) and pubertal status (power = 0.46), indicating limited ability to detect small-to-moderate differences. Therefore, the absence of statistically significant differences should be interpreted with caution. In addition, genetic analyses in this study were limited to the GH1 gene, and pathogenic variants in other genes associated with short stature—such as SHOX, ACAN, NPR2, FGFR3, and components of the GH–IGF-1 axis—were not systematically evaluated in this cohort. The present study does not evaluate the predictive value of the IGFGT test itself but rather describes growth outcomes in children who fulfilled national treatment criteria requiring a positive IGFGT response.

Because a positive response to the IGFGT was required for study inclusion, the cohort represented a selected group of patients with preserved GH sensitivity. In Türkiye, reimbursement for rhGH in non-GHD short stature requires evidence of an adequate IGF-1 increment in the IGFGT, which influenced our study design and likely contributed to the positive outcomes observed. As a result, the cohort represents a biologically selected group with preserved GH sensitivity. However, the absence of a control group of IGFGT non-responders limits the ability to evaluate the predictive value of the test or to determine whether similar outcomes would have been observed in patients who did not undergo IGFGT selection. While the clinical utility of IGFGT has diminished with advances in molecular characterization of GH insensitivity [16, 17], it was used in the present study as part of the national treatment criteria for selecting patients eligible for rhGH therapy. Reported diagnostic specificity ranges from 82 to 100% and sensitivity from 77 to 100%, depending on the protocol used [17, 18]. This suggests that partial GH insensitivity or post-receptor signaling defects may underlie impaired IGF-1 production in some patients, supporting the need for individualized diagnostic and therapeutic strategies [19]. However, the diagnostic value of the IGFGT remains controversial because of its limited reproducibility and variability in IGF-1 responses, and its results should therefore be interpreted with caution [20].

Another methodological aspect concerns the diagnostic criteria used to exclude classical GHD. During the study period, a GH peak response threshold > 10 ng/mL was used, which was widely accepted at that time. Although lower thresholds have been proposed in more recent guidelines, the use of this cut-off ensured that patients with classical GH deficiency were excluded from the cohort. Similarly, GH stimulation protocols included tests such as L-dopa, clonidine, and insulin tolerance tests, which were commonly used during the period when the patients were evaluated but may not always be considered first-line diagnostic tools in current clinical practice. These historical diagnostic approaches should therefore be taken into account when interpreting the results of the present study.

In our cohort, although biologically inactive GH was initially suspected, no GH1 gene variants were identified in the analyzed regions, and patients demonstrated a favorable growth response to rhGH therapy. These findings indicate that the absence of GH1 mutations does not preclude a clinically meaningful response to rhGH therapy. Previous studies have reported similar findings. Pagani et al. [8] observed that children with biologically inactive GH and those with GHD exhibited comparable increases in growth velocity during the first year of treatment, with growth rates equalizing in subsequent years. In contrast, Travaglino et al. [7] reported that biologically inactive GH patients showed catch-up growth only in the first year, after which their growth decelerated. Similarly, in our cohort, growth velocity was highest in the first year of therapy and declined thereafter, but remained higher than pre-treatment levels, suggesting a sustained association with improved growth outcomes. A recent meta-analysis confirmed that rhGH therapy in ISS results in a mean adult Δheight SDS of approximately + 1 SDS (equivalent to + 5.3 cm in boys and + 4.7 cm in girls), with the most pronounced effect occurring in the first treatment year [1]. Consistent with these findings, our patients achieved a mean NFH gain of nearly + 1 SDS. Furthermore, rhGH therapy has been shown to increase final height in ISS in a dose-dependent manner [10–12], emphasizing the importance of personalized dosing regimens.

Several factors emerged as significant predictors of NFH outcomes in our study. Female sex, higher TH, lower Δheight_baseline SDS–TH SDS and Δheight_baseline–PAH SDS, and greater first-year growth velocity SDS were associated with improved height SDS gain. In addition to the analysis based on NFH SDS, we also evaluated predictors of height gain expressed as Δheight SDS. In this model, baseline height SDS emerged as a significant negative predictor, suggesting that children with more severe baseline short stature tended to experience greater catch-up growth during rhGH therapy. This finding is consistent with previous studies indicating that baseline auxological status strongly influences treatment responsiveness in ISS. In contrast, baseline age at treatment initiation and duration of rhGH therapy were not significant predictors of NFH or Δheight SDS in our regression analyses. This may partly reflect the relatively homogeneous age distribution at treatment initiation in our cohort as well as the retrospective design of the study. When attained NFH was compared with baseline PAH, the overall difference was small. This limited difference between NFH and baseline PAH suggests that part of the observed height gain may reflect intrinsic growth potential, including constitutional delay of growth and puberty, rather than treatment effect alone. In addition, relatively rapid advancement of bone age during therapy may also limit the remaining growth potential, which could further contribute to the modest difference between NFH and PAH. Interestingly, females achieved higher NFH relative to PAH than males. In contrast, patients who were pubertal at treatment initiation showed a trend toward lower NFH compared with baseline PAH estimates, although this difference was not statistically significant. This observation may reflect limitations of PAH prediction methods, particularly the Bayley–Pinneau method, which has been reported to overestimate adult height in certain clinical contexts, including children with constitutional delay of growth and puberty or during pubertal development [21]. However, this potential bias alone may not fully explain the limited difference observed between NFH and baseline PAH in our cohort. Therefore, these interpretations should be considered cautiously, as bone age progression was not longitudinally analyzed in the present study.

These findings are consistent with earlier studies highlighting the importance of early growth response and genetic growth potential. Ranke et al. [22] identified younger age, higher mid-parental height SDS, and robust first-year responsiveness as key determinants of final height. Similarly, Soliman et al. [10] reported that younger age, lower baseline height SDS, higher rhGH doses, and longer treatment duration positively influenced height SDS gain. In our cohort, the duration of rhGH therapy varied among patients, reflecting differences in age at treatment initiation as well as the retrospective design of the study. Our data align with findings from larger cohorts, including a multicenter study of peripubertal ISS patients showing that rhGH therapy alone resulted in an average Δheight SDS of + 1.23 SDS, with greater benefit observed in girls and in those treated for ≥ 2 years [23]. Likewise, Wang et al. [24] reported that peri-pubertal boys with ISS and advanced bone age achieved a mean NFH gain of + 1.30 SDS, with adult height approaching their TH. Because pubertal status can substantially influence growth velocity and responsiveness to rhGH therapy, careful assessment of pubertal stage is important when interpreting treatment outcomes. However, detailed biochemical data on gonadotropins and sex steroid concentrations were not consistently available in this retrospective cohort. Therefore, pubertal status was assessed using clinical Tanner staging rather than hormonal measurements, which may limit the interpretation of pubertal influences on rhGH responsiveness.

Although biologically inactive GH was initially considered during the diagnostic evaluation, no GH1 variants were identified in the analyzed regions. However, the absence of GH1 variants does not exclude other molecular mechanisms that may impair GH bioactivity. Importantly, the diagnosis of ISS in the present study was based on clinical criteria after exclusion of known causes of growth failure.

The concept of biologically inactive GH remains relevant in explaining growth failure in patients with normal or elevated GH secretion but impaired biological activity [7, 25]. Although we did not assess GH isoforms in our study, post-transcriptional defects (e.g., abnormal mRNA splicing), post-translational modifications, and altered post-secretory processing may contribute to impaired GH activity despite an intact GH1 gene [26, 27]. These observations support the notion that the absence of GH1 mutations does not definitively exclude altered GH bioactivity as a potential mechanism.

Advances in molecular genetics have shown that a proportion of children previously classified as ISS may actually have identifiable monogenic causes of short stature. In a study by Millar et al. [6], 24 GH1 gene variants were identified in individuals with short stature, including six functionally impaired mutations. However, two of these (T27I and N47D) were also found in individuals with normal height, suggesting variable penetrance and potential modulation by other genetic or environmental factors. Similarly, a cohort of 86 ISS patients without SHOX abnormalities revealed rare heterozygous variants in ACAN, FGFR3, GHRHR, GHR, and IGFALS in 19 patients, while no pathogenic variants were detected in NPR2, GH1, or IGF1 [28]. These findings, together with our results, indicate that GH1 gene mutations are relatively uncommon in patients clinically diagnosed with ISS or biologically inactive GH; therefore, the absence of GH1 mutations in our cohort does not exclude the possibility of other genetic etiologies contributing to growth failure. Nevertheless, Joustra et al. [29] reported a genetic etiology in 81% of Turkish children with short stature born to consanguineous parents, underscoring the importance of broader molecular approaches, such as next-generation sequencing (NGS) and whole-exome sequencing (WES), to identify rare or novel variants contributing to growth disorders in populations with high rates of parental consanguinity.

Although the sample size is modest relative to the prevalence of ISS, the study provides detailed longitudinal data on a well-characterized cohort. However, several limitations should be acknowledged. First, the retrospective design may introduce inherent biases. In addition, the absence of an untreated ISS control group or a group of IGFGT non-responders limits the ability to distinguish the effect of rhGH therapy from the natural growth trajectory of children with ISS. Therefore, the observed Δheight SDS should be interpreted as an association rather than definitive evidence of treatment efficacy. Moreover, part of the observed improvement may reflect the natural variability of growth patterns in children with ISS. Second, NFH was defined according to bone age criteria rather than confirmed epiphyseal closure, which may have led to slight over- or underestimation of Δheight SDS in some patients. Third, treatment adherence was not systematically documented in the medical records and therefore could not be formally evaluated. Finally, comprehensive genetic analyses beyond GH1 sequencing were not performed due to technical constraints. Consequently, pathogenic variants in other genes associated with short stature—such as SHOX, ACAN, NPR2, FGFR3, and components of the GH–IGF-1 axis—were not evaluated. As a result, some patients classified as ISS may in fact have had unrecognized monogenic growth disorders. Future studies incorporating prospective designs and advanced genomic approaches, including NGS, are needed to better elucidate the genetic basis of ISS and refine patient selection for rhGH therapy.

In this retrospective multicenter study, children clinically diagnosed with ISS and treated with rhGH after fulfilling national treatment criteria were observed to have increases in height SDS and NFH. Despite initial suspicion of biologically inactive GH, no GH1 gene mutations were identified, suggesting that other genetic, epigenetic, or post-translational mechanisms may underlie growth failure in these patients. Importantly, these results should be interpreted as outcomes in a clinically selected cohort, rather than as evidence that the IGFGT predicts treatment response. Overall, our findings support a potential clinical benefit of rhGH therapy in selected ISS patients and emphasize the need for further studies employing advanced molecular techniques to elucidate the genetic and biological pathways involved and to optimize patient selection and treatment strategies.

Abbreviations

ISS

Idiopathic short stature

GH

Growth hormone

IGF-1

Insulin-like growth factor-1

GHR

GH receptor

rhGH

Recombinant human growth hormone

NFH

Near-final height

IGFGT

IGF-1 generation test

SDS

Standard deviation score

PAH

Predicted adult height

TH

Target height

Author contributions

Y. Ö. and O. E. were involved in all stages of the study, including concept development, design, data collection, literature review, analysis, and manuscript writing. H. T., M. K., S. Ş., V. N. B., Z. Ş., F. B., Z. A., M. B., B. H., F. D., O. E., and O. E. contributed to clinical and/or surgical management of the patients. B. H. assisted in data collection and processing. H.I.R. and A.T. contributed to the analysis and interpretation of genetic findings. All authors reviewed and approved the final version of the manuscript.

Funding

This study has been funded by unconditional research support from Pfizer Inc. (Grant number WS677816). The funder had no role in the design, data collection, data analysis, and reporting of this study.

Data availability

The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethical approval

This study was conducted in accordance with the ethical standards of the institutional and national research committees and the 1964 Helsinki Declaration and its later amendments. Ethical approval was obtained from the Clinical Research Ethics Committee of Kırıkkale University (Project No: 2009/134).

Informed consent

Participants and parents provided written informed consent.

Competing interests

The authors declare no competing interests.