{'fraction_non_alphanumeric': 0.03837471783295711, 'fraction_numerical': 0.02286779860634017, 'mean_word_length': 4.85295807007467, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
p.R209H GH1 variant challenges short stature assessment

OBJECTIVE
to describe the marked variability in clinical and biochemical patterns that are associated with a p.R209H GH1 missense variant in a large Argentinean pedigree, which makes the diagnosis of GHD elusive.


DESIGN
We describe a non-consanguineous pedigree composed by several individuals with short stature, including 2 pediatric patients with typical diagnosis of isolated growth hormone deficiency (IGHD) and 4 other siblings with severe short stature, low serum IGF-1 and IGFBP-3, but normal stimulated GH levels, suggesting growth hormone insensitivity (GHI) in the latter group.


RESULTS
Patients with classical IGHD phenotype carried a heterozygous variant in GH1: c.626G>A (p.R209H). Data from the extended pedigree suggested GH1 as the initial candidate gene, which showed the same pathogenic heterozygous GH1 variant in the four siblings with short stature and a biochemical pattern of GHI.


CONCLUSIONS
We suggest considering GH1 sequencing in children with short stature associated to low IGF-1 and IGFBP-3 serum levels, even in the context of normal response to growth hormone provocative testing (GHPT).


Introduction
Endocrine causes of short stature include defects in the growth hormone (GH)-insulin-like growth factor (IGF) system and main discrimination must be done between growth hormone deficiency (GHD) and GH insensitivity (GHI) [1]. Patients with GHD usually present severe postnatal short stature, low IGF-I and IGF binding protein-3 (IGFBP-3) levels, subnormal response to GH provocative tests (GHPT) and typical clinical features. GHI is generally characterized by pre-and/or postnatal growth retardation, low IGF-I levels, and normal or even increased circulating GH concentrations; additional features may vary according to its pathogenesis. Nevertheless, confirming the diagnosis of GHD and GHI is a challenge since clinical and biochemical features may overlap between these entities.
GHPT are a useful biochemical tool and have been the gold standard to confirm the diagnosis of GHD. Many limitations still exist such as weak grade of evidence for the cut-off values used and poor test specificity and reproducibility [2]. With the expansion of genomics, genetic evaluation has become an additional tool in aiding the process of obtaining a definitive diagnosis [3].
Isolated GHD (IGHD) of genetic origin is mostly caused by GH1 gene pathogenic variants, and less commonly by variants in GHRH receptor (GHRHR) or Ghrelin receptor (GHSR) genes [4][5][6]. GH1 variants inherited with an autosomal dominant pattern are classified as type II IGHD. Most of them affect GH1 splicing and result in exon 3 skipping, but missense mutations have also been described, usually with a milder phenotype [5,7].
The aim of our study was to describe marked variability in clinical and biochemical pattern due to a p.R209H GH1 missense variant, previously described as p.R183H, in a large Argentinean pedigree, making the diagnosis of IGHD elusive.

Auxological variables
Height, weight and head circumference were measured and expressed in SDS for age and sex, according to Argentinean reference growth charts [8]. Length and height were determined using an infantometer or a wall mounted stadiometer according to age.

Hormonal studies
Serum levels of GH, IGF-I and IGFBP-3 were determined by chemiluminescent immunometric assays (Immulite 2000, Siemens Healthcare Diagnostics, Llamberis, Gwynedd, UK). GH provocative tests were performed with arginine (0.5 g/kg) and clonidine (0.100 mg/m 2 ): GH was measured at baseline and at standard intervals (IS 98/574, Immulite). GHD was defined as a peak GH lower than 4.8 ng/ml in two GHPT [9]. Intraand interassay coefficients of variation were <5.5% for all serum measurements. IGF-I generation test (IGFGT) was performed as follows: subcutaneous rhGH (0.23 mg/kg.day) administered daily for 7 days; IGF-I and IGFBP-3 serum concentrations were measured at baseline, 5 th and 8 th day. We considered a positive response the increment of IGF-I levels to the normal range for the patientś age, sex and pubertal status.
Unexpectedly, GHPT (Arginine and Clonidine) performed in the first patient (IV.2) at 5.75 yrs. showed adequate GH response (GH peak 7.59 ng/ml). As his clinical features were strongly indicative of GHD, GHPT were repeated at 7.4 yrs., showing low GH response (GH peak of 3.62 ng/ml), that confirmed the diagnosis of GHD. The second patient (IV.3) showed an insufficient response to GHPT (GH peak 2.65 ng/ml) at 2.4 yrs. Brain MRI was normal in IV.2 and showed anterior pituitary hypoplasia (2.5 mm) in IV.3. Both responded adequately to recombinant human growth hormone treatment (rhGH, 0.17 mg/kg/wk; with a mean height increment of 1.4 SDS in the 1 st yr of treatment) [10]. Patient IV.2ś mother presented with short stature. Patient IV.3ś parents and brother had normal stature, but the paternal grandmother (II.4) was severely short (134.6 cm, −4.3 SDS) with typical GHD clinical phenotype, undetectable IGF-I, and extremely low IGFBP-3 levels.

Molecular studies
Genomic DNA was isolated from peripheral venous blood by cetyltrimethylammonium bromide lysis buffer and chloroform-isoamyl alcohol extraction [11]. Patient IV.3 was included in a research protocol using a panel of 67 genes associated to combined pituitary hormone deficiency (CPHD) and IGHD in humans and mice [12]. In patients IV.2 and III.29, the whole coding sequence of GH1 gene was PCR amplified [13] and sequenced in a 3500 Genetic Analyzer (Applied Biosystems™, Thermo Fisher Scientific). In patients III.27, III.28, III.30 and other family members (marked as sequenced in figure 1), GH1 exon 5 was PCR amplified and sequenced. The following GH1 reference sequences were used: All subjects were informed of the purpose of the study and their written consent was obtained. The study was approved by the Ethics Committee of Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina.

Results and Discussion
Gene panel NGS analysis revealed a heterozygous c.626G>A transition in exon 5 of the GH1 gene in GHD patient IV.3, which was confirmed by Sanger sequencing. This variant predicts the change of an arginine for a histidine in position 209 (p.R209H). Her father was heterozygous for the GH1 p.R209H variant, and her paternal grandmother was homozygous for this variant.
Sanger sequencing revealed that GHD patient IV.2 and his mother were also heterozygous for p.R209H.
Data from the extended pedigree (III.27, III.28, III.29, III.30) suggested GH1 as the initial candidate gene. The same pathogenic heterozygous GH1 variant was identified in these four siblings and their father. Four of the non-affected siblings and their mother were negative for the variant (Table 1).
Autosomal dominant type II IGHD is caused by heterozygous GH1 mutations, and these patients have variable clinical expression, including low but detectable serum GH levels, wide range of height deficit, and different pattern on brain MRI (normal or anterior pituitary hypoplasia) [5,7,14,15]. The missense p.R209H variant reduces GH secretion due to defects in exocytosis of secretory granules, but without evidence of toxic effect on somatotrophs. Thus, the GH molecule remains fully bioactive but unable to reach its target receptor [16,17]. Hence, some type II IGHD patients may still produce and secrete GH at a reduced level and GHD can be time-dependent [18].
In the large pedigree described here, 10 individuals carried the same GH1 p.R209H variant but presented variable clinical and biochemical phenotype. Firstly, two children presented the classical IGHD phenotype; the genetic disorder was inherited from a short mother in one case, and a normal stature father in the other. Additionally, four children carried the same GH1 p.R209H variant and initially resembled a pattern of GHI: postnatal growth failure with minimal or absence of typical GHD phenotype, normal GHPT response and low basal IGF-I and IGFBP-3 levels. Thereafter, IGFGT was normal. They inherited the variant from the carrier father who achieved a normal adult height.
Homozygosity for p.R209H variant found in II.4 was unexpected. Unfortunately, we were not able to confirm whether the grandmother was hemizygous for the variant (one deleted together with one mutated allele). Thus, further studies are necessary to rule out the presence of hemizygosity.
These findings highlight the existence of a great variability within the clinical and biochemical phenotype associated to GH1 p.R209H variant. Even more, the change in the response to GHPT suggests an evolving process in GH secretion [7].
We describe four siblings showing an elusive clinical and biochemical phenotype of GHD as they presented growth retardation and abnormal surrogates of GH action (IGF-I, IGFBP-3), but normal response to GHPT. In this subgroup of patients, GHPT failed to diagnose GHD.
Similar to other published cases, adult height in affected GH1 p.R209H parents fell within a wide range of −1.0 to −4,5 SDS, showing that normal final height may be obtained, even with low-normal IGF-I levels [7,19]. If this could be due to a time-dependent deficiency or to other factors beyond GH, remains to be answered.

Conclusion
The identification of short stature disorders requires the integration of all clinical, biochemical and radiological data and it is critical to ascertain an extended family history. The wide spectrum of clinical and biochemical presentation in individuals carrying the p.R209H GH1 mutation could hinder the diagnosis of GHD, therefore misleading the genetic diagnosis approach. We suggest considering GH1 sequencing in children with short stature associated to low IGF-I and IGFBP-3 serum levels, even in the context of normal response to GHPT.

•
Growth hormone provocative tests have many limitations.

•
Phenotype between GHD and GHI may overlap.
• Genetic evaluation is an additional tool towards a definitive diagnosis of GHD • GH1 p.R209H variant has markedly variable clinical and biochemical consequences.
• It is advisable to explore GH1 in short children with low IGF-I and IGFBP-3, despite normal GHPT.