Isolated Growth hormone deficiency is an important and treatable cause of short stature. However, it is often difficult to diagnose the condition with certainty due to the lack of a single robust diagnostic test. Short children, other than those with the classical phenotype of immature chubby facies, truncal obesity and micropenis in boys, or those with history of cranial lesions with known association with hypopituitarism, should be evaluated for growth hormone deficiency only after excluding the other more common conditions. These children typically have height markedly below that expected for their midparental height with low height velocity and delayed bone age. Growth hormone levels should be checked by provocative testing, after ensuring that the child is euthyroid, and after priming with sex steroids if indicated. Low levels of Insulin-like growth factor 1 and Insulin-like growth factor binding protein 3 and pituitary abnormalities on neuroimaging provide important corroborative evidence to the diagnosis.

Keywords:Diagnosis,Short stature,Height velocity, Hypopituitarism, IGF1.

Growth hormone (GH) is a polypeptide hormone secreted by the anterior pituitary gland and is the chief driver of statural growth during childhood. Human Growth Hormone (hGH) extracted from cadaver pituitary glands was first isolated in 1956, and by 1959 its clinical use in patients with presumed GH deficiency had started. In 1985, the use of hGH was abruptly halted amidst reports of death due to Creutzfeldt Jacob Disease (CJD) in some recipients [1]. Later in the same year, synthetic recombinant human growth hormone (rhGH) was approved by the United States Food and Drug Administration (USFDA) for clinical use. The indications for GH therapy were sequentially expanded to include chronic renal insufficiency in 1993, Turner syndrome in 1997, Prader Willi syndrome in 2000, small for gestational age in 2001, idiopathic short stature in 2003, and Noonan syndrome in 2007 [2].

However, the unlimited availability of rhGH increased the potential for its misuse; as an anabolic agent by athletes, as a ‘supposed’ anti-ageing agent, and in children who are not short by definition but falling short on parents’ expectation of stature. Lack of a simple one point robust test for diagnosing GH deficiency, compounds this problem further [3]. Erroneous interpretation of a random GH level as low by a physician leaves room for unwarranted prescriptions.

We, herein, present a brief review of the physiology of growth hormone, and discuss the clinical, biochemical, radiological and genetic investigations for diagnosis of growth hormone deficiency in children, with special focus on avoidance of over-diagnosis of this condition.

GH is secreted from the anterior pituitary in a pulsatile manner under hypothalamic regulation and other physiologic regulators. There are approximately 10 daily pulses of GH secretion of about 90 minutes duration in total, each separated by about 128 minutes. The level of GH in between the pulses is nearly undetectable. Therefore single isolated unprovoked GH measurement is of no value, and diagnosis of GH deficiency is made by drawing 4-5 blood samples at half-hourly intervals after administering a pharmacological stimulant [4].

Around 50% of the circulating GH is bound to Growth hormone binding protein (GHBP). GH acts on the liver, muscle and bone, and mediates the production and release of insulin-like growth factor (IGF) 1 and 2 from these organs. Stimulation of linear growth in children is chiefly mediated by IGF1. IGF2 is important for antenatal growth but its role postnatally is not clear. Hepatic IGF1 circulates in blood almost completely bound to IGF binding proteins (IGFBPs), a group of sixstructurally related proteins that bind IGFs with high affinity. Of these, IGFBP3 binds 75% to 90% of the circulating IGF1. This complex is stabilized by Acid Labile Subunit (ALS), which increases the half life of IGF1 [5].

The major congenital and acquired causes of GH deficiency are summarized in Table I [6].

Short stature is defined as height less than -2 standard deviations (SD) for age-and sex- appropriate population norms. This implies that one in every 40 normal children is short. However, the estimated prevalence of GH deficiency is 1 in 4,000 to 1 in 10,000 [7], and hence, GH deficiency is a relatively rare cause of short stature. Before evaluating a short child for GHD, commoner causes such as physiological (familial short stature or constitutional delay of growth and puberty), hypothyroidism, small for gestational age (SGA), chronic systemic disease, celiac disease, Turner syndrome, or skeletal dysplasia need to be considered and appropriately ruled out [7].

Children with GHD have normal anthropometry at birth. Those with congenital hypopitutarism may have hypoglycemia, jaundice and micropenis in the neonatal period. The most common presentation is with complaint of poor growth in childhood. Typically, these children have immature facies, mid-facial hypoplasia, pot belly, low height velocity (<4 cm/ year during childhood) and delayed skeletal maturation.Presence of midline anomalies such as single central incisor may be a pointer to hypopituitarism. Acquired deficiency due to cranial lesions typically presents as slowing or even cessation of linear growth. This may be accompanied by other features such as polyuria and polydipsia (posterior pituitary involvement), visual impairment and headache.

As per the consensus guidelines of the Growth Hormone Research Society [7], investigation for GH deficiency should be considered only in children who fulfil at least one of the criteria listed in Box 1.

A period of fasting is required before all provocative GH testing protocols. It is imperative to ensure that all patients are euthyroid at the time of testing. A summary of the various protocols is given in Table II [4]. It is recommended that in children with suspected isolated GH deficiency, two provocative tests should be done either sequentially or on separate days, and the deficiency should be diagnosed only if the peak GH values in both the tests are below the diagnostic cut-off.

Surveys of pediatric endocrinologists indicate that there is no standard practice for priming peripubertal children with sex steroids [10]. The Pediatric Endocrine Society in its recent guidelines on diagnosis and treatment of GH deficiency has recommended that prepubertal boys >11years and prepubertal girls >10 years of age should undergo priming, especially if their predicted adult height is within -2 SD of the reference population mean. Priming with sex steroids reduces the chances of misdiagnosis of children with constitutional delay of growth and puberty as GHD [11]. Estradiol valerate1-2 mg can be used for both boys and girls on each of the two evenings prior to the test. Alternatively, boys can be primed with intramuscular depot testosterone 50 to 100 mg 1 week prior to the test [11].

Conventionally peak GH values below 7 µg/L or 10 µg/L after provocative tests are considered as indicative of GH deficiency. However, these cut-offs are a matter of debate. In a study that evaluated the secretion of GH after stimulation by clonidine, insulin and arginine in 7- to -18 year-old children with normal stature, it was noted that the mean (SD) peak values for GH were higher in response to clonidine [21.0 (10.7) µg/L], compared to those in response to arginine[13.1 (6.1) µg/L] or insulin [14.2 (6.3) µg/L] [12]. It was also observed that the mean (SD) peak GH level after clonidine stimulation increased from 12.8 (5.1) µg/L in Tanner I girls to 35.5 (5.1) µg/L in Tanner IV/V girls and similarly, from 16.9 (6.7)µg/L in Tanner I boys to 26.5 (12.0) µg/L in Tanner IV/ V boys. In 1996, Ghigo,et al. [13] published a study that assessed the reliability of some of the provocative agents used in GH testing. Ideally, administration of these agents should be able to produce peak GH values above the conventional cut-offs in normal children. In 472 children, including those with short as well as normal stature, but with normal height velocity, normal IGF1 levels, and no delay in bone age, it was observed that using various provocative agents, 23% to 49% of these GH non-deficient subjects failed to achieve peak GH values above 10 µg/L, and 10–24% had peak GH <7 µg/L [13].

The Pediatric Endocrine society (PES) 2016 guidelines also caution against using provocative GH testing as the sole basis for diagnosing GH deficiency [11]. While patients with severe GH deficiency generally have very low values on provocative testing, the threshold that distinguishes partial GHD from normal is not clear. It is also important to remember that obese children have blunted GH peak in response to various stimuli [11]. Data from several big post-marketing surveys of GH therapy suggest that the most significant increase in height velocity and height standard deviations are seen in those children who had a peak GH value <5µg/L at diagnosis [14-16].

In short children with low height velocity, who have a known hypothalamic-pituitary defect, such as a major congenital malformation, history of cranial tumor or irradiation, and deficiency of at least one more pituitary hormone, provocative GH testing is not needed to diagnose GHD. Similarly, a newborn with hypoglycemia, who has a GH level <5 µg/L in a critical blood sample and has the classical triad of hypoplastic anterior pituitary, ectopic posterior pituitary and abnormal stalk on neuroimaging; and/or deficiency of at least one other pituitary hormone can be diagnosed as having GH deficiency [11].

The production of IGF1 and IGFBP3 is dependent on GH. More than 75% of circulating IGF1 is bound to IGFBP3 and this complex has a half life of 16 hours [17]. The two, hence, seem to be very convenient molecules that can be measured for a functional bioassay of GH. However, these also have their own limitations; the foremost being that their levels may be affected by nutritional status, age, thyroid function, degree of sexual maturation, genetic factors and liver function [18]. IGFBP3 is less affected by these factors as compared to IGF1. There is no single cut-off for IGF1 and IGFBP3, and values have to be compared to age, genderand pubertyspecific normative reference data [19-21].

In a retrospective analysis in 33 children with GH deficiency (diagnosed on the basis of short stature, delayed bone age, two failed GH provocative tests, hypothalamo-pituitary anomalies on MRI, height catch-up on GH therapy, and a repeat GH provocative test value <10 µg/L after completion of linear growth) and 56 children with idiopathic short stature, a cut-off value of <10　µg/L to define GH deficiency using provocative tests was 100% sensitive and 57% specific. Decreasing the cut-off to 7 µg/L changed the sensitivity and specificity to 66% and 78%, respectively. Low IGF1 had a sensitivity of 73% and specificity of 95%, low IGFBP3 had a sensitivity of 30% and specificity of 98%, andlow height velocity had a sensitivity of 82% and specificity of 43%. Combination of height velocity and IGF1 had a sensitivity of 95% and specificity of 96% [22].In another similar study, IGFBP3 and IGF1 were evaluated in patients with GH deficiency and idiopathic short stature. It was observed that for diagnosing GHD, low IGF1 (<5th centile) had a sensitivity of 69%. The specificity was 91% in children aged <11 years but only 53% in children aged >11 years.The specificity of low IGFBP3 was 100% for diagnosis of GHD, but sensitivity was less than 50% [23].

In conclusion, low levels of IGF1 and IGFBP3 are reasonably specific for diagnosis of GHD, especially in children younger than 10-11 years of age, but their sensitivity is low, so that normal values are not sufficient to exclude GH deficiency. If height velocity is combined with the laboratory parameters, the sensitivity and specificity of the diagnosis improve.

As per Growth Hormone Research Society Consensus Guidelines, in patients with confirmed isolated GH deficiency or Multiple Pituitary Hormone Deficiency, magnetic resonance imaging (MRI) with 2 mm slices should be done to note the height/volume of anterior pituitary, position of posterior pituitary and anatomy of the stalk. The abnormalities include pituitary hypoplasia (gland height <-2SD for age), ectopic posterior pituitary and stalk abnormalities [7].

In a recent study in 68 children diagnosed with GH deficiency before 4 years of age, MRI abnormalities were noted in 84% patients with isolated GH deficiency, of which 49% had only isolated pituitary hypoplasia; while in patients with multiple hormone deficiencies100% had complex defects [24].

Genetic mutations are identified in a relatively small number of patients with GH deficiency. However, a genetic diagnosis should be established where feasible in children with congenital GH deficiency. It helps in planning follow-up and appropriate evaluation of other family members.GH deficiency can occur due to mutations in transcription factors genes involved in the development of the pituitary gland (summarized in Web Table I), which typically are associated with multiple pituitary hormone deficiencies; or due to mutations in genes encoding growth hormone (GH1), growth hormone releasing hormone (GHRH) or its receptor (GHRHR), which lead to isolated GH deficiency [25-27].

In a study by Desai, et al. [28] in 97 patients with isolated GHD, GH1 gene deletion was noted in 17%, while GHRHR gene mutations were present in 35%. In recent studies from Delhi, among 51 patients with multiple pituitary hormone deficiency, 6% had mutations in PROP1 gene and 14% in POU1F1 gene [29]; while among 116 patients with isolated GH deficiency, mutations in GH1 and GHRHR genes were observed in 7% and 21% patients, respectively [30].

The key messages related to diagnosis of GH deficiency in children are summarized in Box 2.

Contributors: The review was conceptualized by VJ. All thethree authors contributed to literature search and writing the review.

Funding: None; Competing interests: None

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