Accepted Manuscript

Diagnosis of growth hormone deficiency in the paediatric and transitional age

A. Chinoy, MBBS, BSc, MRCPCH, Paediatric Endocrine Registrar, Dr P.G. Murray,

MBChB, BSc, PhD, MRCPCH, Consultant Paediatric Endocrinologist

PII: S1521-690X(16)30068-9
DOI: 10.1016/j.beem.2016.11.002
Reference: YBEEM 1118

To appear in: Best Practice & Research Clinical Endocrinology & Metabolism

Please cite this article as: Chinoy A, Murray P, Diagnosis of growth hormone deficiency in the paediatric
and transitional age, Best Practice & Research Clinical Endocrinology & Metabolism (2016), doi:
10.1016/j.beem.2016.11.002.

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 ACCEPTED MANUSCRIPT

Title
Diagnosis of growth hormone deficiency in the paediatric and transitional age

Authors
A Chinoy, MBBS, BSc, MRCPCH, Paediatric Endocrine Registrara
P G Murray, MBChB, BSc, PhD, MRCPCH, Consultant Paediatric Endocrinologista,b
a
Department of Paediatric Endocrinology, Royal Manchester Children's Hospital,

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Central Manchester Foundation Hospitals NHS Trust, Manchester, UK
b
Centre for Paediatrics and Child Health, Institute of Human Development, University
of Manchester, Manchester, UK

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Corresponding author

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Dr Philip Murray, Department of Paediatric Endocrinology, Royal Manchester
Children’s Hospital, Oxford Road, Manchester, UK, M13 9WL. E-mail:
Philip.Murray@cmft.mhs.uk

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Abbreviations
GH – growth hormone; GHD – growth hormone deficiency; iGHD – isolated growth
hormone deficiency; MPHD – multiple pituitary hormone deficiency; SD – standard
deviation; GHRH – growth hormone releasing hormone; MRI – magnetic resonance
imaging; GHBP – growth hormone binding protein; BMI – body mass index; IGF-1 –
insulin-like growth factor-1; IGFBP-3 – insulin-like growth factor binding protein-3;
ESPE – European Society for Paediatric Endocrinology
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Abstract

Growth hormone deficiency is a rare cause of childhood short stature, but one for
which treatment exists in the form of recombinant human growth hormone. A
diagnosis of growth hormone deficiency is made based on auxology, biochemistry
and imaging. Although no diagnostic gold standard exists, growth hormone
provocation tests are considered the mainstay of diagnostic investigations. However,
these must be interpreted with caution in view of issues with variability and

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reproducibility, as well as the limited evidence-base for cut-off values used to
distinguish growth hormone deficient and non-growth hormone deficient subjects. In
addition, nutritional and pubertal status can affect results, with no consensus on the

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role of priming with sex steroid hormones. Difficulties with assays exist both for
growth hormone as well as insulin-like growth factor-1. Pituitary magnetic resonance
imaging is a useful diagnostic, and possibly prognostic, aid. Although genetic testing

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is not routine, the discovery of more relevant mutations makes it an increasingly
important investigation. Children with growth hormone deficiency are retested
biochemically on completion of growth, to assess whether they remain so into
adulthood.

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Keywords
Growth hormone deficiency
IGF-1
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Short stature
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1. Introduction
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Using a definition of short stature as a height below -2 SD, 2.3% of the population
will have short stature, often causing significant distress to a growing child and their
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family. Short stature is one of the commonest presentations to a paediatric

endocrinologist. Once commoner causes of short stature are ruled out or if the short
stature is severe, growth hormone deficiency (GHD) needs to be considered. GHD is
a rare disorder, with estimated prevalence being approximately 1 in 4000 [1, 2].
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However, it is an important diagnosis to make and do so in a timely manner, as

treatment with recombinant human growth hormone (GH) is highly effective in
improving final height. Conversely, a false positive diagnosis will lead to a childhood
of unnecessary daily injections, exposure to potential adverse effects and a significant
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economic burden.
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The aetiology of GHD can be divided into congenital and acquired. Acquired causes
include midline tumours (craniopharyngioma, optic nerve glioma, germinoma, and
pituitary adenoma), cranial irradiation, traumatic brain injury, central nervous system
infections and inflammatory conditions (sarcoidosis, Langerhan’s cell histiocytosis).
Congenital causes include genetic mutations (both specific to GH such as GHRHR
and GH1, as well as those associated with pituitary development and multiple
pituitary hormone deficiency) and structural brain malformations (holoprosencephaly,
septo-optic dysplasia, agenesis of corpus callosum, Rathke’s cyst and pituitary stalk
interruption syndrome).
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The diagnosis of GHD can be challenging, particularly in idiopathic isolated GHD

(iGHD), i.e. where there is no evidence of multiple pituitary hormone deficiency
(MPHD) or cranial pathology evident on magnetic resonance imaging. The efficacy
of tools used to assist in diagnosing GHD have been the subject of much debate over
the years, due largely to the lack of a gold standard diagnostic test. This article
reviews these diagnostic tools (clinical, biochemical and radiological), as well as their
evidence-base and associated problems.

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2. Initial assessment

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Although growth failure is the major presenting feature of GHD in children, a
thorough history and examination may give supportive evidence of GHD and possible
aetiology, or suggest other causes of short stature. Severe congenital GHD is

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associated with neonatal hypoglycaemia, prolonged hyperbilirubinaemia in the
neonatal period and microphallus [3]. Growth failure typically presents during the
first year of life in such cases. A family history of GHD and consanguinity should be
sought. Children with acquired GHD may have a history of cranial pathology,

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trauma, surgery or irradiation. The typical physical features of GHD are midface
hypoplasia, frontal bossing, "doll-like" facies and increased truncal adiposity.
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Accurate auxological measurements are crucial in identifying when to investigate for
GHD, particularly height, height velocity and mid-parental height. Consensus
guidelines for the diagnosis of GHD in childhood were published in 2000 by the
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Growth Hormone Research Society. These outline the presenting and auxological
criteria that warrant further investigation for GHD (Table 1) [3]. It is important
however to rule out other causes of short stature on initial assessment, such as familial
short stature, constitutional delay in growth and puberty, hypothyroidism, chronic
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disease, Turner's syndrome and skeletal dysplasia, with appropriate investigations.

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Table 1. GH Research Society consensus guidelines for when to consider immediate

investigation for GH deficiency [3]
Severe short stature, defined as a height more than 3 SD below the mean
Height more than 1.5 SD below the mid-parental height
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Height more than 2 SD below the mean, and a height velocity over 1 year more than 1
SD below the mean for chronological age, or a decrease in height SD of more than 0.5
over 1 year in children over 2 years of age
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In the absence of short stature, a height velocity more than 2 SD below the mean over
1 year or more than 1.5 SD sustained over 2 years
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Signs indicative of an intracranial lesion

Signs of multiple pituitary hormone deficiency
Neonatal symptoms and signs of growth hormone deficiency

3. GH provocation tests

GH secretion occurs in a pulsatile nature. During the neonatal period, a random GH

measurement may be clinically useful [4], with Binder et al reporting that values <7
µg/L are 100% sensitive and 98% specific in diagnosing GHD [5]. Beyond the
neonatal period, measurement of random serum GH concentration is of no clinical
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value, as the majority of GH pulses occur overnight. Therefore physiological and

pharmacological GH provocation tests are key to the assessment of GH secretion.
Physical stimuli of GH secretion such as exercise are no longer routinely used due to
poor reproducibility. Pharmacological stimuli in current practice include arginine,
clonidine, glucagon, insulin, levodopa and GH releasing hormone (GHRH). The
latter is not often used in isolation as it can give false negative results in hypothalamic
disorders. However, GHRH is used in combination with arginine [6]. All of the
pharmacological stimuli carry the risk of side effects, including nausea, hypotension

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and hypoglycaemia. The insulin tolerance test in particular needs careful monitoring
and has been associated in rare cases with significant morbidity and even mortality
[7].

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3.1 Selecting a Cut-off Level for the Diagnosis of GHD
The cut-off peak GH concentration from provocation tests to distinguish children with

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GHD from children without GHD has a limited evidence base, and will vary between
centres. With the introduction of GH provocation tests in the 1960s, a peak GH
concentration of <5 µg/L was used to diagnose GHD as this was felt to best correlate
with the GHD phenotype [8]. Dattani et al demonstrated in short prepubertal

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children, comparing cohorts with a height velocity greater than -0.8 SD (likely GH
sufficient) to those with a height velocity less than -0.8 SD (possible growth hormone
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insufficient), that the optimal cut-off for GH concentration for the insulin tolerance
test to diagnose GHD was 13.5 mU/L (~ 5 µg/L) [9]. However, over the years, the
cut-off has increased to 7-10 µg/L with very limited evidence backing this. The lack
of evidence-base for an optimal cut-off is due in part to the lack of a gold standard
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diagnostic tool to identify GHD subjects. A recent study by Wagner et al has
attempted to define evidence-based cut-off limits for peak GH concentrations to
diagnose GHD [10]. Retrospectively re-examining the serum samples of short
children without GHD and children diagnosed with GHD and treated with GH
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(categorised based on previous GH provocation testing using arginine and either

glucagon or insulin tolerance testing), a cut-off peak GH of 7.09 µg/L was concluded.
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The diagnoses of the two cohorts (treated GHD vs. non-GHD) was likely to be
accurate in view of height SDS at the time of provocation and on follow-up being
comparable.
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3.2 Variability and reproducibility

Variability in peak GH concentrations between provocation tests remains a difficult
issue in interpretation. By assessing peak GH concentrations in 472 children without
GHD using various stimuli, Ghigo et al demonstrated that the mean peak GH
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concentrations varied between tests from 9.7 µg/L to 61.8 µg/L [6]. Every individual
stimulus incorrectly classified some children as GHD, with false positive rates
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ranging from 8.9-49% dependent on cut-off used (7 µg/L or 10 µg/L) and stimulus
used. Similarly in 48 children diagnosed with GHD using insulin tolerance and
arginine stimulation tests, 28 (58%) were found to have adequate peak GH levels on
glucagon stimulation testing (cut-off 10 µg/L) [11]. In view of this inter-test
variability and potential for false-positives, current consensus guidelines recommend
the use of two provocation tests for the diagnosis of idiopathic iGHD. One positive
provocation test is sufficient in children with evidence of cranial pathology,
irradiation, MPHD or genetic mutation associated with GHD [3]. It is clear that
different cut-off levels are required with different stimulation tests. Although the
insulin, glucagon and arginine tests are broadly comparable the combined tests such
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as the GHRH-arginine test are very powerful stimuli and require adjustment of the
cut-off level used.

Reproducibility of individual provocation tests has also been questioned. In children

undergoing evaluation for short stature, Hilczer et al repeated either the clonidine test,
glucagon test or both [12]. Correlation of peak GH between the first and second tests
was weak for clonidine (r=0.22) and nil for glucagon (r=0.08), with the within-subject
variability for peak GH being 43.4% and 50.3% respectively. In 61.7% of patients

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(n=60), the first and second provocation test of the same stimulus gave divergent
results (GHD vs. not GHD). Loche et al retested 33 children diagnosed to have GHD
using 2 provocation tests (cut-off <10 µg/L for peak GH) but with normal magnetic

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resonance imaging (MRI) of the hypothalamic-pituitary region 1-6 months after initial
test [9]. 85% of these children had a normal provocation test (peak GH >10 µg/L) on
retesting [13].

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3.3 Assay issues
There exists significant inter-assay variability in the measurement of GH between
different laboratories, which further complicates interpretation of provocation tests.

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This is best demonstrated in an audit by the UK National External Quality
Assessment scheme [14]. Serum samples supplemented with a defined level of GH
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were analysed by 96 clinical laboratories - a geometric coefficient variation of 25%
was reported. Put into context, a sample of GH with a mean concentration of 7 µg/L
could be reported as anything from 5 µg/L to 10 µg/L by different laboratories - the
difference between a positive and negative GH provocation test by any currently
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accepted cut-off. Muller et al have further emphasised this variability between assays
recently by retrospectively testing serum collected during provocation testing from
312 children investigated for GHD using 8 different assays [15]. The mean difference
between assays ranged from 0.35 µg/L to 2.71 µg/L. Using 6 µg/L as a cut-off for
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GHD, in 29% of children the diagnosis would be dependent on the immunoassay

used.
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There are multiple reasons for this inter-assay variability. In serum there are multiple
GH variants – multiple monomeric isoforms, homo- and hetero-polymers, fragments
and complexes with other molecules [16]. The major GH isoform is 22kDa with the
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20kDa isoform generated by alternative splicing and accounting for around 10-20% of
circulating GH. Each immunoassay will differ in the degree to which it detects
different isoforms, GH variants and hetero- and homo-dimers of GH. GH binding
protein (GHBP) can also affect assays. Up to 50% of GH in serum is bound to
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GHBP, and variations in GHBP levels even within the physiological range have been
shown to affect GH concentrations detected [17]. Furthermore, detected GH
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concentrations may also be affected in modern assays using monocloncal antibodies if

the epitope of the antibody is obscured by binding of GH to GHBP. A consensus
statement was published in 2011 on the standardisation of GH assays [18]. It
recommended the use of WHO IRP 98/574, that each assay should specify the degree
of interference by GHBP, GH concentrations should be reported as µg/L rather than
mU/L, and that commutable materials should be used to facilitate quality control
between laboratories. The study by Wagner et al discussed earlier has recently
attempted to identify individual cut-off values for several currently available
commercial immunoassays by regression equations, thus allowing better
interpretation of results from a given immunoassay [10].
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Measuring GH by mass spectrometry offers the potential to overcome some of the

issues associated with immunoassays, mainly as it measures GH by analyte mass
rather than epitope. With this technique, GH measurement has been shown to be
independent of GHBP levels and offers a more reproducible and sustainable cut-off
concentration [19].

3.4 Obesity

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Rates of childhood obesity are rapidly rising globally. UK data estimates that 31% of
children aged 2 to 15 years are classed as overweight or obese [20]. Spontaneous
release of GH is known to be inversely correlated to body mass index (BMI) [21].

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Similarly with GH provocation tests, Stanley et al showed in short children
undergoing evaluation of the GH axis, a peak GH on stimulation testing <7 µg/L was
present in 50% of patients with BMI greater than +1 SD compared to 4% in those

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with BMI between 0 and -1 SD [22]. Given that classical GHD does present as short
stature with increased truncal adiposity, it is perhaps not surprising that the prevalence
of GHD rises with increasing BMI. However, within the same group of patients in
the study by Stanley et al, despite the difference in prevalence of GHD by BMI, there

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was no difference in serum insulin-like growth factor-1 (IGF-1) concentrations
between groups [22]. This would imply that some of the increase in positive
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provocation tests is false positives directly caused by obesity. Within the adult
population, studies have suggested separate peak GH concentration cut-offs in
overweight and obese individuals to minimise false positive diagnoses. Ditchel et al
investigated a cohort of adults with BMI greater than 25 kg/m2 (including subjects
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with known pituitary disease and healthy controls); using a cut-off of 3 µg/L (as
recommended by consensus guidelines for diagnosis of GHD in adulthood), 45% of
the control subjects were incorrectly classified as GHD whilst 95% of patients with
hypopituitarism were correctly identified as GHD [23]. By reducing the cut-off to 1
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µg/L for this obese cohort the rate of incorrect classification for the control subjects
was reduced to 6% whilst 90% of the patients with hypopituitarism were correctly
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classified as GHD. Unfortunately such BMI-specific data is not currently available

within the paediatric population, but is needed to prevent false positive diagnoses of
GHD on provocation testing in the increasing overweight/obese cohort.
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While the mechanisms through which obesity reduces GH secretion remain unclear,
one proposed hypothesis is that high circulating levels of free fatty acids supress GH
secretion via an action on the hypothalamus. Inhibition of lipolysis with niacin
(vitamin B3) has been shown to increase non-stimulated GH levels in obese children
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[24]. This raises the prospect that niacin administration during GH stimulation testing
may improve diagnostic accuracy in obese children.
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3.5 Puberty and priming

During puberty, high levels of sex steroid hormones are thought to augment
endogenous GH secretion and insulin-like growth factor-1 concentrations. Children
with delayed puberty are known to have relatively low growth velocity and are
frequently assessed for short stature. GH provocation testing in this group frequently
yields sub-optimal peak GH concentrations. However, this cohort will often show a
normal peak GH to provocation following puberty, whether treated for GHD or not
[25, 26]. This has led to the suggestion that it is a deficiency in sex steroids in these
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children (usually due to constitutional delay in puberty) that underlies the positive
provocation test results, rather than true GHD.

Priming refers to the administration of sex steroids to pre-pubertal children prior to

GH provocation testing. There is substantial evidence to support this practice. Marin
et al showed that 61% of healthy prepubertal children failed to demonstrate a peak
GH >7 µg/L to three GH provocation tests (exercise, insulin and arginine), but after
administration of oestrogen 95% of these children demonstrated a peak GH >7 µg/L

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[27]. Martinez et al randomised children undergoing GH stimulation testing to
priming with placebo or oral oestradiol [28]. For those children with a clinical
phenotype compatible with GHD, priming had no effect on peak GH concentrations.

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However for the group of healthy short normal children priming increased GH peak.
Follow-up studies have demonstrated that not treating children with subnormal
unprimed GH stimulation tests but normal primed GH stimulation tests does not result

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in an impaired final height - Gonc et al followed 50 such boys and reported a mean
final height of -1.27 SD, similar to the mid-parental height at -1.38 SD [29].

Despite this evidence, practices in sex steroid priming vary. An audit of practice of

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paediatric endocrinologists across Europe suggested that priming prior to GH
provocation tests is utilised by 50% in pre-pubertal boys and 40% in pre-pubertal girls
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[30]. Although the studies above would suggest that priming for GH provocation
tests reduces the rate of false-positive diagnoses of GHD, there are concerns that it
only temporarily increases GH secretion, thereby missing a cohort of children with
transient peri-pubertal GHD who may be responsive to treatment [31]. These
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conflicting views have resulted in three common forms of practice: no priming,
priming for children with pubertal delay only, or priming for all pre-pubertal children
(boys >9 years, girls >8 years). Commonly used protocols for priming include
intramuscular testosterone injections (100 mg) for 7-10 days prior to testing in boys
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and oral oestrogen preparations (for example, 10-20 µg ethinyloestradiol) for 48-72
hours prior to testing in girls. Some centres will use oestrogen preparations to prime
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boys also, to avoid the need for intramuscular injections.

4. GH secretion profiles
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Studies in the 1980s and 1990s assessed measurement of physiological GH secretion

profiles over 12 or 24 hours, as an alternative to GH provocation tests. Although
there was evidence to suggest better reproducibility [32], there appears to be overlap
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in the profiles between GHD and non-GHD subjects with 25% of normally growing
children incorrectly classified as GHD [33]. Sensitivity is also an issue, with 57% of
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children with GHD not identified by GH secretion profiles in one study [34]. Other
disadvantages include the large number of samples (every 20 minutes) and the cost of
an overnight admission to hospital. Therefore it is not a test that is frequently used in
current practice. However it is useful in the diagnosis of GH neurosecretory
dysfunction, defined by a sub-optimal GH secretion profile, clinical features of GHD,
a low IGF-1 but a normal GH provocation test.

5. IGF-1 and IGFBP-3

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Serum IGF-1 is mainly produced in the liver under the control of GH. It circulates
bound to the IGF binding proteins (IGFBPs), of which IGFBP-3 is the major serum
carrier. Both IGF-1 and IGFBP-3 offer the advantage over GH of being present at
relatively constant concentrations in the serum throughout the day, allowing random
measurements to be taken rather than necessitating a provocation test. Values vary
with age and pubertal stage, with normative ranges specific for age and pubertal stage
available [18]. These are important to consider in a child with pubertal delay and low
IGF-1 concentrations for age, but normal when adjusted for Tanner stage. Several

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other factors affect IGF-1 concentrations – poor nutrition, hypothyroidism, chronic
disease, renal failure and diabetes all decrease IGF-I concentrations.

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Similar to GH assays, issues exist with IGF-1 assays also [35, 36]. Foremost amongst
these is that the majority of IGF-1 in serum exists as a ternary (IGF-1/IGFBP-3/Acid
Labile Subunit) complex. IGFBPs interfere with both competitive and non-

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competitive assays, and must be removed in order to allow measurement of total
rather than free IGF-1. The completeness of this dissociation will lead to variability
in IGF-1 concentrations between assays. Furthermore, intra-assay variability within
the same patient can be up to 35% [37]. Although consensus guidelines recommend

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calibration of assays against the WHO IRP 02/254 [18], considerable variation exists
among different commercial assays [36].
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As discussed previously, the lack of a true gold standard makes assessment of the
diagnostic performance of any test for GHD difficult. Nevertheless, IGF-1 has been
suggested to have a high specificity (>90%) but relatively poor sensitivity (50-70%),
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particularly in children under 5 years of age [38-40]. Therefore a low IGF-1
concentration is highly predictive of GHD, whereas normal levels do not exclude
GHD. However, in combination with a measure that has high sensitivity for GHD,
IGF-1 can be a powerful screening tool. This was demonstrated by Cianfarani et al,
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who showed that combination of height velocity assessment and IGF-1 resulted in a
sensitivity of 95% and specificity of 96% [40].
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Measurement of IGFBP-3 concentrations was originally thought to be superior to

IGF-1 concentrations as it is less affected by nutritional state and age. Certainly
initial reports suggested both a high sensitivity (95%) and specificity (97%) [41].
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However these results have not been reproduced in further studies thereafter, which
have suggested predictive values similar to IGF-1 and that it offers no diagnostic
advantage to IGF-1 [39, 42].
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6. Neuroimaging
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Structural abnormalities identified on pituitary MRI in a child being investigated for

GHD, adds significantly to the diagnosis. Within a cohort of children with
biochemically diagnosed GHD, those with hypothalamic pituitary abnormalities on
imaging had more severe biochemical (all had peak GH <5 µg/L) and clinical (shorter
and younger at time of diagnosis) GHD than those with a normal MRI, and displayed
better catch-up growth with GH treatment [43].

The structural abnormalities most commonly encountered in association with

congenital GHD are an ectopic posterior pituitary gland, anterior pituitary hypoplasia
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and/or thinning/interruption of the pituitary stalk. Tillmann et al evaluated MRI scans

and GH status of 110 children undergoing provocation tests - of those with
biochemical GHD, 79% had one of the above MRI abnormalities, whereas 46% of
non-GHD children had MRI abnormalities [44]. They calculated that the presence of
any MRI abnormality as a marker of GHD would generate a sensitivity of 79% and
specificity of 54%. However, the presence of one of the more severe abnormalities
(ectopic posterior pituitary or hypoplastic anterior lobe and stalk) increased specificity
and positive predictive value. Less common findings on MRI are features of septo-

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optic dysplasia, abnormalities of the corpus callosum and holoprosencephaly.
Features in acquired GHD include tumours affecting the hypothalamic-pituitary axis
such as craniopharyngiomas, adenomas and germinomas, as well as pituitary stalk

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thickening suggestive of Langerhans cell histiocytosis.

As would be expected, children with MPHD are more likely to demonstrate MRI

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abnormalities than those with iGHD [45, 46]. Furthermore, children with iGHD who
have MRI abnormalities are more likely to develop MPHD in the future than those
with normal MRI scans [45, 46]. MRI abnormalities are also predictive of the
persistence of GHD into adulthood (discussed further below). Given the diagnostic

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and prognostic importance, it is recommended that all children with biochemical or
clinical evidence of GHD undergo a pituitary MRI scan [3].

7. Genomic evaluation
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The role of genetic investigations in the diagnosis of GHD is currently limited, but is
set to expand with more sophisticated genetic technologies (such as whole exome and
whole genome sequencing) moving from the research setting into the clinical setting.
Many genes are associated with GHD when it is part of MPHD (POU1F1, PROP1,
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LHX3, LHX4, HESX1, OTX2, SOX2, SOX3, GLI2, GLI3, FGFR1, FGF8 and
PROKR2). This is reviewed in greater detail elsewhere [47]. In children with iGHD,
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mutations in GH1 and GHRHR are most common, with more recently mutations in
RNPC3 also being described [48, 49]. Alatzoglou et al screened for mutations in GH1
and GHRHR in 224 patients with iGHD, with mutations identified in 11% of cases,
and in 39% of familial cases [50]. There was no difference in the severity of
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biochemical GHD or MRI abnormalities between those with and without mutations,
although those with mutations presented with more significant growth failure.
Current recommendations suggest consideration for genetic investigations in children
with iGHD who demonstrate early growth failure, severe growth failure (more than
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3SD below the mean), positive family history or consanguinity, and extremely low
peak GH concentrations and IGF-1/IGFBP-3 concentrations [3].
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While the role of assessing for genetic mutation in reaching a diagnosis is well
established, transcriptomics (the global assessment of mRNA) is not currently
extensively used in diagnosis of endocrine disease. Using a machine learning
algorithm (Random Forest Classification) and transcriptome analysis of peripheral
blood mononuclear mRNA Murray et al were able to discriminate GHD from control
subjects with a sensitivity of 100% and specificity of 71% [51]. This study used
normal control children and further assessment against short stature controls is
required.
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8. Re-assessment of GHD at the transitional age

It has long been appreciated that a proportion (45-60%) of children with iGHD are
shown to be growth hormone sufficient on retesting following completion of growth
[38, 52]. On completion of growth, adolescents that have been treated with GH for
GHD should be retested for GHD, having discontinued GH treatment for at least one
month. This identifies those individuals with ongoing severe GHD who would

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benefit from treatment into adulthood for the effects of GH on bone mineral density,
body composition, serum lipids and quality of life. The European Society of
Paediatric Endocrinologists (ESPE) guidelines suggest that those with a high

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likelihood of ongoing GHD (patients with MPHD, a defined genetic cause for GHD,
pituitary abnormalities on MRI, history of cranial tumours and irradiation) should just
have IGF-1 measured in the first instance [53]. If this is low then ongoing GHD is

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confirmed, whereas if this is normal then a GH provocation test is required. In those
with a low likelihood of ongoing GHD (patients with iGHD), both an IGF-1 and GH
provocation test are required.

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Similar to issues within the paediatric population, the cut-off for peak GH on
provocation testing when retesting for GHD at the transitional age is debated. ESPE
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guidelines suggest a cut-off of 5 µg/L [53] and GH Research Society guidelines
suggest a cut-off of 6 µg/L [54, 55]. Both of these are higher than the adult cut-offs
for peak GH on provocation testing, which is 3 µg/L and is adopted by the American
Association of Clinical Endocrinologists guidelines for retesting [56].
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Pituitary abnormalities on MRI, especially an ectopic posterior pituitary gland,
increase the likelihood of persistent GHD into adulthood in children with iGHD [52,
57]. However, Leger et al have shown that even in a cohort of patients with MRI
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abnormalities, 22% of patients achieved peak GH >10 µg/L on retesting, suggesting

that even such individuals should be retested at transitional age [58].
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The reasons why such a large proportion of children with iGHD are shown to be
growth hormone sufficient on retesting following completion of growth are unclear.
It is thought that fallacies in GH provocation tests which have been previously
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discussed may play a role in false diagnosis in childhood, but also that they may have
had a transient form of GHD. In addition the change in cut-off concentrations and sex
steroid augmented increase in GH secretion may play a role.
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9. Summary
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The decision to investigate for a potential diagnosis of GHD in childhood should be

primarily based on clinical presentation and auxology, while the diagnosis is reached
through a combination of clinical, biochemical and radiological assessment. That
many children with iGHD are shown to be GH sufficient on retesting following
completion of growth highlights some of the issues present in the current diagnostic
measures of GHD in children, and why caution is needed in interpretation of results.

10. Practice points

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- There is no gold standard diagnostic test for GHD - assessment should be

multifactorial, involving a combination of clinical, auxological, biochemical,
radiological and genetic tools.
- Clinicians should be aware of their local laboratory's assays for GH, IGF-1 and
IGFBP-3, as well as their limitations.
- The diagnosis of iGHD requires two positive GH provocation tests, due to the
potential for false-positives.

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- The interpretation of GH provocation tests should take into account possible
confounding factors such as obesity and whether priming was utilised.
- IGF-1 and IGFBP-3 are useful screening tools in the assessment of GHD, with high

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specificity but poor sensitivity. Neither in isolation is advantageous over the other.
- Pituitary imaging should take place in all children with GHD, as it carries diagnostic
and prognostic importance.

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- Genetic investigations should be considered in children with severe growth failure,
severe biochemical GHD and a positive family history of GHD.
- All children with iGHD should be retested with GH provocation tests on completion
of growth.

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11. Research agenda
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- There exists limited evidence-based data on the optimal peak GH concentration cut-
off which separates children with GHD from those without GHD. Similarly, specific
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cut-offs for different assays and provocation tests are lacking.
- With evidence that increasing BMI reduces peak GH concentrations, studies are
required to elucidate BMI-specific cut-offs for GH provocation tests.
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Declaration of Interest: PG Murray has received support from Merck and Pfizer.

Funding Source Declaration: This research did not receive any specific grant from
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funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgements: None
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Vitae
- Dr Amish Chinoy is currently a Grid trainee in Paediatric Endocrinology at Royal
Manchester Children’s Hospital. He graduated from King’s College Hospital,
London, and has trained in Paediatrics in Kent, Surrey, Sussex and Manchester.
- Dr Philip Murray, is a Consultant Paediatric Endocrinologist at the Royal
Manchester Children’s Hospital and a honorary Clinical Senior Lecturer at the
University of Manchester. He graduated from Glasgow University and trained in
paediatrics in Manchester, Melbourne and London.
 ACCEPTED MANUSCRIPT

References
[1] Vimpani GV, Vimpani AF, Lidgard GP, et al. Prevalence of severe growth
hormone deficiency. Br Med J. 1977;2(6084):427-30.
[2] Lindsay R, Feldkamp M, Harris D, et al. Utah Growth Study: growth standards
and the prevalence of growth hormone deficiency. J Pediatr. 1994;125(1):29-35.
[3] Growth Hormone Research Society. Consensus guidelines for the diagnosis and
treatment of growth hormone (GH) deficiency in childhood and adolescence:

PT
summary statement of the GH Research Society. GH Research Society. J Clin
Endocrinol Metab. 2000;85(11):3990-3.
[4] Hawkes CP, Grimberg A. Measuring growth hormone and insulin-like growth

RI
factor-1 in infants: What is normal? Pediatr Endocrinol Rev. 2013;11(2):126-146.
[5] Binder G, Weidenkeller G, Blumenstock M et al. Rational approach to the
diagnosis of severe growth hormone deficiency in the newborn. J Clin Endocrinol

SC
Metab. 2010;95(5):2219-2226.
[6] Ghigo E, Bellone J, Aimaretti G, et al. Reliability of provocative tests to assess
growth hormone secretory status. Study in 472 normally growing children. J Clin

U
Endocrinol Metab. 1996;81(9):3323-7.
[7] Shah A, Stanhope R, Matthew D. Hazards of pharmacological tests of growth
AN
hormone secretion in childhood. BMJ. 1992;304(6820):173-4.
[8] Kaplan SL, Abrams CA, Bell JJ, et al. Growth and growth hormone. I. Changes in
serum level of growth hormone following hypoglycemia in 134 children with growth
retardation. Pediatr Res. 1968;2(1):43-63.
M
[9] Dattani MT, Pringle PJ, Hindmarsh PC, et al. What is a normal stimulated growth
hormone concentration? J Endocrinol. 1992;133(3);447-50.
D

[10] Wagner IV, Paetzold C, Gausche R, et al. Clinical evidence-based cutoff limits
for GH stimulation tests in children with a backup of results with reference to mass
spectrometry. Eur J Endocrinol. 2014;171(3):389-97.
TE

[11] Secco A, di Iorgi N, Napoli F, et al. The glucagon test in the diagnosis of growth
hormone deficiency in children with short stature younger than 6 years. J Clin
Endocrinol Metab. 2009;94(11):4251-7.
EP

[12] Hilczer M, Smyczynska J, Stawerska R, et al. Stability of IGF-I concentration

despite divergent results of repeated GH stimulating tests indicates poor
reproducibility of test results. Endocr Regul. 2006;40(2):37-45.
C

[13] Loche S, Bizzarri C, Maghnie M, et al. Results of early reevaluation of growth

hormone secretion in short children with apparent growth hormone deficiency. J
AC

Pediatr. 2002;140(4):445-9.
[14] Seth J, Ellis A, Al-Sadie R. Serum growth hormone measurements in clinical
practice: An audit of performance from the UK National External Quality Assessment
scheme. Horm Res. 1999;51 Suppl 1:13-9.
[15] Müller A, Scholz M, Blankenstein O, et al. Harmonization of growth hormone
measurements with different immunoassays by data adjustment. Clin Chem Lab
Med. 2011;49(7):1135-42.
[16] Baumann G. Growth hormone heterogeneity: genes, isohormones, variants, and
binding proteins. Endocr Rev. 1991;12(4):424-49.
 ACCEPTED MANUSCRIPT

[17] Ebdrup L, Fisker S, Sørensen HH, et al. Variety in growth hormone

determinations due to use of different immunoassays and to the interference of growth
hormone-binding protein. Horm Res. 1999;51 Suppl 1:20-6.
[18] Clemmons DR. Consensus statement on the standardization and evaluation of
growth hormone and insulin-like growth factor assays. Clin Chem. 2011;57(4):555-9.
[19] Arsene CG, Kratzsch J, Henrion A. Mass spectrometry - an alternative in growth
hormone measurement. Bioanalysis. 2014;6(18):2391-402.

PT
[20] Health Survey for England 2014. http://www.hscic.gov.uk/catalogue/PUB19295
(last accessed 26/06/2016).
[21] Martha PM Jr, Gorman KM, Blizzard RM, et al. Endogenous growth hormone

RI
secretion and clearance rates in normal boys, as determined by deconvolution
analysis: relationship to age, pubertal status, and body mass. J Clin Endocrinol
Metab. 1992;74(2):336-44.

SC
[22] Stanley TL, Levitsky LL, Grinspoon SK, et al. Effect of body mass index on
peak growth hormone response to provocative testing in children with short stature. J
Clin Endocrinol Metab. 2009;94(12):4875-81.

U
[23] Dichtel LE, Yuen KC, Bredella MA, et al. Overweight/Obese adults with
pituitary disorders require lower peak growth hormone cutoff values on glucagon

Endocrinol Metab. 2014;99(12):4712-9.AN

stimulation testing to avoid overdiagnosis of growth hormone deficiency. J Clin

[24] Galescu OA, Crocker MK, Altschul AM et al. Effects of niacin administration on
free fatty acid and growth hormone concentrations in obese children. Endocr Rev.
M
2016;37(2 Suppl):OR31-3.
[25] Cacciari E, Tassoni P, Parisi G, et al. Pitfalls in diagnosing impaired growth
hormone (GH) secretion: retesting after replacement therapy of 63 patients defined as
D

GH deficient. J Clin Endocrinol Metab. 1992;74(6):1284-9.

[26] Gourmelen M, Pham-Huu-Trung MT, Girard F. Transient partial hGH deficiency
TE

in prepubertal children with delay of growth. Pediatr Res. 1979;13(4 Pt 1):221-4.

[27] Marin G, Domené HM, Barnes KM, et al. The effects of estrogen priming and
puberty on the growth hormone response to standardized treadmill exercise and
arginine-insulin in normal girls and boys. J Clin Endocrinol Metab. 1994;79(2):537-
EP

41.
[28] Martínez AS, Domené HM, Ropelato MG, et al. Estrogen priming effect on
growth hormone (GH) provocative test: a useful tool for the diagnosis of GH
C

deficiency. J Clin Endocrinol Metab. 2000;85(11):4168-72.

[29] Gonc EN, Kandemir N, Ozon A, et al. Final heights of boys with normal growth
AC

hormone responses to provocative tests following priming. J Pediatr Endocrinol

Metab. 2008;21(10):963-71.
[30] Juul A, Bernasconi S, Clayton PE, et al. European audit of current practice in
diagnosis and treatment of childhood growth hormone deficiency. Horm
Res. 2002;58(5):233-41.
[31] Lazar L, Phillip M. Is sex hormone priming in peripubertal children prior to
growth hormone stimulation tests still appropriate? Horm Res
Paediatr. 2010;73(4):299-302.
 ACCEPTED MANUSCRIPT

[32] Zadik Z, Chalew SA, Gilula Z, et al. Reproducibility of growth hormone testing
procedures: a comparison between 24-hour integrated concentration and
pharmacological stimulation. J Clin Endocrinol Metab. 1990;71(5):1127-30.
[33] Lanes R. Diagnostic limitations of spontaneous growth hormone measurements
in normally growing prepubertal children. Am J Dis Child. 1989;143(11):1284-6.
[34] Rose SR, Ross JL, Uriarte M, et al. The advantage of measuring stimulated as
compared with spontaneous growth hormone levels in the diagnosis of growth

PT
hormone deficiency. N Engl J Med. 1988;319(4):201-7.
[35] Clemmons DR. IGF-I assays: current assay methodologies and their limitations.
Pituitary. 2007;10(2):121-8.

RI
[36] Frystyk J, Freda P, Clemmons DR. The current status of IGF-I assays-a 2009
update. Growth Horm IGF Res. 2010;20(1):8-18.
[37] Milani D, Carmichael JD, Welkowitz J, et al. Variability and reliability of single

SC
serum IGF-I measurements: impact on determining predictability of risk ratios in
disease development. J Clin Endocrinol Metab. 2004;89(5):2271-4.
[38] Juul A, Kastrup KW, Pedersen SA, et al. Growth hormone (GH) provocative

U
retesting of 108 young adults with childhood-onset GH deficiency and the diagnostic
value of insulin-like growth factor I (IGF-I) and IGF-binding protein-3. J Clin
AN
Endocrinol Metab. 1997;82(4):1195-201.
[39] Cianfarani S, Boemi S, Spagnoli A, et al. Is IGF binding protein-3 assessment
helpful for the diagnosis of GH deficiency? Clin Endocrinol (Oxf). 1995;43(1):43-7.
[40] Cianfarani S, Tondinelli T, Spadoni GL, et al. Height velocity and IGF-I
M
assessment in the diagnosis of childhood onset GH insufficiency: do we still need a
second GH stimulation test? Clin Endocrinol (Oxf). 2002;57(2):161-7.
[41] Blum WF, Ranke MB, Kietzmann K, et al. A specific radioimmunoassay for the
D

growth hormone (GH)-dependent somatomedin-binding protein: its use for diagnosis

of GH deficiency. J Clin Endocrinol Metab. 1990;70(5):1292-8.
TE

[42] Nunez SB, Municchi G, Barnes KM, et al. Insulin-like growth factor I (IGF-I)
and IGF-binding protein-3 concentrations compared to stimulated and night growth
hormone in the evaluation of short children--a clinical research center study. J Clin
Endocrinol Metab. 1996;81(5):1927-32.
EP

[43] Coutant R, Rouleau S, Despert F, et al. Growth and adult height in GH-treated
children with nonacquired GH deficiency and idiopathic short stature: the influence of
pituitary magnetic resonance imaging findings. J Clin Endocrinol
C

Metab. 2001;86(10):4649-54.
[44] Tillmann V, Tang VW, Price DA, et al. Magnetic resonance imaging of the
AC

hypothalamic-pituitary axis in the diagnosis of growth hormone deficiency. J Pediatr

Endocrinol Metab. 2000;13(9):1577-83.
[45] Bozzola M, Mengarda F, Sartirana P, et al. Long-term follow-up evaluation of
magnetic resonance imaging in the prognosis of permanent GH deficiency. Eur J
Endocrinol. 2000;143(4):493-6.
[46] Jagtap VS, Acharya SV, Sarathi V, et al. Ectopic posterior pituitary and stalk
abnormality predicts severity and coexisting hormone deficiencies in patients with
congenital growth hormone deficiency. Pituitary. 2012;15(2):243-50.
 ACCEPTED MANUSCRIPT

[47] Bancalari RE, Gregory LC, McCabe MJ, et al. Pituitary gland development: an
update. Endocr Dev. 2012;23:1-15.
[48] Alatzoglou KS, Dattani MT. Genetic causes and treatment of isolated growth
hormone deficiency-an update. Nat Rev Endocrinol. 2010;6(10):562-76.

[49] Argente J, Flores R, Gutierrez-Arumi A, et al. Defective minor spliceosome

mRNA processing results in isolated familial growth hormone deficiency. EMBO

PT
Mol Med. 2014; 6(3): 299–306.
[50] Alatzoglou KS, Turton JP, Kelberman D, et al. Expanding the spectrum of
mutations in GH1 and GHRHR: genetic screening in a large cohort of patients with
congenital isolated growth hormone deficiency. J Clin Endocrinol

RI
Metab. 2009;94(9):3191-9.
[51] Murray PG, Stevens A, Koledova EB, et al. A distinct genomic signature can

SC
differentiate control form growth hormone deficient children and predict severity of
disease. Endocr Rev 2016;37(2 Suppl):SAT-004.
[52] Murray PG, Hague C, Fafoula O, et al. Likelihood of persistent GH deficiency
into late adolescence: relationship to the presence of an ectopic or normally sited

U
posterior pituitary gland. Clin Endocrinol (Oxf). 2009;71(2):215-9.
[53] Clayton PE, Cuneo RC, Juul A, et al. Consensus statement on the management of
AN
the GH-treated adolescent in the transition to adult care. Eur J
Endocrinol. 2005;152(2):165-70.
[54] Ho KK, 2007 GH Deficiency Consensus Workshop Participants. Consensus
M
guidelines for the diagnosis and treatment of adults with GH deficiency II: a statement
of the GH Research Society in association with the European Society for Pediatric
Endocrinology, Lawson Wilkins Society, European Society of Endocrinology, Japan
Endocrine Society, and Endocrine Society of Australia. Eur J
D

Endocrinol. 2007;157(6):695-700.
[55] Maghnie M, Aimaretti G, Bellone S, et al. Diagnosis of GH deficiency in the
TE

transition period: accuracy of insulin tolerance test an insulin-like growth factor-1

measurement. Eur J Endocrinol. 2005;152(4):589-96.
[56] Cook DM, Yuen KC, Biller BM, et al. American Association of Clinical
EP

Endocrinologists medical guidelines for clinical practice for growth hormone use in
growth hormone-deficient adults and transition patients - 2009 update. Endocr
Pract. 2009;15 Suppl 2:1-29.
[57] Maghnie M, Strigazzi C, Tinelli C, et al. Growth hormone (GH) deficiency
C

(GHD) of childhood onset: reassessment of GH status and evaluation of the predictive

criteria for permanent GHD in young adults. J Clin Endocrinol
AC

Metab. 1999;84(4):1324-8.
[58] Léger J, Danner S, Simon D, et al. Do all patients with childhood-onset growth
hormone deficiency (GHD) and ectopic neurohypophysis have persistent GHD in
adulthood? J Clin Endocrinol Metab. 2005;90(2):650-6.