The suggested scheme for laboratory screening tests to exclude general pediatric pathology, including thyroid function, insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3), and karyotype in girls, was proposed in the 2008 ISS Consensus Statement [8]. The practice of performing general pediatric tests was challenged in 2013 by Sisley et al. [9], who reported from the Cincinnati Children's Hospital that of 232 short patients who were healthy and had normal physical appearance, 98.7% had normal screening tests. The message in this publication was that screening tests were not cost-effective in healthy short children. To some extent this view was perpetuated in the recent Growth Hormone Research Society (GRS) 'Guidelines' article [13], which stated that 'Laboratory tests should be guided by clinical features rather than routinely applied to all patients with short stature ... Clinical discretion should be applied to the scope of testing for nonendocrine disease'.

We do not agree with this recommendation. In children with unexplained short stature, it is essential that routine hematological and biochemical screening tests are performed (Fig. 2). Chronic illnesses such as celiac and Crohn diseases need to be excluded, as do sex chromosome defects such as mosaic Turner syndrome [14]. These disorders cannot be excluded by clinical assessment alone. Short stature patients with nonFSS are most likely to have occult illness, being shorter than their parental target height, so pathological causes need to be excluded before endocrine assessment. Recombinant human growth hormone (rhGH) therapy may be prescribed in many children with ISS, and normal renal, liver, and hematological status are important to document before this therapy is initiated. Laboratory screening should consist of full blood count, erythrocyte sedimentation rate, IGF-1, free thyroxine, thyroid-stimulating hormone, a test for celiac disease, electrolyte and renal and liver function tests, calcium, phosphate, and alkaline phosphatase (Fig. 1) [6,8]. A hand/wrist bone age x-ray with examination for anatomical features of skeletal dysplasia should be performed in all referred short children. A karyotype or single nucleotide polymorphism array analysis (for detecting Turner syndrome, and with the latter tool also for copy number variants and uniparental disomy) in all girls with height < -2 SDS or height SDS < target height SDS -1.6 should also be performed [6]. If serum IGF-I is low, a GH stimulation test or an IGF-I generation test (IGFGT) are further options for identification of an endocrine etiology.

In the 1980s and 1990s, the study of childhood linear growth focused on the function of different components of the GH–IGF-I axis, and enormous progress in the understanding of this axis was made [15]. The somatomedin hypothesis first published in 1957 [16] was revisited 50 years later [17] to show that the IGF system plays a key role in growth regulation, with both circulating and peripherally produced IGF-I having individual roles [18]. IGF-1 deficiency was reported to occur in a proportion of short patients with normal GH secretion [19], which placed some ISS patients in an intermediate position between GH deficiency and GH resistance, although some overlap existed.

Some ISS patients have a degree of functional GH insensitivity [20] with a broad range of generation of IGF-I in response to GH. The important 2007 study by Cohen et al. [21] reported that high doses of rhGH were needed to reach a serum IGF-I concentration of +2 SD. Evidence of subnormal generation of IGF-I was also demonstrated in studies by Selva et al. [22] and Buckway et al. [23] on responses in the IGFGT. Compared to normal subjects, ISS patients had basal IGF-1 levels below the normal mean, and after GH stimulation on days 5 and 8 of the IGFGT, IGF-I levels were significantly lower than normal, regardless of GH dose. These hormonal findings challenge the definition of ISS patients having no endocrine abnormality.

Endocrine assessment of GH secretion and the IGF system should follow the guidelines published previously [8] and reviewed in the recent GRS Consensus Statement [13]. Important messages are highlighted in this publication, including the guidance that IGF-I assays with reliable reference data be used alongside age-appropriate pediatric reference ranges and that serum IGF-I is influenced by many factors such as assay methodology, age, nutrition, and chronic illness [24]. An IGF-I level > 0 SDS at any age makes GH deficiency unlikely but not impossible [13.24]. The GH stimulation test (GHST) has been criticized over the years for being a nonphysiological stimulus and for lack of reproducibility. Precision of the timing of peak GH in certain tests such as the arginine and levodopa tests will increase their sensitivity [25].

The GHST remains of key importance in the assessment of short children with undefined etiology [4]. An abnormal GHST, with a peak GH level of <7 or <10 μg/L in the United States [13] and auxological features consistent with GHD, removes a patient from the 'ISS' category. A low IGF-I value without knowledge of GH status cannot distinguish GH deficiency from GH insensitivity [19]. Longitudinal observation and monitoring may lead to re-evaluation of the GH-IGF-I axis in some slowly growing children that may involve a repeat of the GHST. Once the distinction between GHD and non-GHD short stature has been made, further investigations to define etiology such as measurements of IGFBP-3, acid labile subunit, and an IGFGT, with its recognized disadvantages [26], are options.

Short stature patients may have variable GH sensitivity and IGF-I concentrations [11]. Therefore, it has been suggested, but remains controversial, that fewer deleterious GHR gene defects may cause ISS associated with features of GH insensitivity [4]. Studies of ISS cohorts have reported heterozygous GHR variants occurring with a frequency ranging from 5% to 15.5% [28]. It has also been noted that although GHR sequence changes are common in children with ISS, many were also identified in control subjects and normal stature family members [29].

Since molecular investigations of short stature phenotypes started in the late 1980s, a number of pathogenic variants have been discovered in children labelled as having ISS or SGA. In 2019, Storr et al. [29] published an extensive review of mild or 'nonclassical' abnormalities of GH action. Mild forms of GH insensitivity can be divided into 3 categories: (1) aberrations of GH signaling caused by homozygous or heterozygous variants of genes encoding the GHR or signal transducer and activator of transcription 5B (STAT5B) [29-32]; (2) defects of IGF-I secretion (IGF1), transport (IGFALS), and bioavailability (PAPPA2) [29,33-35]; and (3) IGF-I insensitivity (IGF1R) [36]. Most patients with GH1, GHR, and STAT5B defects are born with a normal birth size, while patients with IGF1 and IGF1R variants are typically born with a low birth size. Paternally-transmitted heterozygous IGF2 variants are one of the recognized genetic causes of Silver-Russell syndrome [37]. A summary of phenotypic and endocrine features of genetic defects in patients with mild to moderate short stature is shown in Table 1.

Genetic defects which impair chondrogenesis are likely to cause some degree of body disproportion; however, this may be mild and not noticed by the clinician, resulting in the child being labelled as ISS or SGA [38]. Out of the multiple reported genes [12], we discuss 4 examples.

Clinically heterogeneous short children may harbor a range of molecular etiologies in up to 40% of cases [51-54]. Selection criteria for genetic testing and methods will continue to evolve. In general, genetic investigations are indicated if there are positive clinical diagnostic clues for a monogenic disorder. Examples of such clues include severe short stature, microcephaly or relative macrocephaly, dysmorphic features, disproportion, a positive family history, and a low birth size (SGA) [6]. In general, patients with a clinical suspicion of monogenic disorders in whom a clear diagnosis will enhance clinical management in terms of genetic counseling are good candidates for genetic testing. Examples include soft dysmorphic features suggestive of Noonan syndrome, patients with possible mild GH insensitivity [29], and patients with borderline body disproportion suggestive of SHOX, ACAN, or NPR2 genetic variants [5,55]. In general, syndromic rather than nonsyndromic cases are more likely to yield positive genetic results.

The principal aim of genetic analysis is to identify monogenic disorders having a significant effect on growth [10]. In children with ISS, one would expect that these defects would be found mainly in the non-FSS subjects, where parents with normal stature may be carriers of a recessive gene or the patient carries a de novo pathogenic gene variant. An example of apparent NFSS of genetic origin is the pseudoexon GHR variant, which can present with mild short stature and normal IGF-1 levels [56]. Many of the FSS patients are likely to have polygenic short stature, having inherited common gene variants of small effect size [51]. However, if dominant inheritance of short stature is traceable in one or more generations, monogenic defects as in NPR2 [51] and SHOX mutations, dominant negative GHR mutations, and Noonan syndrome defects, or an inherited CNV, are likely [4].

In short children born SGA, genetic causes appear to be more frequent than in ISS, although direct comparative studies have yet to be performed. Extreme examples include genetic defects in fundamental cellular processes, which can produce severe global growth deficiencies known as primordial dwarfism, where pre- and postnatal growth is severely affected. An extensive overview of gene variants associated with SGA was recently published by Finken et al. [38] As with ISS, the primary aim of genetic studies in SGA subjects is to identify monogenic defects. These may be predicted by the use of clinical scoring systems, such as the Netchine-Harbison system for Silver-Russell syndrome [57], the recently published scoring system for IGFR mutations [36] and concordance with characteristics of ACAN variants [55] or Madelung deformity, which is pathognomonic of SHOX haploinsufficiency. Positive scores for a recognized disorder can be followed up by candidate gene sequencing or, preferably using a hypothesis-free approach, with a genome-based targeted gene panel, including a check for copy number variations and uniparental disomy.

In a study of 55 unexplained cases of short SGA subjects, genotyping using a targeted gene panel or ES gave a positive diagnostic yield of 15% [58]. Heterozygous pathogenic or likely pathogenic genetic variants in 8 of the 55 patients were identified in genes already associated with growth disorders. Four of the genes were associated with growth plate development, IHH (n=2), NPR2 (n=2), SHOX (n=1), and ACAN (n=1), and 2 with the RAS/MAPK pathway, PTPN11 (n=1) and NF1 (n=1) [58]. In the case of a dysmorphic SGA child where the diagnosis was uncertain, ES was particularly helpful in identifying the mutation in the BLM gene confirming the diagnosis of Bloom syndrome [59]. In special cases where a novel monogenic disorder is suspected, ES in a 'trio,' that is, in the patient and both parents, can be performed. It is likely that future bioinformatic analyses of next-generation sequencing technology will show that in many cases, SGA is caused by a combination of multiple (epi)genetic variants [60].

We believe that a case can be made for the genetic assessment of short children born SGA, because the pretest likelihood of detecting a genetic condition is high (on the order of 30%–40%), particularly if a child presents with additional features as summarized previously. Another benefit of positive genetic analysis is to be sure that the child does not have a syndrome for which rhGH treatment is contraindicated (e.g., Bloom syndrome) or debatable (e.g., neurofibromatosis type 1) [61]. A third reason is that the positive diagnosis of IGF1R haploinsufficiency or genetic syndromes affecting growth plate function (e.g., SHOX haploinsufficiency) implies that a higher dosage of rhGH is indicated to generate effective growth acceleration.

The decision about genetic testing in a short child must be made by the clinician responsible for the child's care by subjectively weighing the diagnostic clues for a primary growth disorder, their severity, and potential impact on management [12]. It is reasonable to assume that when the label of ISS or SGA is given to a child, a detailed auxological examination would have been performed to exclude body disproportion and major dysmorphic features. Similarly, endocrine investigations to exclude GH deficiency and GH insensitivity should have been performed. In general, the greater the severity of short stature, the more likely it is that there is an identifiable genetic defect [13], but the number and severity of any additional congenital anomalies or dysmorphic features and evidence of a skeletal dysplasia, associated intellectual disability, microcephaly, or relative macrocephaly should be added to the interpretation [6,10,51]. Not all short children should have genetic investigations [13]. Specifically, children with mild FSS without any additional abnormal clinical features should not be tested. Similarly, children with delayed bone age with expected adult heights within the normal range with a positive family history for delayed puberty for the family should in general not be tested as they may have CDGP or polygenic FSS.