The human HMGA2 gene has 5 exons and 4 introns and is located on chromosome 12 (12q14.3) (Su et al. 2020). It encodes a small multipotential nuclear protein, the mammalian high-mobility group protein AT-hook2, which belongs to the non-histone chromosomal high-mobility group (HMG). HMGA2 is well conserved in mammals, as the protein sequences in humans and mice are almost identical. Its structure contains 3 “AT-hook” DNA-binding motifs and a negatively charged C-terminal motif. The “AT-hook” motifs specifically bind to the minor groove of AT-rich DNA and induce DNA bending. It has been hypothesized that HMGA2 may function as an architectural transcription factor or an epigenetic regulator (Su et al. 2020; Lee et al. 2022). It appears to affect diverse biological processes, as it interacts with numerous proteins, primarily transcriptional factors, and is involved in the regulation of various genes (Lee et al. 2022). Human and animal studies have reported its high expression during embryogenesis and fetal development, but hardly detectable expression levels in adult tissues (Gorbenko del Blanco et al. 2011; Mercandante et al. 2020). The main functions of HMGA2 are likely related to promoting cell proliferation and differentiation and maintaining the potency of stem cells (Su et al. 2020; Lee et al. 2022). HMGA2 has been linked to diverse processes, including linear growth, adiposity, and tumorigenesis. Overexpression or aberrant expression of HMGA2 is linked to the development and progression of various tumors (benign or malignant), including lung, breast, prostate, or gastric cancers, as well as melanoma or leukemia (Su et al. 2020; Lee et al. 2022; Mercandante et al. 2020).

The HMGA2 gene became a candidate for longitudinal growth regulation as rare severe mutations in this gene alter body size in various species, including mice and humans (Grant et al. 2008; Yang et al. 2010). In murine studies, it has been demonstrated that homozygous deletion leads to dwarfism (Hendriks et al. 2011; Lee et al. 2022).

An essential role of HMGA2 in human height regulation is supported by observations of patients with microdeletion of chromosomal region 12q14, who exhibit similar symptoms, including failure to thrive, short stature, and developmental delay. Severe growth restriction in these patients is thought to be a consequence of HMGA2 gene loss, as it lies within this particular region (Fusco et al. 2016; Lynch et al. 2011). This theory was supported by Buysse et al. (2009), who described a case of submicroscopic intragenic deletion disrupting only HMGA2 in a child with proportionate short stature as an isolated symptom with no other abnormalities. In this case, a cosegregation of this deletion in the child’s family was related to reduced adult height. HMGA2 was also proposed to be included as a candidate gene in Silver-Russel syndrome (SRS) molecular background. Apart from prenatal and postnatal growth failure, this syndrome is additionally related to certain dysmorphic features, like macrocephaly at birth, protruding forehead, or body asymmetry (Mercandante et al. 2020). However, if an effect on expression is low, it could also cause growth restrictions without other symptoms.

More data on the importance of the HMGA2 gene for growth in the general population has been obtained from genome-wide association studies (GWAS). It has been shown that HMGA2 gene variants affect height outcomes in both adults and children. Namely, the rs1042725 variant localized in the 3’ untranslated region (UTR) explains approximately 0.3% of population height variation with an increase of 0.4–0.9 cm in adult height (Weedon et al. 2007, 2008; Yang et al. 2010). In the study of Weedon et al. (2007), this variant was also strongly associated with height in children at the age of 7, 9, 10, and 11. The overall effect was assessed as approximately 0.4 cm increase in the C allele carriers. In the same paper, limb length was also related to the rs1042725 variant, suggesting an impact on the general skeletal longitudinal growth. However, no association was found with the birth length.

Yang et al. (2010), have confirmed the association of the HMGA2 gene with adult human height using data from approx. 11,000 subjects, including Caucasian and Chinese origin, which points to this gene’s importance for height across ethnic boundaries.

It has been shown that, along with the role in determining variability in human height, HMGA2 variants may contribute to susceptibility to idiopathic short stature. It also seems to be involved in the regulation of GH/IGF1 and IGF2 axis (Toni et al. 2024). A known target gene for HMGA2 is IGF2BP2, encoding insulin-like growth factor 2 mRNA-binding protein. IGF2BP2, in turn is engaged in the post-transcriptional regulation of IGF2, one of the important fetal growth factors (Gorbenko del Blanco et al. 2011). Moreover, in some patients with 12q14 microdeletion syndrome, low GH levels were detected (Lynch et al. 2011), while Gorbenko del Blanco et al. (2011), in their research, reported a deletion in the HMGA2 gene that might be related to isolated GHD.

As GH is crucial for normal growth in children, its deficiency usually results in short stature. However, GH is considered to affect the postnatal stage of human growth and children with isolated GHD are typically born with normal weight (Toni et al. 2024). Still, a child born small for gestational age (SGA) may additionally present decreased GH serum levels and fulfill the criteria of GHD.

It is important to note that an endocrine diagnosis of GHD carries a risk of ambiguity, as diagnostic criteria are not uniform across countries. Appropriate peak GH cut-off levels remain a matter of discussion and vary between 5 and 10 ng/ml (Hage et al. 2021). In Poland, deficient GH secretion is diagnosed below 10 ng/ml, while in many countries lower values are used. Furthermore, GHD can be defined as partial with a peak serum GH concentration between 5 and 10 ng/ml, or severe with a GH level below 5 or according to other authors below 3 ng/ml (Majewska et al. 2024; Binder et al. 2019; Allen 2021; Collett-Solberg et al. 2019; Murray et al. 2016; Webb and Dattani 2010; Grimberg et al. 2016). Therefore, due to the above considerations, for the purposes of the study, we used various options for dividing the study group depending on peak GH levels, including additional ranges below 5 and between 5 and 10 ng/ml.

The incidence of congenital form of GHD ranges from 1 in 4000 to 1 in 10,000 live births. Possible causes include genetic factors or structural changes in the area of the hypothalamus and pituitary gland, but most cases are classified as idiopathic. Familial occurrence of GHD concerns 3% to 30% of cases, suggesting a genetic background (Grunauer and Jorge 2018; Hage et al. 2021; Alatzoglou et al. 2014). Mutations in genes encoding growth hormone (GH1) and the receptor for growth hormone-releasing factor (GHRHR) have been proven to cause GHD. However, in the vast majority of patients, these mutations are not detected. Also, in our previous study, where we investigated the GH1 gene in Polish children with short stature and GHD, we did not find it to be a frequent causative factor (Majewska et al. 2020). That, in turn, suggests the involvement of other genes in GHD pathogenesis (Gorbenko del Blanco et al. 2011).

Although the HMGA2 gene’s relation to height regulation seems to be confirmed, its associations with idiopathic short stature or GHD are only suspected. In this research, we compared a cohort of Polish children with short stature to healthy children of normal height. We did not find any sequence variant in the assessed coding region of the HMGA2 gene. Nonetheless, in the flanking intronic sequences of exon 3, we detected two polymorphic variants: rs3834468 and rs73115423. We observed the lack of significant differences in allele frequencies between all patients and controls in both polymorphic sites, which would suggest that the HMGA2 gene is irrelevant in the pathogenesis of short stature in the Polish population. However, a detailed analysis among children with short stature, based on various growth hormone peak serum levels, showed a significant difference in the frequency of the rs73115423 variant between children with normal growth hormone secretion and those with moderate GHD, while differences with the lowest GH secretion did not reach statistical significance. This observation suggests that HMGA2 may be involved in complex mechanisms of GH secretion regulation. The other detected variant, rs3834468, presented no statistically significant results among short stature children, but in the control group, a significant difference was found in terms of hSDS. Children with a heterozygous genotype for the G insertion appeared to be significantly taller than subjects carrying wild type genotype. We may hypothesize this variant to be height-increasing in healthy populations, which has not been described in previous reports. However, our observation was made based on a relatively small number of patients; therefore, confirmation would require further research with a substantially increased cohort of healthy individuals.

Observed intronic variants may affect protein structure and function if they interfere with the normal pre-mRNA splicing process or are in the linkage disequilibrium with another sequence alteration that may influence the protein integrity and function. Moreover, genetic variants within intronic regions may impact regulatory elements, including enhancers and silencers, that are situated in these non-coding sequences (Wrighton 2017). In relation to growth, a known example of an intron-located functional polymorphism was identified in the GH1 gene (the rs2665802 A > T in position + 1169; intron 4). Functional assays revealed that the + 1169 A allele is directly associated with reduced GH1 expression and GH secretion, despite not affecting coding sequences. This variant has been linked to lower circulating GH and IGF-1 levels, as well as altered disease risks for e.g., colorectal cancer, osteoporosis (Millar et al. 2010). These findings highlight the underappreciated role of intronic regulatory elements, which may harbor functional variants influencing splicing, enhancers, and long-range regulation. Such intron-based polymorphisms should thus be given greater consideration in genetic studies.

Diagnostic process in children with growth retardation is complex, multidirectional, and time-consuming. At the same time, the results obtained are not always unambiguous, and the final decision is often a judgment call depending on the experience of the specialist taking care of the patient (Allen 2021). Hence, it is reasonable to search for methods that enable a quick and accurate diagnosis, and at the same time enable the selection of the most appropriate treatment. Precise determination of the genetic causes of short stature in children could potentially help to reduce certain burdensome examinations carrying the risk of complications, such as GH stimulation tests. It could also help to predict the possible effectiveness of available therapies such as rhGH (Vasques et al. 2019). Treatment with rhGH is considered safe and effective, but requires long-term adherence and is cost-consuming, while the degree of therapeutic response differs across patients.

Among the limitations of our study, it is worth noting that GH serum concentrations were evaluated only in children with short stature. Since stimulation tests could not be performed in healthy children from the control group, the assumption of normal GH secretion in these children was based on their normal height, appropriate for their age and sex. Another issue that should be addressed is the disproportion in gender distribution within the study group. Male predominance among patients with short stature is a common observation, consistently reported in studies worldwide and confirmed in our previous research (Collett-Solberg et al. 2019; Grimberg et al. 2008; Majewska et al. 2020, 2024). However, this is not due to a generally higher incidence of short stature in boys, as the diagnosis is established in both genders according to similar criteria, when height is below − 2 SD from the mean for age and gender. By this definition, approximately 2.5% of both girls and boys meet the criteria for short stature. Nevertheless, among patients, i.e., children who are referred for diagnostics, boys commonly predominate. It is assumed to be a consequence of greater social acceptance of shorter stature in girls, whereas there is a greater social expectation for taller stature in boys. This, in turn, may lead to boys being more frequently referred to specialist centers, while girls are more often left undiagnosed, even though they should also be appropriately evaluated and treated (Grimberg et al. 2008; Majewska et al. 2020, 2024).