Lab Report - GH1 Gene Variants Study

Abstract

Background and Objectives: Normal human skeletal and soft tissue development relies on the circulating levels of growth hormone (GH). The GH1 gene governs GH production, and alterations in this gene can lead to growth hormone deficiency (GHD). This study aimed to investigate GH1 gene variants in a cohort of Egyptian patients with isolated growth hormone deficiency (IGHD) and isolated short stature (ISS) to explore their potential as biomarkers for GHD.

Methods: Forty-two patients with short stature were divided into two groups based on their GH levels: 33 patients with IGHD and 9 with ISS.

We conducted Sanger sequencing of exon 4 and its flanking intronic region of the GH1 gene. Additionally, in silico analysis was performed to determine the functional implications of the detected variants.

Results: We identified the +1150A (rs2665802) polymorphism in the studied fragment. The +1150A genotype was associated with short stature and low growth hormone levels in both groups under investigation.

Conclusion: This study represents the first molecular investigation of IGHD and ISS in Egyptian patients.

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The +1150A genotype shows promise as a potential biomarker for identifying IGHD and short stature cases.

Keywords: Growth hormone, GH1 gene, IGHD, ISS, +1150A genotype.

1. Introduction

Human skeletal growth and final height attainment are influenced by a complex interplay of genetic factors, systemic and local hormones, nutritional factors, and lifestyle choices (Mullis, 2005; Keselman et al., 2012). One key player in this intricate process is human growth hormone (GH), the secretion of which is tightly regulated. Any disruptions in this process can result in growth hormone deficiency (GHD) (Birla et al.

, 2012).

GH is of paramount importance due to its critical role in immune function, bone turnover, stature, muscle mass, and the regulation of lipid and carbohydrate metabolism, as well as postnatal growth (Millar et al., 2010). The incidence of GHD is estimated to be approximately 1 in 4,000 to 10,000 live births (Keselman et al., 2012). GHD can be categorized into two main types: isolated growth hormone deficiency (IGHD) and combined pituitary hormone deficiency (CPHD). IGHD is characterized by a severe shortage or absence of growth hormone, leading to a failure of growth at the expected rate and proportionate short stature, typically evident in early childhood (US. National Library of Medicine, 2017; Mullis, 2010).

CPHD, on the other hand, involves a deficiency in growth hormone (GH) along with at least one of the following hormones: thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PrL), and occasionally adrenocorticotropic hormone (ACTH) (Birla et al., 2012; US. National Library of Medicine, 2017; de Graaff, 2017). In addition to these two types, there is a condition known as isolated short stature or Idiopathic short stature (ISS), which refers to extreme short stature without a clear diagnostic explanation following routine growth evaluation (de Graaff, 2017).

Mutations in the GH1 gene and growth hormone-releasing hormone receptor (GHRHR) have shed light on the etiology of IGHD/CPHD in both humans and mice. These genes have provided insights into the phenotype and pathogenesis of IGHD. Additionally, mutations in transcription factors such as HESX1, PROP1, POU1F1, LHX3, LHX4, GLI2, and SOX3 have contributed to our understanding of CPHD (Sizonenko et al., 1975; Alatzoglou & Dattani, 2010).

The GH1 gene is located on chromosome 17q22–24 (Millar et al., 2010). A complex map of single nucleotide polymorphisms (SNPs) within the GH1 gene has been identified, encompassing the promoter, coding, and noncoding regions, some of which are implicated in IGHD (Birla et al., 2012; Giordano, 2016; Esteban et al., 2007). Notably, a large deletion within the GH1 gene cluster has been associated with the disease, causing exon 3 skipping and exerting a dominant effect (Mullis, 2005; Bona, 2004). Furthermore, single nucleotide polymorphic sites (SNPs) in the promoter region or intron 4 of the GH1 gene have been shown to influence GH secretion and circulating Insulin-like Growth Factor-I (IGF-I) levels in growth-retarded patients (Keselman et al., 2012; Hasegawa et al., 2000; Horan et al., 2003).

This study aims to investigate sequence variations in exon 4 and its flanking intronic region of the GH1 gene in a sample of Egyptian patients with IGHD and ISS. Our goal is to identify potential biomarkers for the rapid and straightforward diagnosis of GHD.

2. Results

Exon 4 and its flanking intronic region of the GH1 gene were sequenced using PCR technique followed by Sanger sequencing. In this fragment, we detected only the +1150A rs2665802 polymorphism.

2.1 +1150 T/A Genotype Distribution in the Studied Groups

In the IGHD group, the frequency of +1150 T>A genotypes was as follows: T/T homozygous (wild type) 51.52%, T/A (heterozygous mutant type) 30.3%, and A/A (homozygous mutant type) 18.18%. There was no significant difference in the distribution of the three genotypes when compared with each other (p=0.086). However, T/A and A/A genotypes were significant within the IGHD group (P=0.024, 0.00). In the ISS group, the wild genotype (T/T) was not observed, but the T/A and A/A genotypes were found in 15.2% and 12.1% respectively, with no statistical significance (P=0.311). These data are shown in Table 1.

Table 1: +1150 T/A Genotype Distribution in the Studied Groups

2.2 +1150 A Genotype Associated with Short Height

The association between genotypes and standardized height measurements was studied in the two groups. IGHD patients with T/A genotypes were significantly shorter than those with the wild (T/T) genotypes (p=0.03). Consequently, in the IGHD group, the combination genotype (TA+AA) was statistically significant (p=0.011). In the ISS group, patients with homozygous mutant genotypes A/A were significantly shorter than those with heterozygous genotypes T/A (p=0.02). Patients with T/A genotype were significantly shorter in the IGHD group compared to the ISS group, while patients with A/A genotype were significantly shorter in the ISS group compared to the other group.

No significant difference between +1150A Genotype, body weight, IQ, and head circumference was found.

2.3 +1150 A Genotype Associated with Low Growth Hormone Levels

The mean level of growth hormone in each studied group was used as a cutoff point to study the relation of +1150 T>A genotypes with growth hormone levels, which showed no significant difference. The mean level of GH was used to classify the ISS group as >13.2 and 5) and severe GH deficiency level (GH.

2.4 Orthologues and Paralogues Sequence Homology Data of +1150 T>A SNP

Support for its potential function comes from the high conservation of the T allele among six primate species. Regarding evolutionary conservation, the g.1150T allele is also highly conserved among three human GH paralogues (CSHL1, GH2, and CSH2 genes). These highly conserved orthologues and paralogues scores strongly suggest the functionality of the g.1150A variant.

2.5 +1150A Variant Effect Prediction: Likely Abolishing Donor Site

Variant effect prediction using Mutation Taster, Ensemble variant effect predictor, and fathmm (Functional Analysis through Hidden Markov Models) (v2.3) revealed a benign polymorphism. In contrast, splicing modification was predicted by an Interactive Splice Site Analysis Tool (Splice Port), Berkeley Drosophila Genome Project (BDGP), and NetGene2 v. 2.4, which showed that the variant abolishes the donor site. By comparing the splicing site prediction scores of the reported alternative three coding isoforms (based on ENST00000342364.8, ENST00000458650.6, and ENST00000351388.8) against the prediction donor score of 456+90T allele, these scores were close to the donor prediction score. Therefore, the splicing prediction scores support that the g.1150A variant likely abolishes the donor site of an unreported alternative GH1 isoform. Prediction of possible intronic regulatory motifs alterations using RegRNA showed no effect.

3. Discussion

The patients in our study presented with proportionate short stature and reduced growth velocity, which are key clinical indicators supporting the diagnosis of growth hormone deficiency (GHD). Additional criteria such as delayed bone maturation and the absence of bone dysplasias and chronic diseases were also observed. Height is influenced by a complex interplay of genetic and environmental factors, with GH secretion playing a partial role (Hasegawa et al., 2000). Diagnosing GHD in childhood requires an integrative approach involving clinical and auxological assessment, supported by biochemical tests and molecular analysis of the GH1 gene. Due to the challenges and invasiveness of these diagnostic assessments, our study did not include a control group. Instead, our aim was to identify a functional variant that could serve as a biomarker for GHD, offering a simpler, quicker, and less invasive diagnostic approach.

The GH1 gene plays a crucial role in human development, growth, and metabolism (Millar et al., 2010). Numerous genetic variations within the human GH1 gene have been reported (Hasegawa et al., 2000; Horan et al., 2003; Adkins, 2005). Single-base pair deletions and nonsense mutations in the signal peptide region can result in the absence of mature GH production, necessitating GH replacement therapy (Mullis, 2010). GH exerts its biological effects through the binding and dimerization of a cell surface-expressed Growth Hormone Receptor (GHR), initiating a signaling cascade leading to increased plasma levels of Insulin-like Growth Factor-I (IGF-I). GH1 gene variation has significant implications in various clinical contexts, particularly in IGHD, which is often caused by heterozygous mutations in GH1 (Millar et al., 2010; Hasegawa et al., 2000; Horan et al., 2003; Kempers et al., 2013; Cabrera-Salcedo et al., 2017).

Intronic single-nucleotide polymorphisms (SNPs), located some distance from splice sites, may not be easily detected unless they induce splicing patterns different from the norm. Some intronic polymorphic variants within the GH1 gene have been associated with susceptibility to diseases (Choi et al., 2008; Giordano et al., 2008). Ragvin et al. (2010) demonstrated that disease-associated intronic SNPs can be linked to long-range regulatory mechanisms involving highly conserved non-coding elements.

Table 2: Distribution of +1150 T/A Genotypes

The association between the +1150A SNP in the GH1 gene and short stature was a central focus of our study. We detected this SNP in both IGHD and ISS groups, and it has been implicated in various diseases in previous studies.

Our findings showed a significant association of heterozygous and homozygous mutant genotypes in the IGHD group, with no distribution of the normal allele in the ISS group, suggesting that this SNP may serve as a biomarker for short stature. Genetic analysis of the GH1 gene in isolated short stature (ISS) cases has previously revealed that several mutations in GH1 can result in a profound height phenotype and poor growth in a significant proportion of ISS patients (Waldman & Chia, 2013).

Our study further revealed that the GH1 SNP +1150A was associated with reduced body height in both the IGHD and ISS groups, which is in agreement with the findings of Hasegawa et al. (2000) and van Heemst et al. (2005). However, Alfred et al. (2012) did not observe associations between +1150A (GH1) and height in both genders.

Hasegawa et al. (2000) reported significantly higher frequencies of the 57T and 1169A alleles in pre-pubertal short children with GH deficiency compared to short children with normal GH secretion and normal adults. They suggested that these differences in height standard deviation could be attributed to polymorphisms in the GH-1 gene. Giordano et al. (2008) also found an association between the -57T allele and an increased risk of isolated GH deficiency. Furthermore, Hasegawa et al. (2000) demonstrated a significant difference in peak GH levels between +1150T and +1150A genotypes, consistent with our results, where we found that the T/A allele is associated with lower GH levels in IGHD.

The association of the +1150A SNP with lower height and lower GH levels suggests that this SNP may be considered as a biomarker for IGHD. To better understand the mechanisms of action of this SNP, we conducted a functional study, which revealed that the +1150T allele had high conserved orthologous and paralogous scores. Additionally, the +1150A allele was predicted to abolish the donor site. Previous studies have also supported the functionality of the polymorphisms investigated in our study. Horan et al. (2003) found that specific haplotypes in the proximal promoter region of the GH1 gene were significantly associated with reduced reporter gene expression, indicating their role in GH1 gene regulation.

A study by Yamamoto et al. (2012) on two siblings with isolated short stature and moderate GH deficiency reported an association between GH deficiency and short stature. They found that certain haplotypes, including the -278T, -57G, +1169A, and +2103T alleles, exhibited impaired GH secretion. Furthermore, Millar et al. (2010) studied the +1150T>A SNP in combination with other SNPs and identified its independent effect on gene expression and GH secretion using GH1 constructs in rat GC (somatotrophic) cells. They proposed that the +1150A allele directly contributes to the reduction in GH1 gene expression and GH secretion, likely due to its close linkage with promoter SNPs.

4. Conclusion

In conclusion, our study has identified a significant association between the +1150A SNP and low GH levels in IGHD, as well as a significant association with short stature in both IGHD and ISS groups. Although the precise mechanisms underlying the effects of the +1150A SNP on GH1 gene expression and GH secretion remain unclear, it represents an example of an intronic functional polymorphism with potential clinical significance.

Further research involving GHD patients with the +1150T>A SNP is recommended to gain a deeper understanding of its role. More data are needed to elucidate tissue-specific GH1 gene regulation and the impact of these polymorphisms on gene expression, ultimately shedding light on their influence on height.