Ana Cláudia Ribeiro
Omneya Magdy Omar
Ebtesam Abdalla
Manuel Carlos Lemos- 1RISE-Health, Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal
- 2Department of Pediatrics, Faculty of Medicine, Alexandria University, Alexandria, Egypt
- 3Department of Human Genetics, Medical Research Institute, Alexandria University, Alexandria, Egypt
Introduction: Growth Hormone (GH), secreted by the anterior pituitary gland, is a key regulator of postnatal growth. Mutations in the GH1 gene can lead to isolated GH deficiency (IGHD), a rare disorder characterized by growth failure and severe short stature. The aim of this study was to identify the genetic basis of IGHD in three siblings born to consanguineous parents.
Methods: Three siblings were diagnosed with short stature due to GH deficiency (stimulated GH peak levels between 0.07 and 0.77 µg/L). To identify their genetic cause, whole-exome sequencing (WES), multiplex ligation-dependent probe amplification (MLPA), and targeted GH1 sequencing was performed.
Results: A shared homozygous GH1 haplotype comprising nine single nucleotide polymorphisms (SNPs), spanning the promoter, coding, and 3’ flanking regions, was revealed. The parents were heterozygous carriers of this haplotype. This rare SNP combination (with less than 1% population frequency) overlaps with transcriptional regulatory elements and has previously been associated with significantly reduced promoter activity (58% promoter activation relative to wild-type). No pathogenic coding mutations or deletions were identified.
Conclusion: Our findings suggest that this haplotype likely underlies the GH deficiency observed in the affected siblings. This represents the first report linking a homozygous GH1 promoter haplotype to IGHD, underscoring the role of noncoding variants in endocrine disease.
Introduction
Growth Hormone (GH) plays a fundamental role in human development, growth, and metabolism (1, 2). Its production and secretion by the anterior pituitary gland are highly regulated and complex processes; therefore, any disruption can result in GH deficiency (3). In children, congenital GH deficiency typically presents as decelerated linear growth and is frequently classified as idiopathic (2–4). However, some patients have a genetic cause for their disorder, due to mutations in one of several genes known to cause isolated (IGHD) or combined pituitary hormone deficiency (5, 6).
GH is encoded by the GH1 gene, located on chromosome 17q23.3, within a cluster of five related genes (7). Its expression is regulated by upstream enhancer elements and a promoter region containing multiple transcription factor binding sites (8). The GH1 regulatory and coding regions are highly polymorphic, with over 16 single nucleotide polymorphisms (SNPs) that can form haplotypes influencing transcriptional activity (9, 10). These polymorphisms span the promoter, coding, and noncoding regions, complicating the genetic architecture of GH1 (11).
Mutations in the GH1 coding sequence and whole gene deletions are well-established causes of inherited forms of IGHD, which typically occur without deficiencies of other pituitary hormones (5, 6). Despite extensive studies of GH1 mutations, the cumulative effect of multiple promoter variants has not been linked to monogenic IGHD. Indeed, non-coding regulatory SNPs are often under-investigated, as their precise effect is difficult to determine, requiring extensive functional analysis, which is often not feasible in clinical routine (12). The aim of our study was to determine the genetic cause of IGHD in three siblings born to consanguineous parents.
Material and methods
Patients
We studied five individuals from a consanguineous Egyptian family, including three siblings diagnosed with IGHD and their unaffected parents, who were first cousins. The siblings presented normal motor and cognitive development but exhibited growth retardation and short stature. GH stimulation testing using clonidine and levodopa confirmed GH deficiency in the siblings (Table 1). Other pituitary hormone levels were normal, and brain magnetic resonance imaging (MRI) revealed no structural abnormalities. The two older siblings responded positively to recombinant GH therapy. Clinical data are summarized in Table 1.
Table 1. Clinical characteristics of the affected siblings.
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Faculty of Health Sciences, University of Beira Interior (Ref: CE-FCS-2012-012). Informed consent was obtained from all subjects involved in this research.
Genetic analysis
Genomic deoxyribonucleic acid (DNA) was extracted from peripheral blood leucocytes using previously described methods (13) and used with polymerase chain reaction (PCR) primers to amplify the GH1 promoter and coding regions (NM_000515.5) (primer sequences and PCR conditions available upon request). Bidirectional sequencing of the PCR products was performed using a semi-automated capillary DNA sequencer (STAB VIDA, Caparica, Portugal; ABI 3730XL, Applied Biosystems; Thermo Fisher Scientific, Waltham, MA, USA). In order to exclude other genetic causes of GH deficiency, we applied additional steps of multiplex ligation-dependent probe amplification (MLPA) and whole-exome sequencing (WES). Specifically, in sibling 2, we analysed copy number variations, through an MLPA assay, using the SALSA MLPA probemix P216-C1 (MRC Holland, Amsterdam, Netherlands), according to the manufacturer’s guidelines. This kit includes probes for GH1, as well as for other GH deficiency-related genes (POU1F1, PROP1, GHRHR, LHX3, LHX4, and HESX1). Fragment analysis was performed with the DNA sequencer described above. In addition, WES was carried out on siblings 1 and 2, as previously described (14). Genetic sequence variants were filtered according to the following cumulative criteria: i) found in both siblings; ii) non-synonymous or located within ten intronic nucleotides adjacent to coding exons; iii) absent or rare (maximum allele frequency < 0.01) in the Genome Aggregation Database (v.4.1.0) (15); and iv) located in genes associated with GH deficiency in the Online Mendelian Inheritance in Man database (16). Variants were classified according to American College of Medical Genetics and Genomics/Association for Molecular Pathology criteria guidelines (17).
Results
Based on the suspicion of autosomal recessive inheritance due to the parental consanguinity, we initially analysed the GH1 gene in the three siblings. No pathogenic mutations were identified in the coding regions or canonical splice sites (Figure 1). However, all three siblings were homozygous for a shared set of nine previously reported SNPs across the GH1 locus: −278T, −123C, −75G, −57G, −6G, +59G, +69G, +1169A, and +2103T, as numbered according to Horan et al. (10) (Figure 2). The population frequency of this haplotype has been estimated to be <1% (10). Both parents were heterozygous for this haplotype. Additional analyses, including MLPA and WES, did not reveal any other potentially pathogenic variants associated with GH deficiency (Figure 1).
Figure 1. Flowchart of the genetic testing of a family with isolated growth hormone deficiency (IGHD). To identify the genetic cause in the siblings, targeted GH1 sequencing, multiplex ligation-dependent probe amplification (MLPA) and whole-exome sequencing (WES) was performed. No pathogenic coding mutations, duplications or deletions were identified. The three siblings were homozygous for a shared set of polymorphisms across the GH1 locus, and both parents were heterozygous for this haplotype. UTRs, untranslated regions; OMIM, Online Mendelian Inheritance in Man; GH, Growth Hormone; ACMG/AMP, American College of Medical Genetics and Genomics and Association for Molecular Pathology.
Figure 2. Genetic analysis of GH1 in a family with isolated growth hormone deficiency. (A) Schematic representation of the GH1 gene showing the positions of 20 known single nucleotide polymorphisms (SNPs) from –476 to +2103, numbered relative to the transcriptional start site, as described by Horan et al., 2003 (10). SNPs identified in the three affected siblings are highlighted in red. Exons are depicted as grey boxes. Coloured boxes indicate transcription factor binding sites: nuclear factor 1(NF1, in green), pituitary transcription factor 1 (PIT1, in blue), vitamin D receptor (VDR, in brown), and the TATA box (orange). (B) Sequence chromatograms of the nine SNP positions for all five family members. Asterisks (*) indicate the position of each SNP. The three affected siblings were homozygous for the rare haplotype, while both parents were heterozygous, consistent with autosomal recessive inheritance.
Discussion
Previous studies have demonstrated that polymorphisms in the GH1 promoter can modulate gene expression and influence circulating GH levels (8, 10, 11, 18–23). To date, no homozygous haplotype has been clearly implicated in monogenic IGHD. In this study, we report three siblings with IGHD who share a homozygous GH1 haplotype consisting of nine SNPs, distributed from the promoter to the 3′ flanking region.
Five of the SNPs (−278T, −75G, −57G, −6G, and +59G) comprise the promoter haplotype 21 described by Horan et al. (10). These authors identified 36 different GH1 haplotypes from 15 SNPs (10, 24, 25) in a control population. Haplotype 21 had a population frequency of <1% and demonstrated only 58% promoter activation relative to wild-type in functional assays (10). These SNPs are located in regions essential for GH1 transcriptional regulation (22). The -278T is located within the nuclear factor 1 (NF1) binding site, the -75G in the proximal pituitary transcription factor 1 (PIT1) binding site, the -57G in the vitamin D receptor (VDR) response element region, and the -6G at the transcriptional start site (22). Giordano et al. (19) specifically studied the -75G polymorphism and found reduced GH1 transcription due to decreased PIT1 binding affinity. However, GH stimulation studies in individuals carrying this variant did not confirm a functional effect, likely due to insufficient homozygous sample size (19). These findings support the hypothesis that cumulative effects of multiple regulatory SNPs may reduce GH1 transcription. Indeed, the impaired binding of essential transcription factors, caused by these SNPs, can weaken the promoter’s ability to recruit the necessary machinery for transcription (22).
Our patients also carried four additional SNPs (-123C, +69G, +1169A, and +2103T). The −123C SNP, located in a conserved Specificity Protein 1 (SP1) and PIT1 binding site, has been shown to have no significant impact on transcription in luciferase reporter assays (18, 23). The +69G SNP (p.Thr3Ala) is a missense variant located in the GH signal peptide. Though initially reported in GH-deficient patients (26), it is now considered a benign polymorphism (11, 18, 26, 27). Importantly, the intronic +1169A SNP, in cis with −278T and −57G, has been associated with reduced GH secretion and lower insulin-like growth factor 1 (IGF1) levels (28). Functional studies have demonstrated impaired GH1 expression and GH secretion linked to this variant, although the effect may be influenced by adjacent regulatory SNPs (29). The +2103T SNP, located in the 3′ flanking region, has also been implicated in reduced GH secretion when present with −278T, −57G, and +1169A (29). Notably, Yamamoto et al. (30) identified this four-SNP combination in GH-deficient siblings, although their homozygous mother had normal stature, suggesting these variants alone may be insufficient to cause disease.
In our study, the homozygous co-occurrence of all nine variants — many with individually demonstrated or suspected functional impact — strongly suggests a cumulative regulatory effect on GH1 expression. This may explain the phenotype observed in our patients and supports a role for the genetic screening of GH1 promoter haplotypes in cases of IGHD lacking coding mutations.
Limitations of our study include the absence of in vivo functional data confirming reduced GH secretion associated with this haplotype. Such functional assays would be useful to validate our findings. While MLPA and WES excluded known GH deficiency-associated variants, we cannot rule out the involvement of unknown regulatory elements or genes. Nonetheless, the identification of a shared homozygous rare haplotype in three affected siblings provides compelling evidence for its pathogenicity.
In conclusion, we identified a rare homozygous GH1 haplotype comprising nine regulatory and coding SNPs that may collectively impair GH1 expression, representing the likely cause of IGHD in three siblings from a consanguineous family.
Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.
Ethics statement
The studies involving humans were approved by Ethics Committee of the Faculty of Health Sciences, University of Beira Interior (Ref: CE-FCS-2012-012). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin. Written informed consent was obtained from the minor(s)’ legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.
Author contributions
AR: Writing – original draft, Formal analysis, Investigation, Methodology. OO: Writing – review & editing, Resources. EA: Resources, Writing – review & editing. ML: Writing – review & editing, Conceptualization, Funding acquisition, Supervision.
Funding
The author(s) declared that financial support was received for work and/or its publication.This work was funded by the Fundação para a Ciência e a Tecnologia (grant numbers PTDC/SAU-GMG/098419/2008, UIDB/00709/2020, and UI/BD/151021/2021). Open-acess fees were funded by the UBI/CGD Research Project Funding Support Program.
Conflict of interest
The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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The author(s) declared that generative AI was not used in the creation of this manuscript.
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References
1. Moller N and Jorgensen JO. Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects. Endocr Rev. (2009) 30:152–77. doi: 10.1210/er.2008-0027
2. Ranke MB and Wit JM. Growth hormone - past, present and future. Nat Rev Endocrinol. (2018) 14:285–300. doi: 10.1038/nrendo.2018.22
3. Di Iorgi N, Morana G, Allegri AE, Napoli F, Gastaldi R, Calcagno A, et al. Classical and non-classical causes of GH deficiency in the paediatric age. Best Pract Res Clin Endocrinol Metab. (2016) 30:705–36. doi: 10.1016/j.beem.2016.11.008
4. Alatzoglou KS, Webb EA, Le Tissier P, and Dattani MT. Isolated growth hormone deficiency (GHD) in childhood and adolescence: recent advances. Endocr Rev. (2014) 35:376–432. doi: 10.1210/er.2013-1067
5. Giordano M. Genetic causes of isolated and combined pituitary hormone deficiency. Best Pract Res Clin Endocrinol Metab. (2016) 30:679–91. doi: 10.1016/j.beem.2016.09.005
6. Vasques GA, Andrade NLM, Correa FA, and Jorge AAL. Update on new GH-IGF axis genetic defects. Arch Endocrinol Metab. (2019) 63:608–17. doi: 10.20945/2359-3997000000191
7. Barsh GS, Seeburg PH, and Gelinas RE. The human growth hormone gene family: structure and evolution of the chromosomal locus. Nucleic Acids Res. (1983) 11:3939–58. doi: 10.1093/nar/11.12.3939
8. Birla S, Jyotsana PV, Sharma A, Khadgawat R, Garg M, and Jain V. Genetic characterization of growth hormone 1 gene in patients with isolated growth hormone deficiency. Indian J Endocrinol Metab. (2012) 16:S310–2. doi: 10.4103/2230-8210.104071
9. Dennison EM, Syddall HE, Rodriguez S, Voropanov A, Day IN, Cooper C, et al. Polymorphism in the growth hormone gene, weight in infancy, and adult bone mass. J Clin Endocrinol Metab. (2004) 89:4898–903. doi: 10.1210/jc.2004-0151
10. Horan M, Millar DS, Hedderich J, Lewis G, Newsway V, Mo N, et al. Human growth hormone 1 (GH1) gene expression: complex haplotype-dependent influence of polymorphic variation in the proximal promoter and locus control region. Hum Mutat. (2003) 21:408–23. doi: 10.1002/humu.10167
11. Esteban C, Audi L, Carrascosa A, Fernandez-Cancio M, Perez-Arroyo A, Ulied A, et al. Human growth hormone (GH1) gene polymorphism map in a normal-statured adult population. Clin Endocrinol (Oxf). (2007) 66:258–68. doi: 10.1111/j.1365-2265.2006.02718.x
12. Ellingford JM, Ahn JW, Bagnall RD, Baralle D, Barton S, Campbell C, et al. Recommendations for clinical interpretation of variants found in non-coding regions of the genome. Genome Med. (2022) 14:73. doi: 10.1186/s13073-022-01073-3
13. Miller SA, Dykes DD, and Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. (1988) 16:1215. doi: 10.1093/nar/16.3.1215
14. Goncalves CI, Carrico J, Bastos M, and Lemos MC. Disorder of sex development due to 17-beta-hydroxysteroid dehydrogenase type 3 deficiency: A case report and review of 70 different HSD17B3 mutations reported in 239 patients. Int J Mol Sci. (2022) 23:10026. doi: 10.3390/ijms231710026
15. Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alfoldi J, Wang Q, et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature. (2020) 581:434–43. doi: 10.1038/s41586-020-2308-7
16. Amberger JS and Hamosh A. Searching online Mendelian inheritance in man (OMIM): A knowledgebase of human genes and genetic phenotypes. Curr Protoc Bioinf. (2017) 58:121–212. doi: 10.1002/cpbi.27
17. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. (2015) 17:405–24. doi: 10.1038/gim.2015.30
18. Millar DS, Lewis MD, Horan M, Newsway V, Easter TE, Gregory JW, et al. Novel mutations of the growth hormone 1 (GH1) gene disclosed by modulation of the clinical selection criteria for individuals with short stature. Hum Mutat. (2003) 21:424–40. doi: 10.1002/humu.10168
19. Giordano M, Godi M, Giacopelli F, Lessi M, Mellone S, Paracchini R, et al. A variation in a Pit-1 site in the growth hormone gene (GH1) promoter induces a differential transcriptional activity. Mol Cell Endocrinol. (2006) 249:51–7. doi: 10.1016/j.mce.2006.01.006
20. Giordano M, Godi M, Mellone S, Petri A, Vivenza D, Tiradani L, et al. A functional common polymorphism in the vitamin D-responsive element of the GH1 promoter contributes to isolated growth hormone deficiency. J Clin Endocrinol Metab. (2008) 93:1005–12. doi: 10.1210/jc.2007-1918
21. Rojas-Gil AP, Ziros PG, Kanetsis E, Papathanassopoulou V, Nikolakopoulou NM, He K, et al. Combined effect of mutations of the GH1 gene and its proximal promoter region in a child with growth hormone neurosecretory dysfunction (GHND). J Mol Med (Berl). (2007) 85:1005–13. doi: 10.1007/s00109-007-0200-7
22. de Graaff LC, Argente J, van Meurs JB, Uitterlinden AG, and Hokken-Koelega AC. Genetic polymorphisms in the locus control region and promoter of GH1 are related to serum IGF-I levels and height in patients with isolated growth hormone deficiency and healthy controls. Horm Res Paediatr. (2010) 73:25–34. doi: 10.1159/000271913
23. Madeira JL, Jorge AA, Martin RM, Montenegro LR, Franca MM, Costalonga EF, et al. A homozygous point mutation in the GH1 promoter (c.-223C>T) leads to reduced GH1 expression in siblings with isolated GH deficiency (IGHD). Eur J Endocrinol. (2016) 175:K7–K15. doi: 10.1530/EJE-15-0149
24. Giordano M, Marchetti C, Chiorboli E, Bona G, and Momigliano Richiardi P. Evidence for gene conversion in the generation of extensive polymorphism in the promoter of the growth hormone gene. Hum Genet. (1997) 100:249–55. doi: 10.1007/s004390050500
25. Wagner JK, Eble A, Cogan JD, Prince MA, Phillips JA 3rd, and Mullis PE. Allelic variations in the human growth hormone-1 gene promoter of growth hormone-deficient patients and normal controls. Eur J Endocrinol. (1997) 137:474–81. doi: 10.1530/eje.0.1370474
26. Miyata I, Cogan JD, Prince MA, Kamijo T, Ogawa M, and Phillips JA 3rd. Detection of growth hormone gene defects by dideoxy fingerprinting (ddF). Endocr J. (1997) 44:149–54. doi: 10.1507/endocrj.44.149
27. Millar DS, Lewis MD, Horan M, Newsway V, Rees DA, Easter TE, et al. Growth hormone (GH1) gene variation and the growth hormone receptor (GHR) exon 3 deletion polymorphism in a West-African population. Mol Cell Endocrinol. (2008) 296:18–25. doi: 10.1016/j.mce.2008.09.023
28. Hasegawa Y, Fujii K, Yamada M, Igarashi Y, Tachibana K, Tanaka T, et al. Identification of novel human GH-1 gene polymorphisms that are associated with growth hormone secretion and height. J Clin Endocrinol Metab. (2000) 85:1290–5. doi: 10.1210/jcem.85.3.6468
29. Millar DS, Horan M, Chuzhanova NA, and Cooper DN. Characterisation of a functional intronic polymorphism in the human growth hormone (GH1) gene. Hum Genomics. (2010) 4:289–301. doi: 10.1186/1479-7364-4-5-289
Keywords: growth hormone deficiency, GH deficiency, IGHD, GH1, haplotype, promoter polymorphism, transcriptional regulation
Citation: Ribeiro AC, Omar OM, Abdalla E and Lemos MC (2026) Growth hormone deficiency in three siblings homozygous for a rare GH1 haplotype. Front. Endocrinol. 16:1704842. doi: 10.3389/fendo.2025.1704842
Received: 14 September 2025; Accepted: 08 December 2025; Revised: 06 November 2025;
Published: 05 January 2026.
Edited by:
Zhaoxiang Liu, Beijing Tsinghua Changgung Hospital, ChinaReviewed by:
Rana Muhammad Kamran Shabbir, Quaid-i-Azam University, PakistanSara Mumtaz, National University of Medical Sciences (NUMS), Pakistan
Copyright © 2026 Ribeiro, Omar, Abdalla and Lemos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Manuel Carlos Lemos, bWNsZW1vc0BmY3NhdWRlLnViaS5wdA==
†ORCID: Manuel Carlos Lemos, orcid.org/0000-0001-9326-8900, Ana Cláudia Ribeiro, orcid.org/0000-0002-3264-4156, Omneya Magdy Omar, orcid.org/0000-0003-1967-8893, Ebtesam Abdalla, orcid.org/0000-0002-2486-8782, Omneya Magdy Omar, orcid.org/0000-0003-1967-8893