Gigantism: X-linked acrogigantism and GPR101 mutations

    Gigantism: X-linked acrogigantism and GPR101 mutations Donato Iacovazzo, M´arta Korbonits PII: DOI: Reference:

S1096-6374(16)30058-2 doi:10.1016/j.ghir.2016.09.007 YGHIR 1151

To appear in:

Growth Hormone & IGF Research

Received date: Revised date: Accepted date:

28 August 2016 24 September 2016 28 September 2016

Please cite this article as: Donato Iacovazzo, M´arta Korbonits, Gigantism: Xlinked acrogigantism and GPR101 mutations, Growth Hormone & IGF Research (2016), doi:10.1016/j.ghir.2016.09.007

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ACCEPTED MANUSCRIPT Gigantism: X-linked acrogigantism and GPR101 mutations Donato Iacovazzo1 and Márta Korbonits1 1

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Centre for Endocrinology, Barts and The London School of Medicine, Queen Mary University of London, London, EC1M 6BQ, UK

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Keywords: GPR101, XLAG, gigantism, pituitary, mutation

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Word count: 2545

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Number of figures: 2 Number of tables: 1

Corresponding author and person to whom reprint requests should be addressed: Márta Korbonits, MD, PhD Professor of Endocrinology and Metabolism. Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary University of London. Charterhouse square, London EC1M 6BQ, UK. Tel:

+44

20

7882

8284

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[email protected] 1

ACCEPTED MANUSCRIPT Abstract X-linked acrogigantism (XLAG) is a recently identified condition of early-onset GH excess resulting

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from the germline or somatic duplication of the GPR101 gene on chromosome Xq26.3. Thirty patients

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have been formally reported so far. The disease affects mostly females, occurs usually sporadically, and is characterised by early onset and marked overgrowth. Most patients present with concomitant

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hyperprolactinaemia. Histopathology shows pituitary hyperplasia or pituitary adenoma with or without

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associated hyperplasia. XLAG-related pituitary adenomas present peculiar histopathological features that should contribute to raise the suspicion of this rare condition. Treatment is frequently challenging

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and multi-modal. While females present with germline mutations, the sporadic male patients reported so far were somatic mosaics with variable levels of mosaicism, although no differences in the clinical

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phenotype were observed between patients with germline or somatic duplication. The GPR101 gene

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encodes an orphan G protein-coupled receptor normally expressed in the central nervous system, and at

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particularly high levels in the hypothalamus. While the physiological function and the endogenous ligand of GPR101 are unknown, the high expression of GPR101 in the arcuate nucleus and the occurrence of increased circulating GHRH levels in some patients with XLAG, suggest that increased hypothalamic GHRH secretion could play a role in the pathogenesis of this condition. In this review, we summarise the published evidence on XLAG and GPR101 and discuss the results of recent studies that have investigated the potential role of GPR101 variants in the pathogenesis of pituitary adenomas.

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ACCEPTED MANUSCRIPT Abbreviations X-linked acrogigantism, XLAG

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G protein-coupled receptor, GPCR

Fork

stalling

and

template

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Growth hormone releasing hormone receptor, GHRHR

switching/microhomology-mediated

replication,

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FoSTeS/MMBIR

break-induced

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Comparative genomic hybridization array, aCGH

Droplet digital PCR, ddPCR Exome Aggregation Consortium, ExAC

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cAMP response element, CRE

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G inhibitory protein, Gi

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G stimulatory protein, Gs

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High-density comparative genomic hybridization array, HD-aCGH

Epidermal growth factor, EGF

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ACCEPTED MANUSCRIPT Introduction X-linked acrogigantism (XLAG) is a recently identified condition of early-onset pituitary gigantism

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due to pituitary hyperplasia or mixed somatotroph/lactotroph adenomas [1-3]. The initially published

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patients harboured microduplications of Xq26.3 spanning an area of approximately 500kb encompassing four genes (CD40LG, ARHGEF6, RBMX and GPR101) [1, 2]. Among these, only one,

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GPR101, encoding an orphan G protein-coupled receptor (GPCR), was found to be significantly

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overexpressed in these patients’ pituitary samples. More recently, we have described one patient with a typical phenotype whose microduplication allowed to define a new smallest region of overlap of

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duplications to an area encompassing GPR101 only [3], while the other three genes involved in

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previously published patients were not duplicated, thus proving the causative role of GPR101.

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Clinical features

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In two recently published large series of patients with pituitary gigantism, XLAG accounted for 7.8% and 10% of all cases respectively [3, 4]. These patients had been screened for mutations in the AIP gene as well, allowing a comparison of the clinical features of XLAG patients with AIP mutation carriers and patients without an identified genetic predisposing mutation. XLAG patients were predominantly females, were significantly younger and presented with higher height Z-scores at diagnosis compared with AIP mutation carriers and genetically negative patients [3, 4]. Moreover, in one of these studies, XLAG patients had a lower rate of pituitary adenoma invasion and extension compared with the two other groups of patients [4]. There was no statistically significant difference in tumour size, although none of the XLAG patients presented with giant adenomas, as opposed to 1129% of patients in the other two groups [3, 4]. Hyperprolactinaemia was significantly more prevalent among XLAG patients [3, 4].

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ACCEPTED MANUSCRIPT To date, 30 XLAG patients have been described [1-3, 5-7]. Available clinical data from most patients are summarised in Table 1. The clinical phenotype of these patients is remarkably distinct. In all cases,

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disease onset was before the age of four. Most affected subjects were females (23/30, 76.7%), and the

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disease occurred mostly sporadically, with only two families reported so far [1]. Overgrowth was the most common clinical sign, followed by acral enlargement and coarse facial features [2, 3]. Increased

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appetite was reported in approximately a third of the patients [2, 3], and BMI was also frequently

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increased at diagnosis. All patients had marked hypersecretion of GH. Among patients with available data, none showed suppression of GH levels following the oral glucose tolerance test [2, 3]. At least six

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patients presented a paradoxical rise following the glucose load [2, 3], while a paradoxical rise of GH levels in response to TRH was reported in two cases [6, 8]. Hyperprolactinaemia at diagnosis was

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present in all patients with available data except two, whose prolactin levels were repeatedly normal

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during follow-up [3]. A subset of patients had their circulating GHRH levels measured, and they were

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found to be in the reference range or slightly raised, although in no case they reached levels normally observed in cases of ectopic secretion of GHRH [1-3]. Most patients harboured a pituitary adenoma, while five presented with diffuse hyperplasia without radiological or histopathological evidence of an adenoma [1-3, 5, 6]. With the exemption of one case [2], all adenoma patients presented with macroadenomas, and one patient was found with a giant tumour (maximum diameter over 40mm) after being lost to follow-up [5]. In three patients, adenomas were accompanied by surrounding hyperplasia, suggesting a progression from diffuse hyperplasia to the development of pituitary tumours [1, 2, 6]. Treatment was frequently multi-modal, requiring, in most cases, a combination of surgery, medical treatment and radiotherapy (Table 1) [2, 3]. Despite a widespread expression of somatostatin receptors [2, 3], somatostatin analogues were generally poorly effective. The GH receptor antagonist pegvisomant was effective in controlling the disease in most cases for whom it has been employed [2, 3]. As a result of extensive treatment, permanent hypopituitarism was common and occurred in at least 5

ACCEPTED MANUSCRIPT 21/30 patients [2, 3, 5, 6]. In the absence of prompt and effective treatment, the disease can lead to markedly high stature (Figure 1). The oldest patient in our series was 50 year-old at the time of the last

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follow-up, and has not developed any other disease manifestation [3].

Histopathology

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Among patients with hyperplasia, histopathology showed marked expansion of somatotrophs and

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lactotrophs, together with a large population of mammosomatotrophs, cells expressing both GH and prolactin [7, 8]. XLAG-related adenomas were remarkably similar in their features. They presented a

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rather lobular architecture, pseudo-follicles containing colloid-like material, and calcifications were frequently seen [3]. Densely granulated somatotroph cells represented the main cell population in

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XLAG adenomas. A smaller population was represented by chromophobe cells, either expressing

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prolactin or containing discernible fibrous bodies (intracytoplasmic globular aggregates of cytokeratin)

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representing sparsely granulated somatotrophs [3]. The presence of both densely and sparsely granulated somatotroph cells in mixed somatotroph/lactotroph adenomas is a rare finding and had not been formally described before. This set of peculiar histopathological features should raise the suspicion of XLAG, especially in very young-onset patients with GH excess. Most XLAG-related pituitary tumours had a low proliferation index, estimated by a Ki-67 labelling index lower than 3% [2, 3]. Only two patients, an 11 year-old boy with a large and invasive tumour treated with a significant delay from the onset of first symptoms [5], and a 3 year-old girl with typical phenotype [6], presented with atypical adenomas, with a Ki-67 labelling index of 3.5% and 5% respectively.

Genetics All XLAG patients harbour submicroscopic duplications at chromosome Xq26.3, and the smallest region of overlap between these duplications encompasses the GPR101 gene [1, 3]. These duplications 6

ACCEPTED MANUSCRIPT have unique boundaries, and their features are in keeping, in most cases, with fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR) as the underlying

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mechanism [1, 3]. In one patient, the duplication was generated through an Alu-Alu mediated

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rearrangement [3]. Interestingly, while females carried constitutional mutations, the four sporadic male patients were found to be somatic mosaics with variable levels of mosaicism, meaning that the

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mutation occurred in these patients post-zygotically [3, 7, 9]. The clinical features of these patients

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were not dissimilar from those carrying germline duplications [3, 7, 9]. Among these four patients, analysis of blood-derived DNA allowed the diagnosis of XLAG in three of them, while it showed a

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normal copy number for GPR101 in one case [7], most likely as a result of a low level of mosaicism in the blood cells. In this patient, duplication of GPR101 was in fact identified in DNA isolated from the

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hyperplastic pituitary tissue and in other tissues as well, confirming the somatic mosaicism [7]. This

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case highlights the need to analyse DNA samples isolated from tissues different from blood in male

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patients whose clinical phenotype suggests XLAG but leukocyte-derived DNA tests negative for GPR101 duplication.

While in most cases the duplication spanned an area of approximately 500kb, one patient presented with a complex genomic rearrangement with a proximal duplication followed by a normal-copy segment and a distal duplication encompassing GPR101 only and measuring approximately 200kb (Figure 2) [3]. This patient’s copy number variation was missed by conventional genome-wide comparative genomic hybridization array (aCGH), showing that alternative techniques need to be used to reliably identify all XLAG patients. In order to overcome these limitations, a high-density aCGH (HD-aCGH) was designed for the Xq26.3 region [1]. However, this method is not suitable for the screening of high numbers of patients. Thus, two studies have employed a quantitative droplet digital PCR (ddPCR) to screen large groups of patients with gigantism or acromegaly for GPR101 duplications [3, 9]. This method can reliably identify copy number variation abnormalities, and can 7

ACCEPTED MANUSCRIPT detect low levels of mosaicism with high sensitivity [3, 9, 10], leaving the HD-aCGH for verification of

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the rare cases with indeterminate results.

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GPR101 variants in pituitary adenomas

In the original publication by Trivellin et al. [1], the authors studied the prevalence of GPR101

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sequence variants among patients with acromegaly. The missense GPR101 variant c.924G>C

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(p.E308D) was identified in 4% of a series of 248 patients. When GPR101 with this specific variant was overexpressed in rat pituitary GH3 cells, it led to increased cell proliferation and GH release as

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compared with wild type GPR101, suggesting a pathogenic role [1]. The prevalence of GPR101 variants in patients with acromegaly has been investigated in further studies. In a large series of almost

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600 patients with either sporadic or familial acromegaly [3], the germline c.924G>C (p.E308D)

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GPR101 variant was only found in four patients (0.69%), and the allele frequency was not significantly

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different from what has been reported in the Exome Aggregation Consortium (ExAC) database (0.39%). No other rare or novel variants were identified, either at the germline or somatic level. Lecoq et al. sequenced the entire coding region of GPR101 in a series of patients with sporadic pituitary adenomas of all subtypes in constitutional DNA samples [11]. Six patients were found to carry the c.924G>C (p.E308D) variant (0.8%). Six other rare or novel variants (four missense and two synonymous) were also identified at a low frequency (one patient each). Ferraù et al. have looked at the frequency of the germline c.924G>C (p.E308D) GPR101 variant in a cohort of 215 mostly sporadic acromegaly patients [12], and the variant was not identified in any of the patients. Similarly, the prevalence of the p.E308D variant was investigated in 61 Japanese patients with acromegaly, but no carriers were found [13]. GPR101 was also sequenced in a series of 36 paediatric corticotrophinoma patients, revealing a rare heterozygous variant (p.G31S) in one patient [14]. However, in vitro studies did not seem to support a pathogenic role. Thus, the prevalence of GPR101 variants seems very low, 8

ACCEPTED MANUSCRIPT suggesting that these variants do not play a significant role in the pathogenesis of acromegaly or other pituitary tumours.

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Among 41 patients with isolated idiopathic GH deficiency, one novel heterozygous GPR101 variant

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was identified (c.589G>T, p.V197L) [15]. Functional studies did not show significant differences with the wild type in terms of GH secretion or cAMP response. No further mutations were found, including

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copy number variations, meaning that GPR101 mutations do not seem to be frequently involved in the

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GPR101: biological and molecular aspects

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pathogenesis of GH deficiency.

GPR101 encodes a GPCR whose physiological function and endogenous ligand are unknown. The

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human gene was cloned in 2001 [16]. GPR101 shares transmembrane identity of approximately 30%

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with another orphan GPCR, GPR161, and adrenergic and serotonin receptors [16].

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In mice, GPR101 is predominantly expressed in the hypothalamus [17], particularly in the arcuate neurons, where, among others, GHRH neurons are localised. In the human brain, the highest GPR101 mRNA levels were found in the nucleus accumbens [18]. Expression of GPR101 was found in the anterior pituitary of the rhesus monkey and in the rat pituitary gland [18]. During human development, GPR101 appears in the foetal pituitary approximately at the gestational age of 19 weeks, and its levels steadily increased up to 38 weeks, when over 65% of the anterior pituitary cells stained positive for GPR101 [18]. However, GPR101 was absent or expressed at very low levels in the human adult pituitary [1]. In silico prediction indicates that GPR101 is coupled to the G stimulatory protein (Gs) [17], and this prediction is supported by the elevation of cAMP levels and activation of a cAMP response element (CRE) luciferase reporter in HEK293 cells overexpressing human GPR101 [17], as well as in GH3

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ACCEPTED MANUSCRIPT cells [1]. However, inhibition of forskolin-stimulated CRE reporter activity has been recently reported for GPR101, suggesting possible coupling with the G inhibitory protein (Gi) as well [19].

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In mice, food deprivation resulted in increased GPR101 mRNA expression in the posterior

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hypothalamus, while in ob/ob mice the expression was reduced [20]. However, intraperitoneal injection of leptin did not alter GPR101 expression in ob/ob mice, suggesting that the downregulation of

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GPR101 is regulated by factors other than leptin [20]. Moreover, GPR101 was found to be expressed in

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a significant proportion of neurons expressing the anorexigenic peptide POMC [20], suggesting a role for GPR101 in controlling energy homeostasis. Increased GH secretion occurs in humans in response

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to fasting [21], while GH secretion is impaired in obesity, and partially improved following weight reduction [22]. One could speculate whether GPR101 could contribute to determine the different GH

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secretion patterns observed in response to changes in energy status.

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A possible role for GPR101 in mediating cancer progression has also been proposed. In a group of 77

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colorectal cancer patients, methylation of the GPR101 promoter was found in 40% of cases, and correlated with a longer time to disease progression [23]. Moreover, in the Ishikawa endometrial cancer cell line, GPR101 was found to mediate the release of the epidermal growth factor (EGF) and the consequent cell migration and invasion in response to GnRH[1-5], a GnRH metabolite [24, 25]. Whether this putative ligand might play a role at the pituitary level remains uncertain, as in vitro treatment of pituitary cells from an XLAG patient with GnRH[1-5] did not significantly affect the release of GH or prolactin [6]. The pathogenesis of GH excess in XLAG is not understood. The high expression of GPR101 in the hypothalamus and the finding of increased circulating GHRH levels in some patients with XLAG [1, 2, 6] and in other cases with a phenotype closely resembling XLAG [26, 27], suggests that upregulation of hypothalamic GHRH could play a role in the pathogenesis of this condition. Notably, mammosomatotroph hyperplasia occurs in mice transgenic for GHRH [28], and these animals develop 10

ACCEPTED MANUSCRIPT somatotroph adenomas later in life [29]. However, adenomatous transformation is not normally seen in patients with acromegaly due to the ectopic secretion of GHRH [30]. We could speculate that the early

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exposure to increased GHRH levels, possibly starting during foetal life as a result of duplication of the

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dosage-sensitive GPR101 gene, could induce pituitary hyperplasia, and the subsequent adenomatous transformation observed in most XLAG patients. Another factor corroborating the potential role of

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GHRH in the pathogenesis of XLAG is the upregulation of the GHRH receptor (GHRHR) in these

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patients’ pituitary samples [1, 2, 5]. Downregulation of GHRHR has been shown in rats treated with anti-GHRH antibodies [31], suggesting that GHRH is necessary for the expression of its own receptor,

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and that the high levels of GHRHR seen in the XLAG patients could result from increased hypothalamic GHRH secretion. The presence of functional GHRHR is also confirmed by the in vitro

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inhibitory effect of a GHRHR antagonist on GH and prolactin secretion in an XLAG patient [6],

Conclusions

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patients.

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showing that inhibition of the GHRH signalling could potentially represent a treatment option for these

XLAG is a novel condition of GH excess characterised by female preponderance and early onset, and accounts for a significant proportion of pituitary gigantism cases, especially among females. Most patients present with associated hyperprolactinaemia, and the disease is due to pituitary hyperplasia and, in most cases, to pituitary adenomas showing peculiar histopathological features. XLAG results from either germline or somatic duplication of GPR101, a gene encoding an orphan GPCR highly expressed in the hypothalamus. While microduplications lead to XLAG, variants in the GPR101 gene are rarely found in patients with acromegaly or other pituitary adenoma subtypes and are unlikely to play a role in pituitary tumorigenesis. Further studies are needed to clarify the mechanisms underlying the pathogenesis of GH excess and the role of GPR101 in the regulation of the GHRH-GH axis. 11

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Acknowledgements

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We kindly acknowledge Dr Bo Yuan and Prof James R. Lupski (Baylor College of Medicine, Houston,

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TX, USA) for providing the figure for one of the patients’ aCGH, and Prof Wouter De Herder

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(Erasmus MC, Rotterdam, the Netherlands) for providing pictures from one of the patients.

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A. Saveanu, N. Jullien, R. Reynaud, A. Barlier, V. Bours, T. Brue, A. Beckers, GPR101 mutations are

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not a frequent cause of congenital isolated growth hormone deficiency, Horm. Metab. Res., 48 (2016) 389-393.

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[16] D.K. Lee, T. Nguyen, K.R. Lynch, R. Cheng, W.B. Vanti, O. Arkhitko, T. Lewis, J.F. Evans, S.R. George, B.F. O'Dowd, Discovery and mapping of ten novel G protein-coupled receptor genes, Gene,

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275 (2001) 83-91.

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[17] B. Bates, L. Zhang, S. Nawoschik, S. Kodangattil, E. Tseng, D. Kopsco, A. Kramer, Q. Shan, N.

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Taylor, J. Johnson, Y. Sun, H.M. Chen, M. Blatcher, J.E. Paulsen, M.H. Pausch, Characterization of Gpr101 expression and G-protein coupling selectivity, Brain Res., 1087 (2006) 1-14. [18] G. Trivellin, I. Bjelobaba, A.F. Daly, D.O. Larco, L. Palmeira, F.R. Faucz, A. Thiry, L.F. Leal, L. Rostomyan, M. Quezado, M.H. Schernthaner-Reiter, M.M. Janjic, C. Villa, T.J. Wu, S.S. Stojilkovic, A. Beckers, B. Feldman, C.A. Stratakis, Characterization of GPR101 transcript structure and expression patterns, J. Mol. Endocrinol., 57 (2016) 97-111. [19] A.L. Martin, M.A. Steurer, R.S. Aronstam, Constitutive activity among orphan class-A G protein coupled receptors, PLoS One, 10 (2015) e0138463. [20] K.N. Nilaweera, D. Ozanne, D. Wilson, J.G. Mercer, P.J. Morgan, P. Barrett, G protein-coupled receptor 101 mRNA expression in the mouse brain: altered expression in the posterior hypothalamus and amygdala by energetic challenges, J. Neuroendocrinol., 19 (2007) 34-45.

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ACCEPTED MANUSCRIPT [21] K.Y. Ho, J.D. Veldhuis, M.L. Johnson, R. Furlanetto, W.S. Evans, K.G. Alberti, M.O. Thorner, Fasting enhances growth hormone secretion and amplifies the complex rhythms of growth hormone

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secretion in man, J. Clin. Invest., 81 (1988) 968-975.

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[22] T. Williams, M. Berelowitz, S.N. Joffe, M.O. Thorner, J. Rivier, W. Vale, L.A. Frohman, Impaired growth hormone responses to growth hormone-releasing factor in obesity. A pituitary defect

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reversed with weight reduction, N. Engl. J. Med., 311 (1984) 1403-1407.

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[23] P. Kober, M. Bujko, J. Oledzki, A. Tysarowski, J.A. Siedlecki, Methyl-CpG binding columnbased identification of nine genes hypermethylated in colorectal cancer, Mol. Carcinog., 50 (2011)

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846-856.

[24] M. Cho-Clark, D.O. Larco, N.N. Semsarzadeh, F. Vasta, S.K. Mani, T.J. Wu, GnRH-(1-5)

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Mol. Endocrinol., 28 (2014) 80-98.

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transactivates EGFR in Ishikawa human endometrial cells via an orphan G protein-coupled receptor,

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[25] M. Cho-Clark, D.O. Larco, B.R. Zahn, S.K. Mani, T.J. Wu, GnRH-(1-5) activates matrix metallopeptidase-9 to release epidermal growth factor and promote cellular invasion, Mol. Cell. Endocrinol., 415 (2015) 114-125.

[26] J.M. Dubuis, C.L. Deal, R.T. Drews, C.G. Goodyer, G. Lagace, S.L. Asa, G. Van Vliet, R. Collu, Mammosomatotroph adenoma causing gigantism in an 8-year old boy: a possible pathogenetic mechanism, Clin. Endocrinol. (Oxf.), 42 (1995) 539-549. [27] D. Zimmerman, W.F. Young, Jr., M.J. Ebersold, B.W. Scheithauer, K. Kovacs, E. Horvath, M.D. Whitaker, N.L. Eberhardt, T.R. Downs, L.A. Frohman, Congenital gigantism due to growth hormonereleasing hormone excess and pituitary hyperplasia with adenomatous transformation, J. Clin. Endocrinol. Metab., 76 (1993) 216-222. [28] L. Stefaneanu, K. Kovacs, E. Horvath, S.L. Asa, N.E. Losinski, N. Billestrup, J. Price, W. Vale, Adenohypophysial changes in mice transgenic for human growth hormone-releasing factor: a 17

ACCEPTED MANUSCRIPT histological, immunocytochemical, and electron microscopic investigation, Endocrinology, 125 (1989) 2710-2718.

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[29] S.L. Asa, K. Kovacs, L. Stefaneanu, E. Horvath, N. Billestrup, C. Gonzalez-Manchon, W. Vale,

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Pituitary adenomas in mice transgenic for growth hormone-releasing hormone, Endocrinology, 131 (1992) 2083-2089.

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[30] F. Borson-Chazot, L. Garby, G. Raverot, F. Claustrat, V. Raverot, G. Sassolas, G.T.E. group,

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Acromegaly induced by ectopic secretion of GHRH: a review 30 years after GHRH discovery, Ann. Endocrinol. (Paris), 73 (2012) 497-502.

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[31] R. Horikawa, P. Hellmann, S.G. Cella, A. Torsello, R.N. Day, E.E. Muller, M.O. Thorner, Growth hormone-releasing factor (GRF) regulates expression of its own receptor, Endocrinology, 137 (1996)

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[32] P.D. Lewis, S. Van Noorden, Pituitary abnormalities in acromegaly, Arch. Pathol., 94 (1972) 119-

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[33] J. Rosenstock, F.H. Doyle, R. Hall, K. Mashiter, G.F. Joplin, Childhood acromegaly successfully treated with interstitial irradiation using yttrium-90, Acta Paediatr. Scand., 71 (1982) 851-855. [34] M. Korbonits, Genetics of acromegaly, Endocr. Rev., (2015) S01-01. Abstract. [35] J.H. Davies, T. Cheetham, Investigation and management of tall stature, Arch. Dis. Child., 99 (2014) 772-777. [36] L.H. Behrens, D.P. Barr, Hyperpituitarism beginning in infancy: the Alton giant, Endocrinology, 16 (1932) 120-128.

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ACCEPTED MANUSCRIPT Table 1. Clinical features of XLAG patients reported in the literature with available data. OGTT, oral glucose tolerance test; ULN, upper limit of normal; SSA, somatostatin analogue; DA, dopamine agonist; PEG-V, pegvisomant; Sx, surgery; GK, gamma-knife; DI, diabetes insipidus; PA,

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pituitary adenoma; PRL, prolactin; RT, radiotherapy (conventional); NA, not available; ND, not determined; ↑, 2-10 xULN; ↑↑, 10-50 xULN;

(original

milial

study ID)

somatic

onset

Age at GH

OGTT

IGF-1

Prolactin Tumour/

diagno

suppression (xULN) (xULN)

hyperplasia

(months) sis

3 (III)

F

Sporadic

Sporadic

Germline 12

Germline 24

4.1

3.8

12

↑↑

↑

↑↑

No

No

Sporadic

Germline 9

1.5

↑

AC

F

3.9

Paradoxical 1.2 rise

4 (IV)

NA

No

↑

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Germline 18

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F

Sporadic

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2 (II)

F

Treatment

Histology

Disease

Hypopituitarism Age at References

controlled (axis)

size (mm)

(years)

1 (I)

SC

Sex Sporadic/fa Germline/ Age at

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Case

RI

↑↑↑, >50 xULN.

2.9

Normal

>10

last followup

Intrasellar yttrium

Mixed cell

implants x3, SSA

adenoma

Yes

Yes (ACTH,

50

TSH, LH)

[3, 32, 33]

(acidophilic/ chromophobic) Diffuse

SSA, DA, PEG-V

No surgery

Yes

No

12

[3]

enlargement

NA

13x12

Sx, GK, SSA, DA

NA

Yes

Yes (TSH, LH) 25

[3]

↑

Diffuse

DA, SSA,

Somatotroph,

Yes

Yes (ACTH,

30

[3, 8]

enlargement hemihypophysecto

lactotroph and

TSH, LH, GH,

my, DA, SSA,

mammosomatotrop

DI)

completion of

h hyperplasia

hypophysectomy 5 (V)

F

Sporadic

Germline 30

5.7

↑

NA

3

↑

12x13x15

Sx

PA, GH+ PRL+

Yes

Yes (DI)

8

[3]

6 (VI)

F

Sporadic

Germline 24

7

↑

No

1.9

↑

16x19x12

Sx, GK, SSA, DA

PA, GH+ PRL+

Yes

No

10

[3]

7 (VII)

F

Sporadic

Germline 21

2.8

↑

NA

5

↑↑

32x13x8

Sx, GK, SSA, DA

PA, GH+ PRL+

Yes

No

12

[3]

19

ACCEPTED MANUSCRIPT 8 (VIII)

M Sporadic

Somatic 48

7

NA

NA

NA

Normal

>10

Sx x3, SSA, DA,

PA, GH+ PRL+

No

Sx, RT, GK, PEG-V M Sporadic

Somatic 24

4.7

↑

No

2

↑↑

15x18x13

SSA, DA, PEG-V

33

[3, 34]

TSH, LH) Somatotroph,

Yes

No

11

[3, 7]

Yes

Yes (ACTH,

6

[3]

12

[2, 3, 35]

15

[1-3]

7

[2]

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9 (IX)

Yes (ACTH,

lactotroph and

10 (X)

F

Sporadic

Germline 15

2.7

↑

Paradoxical 2.9

↑

18x15x9

14 (S2)

M Sporadic

F

Sporadic

Germline 7

Somatic 6

Germline 12

3.5

1.6

4.7

3.3

↑↑↑

↑↑↑

↑↑↑

↑

15 (S3)

F

Sporadic

Germline 18

3.5

↑

16 (S4)

F

Sporadic

Germline 2

1.9

↑↑

No

2.2

NA

3.9

No

ND

↑↑↑

↑↑

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Sporadic

Germline 36

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13 (S1)

F

Sporadic

4.4

4.9

CE

12 (XII)

F

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11 (XI)

↑↑↑

↑

SSA, DA, Sx

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rise

SC

RI

mammosomatotrop

>10

SSA, DA, Sx, RT,

h hyperplasia PA, GH+ PRL+, Ki-67 2% PA, GH+ PRL+

TSH, GH, DI) Yes

DA, PEG-V DA, Sx

TSH, LH) PA, GH+ PRL+

Yes

F

Sporadic

Germline 6

3

↑↑↑

DI) 27

Sx, SSA, DA, PEG- PA, GH+ PRL+, V

15

Yes

Ki-67 3%

Sx, DA, SSA, PEG- PA, GH+ PRL+, V, Sx, SSA

Ki-67 <1%

19 (S8)

F

F

Sporadic

Sporadic

Germline 36

Germline 11

5.3

2.8

↑

↑↑↑

Yes (ACTH, TSH, DI)

Yes

Yes (TSH, DI) 18

[2]

No

2.4

↑↑

17

Sx, DA, SSA, GK

PA, GH+ PRL+

No

No

7.5

[2]

ND

5.2

↑↑

17x8x8

DA, SSA, Sx,

PA, GH+ PRL+

Yes

Yes (ACTH,

3.5

[2, 6]

Paradoxical 3.1

↑↑

39

increase 18 (S7)

Yes (ACTH, TSH, LH, GH,

with hyperplasia 17 (S6)

Yes (ACTH,

NA

NA

Paradoxical 3.3

NA

↑

10

18

DA, Sx, SSA, PEG- PA, GH+ PRL+, V

Ki-67 <1%

Sx, SSA, DA, RT,

PA, GH+ FSH+,

Sx, SSA

Ki-67 <1%

DA, Sx, RT, SSA

Eosinophilic PA

TSH) Yes

Yes (DI)

6

[2]

Yes

Yes (TSH)

30

[2]

No

No

8

[2]

increase

20

ACCEPTED MANUSCRIPT 20 (S9)

F

Sporadic

Germline 3

2.8

↑↑

No

3.4

↑↑

18

DA, Sx

Hyperplasia

Yes

Yes (ACTH,

11

[2]

12

[2, 5]

35

[2]

TSH, LH, GH,

21 (S11)

M Sporadic

Somatic 31

5.7

↑↑↑

No

4.4

↑↑

33x24x29

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DI) Sx, SSA, DA, PEG- PA, GH+ PRL+,

F

Sporadic

Germline 10

4

↑

No

NA

↑

25

Sx, SSA, DA, RT,

SC

22 (S12)

Ki-67 3.5%

RI

V

No

PA, GH+ PRL+

TSH, LH, DI) Yes

PEG-V

F

Sporadic

Germline 48

7.6

↑

Paradoxical 2.6

↑

17

↑↑

>10

25 (F1B)

26 (F1C)

F

Familial

M Familial

M Familial

Germline 12

Germline 12

Germline 14

2.6

1.5

1.2

↑↑

↑↑

↑

No

NA

No

3.3

Germline 48

8

↑

No

28 (F2B)

F

Germline NA

22

↑

No

Familial

Hyperplasia

Yes

NA

8

[2]

Eosinophilic PA

Yes

Yes (ACTH,

45

[2]

18

[2]

13

[2]

TSH, GH) SSA, DA, Sx x3

PA with

Yes

Yes (ACTH,

NA

↑

19

Sx

PA, GH+ PRL+

Yes

Yes (DI)

23

[2]

NA

↑

>10

Sx

PA, GH+ PRL+

Yes

Yes (ACTH,

26

[2]

CE

M Familial

AC

27 (F2A)

15

TSH, LH)

Diffuse

Paradoxical 1.9 increase

↑↑↑

Sx

Yes (ACTH,

PT ED

24 (F1A)

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increase

SSA, DA, Sx

NU

23 (S13)

Yes (ACTH,

↑

hyperplasia DA, Sx

enlargement

Microadenoma

TSH, GH) Yes

with hyperplasia

Yes (ACTH, TSH)

TSH, LH)

21

ACCEPTED MANUSCRIPT

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Figure legend

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Figure 1. Marked overgrowth in XLAG. A. The current world’s tallest man was found to be mosaic for an Xq26.3 microduplication

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(case 8; picture kindly provided by Prof De Herder, Erasmus MC, Rotterdam). He was diagnosed with a pituitary macroadenoma at the age of seven, and has received several operations, radiotherapy and medical treatment with somatostatin analogues and, more recently,

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with the GH receptor antagonist pegvisomant. B-C. Based on his clinical phenotype, Robert Wadlow, the Alton Giant, is most likely to

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have suffered from XLAG. His birth weight was 4kg and he immediately started to grow at a rapid rate and, by six months of age, his

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weight was 13kg, which is the 50th centile for a two years and three months old child. Based on the detailed description by Behrens in 1932 [36], his sella turcica measured 2.5cm, suggesting a pituitary adenoma. He received no treatment, reached a final height of 272cm and died at the age of 28 due to an infection. There was no family history of tall stature. As until now all sporadic male patients harboured

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the duplication in a mosaic state, we assume he was mosaic for this genetic abnormality.

Figure 2. Growth curve and mutation analysis of a patient with XLAG. A. A female patient (case 1) was noted to grow rapidly starting from the age of 18 months. She was diagnosed with GH excess at the age of four years in 1970. She underwent several pituitary surgeries in order to place intrasellar Yttrium implants, and was later started on somatostatin analogue treatment. Her final height (175cm) is matching the mid-parental height (174.5cm). B. HD-aCGH showed a complex genomic rearrangement with a proximal duplication followed by a normal-copy segment and a distal duplication, the latter encompassing GPR101 only, while the other three genes

22

ACCEPTED MANUSCRIPT (CD40LG, ARHGEF6 and RBMX) on Xq26.3 were not duplicated. These results suggest that GPR101 is the disease causing gene within

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the Xq26.3 region.

23

ACCEPTED MANUSCRIPT

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CE

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Conflicts of interest: none

24

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ACCEPTED MANUSCRIPT

25

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ACCEPTED MANUSCRIPT

26

ACCEPTED MANUSCRIPT

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Highlights

X-linked acrogigantism (XLAG) is a condition of GH excess resulting from the duplication of the GPR101 gene



GPR101 encodes an orphan G protein-coupled receptor highly expressed in the hypothalamus



XLAG is characterised by early-onset and female preponderance



While GPR101 duplication leads to XLAG, sequence variants in this gene are not frequently found in sporadic pituitary adenoma

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

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patients

27