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Introduction

Cushing's disease (CD) is a severe (and potentially fatal) disease caused by adrenocorticotropic hormone (ACTH)-secreting adenomas of the pituitary gland [often termed pituitary adenomas (PAs)], and constitutes 10–15% of all PAs worldwide (1). Excess secretion of ACTH in CD results in the following symptoms: Central obesity, hirsutism, glucose intolerance and osteoporosis. Since their first description in 1932 (2), the pathogenesis of tumors of the pituitary gland has not been elucidated, which has hindered the early diagnosis of many cases of PA.

Generally, PAs result from clonal expansion of somatic mutated cells (3). Studies have suggested that ~40% of sporadic PAs are associated with somatic mutations of genes (4). Germline mutations in genes also predispose individuals to PAs (5). Thus, tumorigenesis in the pituitary gland may be explained by gene mutation.

Recent studies have demonstrated that mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene, and Carney complex and multiple endocrine neoplasia type 1 (MEN1) are associated with sporadic PAs (6,7). Disruption in the balance of pituitary-secreting hormones as a result of gene mutation may result in severe-to-fatal consequences. Therefore, identification of relevant gene mutations is particularly useful in the early diagnosis of CD, and for genetic counseling of CD patients.

It has been suggested that CD may be a consequence of hereditary disease (8). Previous studies revealed genetic factors to be involved in CD (9,10). Recently, a review by Perez-Rivas and Reincke (11) specified the roles of mutations in ubiquitin-specific protease 8 (USP8) in CD. In the present study, the gene mutations that have been reported in CD patients are reviewed.

Menin 1 (MEN1) mutation

MEN1 is composed of 10 exons, is located in chromosome 11q13 and encodes a 610-amino acid menin protein. Bassett et al (12) identified 47 mutations in the coding exons of MEN1 as follows: 12 Nonsense mutations, 21 deletions, 7 insertions, 1 donor splice-site mutation and 6 missense mutations. The authors suggested that 80% of these mutations were likely to be inactivating (12). Mutations in MEN1 have been found to be associated with pancreatic endocrine tumors (13). Furthermore, inactivating mutations of MEN1 lead to a familial disorder termed MEN1, of which one common component is CD (14). Thus, it has been speculated that mutations in MEN1 may also participate in CD. Stratakis et al (5) reported that two mutations of MEN1 (a deletion mutation and p.Arg415X) were identified in CD patients. Matsuzaki et al (15) reported an R460X mutation in MEN1 in a Brazilian subject with early-onset CD and his sister. A heterozygote C→T transition was detected at codon 460 in exon 10 in MEN1, which converts codon 460 CGA (Arg) to a stop codon TGA (15). Thus, mutations in MEN1 lead to the early clinical manifestations of MEN1, and are involved in CD. Furthermore, these findings confirm the hereditary characteristics of CD.

Nuclear receptor subfamily 3 group C member 1 (NR3C1) mutation

Perfect balance in the requirement and secretion of glucocorticoid hormones (which is maintained by feedback from the hypothalamic-pituitary-adrenal-axis) is critical for the regulation of glucose metabolism and the feedback mechanism in the immune system.

CD patients share the characteristics of resistance to glucocorticoids and unresponsiveness to normal glucocorticoid negative feedback (16). However, somatic mutations of NR3C1, or dysfunction of genes associated with glucocorticoid receptor function, are rarely found in CD (5). However, Karl et al (10) reported a novel heterozygous missense mutation in NR3C1 in a CD patient. The authors found that the mutation occurs in exon 5 of the coding region of NR3C1 in lymphoblasts and fibroblasts, as well in 50% of sperm. As a result, the neutral and polar asparagine at codon 559 substitutes the neutral and hydrophobic amino acid, isoleucine. The authors also suggested that the mutation contributed to severe, sporadic, generalized glucocorticoid resistance. Further investigation demonstrated that the mutation was not detected in the patient's parents or seven siblings. Therefore, it was concluded that this novel mutation was de novo and present in the germline (10). That is, the mutation in NR3C1 may be involved in CD.

Aryl hydrocarbon receptor-interacting protein (AIP) mutation

AIP is a protein of 330 amino acids and acts as a tumor suppressor (17). Studies have demonstrated that AIP combines with the aryl hydrocarbon receptor on the cell surface, and probably exerts its effects by regulating integrin function. More than 100 variants in AIP have been identified, of which the most frequent mutation occurs in the p.R304 locus (18). Approximately 15–30% of familial isolated PAs harbor germline mutations in AIP (19,20). PAs with mutations in AIP are predominantly somatotropinomas and prolactinomas; however, studies have revealed that AIP mutations may also occur in CD. Georgitsi et al (9) found a heterozygous c.696G>C (which leads to the silencing of p.P232P in exon 5) in a CD patient in Poland. Furthermore, Stratakis et al (5) reported a novel germline AIP mutation, c.308A>G/p.Lys103Arg, in the heterozygotic state in one pediatric patient with recurrent CD, although the authors suggested that the overall prevalence of AIP mutation was very low. These findings indicate that AIP mutations may be directly involved in the molecular pathogenesis of CD, but that screening for mutations may not be an effective method for the diagnosis of CD.

Tumor protein p53 (TP53) mutation

The p53 protein is encoded by a tumor-suppressor gene termed TP53, which is located on chromosome 17. p53 inhibits the G1→S transition of the cell cycle, and is significant in suppression of tumorigenesis. Studies have suggested that TP53 mutations are associated with the pathogenesis of ~50% of human cancers, including those in the central nervous system (21,22). However, the role of p53 in tumors of the pituitary gland is controversial. Oliveira et al (23) demonstrated p53 protein to be positive in only two of 148 PA patients, suggesting that p53 may not be a biomarker for tumors of the pituitary gland. Other studies have indicated that p53 expression in tumors of the pituitary gland cannot be detected, including ACTH-secreting adenomas (24–26). By contrast, Buckley et al (27) found that abnormal expression of p53 was involved in the development of invasive pituitary tumors. The common alterations associated with TP53 in human tumors are inactivating mutations, which occur between exon 5 to 8 (28). Levy et al (29) and Herman et al (30) failed to identify mutations in TP53 in PAs, although Kawashima et al (31) reported that a somatic mutation of TP53 contributed to a case of atypical PA that caused CD. The authors sequenced the region of exon 5 through to exon 8 of TP53 and identified a missense mutation of CTG>CGG on codon 145 (L145R). The study indicated that the mutation was detected in tumor tissues, but not in peripheral blood (31). These studies imply that a somatic mutation of TP53 may contribute to the pathogenesis of CD.

Nuclear receptor subfamily 0 group B member 1 (NR0B1) mutation

Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1) is critical in steroidogenic development and sex determination. Studies have demonstrated that DAX-1 suppresses the transcription of various genes expressed in the adrenal cortex and hypothalamic-pituitary-gonadal axis, such as steroidogenic factor-1 (32,33). DAX-1 is encoded by NR0B1, which comprises two exons separated by a 3.4-kb intron. Studies have shown involvement of mutations of NR0B1 in hypogonadotropic hypogonadism and X-linked adrenal hypoplasia congenital (34,35). However, the role of DAX-1 in ACTH-secreting PAs remains poorly understood. Suzuki et al (36) analyzed the regulatory mechanisms of differentiation of pituitary cells in 89 corticotroph adenomas. DAX-1 was found to be positive in all subjects, indicating that DAX-1 is essential for the genesis of ACTH-secreting PAs. Furthermore, De Menis et al (37) described a novel mutation of NR0B1 in a patient with ACTH-secreting PA and X-linked adrenal hypoplasia congenita and his mother [a 4-bp insertion (AGCG) at nucleotide 259 in exon 1 of NR0B1] leading to premature termination of transcription. This evidence indicates that monitoring of mutations in NR0B1 may be beneficial for early diagnosis in patients with CD and X-linked adrenal hypoplasia congenita.

Ubiquitin-specific peptidase 8 (USP8) mutation

USP8 is a member of the USP family. The specific roles of USP8 mutations in CD have been reviewed by Perez-Rivas and Reincke (11). Here, three studies published recently, which reported mutations of USP8 in CD, are reviewed.

Reincke et al (38) found that mutations of p.Ser718Cys, p.Pro720Arg and p.Ser718del in the 14-3-3 protein binding motif promoted the proteolytic cleavage and catalytic activity of USP8, which inhibited epidermal growth factor receptor (EGFR) degradation and prolonged EGF signaling, resulting in increased activity in the proopiomelanocortin (POMC) promoter and transcription, as well as causing CD. Perez-Rivas et al (39) demonstrated that somatic mutations comprising p.718Ser>Pro, Ser718del, p.720Pro>Gln, and p.720Pro>Arg in USP8 diminished EGFR ubiquitination and induced the activity of the POMC promoter. Ma et al (40) reported the significant clinical relevance of three somatic mutations (c.CTC2151-2153del/p.S718del, c.C2159G/p.P720R and c.T2152C/p.S718P) of USP8 and CD. It was revealed that mutations in exon 14 of USP8 disrupt the interaction between USP8 and 14-3-3 protein, leading to protection of the EGFR from lysosomal degradation. Mutations in USP8 sustain EGFR-mitogen-activated protein kinase signaling to promote ACTH production in CD (40). These findings clearly demonstrate that mutations in USP8 contribute to CD.

Dicer 1, ribonuclease (RNase) III (DICER1) mutation

DICER1 is a highly conserved RNase III enzyme, the functions of which are predominantly associated with RNA interference pathways, including the processing of microRNA precursors into mature microRNAs (41,42). DICER1 is associated with various tumors, such as pulmonary adenomas and pleuropulmonary blastomas (41,43). Hill et al (44) suggested that almost all patients with pleuropulmonary blastoma exhibit germline mutations of DICER1. In addition, mutations in DICER1 are involved in the development of cystic nephroma (45). As mutated DICER1 participates in diverse types of tumors, previous studies have indicated that DICER1 mutations result in tumors of the pituitary gland. Wildi-Runge et al (46) reported a germline heterozygous DICER1 mutation in a blastoma of the pituitary gland in an infant, suggesting a role for DICER1 mutations in tumors of the pituitary gland. Furthermore, Sahakitrungruang et al (47) described two novel DICER1 mutations in a one-year-old female with a blastoma of the pituitary gland presenting with CD. The results showed that a novel heterozygous c.3046delA (p.S1016VfsX1065) mutation in DICER1 was identified by whole-exome sequencing of leukocytes and pituitary blastoma tumor tissues, and another somatic missense c.5538A->T (p.E1813V) mutation was identified in tumor tissues only (47). These findings indicate that DICER1 mutations may facilitate with understanding the pathogenesis of CD.

Cytochrome P450 family 21 subfamily A member 2 (CYP21A2) mutation

CYP21A2 encodes active steroid 21-hydroxylase enzyme. Studies have suggested that 21-hydroxylase-deficient mice show failure of inhibition of the hypothalamic-pituitary-adrenal axis (48). Mutations in CYP21A2 are responsible for congenital adrenal hyperplasia (CAH), which is associated with CD in certain cases. Haase et al (49) found that a homozygous mutation in exon 7 of CYP21A2 (CTG>TTG, p.V281L) may have contributed to CD in a female patient with CAH. Boronat et al (50) reported that a 39-year-old female patient with an ACTH-producing PA carried two point mutations in CYP21A2: A severe splicing 655G mutation at intron 2 and a mild V28L mutation at exon 7. Concurrently, a severe 8-bp deletion mutation was found at exon 3 of the CYP21A2 gene, which caused the 21-hydroxylase deficiency, in a 21-year-old CD patient (50). Although 21-hydroxylase deficiency is rarely observed in CD patients, CYP21A2 mutations may (at least in part) contribute to CD.

GNAS complex locus (GNAS) mutation

GNAS1 (also termed gsp oncogene) comprises 13 exons and is located on chromosome 20q13. GNAS encodes various proteins, including the α subunit of the stimulatory G protein (Gsα), extra-large αs and 55-kDa neuroendocrine secretory protein. The activating and inactivating mutations of GNAS have previously been identified (51,52). GNAS mutations have been found to be involved in certain endocrine diseases. Patten et al (53) demonstrated that the A→G point mutation in GNAS (which causes reduced immunoactivity in the Gsα protein) is associated with Albright's hereditary osteodystrophy. Other studies have reported that ~40% of patients with functional PAs exhibit somatic mutations of GNAS, which often occur at codons R201 and Q227. Williamson et al (54) identified mutations of CAG→CGG and CAG→CAC/T at codon Q227 in only two of 32 ACTH-secreting PAs, and suggested that these mutations are an uncommon abnormality in CD. Riminucci et al (55) observed an R201H mutation of GNAS in a child with CD, thereby extending the disease spectrum of the R201 mutation of GNAS. Therefore, an association between GNAS mutations and CD may improve the understanding of CD pathogenesis.

Leukemia inhibitory factor (LIF) mutation and cyclin-dependent kinase inhibitor 1B (CDKN1B) mutation

LIF is the most pleiotropic member of the interleukin-6 family. LIF is essential in activation of the hypothalamo-pituitary-adrenal axis during inflammation (51). In LIF knockout mice, the ACTH response to stress is reduced, whereas LIF overexpression in transgenic mice leads to corticotroph cell hyperplasia and hypercortisolism (56,57). LIF exerts a regulatory function by binding to the LIF receptor (LIF-R) and gp130 (58). These studies indicate that LIF promotes ACTH secretion, and that mutations in LIF- or LIF-R-encoding genes may contribute to CD pathogenesis. However, Heutling et al (59) did not observe mutations in LIF-R in ACTH-secreting adenomas, and suggested that mutations in LIF-R were an unlikely cause for CD development (59).

CDKN1B, also known as MEN4 and p27/kip1, maps to chromosome 12p13 and encodes a CDK inhibitor, which restricts cell cycle progression at G1. Lack of p27/kip1 function leads to the development of PAs (60). Furthermore, Liu et al (61) found that a selective inhibitor of CDK markedly suppressed ACTH levels and restrained growth of ACTH-secreting PAs in mice. One study revealed that germline CDKN1B mutations rendered individuals more susceptible to MEN1 (62). Thus, it is speculated that CDKN1B mutations may also participate in CD. However, Dahia et al (63) proposed that p27/kip1 mutations were not a feature of corticotroph tumors.

Conclusions

The majority of tumors of the pituitary gland appear to arise from a single mutated cell due to expansion of monoclonal cells. Therefore, distinct genetic changes are probably one of the most important events within tumorigenesis in the pituitary gland, including in ACTH-secreting corticotroph tumors. The current study reviewed previous investigations, which showed that various gene mutations are involved in CD (Table I). Cases are predominantly sporadic, therefore, the regulatory mechanisms of the gene mutations in CD are rarely investigated, although USP8 mutations have been more extensively evaluated. Thus, current treatment of CD patients has not progressed as a result of the identification of gene mutations that are associated with CD. However, the findings of the present review offer potential benefits regarding genetic counseling and early diagnosis of CD.

Table I. Gene mutations associated with CD.

Table I.

Gene mutations associated with CD.

Author, year	Gene	Mutation	Codon change	Mutation type	Location	Disease	Refs.
Stratakis, 2010	MEN1	–	–	Deletion mutation	–	Pediatric familial/syndromic CD	(5)
		p.R415X	–	Nonsense mutation	–	Pediatric familial/syndromic CD
Matsuzaki, 2004		p. R460X	CGA>TGA	Nonsense mutation	Exon 10	MEN1 with CD	(15)
Karl, 1996	NR3C1	p.I559N	ATC>AAC	Missense mutation	Exon 5	CD	(10)
Georgitsi, 2007	AIP	c.696G>C/p.P232P	–	Silence mutation	Exon 5	CD	(9)
Stratakis, 2010		c.308A>G/p.K103R	–	Missense mutation	–	Recurrent CD	(5)
Kawashima, 2009	TP53	p.L145R	CTG>CGG	Missense mutation	–	Atypical PA causing CD	(31)
De Menis, 2005	NR0B1	g.259_260insAGCG	–	Insertion mutation	Exon 1	ACTH-secreting PA and X-linked adrenal hypoplasia congenita	(37)
Reincke, 2015	USP8	p.S718C	–	Missense mutation	Exon 14	CD	(38)
		p.P720R		Missense mutation
Perez-Rivas, 2015		p.S718del		Deletion mutation			(39)
Ma, 2015		p. S718P		Missense mutation			(40)
		p. P720Q		Missense mutation
Sahakitrungruang, 2014	DICER1	c.3046delA/p.S1016VfsX1065	–	Frameshift mutation	–	Pituitary blastoma presenting with CD	(47)
		c.5538A>T/p.E1813V		Missense mutation	–
Haase, 2011	CYP21A2	p.V281L	CTG>TTG	Missense mutation	Exon 7	CD with CAH	(49)
		–	–	Splicing mutation	Intron 2	ACTH-producing PA
Boronat, 2004		–	–	Deletion mutation	Exon 3		(50)
Williamson, 1995	GNAS	p.R179G	CGC>GGC	Missense mutation	–	Corticotroph adenomas	(54)
Riminucci, 2002		p.Q227R	CAG>CGG	Missense mutation	–		(55)
		p.Q227H	CAG>CAC/T	Missense mutation	–
		p.R201H	–	Missense mutation	Exon 8	CD

[i] MEN1, menin 1; NR3C1, nuclear receptor subfamily 3 group C member 1; AIP, aryl hydrocarbon receptor-interacting protein; TP53, tumor protein p53; NR0B1, nuclear receptor subfamily 0 group B member 1; USP8, ubiquitin-specific peptidase 8; DICER1, Dicer 1, ribonuclease III; CYP21A2, cytochrome P450 family 21 subfamily A member 2; GNAS, GNAS complex locus; -, not mentioned; CD, Cushing's disease; MEN1, multiple endocrine neoplasia type I; PA, pituitary adenomas; ACTH, adrenocorticotropic hormone; CAH, congenital adrenal hyperplasia.

Acknowledgements

The present study was supported by the National Natural Science Foundation of China (grant no. 81373150).

References