Introduction

The development of the hypothalamic-pituitary-gonadal (HPG) axis is both complex and fascinating, as GnRH neurons develop in metazoan embryos outside of the central nervous system and migrate alongside vomeronasal neurons during fetal life. Immature GnRH precursor neurons are first detectable in the olfactory placode in the nose from an early embryological stage, and then begin a complex journey towards the hypothalamus (1).

The HPG axis is active in fetal and then in early infant life, the so-called ‘mini-puberty’, but thereafter becomes dormant between the age of one and eight-to-nine years (2). Development of the clinical features of puberty is initiated by the reactivation of the HPG axis after this relative quiescence during childhood. What drives this suppression of the axis during childhood, and what controls the release of this ‘brake’ and the timing at which this occurs, is poorly understood.

Despite the demonstrated importance of environmental factors such as body mass, psychosocial stressors and endocrine disrupting chemicals (EDCs) (3), genetic influence on the timing of puberty is clearly fundamental. Whilst the timing of pubertal onset varies within and between different populations, it is a highly heritable trait. Twin studies demonstrate that the timing of sexual maturation is highly correlated between highly related individuals, suggesting strong genetic determinants (4). Previous studies estimate that 60-80% of the variation in pubertal onset is under genetic regulation (5, 6). However, despite this strong heritability, the key genetic factors that determine human pubertal timing in the normal population and in cases of disturbed pubertal timing remain mostly unknown (7).

Pubertal reactivation of the hypothalamic-pituitary axis

Kisspeptin, an excitatory neuropeptide, was identified as a permissive factor in puberty onset by the discovery of patients with GnRH deficiency with loss-of-function mutations in the KISS1 receptor, KISS1R (previously known as GPR54) (8, 9). Mice with knockout of Kiss1r were simultaneously discovered to be infertile despite anatomically normal GnRH neurons and normal hypothalamic GnRH levels (9), with a phenotype consistent with normosmic GnRH deficiency. However, despite a large body of evidence for kisspeptin as one of the most important gatekeepers of GnRH neuronal function, only very rarely have human mutations in KISS1 been found in patients with delayed or absent puberty (10).

The excitatory neuropeptide, neurokinin b, also plays a role in the upstream control of GnRH secretion. Identification of this pathway was also via discovery of loss-of-function mutations in TAC3, encoding neurokinin b, and its receptor TACR3, in patients with normosmic GnRH deficiency and pubertal failure (11). Kisspeptin, neurokinin b and dynorphin are coexpressed in KNDy neurons of the arcuate nucleus of the hypothalamus (12), which project to and directly interact with GnRH neurons. Their expression is downregulated by oestrogen and testosterone as part of the negative feedback regulation of gonadotropin secretion (13, 14). However, administration of neurokinin b agonists failed to stimulate GnRH release in rodents, and Tacr3 knockout mice have grossly normal fertility (15, 16). Of 50 self-limited DP patients investigated for mutations in TAC3 and TAC3R, only one mutation in a single patient was found in the latter gene (17).

The inhibitory role of GABAergic neurotransmission has been clearly shown in primates (18) but is more ambiguous in rodents. Opioid peptides provide additional inhibitory input but this appears to be less critical than the GABAergic signals in restraining the initiation of puberty (19). Additionally, RFamide-related peptide gene (RFRP), the mammalian ortholog of the avian peptide gonadotrophin-inhibiting hormone (GnIH), has been identified as a further inhibitory regulator of GnRH neuronal activity in mice (20). Glial inputs appear to be predominantly facilitatory during puberty and consist of growth factors and small diffusible molecules, including TGFβ1, IGF-1 and neuregulins, that directly or indirectly stimulate GnRH secretion (21).

Upstream regulation of GnRH transcription is less well established. Candidate transcriptional regulators identified from a systems biology approach and animal models include Oct-2, TTF-1 and EAP1 (22) (Figure 1). Oct-2 mRNA is upregulated in the hypothalamus in juvenile rodents, blockage of Oct-2 synthesis delays age at first ovulation whilst activation of Oct-2 expression (e.g. hamartomas) induces precocious puberty (23). TTF-1 (thyroid transcription factor-1) enhances GnRH expression, with increased expression in pubertal rhesus monkeys (24). EAP1 mRNA levels also increase in primate and rodent hypothalamus during puberty. EAP1 transactivates the GnRH promoter, and EAP1 knockdown with siRNA caused DP and disrupted estrous cyclicity in a rodent model (25). However, to date no mutations in these upstream or regulatory factors have been reported in patients with DP.

Epigenetic regulators are potential modulators of pubertal timing. Recent evidence highlights the importance in mice of microRNAs (particularly the miR-200/429 family and miR-155) in the epigenetic up-regulation of GnRH transcription during the critical period (murine equivalent of the mini-puberty) (26). Moreover, miR-7a2, has been demonstrated to be essential for normal pituitary development and HPG function, with deletion in mice leading to hypogonadotropic infertility (27). The effects of environmental changes on the hypothalamic regulation of puberty may also be mediated via epigenetic mechanisms. In particular, epigenetic changes during foetal life are a potential mechanism for the effects of EDCs in utero (28).

Etiologies underlying Delayed Puberty

Self-limited delayed puberty (DP), also known as constitutional delay of growth and puberty (CDGP), represents the commonest cause of DP in both sexes. Up to 83% of boys with pubertal delay have self-limited DP (29-32). Individuals with self-limited DP lie at the extreme end of normal pubertal timing, with the absence of testicular enlargement in boys or breast development in girls at an age that is 2 to 2.5 standard deviations (SD) later than the population mean (7). In addition, self-limited DP may also encompass older children with delayed pubertal progression, a diagnosis that is aided by the use of puberty normograms (31). Self-limited DP is associated with adverse health outcomes including short stature, reduced bone mineral density and compromised psychosocial health (33).

There are three main groups of differential diagnosis of self-limited DP (Table 1) (7, 29), although up to 30 different etiologies underlying DP have been identified (32): hypergonadotropic hypogonadism, with primary gonadal failure leading to elevated gonadotropin levels due to lack of negative feedback; functional hypogonadotropic hypogonadism, where late pubertal development is due to maturational delay in the HPG axis secondary to chronic disease, malnourishment, excessive exercise, psychological or emotional stress; and permanent hypogonadotropic hypogonadism, characterized by low LH and FSH levels. This last condition is due to congenital hypothalamic or pituitary disorders, or an acquired central dysfunction secondary to irradiation, tumour or vascular lesion.

The Inheritance and Genetic Background of Self-Limited Delayed Puberty

Self-limited DP segregates within families with complex patterns of inheritance including autosomal dominant, autosomal recessive, bilineal and X-linked (34), although sporadic cases are also observed (Figure 2). The majority of families display an autosomal dominant pattern of inheritance (with or without complete penetrance) (4, 34).  50 to 75% of subjects with self-limited DP have a family history of delayed pubertal onset (34).

Some insights into the genetic mutations that lead to familial self-limited DP have come from sequencing genes known to cause GnRH deficiency, most recently via next generation sequencing. Linkage analysis and targeted sequencing strategies have been mostly superseded by whole exome and genome sequencing strategies to identify novel candidate genes.

Other candidates have been identified from large-scale genome wide association studies in the general population. Analysis of self-limited DP families is complicated by the fact that this phenotype represents the tail of a normally distributed trait within the population, so it is expected that variants that govern the inheritance of this condition will also be present in the general population at a low level. Thus, the absence of these variants in population databases cannot be used as an exclusion criterion during filtering of sequencing data. Instead, a comparison of prevalence of such variants must be made to identify those that are enriched in patients compared to the general population. In the majority of patients with DP the neuroendocrine pathophysiology and its genetic regulation remain unclear.

Loss-of-function mutations in a member of the immunoglobulin superfamily, IGSF1, have been identified in patients with X-linked central hypothyroidism (35). Notably, male patients with IGSF1 mutations have a late increase in testosterone levels with a delayed pubertal growth spurt. However, pathogenic mutations in IGSF1 have not been conclusively found in patients with isolated DP (36).

More recently, whole exome and targeted resequencing methods have implicated two pathogenic mutations in IGSF10 as the causal factor for late puberty in six unrelated families from a large Finnish cohort with familial DP (37). A further two rare variants of unknown significance were identified in four additional families from the cohort. Mutations in IGSF10 appear to cause a dysregulation of GnRH neuronal migration during embryonic development (Figure 3). An intact GnRH neurosecretory network is necessary for the correct temporal pacing of puberty. Pathogenic IGSF10 mutations leading to disrupted IGSF10 signalling potentially result in reduced numbers or mis-timed arrival of GnRH neurons at the hypothalamus; producing a functional defect in the GnRH neuroendocrine network. With this impaired GnRH system there would follow an increased ‘threshold’ for the onset of puberty, with an ensuing delay in pubertal timing. IGSF10 loss-of-function mutations were also discovered in patients with a hypothalamic amenorrhoea-like phenotype. These findings represent a new fetal origin of self-limited DP, and reveal a potential shared pathophysiology between DP and other forms of functional hypogonadism.

Genetics of GnRH deficiency and relevance to DP

At the extreme end of the spectrum of DP are conditions of GnRH deficiency including congenital hypogonadotropic hypogonadism (CHH), with complete failure to enter puberty. The condition may be due to failure of development of GnRH neurons, lack of activation of GnRH secretion or disrupted GnRH signalling. Because of different causes and incomplete penetrance, there is a wide spectrum of phenotypes, ranging from complete CHH with lack of pubertal development to a partial hypogonadism with an arrest of pubertal development, and reversible CHH in up to 20% of patients post treatment (38-40). Despite recent advances, with over forty genes linked to this disorder identified, the pathophysiological basis of CHH in approximately 50% of individuals remains unclear (Figure 4) (2).

Various malformations affecting the development of the prosencephalon may cause GnRH deficiency combined with deficiency of any or all other pituitary hormones. Other congenital midline defects, which may range from holoprosencephaly to cleft lip and palate, can also be associated with variable hypothalamic–pituitary dysfunction.

Genetic defects affecting the development of the anterior pituitary may cause GnRH deficiency. The pituitary transcription factors LHX3, SOX2 and HESX1 are vital for early patterning of the forebrain and pituitary, and mutations in these developmental genes result in syndromic hypopituitarism with gonadotropin deficiency in humans (41). PROP1 is important for the development of gonadotropin-secreting cells and mutations in this gene are the most common cause of combined pituitary hormone deficiency in humans (42). Patients with PROP1 mutations have variable GnRH deficiency ranging from DP to CHH (41). Mutations in DAX1 cause X-linked adrenal hypoplasia congenita with associated HH, but have not been found in isolated DP (43).

GnRH deficiency may also be associated with other conditions, particularly with neurological phenotypes. Mutations in POLR3A/B result in the 4H syndrome (Hypomyelination, Hypodontia and Hypogonadotropic Hypogonadism) (44) whilst those in RNF216, OTUD4 and PNPLA6 produce the phenotypic combination of HH and ataxia (also known as Gordon-Holmes syndrome) (45, 46). DMXL2 mutations are associated with congenital HH, other endocrine deficiencies and polyneuropathies (47). Dysregulation of the RAB3 cycle, such as with mutations in RAB3GAP1, lead to Warburg Micro syndrome with ocular, neurodevelopmental and central reproductive defects (48, 49).

Loss-of-function mutations within the GnRH receptor are the most frequent cause of autosomal recessive CHH, accounting for 16% to 40% of patients. Mutations have been found within the extracellular, transmembrane and intracellular domains of the receptor leading to impaired GnRH action (50). A homozygous partial loss-of-function mutation in GNRHR was found in two brothers, one with self-limited DP and one with idiopathic HH (51), and a further heterozygous mutation found in one male with self-limited DP (52). A small cohort of 31 patients was analysed for mutations in GHSR and 5 patients were found to have point mutations in this gene (53).

Downstream mutations in the GnRH signalling pathway can also present with DP. LH and FSH are glycoprotein hormones encoded by a common α-subunit gene and a specific β-subunit gene. Mutations of the β-subunits genes of LH or FSH are extremely rare causes of pubertal abnormalities (54). Males with inactivating mutations of the LHB have absent pubertal development with Leydig cell hypoplasia leading to T deficiency and azoospermia. Females with inactivating mutations of LHB present with onset of normal puberty, but with normal or late menarche followed by infertility due to lack of ovulation (54). Individuals with inactivating FSHB mutations present with incomplete pubertal development and azoospermia in males and primary amenorrhea in females (55).

In view of the possible overlap between the pathophysiology of DP and conditions of GnRH deficiency, a few studies have specifically examined the contribution of mutations in CHH genes to the phenotype of self-limited DP. Mutations in HS6ST1, FGFR1 and newly in KLB have been found in a small number of kindreds of CHH patients and their relatives with DP (56-58). Variants in several HH genes including GNRHR, TAC3, TACR3, IL17RD and SEMA3A have been identified by whole exome sequencing in some cases of DP, including self-limited DP (59). However, these variants have not been tested in vitro or in vivo for pathogenicity and thus may be an over-estimation. Overall, the current picture indicates that the genetic background of HH and DP may be largely different, or shared by as yet undiscovered genes (52).

Genetics of pubertal timing in the general population

Attempts to identify key genetic regulators of the timing of puberty in humans have led to several large genome wide association studies (GWAS) of age-at-menarche, examining pubertal timing in healthy women (60-62). These studies demonstrate genetic heterogeneity in pubertal timing, with the observation that the genetic architecture of the timing of puberty in healthy subjects is likely to involve at least hundreds of common variants. The first of many loci associated with age of menarche was the gene LIN28B (63). LIN28B is a human ortholog of the gene that controls, through microRNAs, developmental timing in the Caenorhabditis elegans. However, mutations in LIN28B have not to date been identified in human patients with DP (64) or precocious puberty (65).

The largest study of this type comprises 1000 Genomes Project-imputed genotype data in up to ∼370,000 women, and identifies 389 independent signals (P < 5 × 10^-8) for age at menarche (66). Per-allele effect sizes ranged from 1 week to five months. These signals explain ∼7.4% of the population variance in age at menarche, corresponding to ∼25% of the estimated heritability. Many of these signals have concordant effects on the age at voice breaking, a corresponding milestone in males. However, in women the signals identified had stronger effects on early than on late age of menarche, but in contrast had larger effect estimates for relatively late than relatively early voice breaking in males (66).

Around 250 genes were identified via coding variation or associated expression, particularly those expressed in neural tissues. Importantly, genes already implicated in rare disorders of puberty were identified, including LEPR, GNRH1, KISS1, TACR3. Two imprinted genes were also reported: MKRN3, paternally-inherited mutations in which have been identified as causal in pedigrees of central precocious puberty (CPP) (67); and DLK1 (68). MKRN3 is the third, and to date the most frequently mutated, gene in pedigrees with CPP (67), the others being KISS1 (69) and its receptor GPR54 (70) which have been reported only rarely. MKRN3 is thought to contribute to the puberty ‘brake’ restraining the HPG axis via inhibition of GnRH release. Very recently a complex defect in DLK1 has been identified in one pedigree with CPP (68), and a gain-of-function mutation in PROKR2 reported in a girl with sporadic CPP (71). However, neither MKRN3 nor DLK1 mutations have been implicated in the pathogenesis of DP (Figure 4).

Delayed puberty due to primary gonadal failure

In gonadal dysgenesis in both males and females, pubertal development may be delayed or entirely absent. In Turner syndrome, the most common form of hypergonadotropic hypogonadism in females, puberty is delayed and usually followed by progressive ovarian failure (72). Importantly, however, up to 30% of girls will undergo spontaneous pubertal development and 2 to 5% will have spontaneous menses (73). About half of girls with Turner syndrome have the 45,X karyotype. Other causes of ovarian dysgenesis include X isochromosome, where abnormal chromosome division results in duplication of identical chromosome arms, most commonly of the long (q) arm. These patients have streak gonads and a similar phenotype to Turner syndrome. Various microdeletions on the short and long arm of the X chromosome are also found in women with primary ovarian insufficiency, with several genes implicated including POF1, POF2, DIAPH2, FOXL2 and BMF15 (74). XX gonadal dysgenesis can occur in combination with cerebellar ataxia and learning difficulties, or with multiple malformation syndromes with a range of associated features including microcephaly, limb abnormalities, facial and cardiac defects. Point mutations in the extra-cellular domain of the FSH receptor with subsequent inactivation of the receptor function result in raised FSH levels, variable development of secondary sex characteristics and primary or secondary amenorrhea (75). Whilst up to 40% of Finnish patients have such a mutation, these appear to be rare in other populations.

In males, testicular abnormalities are characterized by elevated gonadotropin and low inhibin-B concentrations, and may present as pubertal delay. The commonest condition underlying hypergonadotropic hypogonadism in males is Klinefelter syndrome (47, XXY), with a prevalence of 1 in 667 live births. The majority of those affected will enter puberty spontaneously at a normal age (76), although DP may be seen in those with a more complex karyotype (48, XXYY, 48, XXXY, 49, XXXXY). In the majority of patients with Klinefelter syndrome testosterone levels become increasingly deficient by Tanner stages 4-5, possibly as a result of secondary regression. 

Metabolism and timing of puberty

Nutritional changes play an important role in the observed secular trend towards an earlier age of pubertal onset in the developed world (77), as shown by the positive correlation between age at puberty onset and childhood body size, particularly in girls (78). In contrast, under-nutrition in females, for example in chronic disease or anorexia nervosa, can result in delay in both the onset and tempo of puberty. (79)

This relationship between fat mass and pubertal timing is mediated, at least in part, through the permissive actions of the metabolic hormone leptin, a key regulator of body mass, produced from white adipose tissue (80). Humans and mice lacking leptin (Lep ob/ob) or the leptin receptor (LepR db/db) fail to complete puberty and are infertile (81). GWAS studies of pubertal timing found, in addition to leptin signalling, overlap with several genes implicated in body mass index including FTO, SEC16B, TMEM18, and NEGR1 (66). However, whilst self-limited DP in boys is associated with hypoleptinaemia (82), there have been no identified association of specific leptin or leptin receptor polymorphisms with DP (83). Ghrelin and other gut-derived peptides may also form part of the mechanism by which energy homeostasis regulates reproductive development (84). Notably, children with CDGP have a dual phenotype of slow growth in childhood with DP. In contrast, both low birth weight and prematurity are associated with earlier onset of puberty (85), particularly in those children with rapid increase in length or weight in the first two years of life (86). It remains unclear, however, if childhood obesity, insulin resistance, excess androgens or underlying genetic or epigenetic factors may explain this association (87).

Conclusions

A wide variety of genetic and epigenetic defects affecting different aspects of the HPG axis at different time periods in fetal and postnatal life may result in delayed and disordered puberty. Although our understanding of the highly complex underlying biological network remains imperfect, results to date demonstrate the importance of defects in GnRH neuronal development and function, GnRH receptor and LH/FSH abnormalities, transcriptional regulation of the HPG axis and metabolic and energy homeostasis derangements in the control of pubertal timing.

Clinically it is important to distinguish between the conditions of DP and idiopathic CHH in adolescents presenting with DP. However, this diagnosis is often a difficult one as both disorders can present with a picture of functional hypogonadotropism and can share an underlying pathophysiology. There is still no definitive test to accurately discriminate between the two diagnoses. More complex and involved management is required in patients with CHH to achieve both development of secondary sexual characteristics and to maximize the potential for fertility (88). Genetic testing may inform diagnosis of associated syndromic features, likelihood of reversal and inheritance in family members. Rapid and efficient diagnosis of patients in clinic would represent a huge leap forward in patient care and a likely significant economic advantage. While presently next generation sequencing in individuals presenting with delayed or incomplete pubertal development is only a reasonable option in a research setting, future progress in gene discovery and technical developments may facilitate the availability of genetic diagnosis as part of clinical care for patients with both GnRH deficiency and self-limiting DP.

Disclosure: no potential conflict of interest in relation to this presentation. 

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