The Genetic Basis of Delayed Puberty

Puberty is the maturational process of the reproductive endocrine system that results in achievement of adult height and body proportion, in addition to development of the genital organs and the capacity to reproduce. The onset of puberty is driven by an increase in the pulsatile release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. This activation results in increased luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release from the anterior pituitary; both hormones act on the gonads to stimulate their development, gametogenesis, and sex steroid production.

The development of the hypothalamic-pituitary-gonadal (HPG) axis is exceptional in that GnRH neurons develop in metazoan embryos outside of the central nervous system. 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 axis is active in fetal and in early infant life, the so-called “mini-puberty,” and then becomes dormant between the age of 1 and 8-9 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 little understood.

Despite the demonstrated importance of environmental factors such as body mass, psychosocial stressors, and endocrine disrupting chemicals [3], the 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. The timing of sexual maturation is highly correlated within families and in twin studies, suggesting strong genetic determinants [4]. Previous epidemiological studies and genetic approaches estimate that 60-80% of the variation in pubertal onset is under genetic regulation [5,6]. However, despite this strong heritability, little is known about the genetic control of human puberty, either in the normal population or in cases of disturbed pubertal timing [7].

Self-limited delayed puberty (DP), also known as constitutional delay of growth and puberty, represents the extreme end of normal pubertal timing, and is defined as the absence of testicular enlargement in boys or breast development in girls at an age that is 2 to 2.5 standard deviations later than the population mean [7]. In addition, cases of self-limited DP may also encompass older children with delayed pubertal progression, a diagnosis that is aided by the use of puberty nomograms [8]. Self-limited DP is associated with adverse health outcomes including short stature, reduced bone mineral density, and compromised psychosocial health [9].

Self-limited DP represents the commonest cause of DP in both sexes. Up to 63% of boys with pubertal delay have self-limited DP [10]. Self-limited DP segregates within families with complex patterns of inheritance including autosomal dominant, autosomal recessive, bilineal, and X-linked [11], although sporadic cases are also observed (Fig. 1). The majority of families display an autosomal dominant pattern of inheritance (with or without complete penetrance) [4,11]. Fifty to 75% of subjects with self-limited DP have a family history of delayed pubertal onset [11].

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. Instead, a comparison of prevalence rates 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. Linkage analysis and targeted sequencing strategies appear to have been superseded by whole exome and genome sequencing strategies to identify novel candidate genes.

Attempts to identify key genetic regulators of the timing of puberty in humans have led to several large genome-wide association studies of age at menarche, examining pubertal timing in healthy women [12,13,14]. 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 at menarche was the gene LIN28B [15]. LIN28B is a human orthologue of the gene that controls, through microRNAs, developmental timing in Caenorhabditis elegans. However, mutations in LIN28B have not to date been identified in human patients with DP [16] or precocious puberty [17].

The most recent study of this type comprises 1000 Genomes Project-imputed genotype data from up to ∼370,000 women, and identifies 389 independent signals (p < 5 × 10^-8) for age at menarche [18]. The per-allele effect sizes ranged from 1 week to 5 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 at menarche, but in contrast had larger effect estimates for relatively late than relatively early voice breaking in males [18].

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, and 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) [19], and DLK1 [20]. MKRN3 is the third, and to date the most frequently mutated, gene in pedigrees with CPP [19], the others being KISS1 [21] and its receptor GPR54 [22], 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 [20]. However, neither MKRN3 nor DLK1 mutations have been implicated in the pathogenesis of DP (Fig. 2).

At the extreme end of the spectrum of DP are conditions of GnRH deficiency including hypogonadotropic hypogonadism (HH), 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 HH with lack of pubertal development to a partial hypogonadism with an arrest of pubertal development, and even reversible HH in some patients after treatment [23,24]. Despite recent advances, with over 20 genes linked to this disorder identified, the pathophysiological basis of HH in approximately 50% of individuals remains unclear [2]. In view of the possible overlap between the pathophysiology of DP and conditions of GnRH deficiency, a few studies have examined the contribution of mutations in HH genes to the phenotype of self-limited DP.

Loss-of-function mutations within the GnRH receptor are the most frequent cause of autosomal recessive idiopathic HH, accounting for 16-40% of patients. Mutations have been found within the extracellular, transmembrane, and intracellular domains of the receptor, leading to impaired GnRH action [25]. A homozygous partial loss-of-function mutation in GNRHR was found in 2 brothers, 1 with self-limited DP and 1 with idiopathic HH [26], and a further heterozygous mutation was found in 1 male with self-limited DP [27]. A small cohort of 31 patients was analysed for mutations in GHSR, and 5 patients were found to have point mutations in this gene [28]. Additionally, mutations in HS6ST1, FGFR1, and newly in KLB have been found in a small number of kindreds of HH patients and their relatives with DP [29,30,31]. Recently, 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 [32]. However, these variants have not been tested in vitro or in vivo for pathogenicity and thus may be an overestimation. However, the current picture indicates that the genetic background of HH and DP may be largely different, or shared by as yet undiscovered genes [27].

Genetic defects affecting the development of the anterior pituitary may also 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 [33]. 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 [34]. Patients with PROP1 mutations have variable GnRH deficiency ranging from DP to congenital HH [33]. Mutations in DAX1 cause X-linked adrenal hypoplasia congenita with associated HH, but have not been found in cases of isolated DP [35].

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) [36], whilst those in RNF216, OTUD4, and PNPLA6 produce the phenotypic combination of HH and ataxia (also known as Gordon Holmes syndrome) [37,38]. DMXL2 mutations are associated with congenital HH, other endocrine deficiencies, and polyneuropathies [39]. Dysregulation of the RAB3 cycle, such as with mutations in RAB3GAP1, leads to Warburg Micro syndrome with ocular, neurodevelopmental, and central reproductive defects [40,41].

Downstream mutations in the GnRH signalling pathway can also present as DP. LH and FSH are glycoprotein hormones encoded by a common α-subunit gene and a specific β-subunit gene. Mutations of the β-subunit genes of LH or FSH are extremely rare causes of pubertal abnormalities [42]. 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 [42]. Individuals with inactivating FSHB mutations present with incomplete pubertal development and azoospermia if they are male, and with primary amenorrhoea if they are female [43].

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) [44,45]. Mice with knockout of Kiss1r were simultaneously discovered to be infertile despite anatomically normal GnRH neurons and normal hypothalamic GnRH levels [45], with a phenotype consistent with normosmic GnRH deficiency. However, to date, only very rarely have human mutations in KISS1 been found in patients with delayed or absent puberty [46].

The excitatory neuropeptide neurokinin B also plays a role in the upstream control of GnRH secretion. Identification of this pathway occurred 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 [47]. Kisspeptin, neurokinin B, and dynorphin are coexpressed in KNDy neurons of the arcuate nucleus of the hypothalamus [48], 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 [49,50]. However, administration of neurokinin B agonists failed to stimulate GnRH release in rodents, and Tacr3 knockout mice have grossly normal fertility [51,52]. Among 50 self-limited DP patients investigated for mutations in TAC3 and TACR3, only 1 mutation in a single patient was found in the latter gene [53].

The inhibitory role of GABAergic neurotransmission has been clearly shown in primates [54] 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 [55]. Additionally, RFamide-related peptide gene (RFRP), the mammalian orthologue of the avian peptide gonadotropin-inhibiting hormone (GnIH), has been identified as a further inhibitory regulator of GnRH neuronal activity in mice [56]. Glial inputs appear to be predominantly facilitatory during puberty and consist of growth factors and small diffusible molecules, including TGF-β₁, IGF-1, and neuregulins, that directly or indirectly stimulate GnRH secretion [57].

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 [58]. Oct-2 mRNA is upregulated in the hypothalamus in juvenile rodents, and blockage of Oct-2 synthesis delays the age at first ovulation, whilst activation of Oct-2 expression (e.g., hamartomas) induces precocious puberty [59]. TTF-1 (thyroid transcription factor 1) enhances GnRH expression, with increased expression in pubertal rhesus monkeys [60]. EAP1 mRNA levels also increase in the hypothalamus of primates and rodents during puberty. EAP1 transactivates the GnRH promoter, and EAP1 knockdown with siRNA caused DP and disrupted oestrous cyclicity in a rodent model [61]. However, to date no mutations in these upstream or regulatory factors have been reported in patients with DP.

Epigenetic regulators are potential mediators of the effects of the environment on the hypothalamic regulation of puberty. However, whilst experimental data from rats give evidence for changes in histone acetylation and gene methylation leading to altered gene expression during puberty, the link between environmental factors and epigenetic control of puberty via the hypothalamus has not been established. Recent evidence highlights the importance in mice of microRNAs (particularly the miR-200/429 family and miR-155) in the epigenetic upregulation of GnRH transcription during the critical period (murine equivalent of the mini-puberty) [62]. Epigenetic changes during fetal life are also a potential mechanism for the effects of endocrine disrupting chemicals in utero [63].

Nutritional changes play an important role in the observed secular trend towards an earlier age of pubertal onset in the developed world [64], as shown by the positive correlation between age at puberty onset and childhood body size, particularly in girls [65]. In contrast, undernutrition in females - for example, in chronic disease or anorexia nervosa - can result in delay in both the onset and the tempo of puberty [66].

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 [67]. Humans and mice lacking leptin (Lep ob/ob) or the leptin receptor (LepR db/db) fail to complete puberty and are infertile [68]. Genome-wide association 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 [18]. However, whilst self-limited DP in boys is associated with hypoleptinaemia [69], there have been no identified associations of specific leptin or leptin receptor polymorphisms with DP [70]. Ghrelin and other gut-derived peptides may also form part of the mechanism by which energy homeostasis regulates reproductive development [71]. Notably, children with constitutional delay of growth and puberty 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 [72], particularly in those children with a rapid increase in length or weight in the first 2 years of life [73]. It remains unclear, however, whether childhood obesity, insulin resistance, excess androgens, or underlying genetic or epigenetic factors may explain this association [74].

Loss-of-function mutations in a member of the immunoglobulin superfamily, IGSF1, have been identified in patients with X-linked central hypothyroidism [75]. 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 [76].

More recently, whole exome and targeted resequencing methods have implicated 2 pathogenic mutations in IGSF10 as the causal factor for late puberty in 6 unrelated families from a large Finnish cohort with familial DP [77]. A further 2 rare variants of unknown significance were identified in 4 additional families from the cohort. Mutations in IGSF10 appear to cause a dysregulation of GnRH neuronal migration during embryonic development (Fig. 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 mistimed 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.

This review serves to highlight the fascinating heterogeneity of genetic defects resulting in delayed and disordered puberty. Although our understanding of the highly complex underlying biological network remains imperfect, results to date have demonstrated 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 homeostatic derangements - in the control of pubertal timing.

Clinically the distinction in adolescence between the conditions of DP and congenital idiopathic HH is an important but difficult one. Both disorders can present with a picture of functional hypogonadotropism and in some cases may share an underlying pathophysiology. There remains no definitive test to accurately discriminate between the two diagnoses. More complex and involved management is required in patients with hypogonadism to achieve both development of secondary sexual characteristics and to maximize the potential for fertility [78]. A rapid and efficient diagnosis of patients in the 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.