A wide-ranging look at puberty and the many mysteries that surround it as well as the clinical management, new genetic tools, and how a variety of factors could influence the timing of this transition into adulthood.
In 2005, the editors of Science, celebrating the journal’s 125th anniversary, posed 125 unanswered questions that it predicted would fuel active research in the following century. High on the list of mysteries was “what triggers puberty?” Indeed, this question has perplexed clinicians, scientists, and parents for centuries.
Though we still lack a clear answer, technological and theoretical advances over the past 10 years have gotten us closer than we have ever been to a solution. Indeed, next generation sequencing approaches have uncovered genes never before implicated in the neuroendocrine control of puberty. At the same time, our understanding of gene regulation beyond the DNA sequence, so called epigenetics, has revealed insights into molecular switches in the brain that both silence and activate activity of GnRH and associated neurons.
In this Tri-Point, a pediatric endocrinologist discusses the discovery of novel genetic causes of both delayed and precocious puberty. Next, two clinician-scientists describe how new genetic tools have uncovered a suite of genes regulating the development and function of GnRH neurons. Finally, a pair of basic scientists describe new advances in our understanding of epigenetic regulation of GnRH secretion, shedding light on how genetic polymorphisms, nutrition, and environmental exposures may affect the timing of puberty.
A Clinical Practitioner’s Perspective
- Congenital hypogonadotropic hypogonadism is genetically heterogeneous (>25 different genes) with several modes of inheritance.
- Loss-of-function mutations of MKRN3 gene represent a frequent cause of familial central precocious puberty (40% of the studied cases).
- Genetic testing is useful for diagnosis, prognosis, and genetic counseling in human pubertal disorders.
Pubertal timing is influenced by complex interactions among genetic, nutritional, environmental and socioeconomic factors. The timing and onset of puberty varies widely in the general population. Early or delayed timing of puberty in girls or boys is associated with increased risks for several adverse outcomes across a range of oncogenic, cardiometabolic, gynecological or obstetric, gastrointestinal, musculoskeletal, and neurocognitive conditions. Frequently, my patients with distinct pubertal disorders have questioned me as to why do they have this problem. Fortunately, understanding of the molecular genetics of human pubertal disorders, such as congenital hypogonadotropic hypogonadism and central precocious puberty, has advanced tremendously in the past 20 years, and I have been able to give them some of the answers.
Congenital Hypogonadotropic Hypogonadism
Congenital Hypogonadotropic Hypogonadism (CHH) also known as isolated Gonadatropin Releasing Hormone (GnRH) deficiency, (IGD) is genetically heterogeneous, with both sporadic and familial cases. Several modes of inheritance were identified in this condition, including X chromosome-linked recessive, autosomal recessive, and dominant. To date, more than 25 different genes have been implicated in CHH; in patients with CHH, genetic testing is useful for diagnosis, prognosis, and genetic counseling. Inheritance pattern and presence of additional phenotypic features such as anosmia, cleft lip or palate, dental agenesis, ear anomalies, congenital hearing impairment, renal agenesis, bimanual synkinesis, skeletal anomalies or early onset of morbid obesity might guide genetic testing.
Combining CHH with specific associated phenotypes can increase the probability of finding causal mutations by targeted gene sequencing. However, oligogenic forms (mutations in several genes, <6) have been identified in CHH. Genetic counseling can be difficult and the transmission risk may be variable when CHH patients carry several mutations in different genes. More recently, we have used pooled targeted sequencing of known genes using next-generation DNA sequencing technology for better and complete genetic diagnosis of CHH. Notably, reversibility of CHH can occur in both male and female cases (approximately 10% – 15%). This phenomenon highlights the importance of environmental (epigenetic) factors such as sex steroid treatment on the reproductive axis in modifying the phenotype. Interestingly, rare loss-of-function mutations in IGSF10, a gene that codifies a factor involved in embryonic migration of immature GnRH neurons, were recently identified in several unrelated families with self-limited delayed puberty using exome and candidate gene sequencing, indicating the role of genetic factors in a wide spectrum of conditions characterized by delayed puberty, from self-limited delayed puberty to functional or permanent hypogonadotropic hypogonadism.
Central Precocious Puberty
I have always been surprised by the high prevalence of idiopathic Central Precocious Puberty (CPP) cases, especially in girls. The evidence of familial CPP (up to 20%) as well as my previous work in genetic causes of peripheral precocious puberty, such as testotoxicosis, stimulated my research group to investigate the potential role of genetic factors in these families. Recently, using whole-exome sequencing, we identified a gene, MKRN3, in the premature reactivation of GnRH secretion leading to CPP in nonsyndromic children. After this discovery, a growing list of loss-of-function mutations of MKRN3 was described in patients with CPP from both sexes and different ethnic backgrounds. Indeed, these mutations now represent a frequent cause of familial CPP (around 40% of the studied cases). MKRN3, an imprinted gene located on the long arm of chromosome 15 (Prader Willi critical region), encodes makorin ring finger protein 3, which is the first factor with an inhibitory effect on GnRH secretion. MKRN3 protein is derived exclusively from RNA transcribed from the paternally inherited copy of the gene due to maternal imprinting. Segregation analysis of the families with CPP due to MKRN3 defects clearly demonstrated an autosomal dominant inheritance with complete penetrance. Because of the imprinting pattern (maternally silenced) of MKRN3, the CPP phenotype can be inherited from an asymptomatic father who carries a MKRN3 defect.
“I have always been surprised by the high prevalence of idiopathic Central Precocious Puberty (CPP) cases, especially in girls.”
Our studies showed that the familial nature of CPP is likely under-recognized due to the difficulty of obtaining a precise family history from the father’s side and the likelihood for under diagnosis of early testicular enlargement. Interestingly, the identification of the MKRN3 loss-of-function mutations as a cause of CPP has impacted current clinical investigation of this common pediatric condition. For instance, routine screening by brain MRI is not useful in patients with a clear family history, such as two siblings with CPP. In these familial cases, genetic studies should precede brain MRIs, which might be postponed (in non-mutant cases) or even avoided in those patients with identified loss-of-function MKRN3 mutations. In addition, the financial costs to perform the genetic analysis of MKRN3, an intronless gene, are significantly lower than a brain MRI scan in children, who usually need anesthesia support.
Finally, next generation sequencing analysis, including pooled target sequencing, whole-exome, or whole-genome sequencing, will improve the molecular genetics diagnosis of human pubertal disorders, promoting earlier diagnosis and personalized approaches to genetic counseling. The clinical use of next generation sequencing is expected to increase rapidly in the coming years with decreasing costs, increasing availability, and high diagnostic yield. New genetic findings affecting regulatory genomic regions, microRNAs, or methylation sites (epigenetic phenomena), leading consequently to abnormal gene expression, are also expected to be discovered as causes of human pubertal disorders in the near future.
Clinical Scientists’ Perspective
- Genetic studies in humans with IGD have provided unprecedented insights into the genes and pathways that govern GnRH neurogenesis, migration, secretion and function.
- The advent of next-generation sequencing will hasten the genetic discovery process and likely to unravel novel insights into the genetic and epigenetic control of puberty.
- These discoveries will provide opportunities to translate these findings to help diagnosis and therapy of both rare and common reproductive conditions.
GnRH Neurons: The Master Regulator of Human Puberty
Developmentally, a series of dynamic and complex genetic networks oversee the ontogeny of the GnRH neurons and their ability to coordinate the hypothalamic secretion of GnRH that eventually triggers puberty and sustains the reproductive axis throughout adulthood. Despite growing understanding of the genes controlling the physiology of GnRH’s secretion and action, the full constituency of the genetic and epigenetic networks that are responsible for the pubertal transition remains incomplete. Given the evolutionary importance of reproduction in speciation, these pathways must have had to continually adapt and evolve to overcome the myriad of powerful adverse environmental challenges that each organism must have faced.
The need for such tightly regulated controls argues strongly for the presence of a highly conserved, multi-tiered network of neurodevelopmental and neuroendocrine genes controlling GnRH. In keeping with this notion, the study of humans with Isolated GnRH deficiency [IGD] (also referred to in the literature as congenital hypogonadotropic hypogonadism), representing a “human knock out” model for GnRH deficiency, has been prismatic in unraveling the fascinating ontogeny of GnRH neurons. Combined with the study of animal models of hypogonadotropic hypogonadism in which interventions can be induced, research over the last three decades has enabled an explosion in the discovery of novel genes governing puberty.
Developmentally, a series of dynamic and complex genetic networks oversee the ontogeny of the GnRH neurons and their ability to coordinate the hypothalmic secretion of GnRH that eventually triggers puberty and sustains the reproductive axis throughout adulthood.
Genes and Pathways that Govern Puberty in Humans
The initial insights into the genetic control of puberty came from the study of humans with Kallmann’s Syndrome (KS). KS patients display complex syndromic phenotypes and were subsequently found to harbor deletions and mutations in the KAL1 gene (now called ANOS1). A series of fascinating clinical investigative studies in these patients subsequently confirmed what had previously been demonstrated in fetal mice by Donald Pfaff and his colleagues at Rockefeller University: GnRH neurons had embryonic origins from outside the central nervous system, emanating from the olfactory placode! Following these initial seminal observations, >25 genes have been shown to cause IGD, each of which either disrupts embryonic migration of GnRH neurons (neurodevelopmental genes, causing the anosmic form of IGD or KS) or affects GnRH secretion/action (neuroendocrine genes, causing the normosmic form of IGD [nIGD]) or play a role in both migration as well as function (overlap genes, causing either KS or nIGD or in some cases both phenotypes in a single family).
To uncover the above genes, clinical investigators have used several complementary approaches:
- homozygosity mapping in endogamous pedigrees (KISS1, KISS1R, TAC3, TACR3);
- mapping genes in genomic alterations/contiguous gene syndrome/copy number changes (FGFR1, WDR11, SEMA3A);
- bioinformatic pathway approaches (IL17RD, FGF17, DUSP6, FLRT3, SPRY4);
- candidate gene approaches (GNRH1, GNRHR, FGF8, PROK2, PROKR2, NSMF);
- study of syndromic IGD phenotypes (CHD7, SOX10, HS6ST1, LEP, LEPR, PCSK1, NROB1, RNF216, OTUD4, TUBB3, POLR3A, POLR3B, PNPLA6, DMXL2); and
- more recently exome sequencing +/- homozygosity mapping (FEZF1, CCDC141, SEMA3E).
Translation of Genetic Findings from IGD: Bench to Bedside
These discoveries have not only shed remarkable new light on the origins and functions of GnRH neurons that could not have been obtained in other ways, but, perhaps more importantly, they have initiated novel ‘therapeutic cascades’ that ultimately may lead to new diagnostic and therapeutic opportunities. In particular, two novel hypothalamic neuropeptide signaling systems (kisspeptin and neurokinin B), now known to lie upstream of GnRH and govern its secretion, are at the forefront. Promising investigations to determine kisspeptin’s potential diagnostic/therapeutic role in several reproductive disorders are currently underway with several pharmaceutical firms beginning to develop kisspeptin analogues. Likewise, identification of novel neurodevelopment genes encoding the neural crest pathway (SOX10, CHD7) have begun to improve our fundamental understanding of the embryonic origins of GnRH neurons in ways that were not possible prior to the The Human Genome Project. Collectively, these studies now suggest a contribution from the neural crest cells in addition to the known contribution from the placodal cells.
Genetic Crossroads: Next-Generation Sequencing, Genome-Wide Association Studies and the Role of Epigenetics in Puberty
In addition to the known Mendelian inheritance of IGD, a previously unappreciated genetic feature of IGD has also emerged. A systematic search of eight IGD genes in nearly 400 IGD cases showed that ~3% of these cases unexpectedly harbored mutations in >2 known IGD genes, thus establishing the presence of ‘modifying genes’ or oligogenicity as a novel characteristic of IGD’s genetic architecture. Fascinatingly, as opposed to most Mendelian mutations that are unique or ‘private,’ some mutations that occur in IGD patients are recurrent, occurring in IGD individuals from different geographical regions and varied ancestry. More detailed studies of the origin of these recurrent mutations have revealed that some represent ancient founder alleles. For example, one recurrent PROKR2 mutation (L173R) represents a 9,000-year-old loss of function mutation in this reproductive controlling gene! These founder alleles argue for the possibility of variants contributing to some as-yet-unknown survival benefit and consequently able to undergo positive selection across populations and time. In some cases, KS and normosmic IGD occur within a single family sharing a similar genetic milieu. In other rare instances, monozygotic twins had been known to be discordant for IGD, suggesting that additional genetic, epigenetic, or environmental factors may also influence the expression of the IGD phenotype. The role of epigenetics is also suggested from the fact that some IGD patients with known mutations may recover spontaneously later in life, signaling a reversal of GnRH deficiency.
Despite this remarkable progress, it is clear that several novel genes remain to be discovered and the collaboration between clinicians seeing these patients and clinical investigators investigating their genetics will remain crucial in the future. The growing genetic, clinical, and molecular complexity of IGD clearly warrants an integrated investigatory approach that combines the deep phenotyping of IGD patients, the use of emerging next generation sequencing (NGS) to aid novel gene discovery, and the use of bioinformatics to expand the biology of these evolving genes and their related signaling pathways. With reducing sequencing costs and increasing access to whole exome and whole genome technologies, further novel genes, potential regulatory and non-coding variants, and epigenetic modifications that cause IGD are likely to be discovered.
However, these approaches will also lead to a surge in data and the need for prioritization and functional validation, which are two key challenges to avoid false positive associations. Finally, several of the Mendelian genes that govern puberty are also being detected via genome-wide association studies (GWAS) of common reproductive complex traits in large populations. The results of these studies suggest that the genetic control of reproduction represents a continuum ranging from common variants with low effect size that underlie many common reproductive conditions (such as hypothalamic amenorrhea) to rare variants with high effect size that result in extreme phenotypes such as KS. The precise biological basis of the onset of human puberty has been cited as one of the critical unanswered scientific questions in the 21st century. The prismatic human model of IGD and concurrent advances in genomic technologies now provide an unprecedented opportunity to unravel the black box of puberty.
Basic Researchers’ Perspective
- The timing of puberty is controlled by epigenetic mechanisms affecting the activity of hypothalamic neurons required for puberty to occur.
- The epigenetic control of puberty depends on a balance between transcriptional repression and activation of gene expression.
- The repressive control of puberty is provided by the Polycomb group of transcriptional silencers that restrains gene expression directly, and by Zinc finger proteins that reduce gene expression by antagonizing the effect of the Trithorax group of transcriptional activators.
- The transcriptional activation of puberty is provided by the Trithorax group of transcriptional activators, which counterbalances the repressive effect of the Polycomb group.
By identifying genes required for puberty, genetic approaches have led to magnificent breakthroughs in understanding the pubertal process in humans. Genome-wide association studies have, however, hinted at a much greater complexity by demonstrating the existence of more than 100 polymorphisms associated with the timing of puberty in human females. And yet, all of these modifications in DNA sequence together explain less than 5% of pubertal disorders, including idiopathic precocious puberty and delayed puberty. The inescapable conclusion is that information other than that provided by DNA sequence must play an important role not only in the etiology of pubertal disorders, but also in defining normal variability in the timing of puberty. At the risk of oversimplification, we submit that epigenetics provides the most robust means of integrating cues and coordinating gene networks involved in the neuroendocrine control of puberty.
Epigenetic Control of Gene Regulatory Networks
The concept that epigenetics play a major role in the neuroendocrine process controlling the timing of puberty has been established in the last few years. Work from other fields provides unquestionable support for the idea that entire gene networks are subject to epigenetic regulation, and that this regulation is furnished by a myriad of molecules. These include enzymes that modify the methylation status of DNA, and others that post-translationally modify histones in regulatory genomic regions. More recently, the category of epigenetic regulators has been expanded to include a vast array of microRNAs, long noncoding RNAs, and methyl adenosine-modified messenger RNAs. It is also now clear that complex biological processes are controlled by a diversity of genes that operate within the constraints of a hierarchy.
The inescapable conclusion is that information other than that provided by DNA sequence must play an important role not only in the etiology of pubertal disorders, but also in defining normal variability in the timing of puberty.
Epigenetic Changes Associated to Puberty
During normal peripubertal development, an increase in GnRH secretion is accompanied by reduced DNA methylation of the GnRH promoter. Another epigenetic change that appears to occur as puberty progresses is the deposition of “activating” histone marks at the promoter of the Kiss1 gene in kisspeptin neurons involved in mediating the positive feedback of estrogen on gonadotropin secretion.
Epigenetic Control of the Timing of Puberty
The epigenetic control of the timing of puberty involves lifting of a repressive tone exerted by the Polycomb group (PcG) of transcriptional silencers on the Kiss1 gene expressed in neurons of the hypothalamic arcuate nucleus (ARC), known as KNDy neurons because they produce the peptides kisspeptin, neurokinin B and dynorphin. KNDy neurons are directly involved in the control of pulsatile GnRH release, which increases in a diurnal fashion with the advent of puberty. We also observed that these changes in PcG-mediated repression are complemented by post-translational histone modifications catalyzed by, or associated with, the Trithorax group (TrxG) of transcriptional activators. The presence of both systems of epigenetic regulation in KNDy neurons suggests that a switch from epigenetic repression to activation within these neurons underlies the developmental process by which pulsatile GnRH release is first kept in check before puberty, and then increases by late juvenile development to bring about the pubertal process.
An additional layer of repressive control appears to prevent the precocious activation of the GnRH pulse generator that could result from premature loss of PcG-mediated repression or an untimely surge of TrxG-dependent transcriptional activation. This repressive effect is exerted by members of a large family of more than 800 transcriptional repressors known as zinc finger proteins (ZNFs) because they contain multiple zinc finger motifs. Studying one of these proteins, GATAD1, suggested that ZNFs may keep puberty in check by promoting the erasure of TrxG-dependent histone marks associated with gene activation. GATAD1 exerts this effect on the promoter of downstream genes required for puberty to occur (such as KISS1 and TAC3). Because this inhibitory influence decreases at puberty, it seems reasonable to conclude that once ZNF-dependent interference is lifted and PcG-mediated repression is lost, transcriptional activation can proceed unimpeded, setting in motion the pubertal process.
Though exciting, these studies represent only the initial steps in the quest to unravel the role of epigenetics in the regulation of puberty. We envision that in the near future, new studies will identify epigenetic pathways linking alterations in nutritional availability, circadian cues, and man-made environmental toxins (including endocrine disruptors) with the timing, progression, and completion of the pubertal process. Furthermore, we predict that in a not-too-distant future, alterations in epigenetic regulation will be uncovered as an underlying cause of the intractable disorders of idiopathic precocious puberty and constitutionally delayed puberty.