Lauren C. Passby1 , Kavitha Rozario2 and Jyothis T. George3, 4
Medical Sciences Division, University of Oxford, John Radcliffe Hospital, 43 Woodstock Road, Oxford, OX2 6HG, Oxfordshire, UK
Department of Paediatric Endocrinology, Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Headley Way, Oxford, OX3 9DU, UK
Royal Berkshire Hospital, Reading, UK
Boehringer Ingelheim, Bracknell, UK
Lauren C. Passby
Kavitha Rozario (Corresponding author)
GnRH pulsatilityKallmann syndromeNormosmic idiopathic hypogonadotropic hypogonadismOligogenicityCentral precious pubertyConstitutional delay of puberty
Timely upregulation of gonadotropin-releasing hormone (GnRH) pulsatility is the fundamental neuroendocrine process underpinning puberty. GnRH is released from a population of approximately 1500 sparse and widely distributed hypothalamic neurons located predominantly in the median eminence of the hypothalamus. Input from rostral periventricular area of the third ventricle (RP3V) neurons controls GnRH secretion from nerve terminals into the hypophyseal portal circulation, which delivers the hormone to its cognate receptor located on the gonadotrope cells of the anterior pituitary. Binding of GnRH to its receptor stimulates the synthesis and release of luteinising hormone (LH) and follicle-stimulating hormone (FSH) , which in turn regulate sex hormones from the gonads.
The ‘Mini-Puberty ’
In the first and second postnatal weeks, the GnRH pulse generator is disinhibited, resulting in the ‘mini-puberty’ of infancy. In males, this peaks between weeks 4–10 postnatally and is predominantly LH driven, before becoming quiescent by 6 months. In females, this mini-puberty is longer, with GnRH secretion remaining active for up to 3 years.
In males, this mini-puberty is thought to facilitate expansion of the Sertoli cell population, with a subsequent increase in germ cell numbers. Exposure to sex steroids during this period potentially has important immediate and future developmental consequences, such as in phallic development and testicular descent—microphallus/micropenis and cryptorchidism are biological sequelae in which this brief window of gonadal axis activation is defective (as in isolated GnRH deficiency). Following the end of this mini-puberty, childhood is a period of reproductive quiescence, wherein there are very low amplitude secretions of GnRH, gonadotropins and sex steroids.
The commencement of puberty is controlled by reactivation of the pulsatile release of GnRH. The onset of puberty is characterised in boys by testicular enlargement, penile growth and the development of pubic hair, typically occurring between the ages of 9 and 14. In girls, puberty usually takes place between the ages of 8 and 13 and begins with thelarche—breast bud development, pubic hair growth and menarche.
This reawakening of the hypothalamic-pituitary-gonadal (HPG) axis heralds the start of puberty, though the precise genetic basis of the onset of this pulsatile GnRH release remains one of human biology’s greatest mysteries. The prismatic model of human GnRH deficiency presents an opportunity to unravel molecular mechanisms and to better understand the arousal of the GnRH pulse generator that signals puberty. The study of individuals with GnRH deficiency has made it possible to untangle some components of the neuroendocrine network, generating improved understanding of the ontogeny of GnRH-releasing hypothalamic neurons (Fig. 12.1).
Functional categorisation of defective pubertal development
GnRH deficiency has numerous genetic causes and, correspondingly, numerous possible phenotypes. Functionally, GnRH deficiency can be classified into three categories, which are discussed below.
Upstream Inhibition of GnRH Release
Mutations affecting the one or several of the upstream regulatory pathways of GnRH can result in hypogonadotropic hypogonadism.
Linkage analysis of consanguineous families facilitated the discovery of mutations in GPR54  (now known as KISS1R), a G protein-coupled receptor, and its endogenous ligands: kisspeptins. Kisspeptins stimulate gonadotropin release from hypothalamic neurons, thereby functioning as upstream gatekeepers to GnRH release. Defects in this pathway can thus result in GnRH deficiency. A number of mutations in the KISS1R gene have been reported in individuals with normosmic idiopathic hypogonadotropic hypogonadism (nIHH) .
Mutations in KISS1 (the gene encoding kisspeptin) have been reported in individuals with a functional GnRH deficiency, though mutations have also been found in control populations in whom there is no evidence of pubertal failure (suggesting a role for other genes in contributing to the GnRH-deficient phenotype) . KISS1/KISS1R mutations represent less than 5 % of cases of GnRH deficiency, possibly reflecting the evolutionary importance of this pathway in initiating reproductive capacity.
Further study of consanguineous families with nIHH led to the discovery of mutations in the gene encoding neurokinin B (TAC3) and its receptor (TAC3R) . Male individuals with such mutations typically have a micropenis and fail to undergo puberty, implicating a role for neurokinin B signalling in the ‘mini-puberty’ of infancy as well as the initiation of puberty at adolescence. There is some evidence of HPG axis functional recovery during adulthood in nIHH patients with TAC3/TAC3R mutations , suggesting that the primary role for this pathway is during the neonatal period and puberty, with a reduced dependence upon this pathway in adulthood.
Adult-onset inhibition of GnRH secretion can also be seen in individuals with hypothalamic amenorrhoea (HA) . This is a functionally reversible deficiency occurring in otherwise normal females, which is precipitated by stress, undernutrition and/or over-exercise in females. Upon removal of the precipitant, GnRH secretion is resumed, and periods are restored. Whilst the role of the above-mentioned environmental factors on HA is well documented, recent studies have also advocated a role for an underlying genetic susceptibility in this mildest form of functional GnRH deficiency .
Defective GnRH Neuron Development
KAL1 was the first human gene to be identified in patients with Kallmann syndrome (KS) —a syndrome of hypogonadotropic hypogonadism with anosmia [7, 8]. The KAL1 gene comprises 14 exons, which produce glycoprotein anosmin-1. Anosmin-1 is required for the formation of the olfactory guidance platform that facilitates GnRH-secreting neuronal migration embryologically. Anosmin-1 plays a key role in axonal elongation of the intracerebral olfactory tract, as well as in attracting olfactory axons towards the forebrain. There is subsequent migration of GnRH neurons along this tract. This explains how a lack of Anosmin-1 in KS patients carrying KAL1 deletions or mutations results in a loss of this trajectory and a failure for GnRH neurons to reach the hypothalamus. Deletions and mutations in KAL1 account for 10–14 % of familial KS and 8–11 % of sporadic KS .
Some patients with KS and nIHH have been found to have loss-of-function mutations in fibroblast growth factor receptor 1 (FGFR1 or KAL2) . There is variation in the specific effects that different mutations have some impair expression of the receptor , whereas others impair the ligand-receptor interaction. Mutations in FGFR1 account for up to 10 % of cases of both KS and nIHH.
Following identification of mutations in FGFR1, subsequent mutations in the ligands for this receptor have been found in patients with KS/nIHH. Fibroblast growth factor 8 (FGF8) binds FGFR1, and decreased FGF8 signalling has been shown to cause a deficiency of GnRH in humans and mice , with mutations in FGF8 being found in patients with GnRH deficiency. FGFR1 is also known to be associated with anosmin-1, offering further evidence of the oligogenic nature of GnRH deficiency.
In mice, NELF (nasal embryonic LHRH factor) encodes a guidance molecule which has been shown to be associated with the guidance of olfactory and GnRH neuronal axons . A unique rare sequence variant in NELF has been reported in human GnRH deficiency, though the biology of NELF remains unclear . Given the role that this gene plays in mice, there is reason to believe that alterations in gene may contribute to GnRH deficiency in humans.
Another gene PROK2 and its receptor PROKR2 are known regulators of the GnRH system, and loss-of-function mutations have been found in PROK2 and PROKR2 in individuals with KS/nIHH . As with FGF8/FGFR1mutations, there is considerable phenotypic heterogeneity, and variable penetrance, further suggesting oligogenicity to underlie GnRH deficiency.
Defective GnRH Signalling
Mutations affecting GnRH signalling—either its production or its actions at its receptor—can also result in KS/nIHH .
GNRH1 is located on chromosome 8 and encodes the preprohormone that is sequentially processed to produce GnRH. The mouse hypogonadal (hpg) model carries a homozygous deletion of GNRH1 and has hypogonadotropic hypogonadism. Supporting a role for pulsatile GnRH release in the ‘mini-puberty’ of early infancy, male patients with homozygous frameshift GNRH1 mutations have microphallus . Individuals carrying heterozygous mutations have also been reported, in whom there is phenotypic heterogeneity . The presence of a GnRH-deficient phenotype in these individuals may be dependent upon the synergistic action of other rare variant alleles (oligogenicity) that, when present together, result in a hypogonadotropic hypogonadal phenotype.
Mutations in GNRHR, the gene encoding the receptor of GnRH, were first documented in patients with nIHH . The effects of these mutations range from a reduction in ligand affinity to impairment of downstream signal transduction. GNRHR mutations account for up to 40 % of familial cases of nIHH and around 17 % of sporadic cases . Reproductive symptoms are heterogeneous implicating oligogenicity underlying the pathology, as well as implicating a role for epigenetic changes in response to environmental factors.
Rationale for Genetic Underpinning of Puberty Timing
A Role for Genetics in Pubertal Timing
Timing of pubertal onset is a normally distributed trait. Other complex traits, such as height, show similar distributions and are known to be genetically influenced—which makes it likely that pubertal timing is heritable. Furthermore, several genes that play a role in puberty have been identified in individuals with pubertal disorders, suggesting that there is a strong genetic influence in determining pubertal timing.
Mothers and daughters tend to be of a similar age at time of menarche (which, whilst not the first indication of the onset of puberty, is a clear milestone in female development), despite there being a regression towards the mean in daughters (potentially due to the combined effects of genetics and important environmental influences).
Whilst the pedigrees of families carrying mutations that result in GnRH-deficient phenotypes are characteristically and expectedly small, studies of families situated at the other end of the pubertal-timing normal distribution curve—those with central precocious puberty (CPP) —have shown evidence for familial inheritance of the condition, thus a clear role for genetics in determining the precocious onset of puberty.
A Role for Environmental Factors in Pubertal Timing
Studies in migrant children have shown an increased incidence of CPP in girls migrating for foreign adoption in several Western European countries . This mirrors a secular trend seen in the USA, where the age of menarche decreased from 17 in the mid-nineteenth century to less than 14 by the mid-twentieth century. This occurred in response to improved living conditions, smaller family sizes, decreased infectious disease and better nutrition and may offer the explanation to the observed phenomenon in migrant children.
Precocity in migrant children may be linked to improved nutrition and catch up growth priming maturation for puberty, though CPP is also seen in some non-deprived migrating children. Comparison of CPP in adopted migrating children and non-adopted migrating children (who migrated with their families) showed similar increased frequencies of precocity, suggesting that factors related to migration itself—such as removal of stressful former living conditions or nutritional changes—may be related to precocity. Geographical factors such as altitude, temperature, humidity and lighting levels are known to impact upon various neuronal networks and may similarly feed in to disrupt the neuroendocrine axis that triggers the initiation of puberty. A role for psychological factors also cannot be excluded, though it is difficult to quantify the effects of any of the stresses (both psychological and physical) encountered in these chronic situations.
Intrauterine growth retardation (IUGR) is known to have several effects on development throughout later life, so it is possible that this may also impact upon pubertal timing. Whilst it is convenient to postulate that many of the adopted children may have experienced IUGR due to being conceived in nutrient-deprived areas, there is a dearth of data to support such claims.
The study of migrant children permits for environmental and peripheral signals , which otherwise play minor modulatory roles in the timing of pubertal onset, to be studied in specific situations wherein their effects may play a crucial role in the onset of puberty. Though focused upon CPP, it is easy to see how nutrition and the environment can have important effects on the timing of puberty, alongside the actions of genes.
Studies into the timing of menarche, and twin studies, offer strong evidence for a genetic input into pubertal timing and provide further support for the claim that 50–80 % of the variation in pubertal onset can be attributed to genetic influence. At the same time, migration studies in populations in whom genetics remain unchanged have shown marked environmental effects upon the timing of puberty, which should not be underappreciated when investigating individuals with pubertal delay.
Genetics of Pubertal Delay
Pubertal delay arises when there is a delay in the reawakening of GnRH secretion. The most severe form of this is a total absence of GnRH secretion from the mini-puberty of infancy (resulting in micropenis/cryptorchidism ) as well as a failure to spontaneously commence puberty. There is, however, a phenotypic spectrum of GnRH deficiency, resulting in heterogeneity in the signs and symptoms that individuals may present with. Baseline clinical presentations may vary in both sexes with respect to the presence or absence of anosmia; the degree, severity and timing of reproductive defects; patterns of endogenous GnRH secretion; and critical presence or absence of other, nonreproductive defects.
Constitutional delay of puberty (CDP) is the mildest defect in GnRH secretion and is defined as the failure to initiate puberty beyond 2SDs of the population mean age. Individuals spontaneously enter puberty eventually and characteristically remain reproductively normal thereafter. This phenotypic ‘recovery’ is in contrast to individuals with more complete forms of GnRH deficiency, in which there is a failure to initiate or complete puberty, resulting in either Kallmann syndrome (KS) or normosmic idiopathic hypogonadotropic hypogonadism (nIHH) .
Kallmann syndrome describes isolated GnRH deficiency accompanied with anosmia (lack of sense of smell), whereas nIHH describes normosmic idiopathic hypogonadotropic hypogonadism, in which the sense of smell is preserved.
Adult-onset idiopathic hypogonadotropic hypogonadism describes a cessation of GnRH secretion following the completion of sexual development and puberty and typically responds to exogenous GnRH administration—demonstrating the hypothalamic nature of the defect. The defect in adult-onset idiopathic hypogonadotropic hypogonadism has been shown to be permanent, in contrast to the reversibility of hypothalamic amenorrhoea (HA) .
HA represents a functional reversible deficiency that occurs in otherwise normal adult females and is the most common reproductive deficiency. Defects in GnRH secretion are precipitated by stress, undernutrition and/or over-exercise in females who otherwise underwent normal sexual and pubertal development, with complete recovery upon removal of the precipitant.
Nonreproductive features may also be present in patients presenting with pubertal delay or failure, which may serve to provide clues as to the underlying genetic defect. Well-described associations include:
· Craniofacial defects (cleft lip/palate, high arched palate, coloboma, choanal atresia)
· Renal agenesis, horseshoe kidney and GU duplications (e.g. bifid ureter)
· Skeletal defects
· Digital anomalies (short fourth metacarpals, campylodactyly, syndactyly, clinodactyly)
· Sensorineural deafness
· Dental agenesis
· Oculomotor abnormalities
· Bimanual synkinesis
· Cerebellar ataxia
The diversity in the nonreproductive features that may accompany GnRH deficiency is important to recognise, and detailed phenotyping of such patients is important in the targeting of genetic testing .
Given the diversity in the phenotypes seen in individuals with GnRH deficiency , it is clear that numerous genetic defects can contribute towards GnRH deficiency. Due to the variable degree of infertility that GnRH deficiency impacts upon individuals, most patients with GnRH deficiency present with sporadic genetic mutations. The genetic basis for 40–50 % of cases of GnRH deficiency is now known, though each of the genes identified individually account for a small percentage of cases. The genes found are involved in various functions and have been described above.
· Genes affecting GnRH development and migration (KAL1, NELF)
· Neuroendocrine genes (GNRH1, GNRHR, KISS1, KISS1R, TAC3, TACR3)
· Genes with neurodevelopment and neuroendocrine functions (FGF8, FGFR1, PROK2, PROKR2) (Fig. 12.2)
Genetic causes of isolated GnRH deficiency : a historical perspective. Genetic aetiology of isolated GnRH deficiency with relative percentage contribution from each identified gene to the heritability of the syndrome from 1995 to 2011. From only two genes that were known in 1995, the number of genes discovered in subjects with isolated GnRH deficiency has steadily increased through 1995–2011. In 2011, the genetic cause of a nearly half of the subjects is known, whilst in the remaining half, the genes are yet to be identified (Data from the Harvard Reproductive Endocrine Sciences Center, Massachusetts General Hospital, Boston, MA) (Reprinted from Balasubramanian R, Crowley WF. Isolated GnRH deficiency: A disease model serving as a unique prism into the systems biology of the GnRH neuronal network. Molecular and Cellular Endocrinology 2011; 346(1–2): 4–12. With permission from Elsevier)
What remains unclear with the as yet-undiscovered genes is whether they will map on to already-known pathways of GnRH neuronal development and function or whether entirely novel pathways will be uncovered.
Making Use of Phenotypic Clues
Phenotypic clues can give hints to the specific underlying genetic mutations in conditions such as nIHH/KS. The incomplete penetrance of many of the identified genes involved in KS and nIHH emphasises the need to detect underlying genetic anomalies when offering genetic counselling to families, and use of specific phenotypic clues can be a cost-effective means of directing this process. When penetrant, there is a tendency for mutations in genes that are responsible for early embryonic development to manifest as KS (e.g. KAL1, FGF8, FGFR1, NELF, CHD7, PROK2 and PROKR2), whereas genes controlling GnRH and its actions in puberty (e.g. KISS1, KISS1R, GnRH1, GnRHR, TAC3, and TAC3R) tend to present as nIHH. Furthermore, there are complex syndromic presentations of GnRH deficiency that are associated with particularly genes, such as LEP/LEPR (leptin and leptin receptor, respectively, giving nIHH associated with severe obesity) and CHD7 (chromodomain-helicase-DNA-binding protein 7, associated with CHARGE syndrome), highlighting how the genetic heterogeneity underlying GnRH deficiency manifests in a similar heterogeneous fashion.
Leptin (LEP) and signalling through its receptor (LEPR) are known to play a key role in stimulating GnRH release from the hypothalamus. Leptin is a hormone produced by adipose tissue that influences the HPA axis and its downstream effects on satiety and energy expenditure. Mutations in the leptin gene result in early obesity, whilst individuals with mutations in the leptin receptor exhibit this early onset obesity as well as a failure of pubertal development . This reflects a widespread reduction in pituitary function, which extends to reduced growth hormone and thyrotropin secretion, generating a distinct clinical picture for this cause of nIHH.
Mutations in DAX1 have been found in patients with X-linked adrenal hypoplasia congenital (AHC) and nIHH . DAX1 encodes an orphan nuclear receptor that is expressed in the adrenal cortex, gonads, hypothalamus and anterior pituitary. Whilst the adrenal failure is clinically apparent at or shortly after birth, the hypogonadotropic hypogonadism of this X-linked form of AHC is often not recognised until the expected time of puberty. Mouse studies and case reports have further indicated that DAX1 plays a role in spermatogenesis, adding further clues that can be investigated when trying to determine the genetic basis of a case of nIHH.
SOX10 is a transcription factor in which mutations are associated with Waardenburg syndrome (WS) . WS is a rare disorder characterised by pigmentation and deafness, though may also include olfactory agenesis. Loss-of-function mutations of SOX10 have been found in approximately one third of individuals with Kallmann syndrome with deafness, indicating a clear role for this gene in this particular phenotype . In patients presenting with Kallmann syndrome and deafness, there is thus the rationale to look for genetic mutations on and around the region of the SOX10 gene.
A relationship between FGFR1 mutations and split hand/foot malformation (SHFM) has also been in a small patient sample . A particular mutation in the receptor was found to be associated with GnRH deficiency and SHFM in patients with CHH both Kallmann Syndrome (KS) and Normosmic Idiopathic Hypogonadotropic Hypogonadism (niHH) seven out of eight patients described carried a mutation in FGFR1. FGFR1 mutations account for around 10 % of all cases of CHH, though in the patients sampled in this study, this increased to 88 % of cases of CHH where SHFM was also seen, suggesting that this phenotypic clue can be informative in identifying the underlying genetic cause of the CHH.
PROK2 and its receptor PROKR2 are known regulators of the GnRH system, as discussed above, and mutations in these genes are known to result in KS . Study of families carrying mutations in PROK2 or PROKR2 show intriguing pedigrees, with some homozygous individuals having KS and others having nIHH. The prokineticin 2 pathway is known to play a role in olfactory bulb neurogenesis, so the KS phenotype is expected. The nIHH phenotype, however, indicates a further role for this pathway in regulation of either GnRH synthesis, secretion and/or action. Family members carrying identical mutations can, however, show a wide spectrum of reproductive and nonreproductive phenotypes, suggesting a role for oligogenicity in the manifestation of their condition. These individuals highlight the potential pitfalls of relying upon phenotypic clues to direct the search for a genetic diagnosis.
Monogenicity vs. Oligogenicity
Some of the genes identified as playing a role in GnRH deficiency are thought to be dominant, and pedigrees show full penetration of the reproductive and olfactory phenotypes (e.g. in males with KAL1mutations). Likewise, individuals with homozygous mutations in several genes known to play a role in GnRH deficiency have a concordance of reproductive and nonreproductive phenotypes (e.g. in GNRH1, RnRHR, TAC3, TAC3R, PROK2, PROKR2, KISS1R mutations).
In documented pedigrees where the mutation is heterozygous, however, there is often considerable phenotypic variability amongst family members in whom the mutant allele is identical (seen in FGFR1, FGF8, PROK2, PROKR2 and GNRHR mutations). Oligogenicity is the phenomenon in which two individually rare genetic variants synergise, or act in a concerted fashion, resulting in the pathogenic phenotype. So, in the case of GnRH deficiency and phenotypic variability, some family members inherit the mutated gene associated with GnRH deficiency, as well as mutated forms of as-yet unidentified genes, which synergise to give the GnRH deficiency phenotype. Other family members will inherit only one of the genes required to act in synergism to produce the phenotype, thus will be phenotypically normal. Large studies of 400 GnRH-deficient families have shown that digenicity occurs in almost a quarter of all those in whom rare sequence variants could be identified . The prevalence of oligogenicity in patients with GnRH deficiency is known to be at least 15 %, and this is likely to increase when the genetic causes of GnRH deficiency are better understood (currently only 40–50 % of genetic causes are known). Oligogenicity explains the hitherto unexplained phenotypic variability seen in GnRH deficiency, which flouts the laws of strict Mendelian inheritance.
Clinical Utility of Genetic Diagnosis
The majority of the genetic causes of GnRH deficiency have yet to be identified. As there is this considerable phenotypic heterogeneity, enabling individuals to receive a specific genetic diagnosis could facilitate better management of other phenotype features seen in their particular instance of GnRH deficiency. In young adults, it might also be necessary to distinguish a permanent failure of GnRH release, from a constitutional delay, allowing for better management of the physical and psychological sequelae of either outcome.
Differentiation from Constitutional Pubertal Delay
Investigating the genetic cause underlying an individual’s failure to spontaneously enter puberty potentially allows for the distinction to be made between a constitutional delay of puberty (CDP) and a failure of puberty. CDP is the mildest form of GnRH deficiency, and individuals typically spontaneously enter puberty without any intervention being required. In contrast, a failure to enter puberty altogether will require medical management, and providing an early genetic diagnosis permits for earlier recognition of this.
Identifying Pubertal Prognosis
If a defect in a pathway known to be involved in regulating puberty is identified, a pubertal prognosis can be generated. It is unclear whether patients presenting with a pubertal delay will eventually enter into puberty (CDP), or whether there is a complete failure to enter puberty (KS/nIHH). When even the mildest manifestation of GnRH deficiency—the constitutional delay of puberty—can result in a psychologically challenging phenotype, there is a great need to identify the specific genetic diagnosis such that appropriate counselling and treatment options can be provided, dependent upon the pubertal prognosis.
Differing manifestations of GnRH deficiency have different pubertal prognoses. Individuals with CDP eventually enter puberty spontaneously and characteristically remain reproductively normal thereafter. This contrasts with the pubertal prognoses for individuals in whom the deficiency of GnRH is more complete (in which there is a failure to initiate or complete puberty, resulting in either KS or nIHH).
Allelic heterogeneity further complicates the phenotype of individuals with GnRH deficiency—different family members carrying the same disease mutations may have different phenotypes as a result of oligogenicity. Along with oligogenicity, there is also a role for epigenetic modifications in contributing to the phenotypic heterogeneity of GnRH deficiency.
Whilst work in recent decades has identified several genes involved in GnRH deficiency, those discovered are responsible for a few per cent of cases each and represent the rare causes of GnRH deficiency. These historic efforts to targeted candidate-gene searching have not been very successful, given our limited understanding of the physiological neuroendocrine networks being perturbed. This may be overcome with exome or genome sequencing, and with the costs of these ever decreasing, the wealth of data that can be gathered from individuals with GnRH deficiency has never been greater. As with all big data endeavours, however, limitations lie within the development and proper use of bioinformatic platforms with which to interpret the data. Challenges also lie within understanding the functional significance of each datum gathered from each individual, for which large collaborative efforts will be required to untangle. To better facilitate this, the COST research consortium—Cooperation on Science and Technology organisation —funded formation of a European-wide research consortium in 2011, with the aims of promoting international collaboration.
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