0163-769X/92/1304-0635$03.00/0 Endocrine Reviews Copyright © 1992 by The Endocrine Society
Vol. 13, No. 4 Printed in U.S.A.
CLINICAL COUNTERPOINT: Gonadotropin-Releasing Hormone Deficiency: Perspectives from Clinical Investigation* WILLIAM F. CROWLEY, JR., AND J. LARRY JAMESON Reproductive Endocrine (W.F.C.) and Thyroid (J.L.J.) Units of the Department of Medicine of the Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
I. Introduction II. Historical Overview of Kallmann Syndrome III. Use of Human GnRH-Deficient Human Models to Unravel the Physiology of Pulsatile GnRH IV. Genetic Basis for GnRH Deficiency in the Hypogonadal (hpg) Mouse V. Evidence for Developmental Abnormalities in Migration of GnRH-Producing Neurons VI. Identification of an X-Linked Gene Defect in Kallmann Syndrome VII. Questions Remaining in the Human Model VIII. Summary and Conclusions
I. Introduction
T
HESE are exciting times for clinicians and investigators interested in how the brain controls reproduction in the human. Patients with GnRH deficiency present to the physician with a failure to undergo puberty, low gonadotropin levels, and an absence of a discernible anatomical defect in their hypothalamus or anterior pituitary gland. Until the advent of GnRH as a therapeutic modality, the only treatments available to these patients were administration of sex steroids to provide secondary sexual characteristics or exogenous gonadotropins to induce fertility. However, several aspects of the central nervous system (CNS) control of reproduction have been elucidated by examining the pathophysiology of GnRH-deficient subjects and animal models, utilizing the tools of clinical investigation as well as more basic approaches of developmental and molecular biology (see accompanying review by SchwanzelFukuda, et al.). Taken together, these studies have not Address requests for reprints to: William F. Crowley, Jr., M.D., Reproductive Endocrine Unit, Bartlett Hall Extension 5, Massachusetts General Hospital, Boston, Massachusetts 02114. *Performed by the National Center for Infertility Research at the Massachusetts General Hospital as part of the NICHD's National Cooperative Program on Infertility Research as supported by Grant HD-29164.
only led to improved therapies for individual patients with these disorders, but also provided important insights into how the central mechanisms of GnRH secretion control reproduction. To put these recent advances into perspective, it is helpful to review the history of these disorders. II. Historical Overview of Kallmann Syndrome In 1943, Kallmann reported the initial descriptions of patients with hypogonadism and anosmia. Even in this early report, the first hints of a wider spectrum of clinical defects began to emerge (1). He made several important observations regarding this syndrome in his report including its: 1) occurrence in both men and women; 2) familial nature; and 3) association with other previously unrecognized defects including several midline congenital anomalies. Pathologists subsequently documented several neuroanatomical defects in these patients including agenesis of the olfactory bulb and tract as well as other midline neuroanatomical defects leading to the term olfactory-genital dysplasia (2). The syndrome occurring in anosmic patients with hypogonadism (who were later shown to be hypogonadotropic) has subsequently been referred to as Kallmann syndrome. What is often not widely appreciated is that many patients with hypogonadotropic hypogonadism lack several of these classical clinical features characteristic of Kallmann syndrome. For example, based upon the Massachusetts General Hospital series of nearly 200 individuals with hypogonadotropic hypogonadism, roughly 50-60% do not have a positive family history or the presence of anosmia (3). This nonanosmic group has been referred to as idiopathic hypogonadotropic hypogonadism (IHH). Until the discovery in 1971 of GnRH, the hypothalamic peptide responsible for gonadotropin secretion, it was not possible to determine whether the defect in patients with Kallmann syndrome resided in the hypothalamus or pituitary gland. Hence, several early reports 635
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referred to this disorder as isolated gonadotropin deficiency (4). Even after the discovery of GnRH, the location of the hormone deficiency was initially ambiguous as the majority of patients with hypogonadotropic hypogonadism failed to secrete gonadotropins in response to single bolus GnRH testing (5). It soon became clear, however, that the defect was hypothalamic in virtually all of these subjects. With repeated injections and eventually pulsatile administration of GnRH, the pituitarygonadal axis in these patients could be normalized by a physiologically designed replacement program of exogenous GnRH (6). Moreover, these paradigms eliminated a defect in the GnRH receptor or at a postreceptor step as the basis for gonadotropin deficiency.
III. Use of Human GnRH-Deficient Models to Unravel the Physiology of Pulsatile GnRH Once it became clear that patients with hypogonadotropic hypogonadism represented an isolated deficiency of GnRH, a series of clinical investigations using these patients as a model of normal reproduction contributed considerably to our understanding of the control of GnRH secretion. By varying the dose, frequency, timing, and interpulse interval of pulsatile exogenous GnRH administration to these subjects, each of these critical components of the GnRH secretory program from the hypothalamus could be isolated and examined experimentally (7). Hence, the hypothalamic or CNS input to human reproduction could be investigated in an isolated fashion not previously possible in normal men and women with an intact and free-running hypothalamicpituitary axis. Because input from GnRH could be controlled experimentally, responses in this 'hypothalamic' clamp model are mediated solely at the level of the pituitary-gonadal axis. Thus, by the tandem study of normal subjects, whose integrated responses incorporate both hypothalamic and pituitary adaptations, and GnRH-deficient subjects receiving physiologically fixed regimens of exogenous GnRH, the separate roles of the hypothalamus vs. pituitary feedback can be elucidated (8, 9). By using GnRH-deficient patients, it was determined that continuous occupancy of the GnRH receptor by long-acting GnRH agonists resulted in complete desensitization of gonadotropin secretion whereas only pulsatile administration could affect physiological levels and patterns of gonadotropin and gonadal sex steroid secretion (10). These results confirmed the earlier findings of Knobil's group utilizing continuous vs. pulsatile administration of natural sequence GnRH to castrated, GnRHdeficient (via anatomical lesions in the arcuate nucleus) Rhesus monkeys (11). Thus, the absolute requirement of the gonadotrope for an intermittent pattern of GnRH
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stimulation was first established utilizing GnRH-deficient primate and human models. Similarly, the frequency dependence of LH and FSH secretion was clarified using men with Kallmann syndrome whose GnRH dose could first be established to be physiological (12) and then varied in a systematic spectrum of increasing and decreasing frequencies (13, 14 ). While each of these previous experiments employed experimentally determinable doses and/or frequencies of exogenous GnRH administration to Kallmann or IHH men whose pituitary-gonadal axes were monitored, the ability to examine variabilities of GnRH interpulse intervals about a mean frequency of 2 h (15, 16) illustrates some of the more complicated neuroendocrine issues that can now be approached using this valuable human model. Whereas recreation of the normal spectrum of GnRH interpulse intervals was possible in GnRH-deficient men (15), the spectrum of resulting LH amplitudes varied from that encountered in normals. These findings inferred that the normal hypothalamus could vary the frequency as well as the dose of hypothalamic GnRH secretion, with the shorter interpulse intervals being associated with lower doses of GnRH secretion. On the other hand, the higher amplitude of LH pulses observed after the longer intervals between pulses of GnRH secretion does not appear to require higher doses of GnRH input; rather, merely lengthening the interpulse interval of GnRH secretion is sufficient to explain the higher LH amplitudes observed after long secretory pauses of hypothalamic GnRH secretion. Finally, the dissection of hypothalamic from pituitary sites of feedback effects of gonadal steroids has been addressed for the first time using this GnRH-deficient human model system (8, 9). This was previously an unapproachable issue in humans whose hypothalamicpituitary responses could only be monitored as an integrated response. Such studies have revealed a previously unappreciated pituitary site of androgen feedback of the pituitary level via aromatization. Thus, much of our updated understanding of the hypothalamic component of reproduction in the human has been considerably enhanced by clinical investigations of this most valuable model.
IV. Genetic Basis for GnRH Deficiency in the Hypogonadal (hpg) Mouse In a murine model of hypogonadotropic hypogonadism (the hpg mouse), GnRH deficiency was found to be caused by a deletion of the GnRH gene (17). Transgenic insertion of a normal GnRH gene restored reproductive function in the hpg mouse, confirming that the GnRH gene deletion was the cause of hypogonadism, and providing a rather dramatic example of gene replacement
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CLINICAL COUNTERPOINT: GnRH DEFICIENCY
therapy (17). In contrast, humans with GnRH deficiency do not have analogous deletions of the GnRH gene (18, 19). Using Southern blot analyses, the GnRH gene, which is located on chromosome 8 in humans (8p21pll.2), was found to be intact in several subsets of IHH patients, including males and females and patients with and without anosmia (18,19). Moreover, point mutations or frame shifts in the coding sequence have been eliminated as a frequent genetic cause of IHH in humans by actual sequencing of the GnRH gene in several affected individuals (19, 20). V. Evidence for Developmental Abnormalities in Migration of GnRH-Producing Neurons The genetic basis of Kallmann syndrome remained unexplained until recently when two separate lines of evidence defined a locus on the X chromosome that appears responsible for at least some cases of the disorder (21, 22). The first line of evidence came from studies of the developmental biology of GnRH-secreting neurons (23, 24). These investigators found that the hypothalamic GnRH neurons are not of CNS origin embryonically. Rather, they migrate into the hypothalamus from an epithelial cluster of cells derived from the olfactory placode outside of the developing CNS. The GnRH neurons must migrate across and eventually populate the olfactory bulb and the olfactory tract before entering the hypothalamus, at which point a sharp turn is required to localize laterally in the mediobasal hypothalamus. This astounding trek requires transportation across several centimeters of the brain and its regulatory mechanisms remain poorly understood. These studies of GnRH neuronal migration fit very nicely with other innovative animal studies demonstrating that GnRH neurons from a normal fetal hypothalamus, when transplanted into the floor of the third ventricle of the GnRH-deficient (hpg) mice, eventually make their way to the hypothalamus, take up residence in their normal anatomical location in the arcuate nucleus, and send projections to the median eminence (25). Not only do the neurons from these fetal transplants migrate to the correct anatomical location, but they apparently function normally, secreting GnRH in a pulsatile fashion to restore fertility to these GnRH-deficient animals (25). VI. Identification of an X-Linked Gene Defect in Kallmann Syndrome The other observation of crucial importance to understanding the basis of GnRH deficiency was made by investigators examining the X chromosome for defects in a variety of genetic disorders including steroid sulfatase deficiency and ichthyosis (26, 27). These geneticists
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noted that a subset of patients with ichthyosis, known to be due to a steroid sulfatase gene deletion on the distal portion of the Xp region, also had Kallmann syndrome. Using the concept of contiguous gene syndromes in which progressively larger chromosomal deletions eliminate additional neighboring genes to expand the clinical phenotype, they were able to localize steroid sulfatase and Kallmann gene defects to adjacent regions of the X chromosome, near the tip of the short arm (Xp22.3). At this point, these two lines of investigation converged in the study of a male child born with agenesis of the olfactory tract, sexual infantilism, severe ichthyosis, and a variety of other congenital abnormalities that eventually proved fatal (21). Since this patient had a large and visible deletion of the distal tip of his X chromosome by karyotype, it was thought likely that his abnormalities could be attributed to the loss of several genes located on the distal X chromosome. When his mother subsequently conceived again, an amniocentesis demonstrated that the second child was similarly affected. Neuroanatomical studies of this aborted fetus revealed an arrest of the migration of the GnRH neurons at the cribriform plate of the ethmoid sinus, as compared to a normal fetus of a similar gestational age, who demonstrated that the GnRH neurons should have already completed their migration into the hypothalamus by this time of development. At this point, the hypothesis was enjoined that the failure of migration of the GnRH neurons in this family was linked to the X chromosome deletion and that some protein, coded for by a gene controlling the normal migration of GnRH neurons, was deleted in patients with Kallmann syndrome. This hypothesis has now been confirmed by two groups who have demonstrated that the deleted gene in the Xp22.3 region, referred to as KAL, encodes a protein that is homologous to members of the fibronectin gene family, known for having important functions in neural chemotaxis, cell adhesion, and now presumably in directing GnRH neuronal migration during embryogenesis (26, 27). Franco et al. (26) found that the KAL gene was deleted or interrupted in each of seven patients with X chromosomal deletions or translocations associated with Kallmann syndrome, whereas the gene was present in other cases with partial X chromosome deletions in which Kallmann syndrome was absent. Similarly, Legouis et al. (27) established that the chromosomal breakpoint in a Kallmann patient with an Xp22.3 to Yqll translocation occurred near the 3'-end of the coding region of the KAL gene, thereby interrupting the gene directly. Importantly, both groups have found that the KAL gene is present by PCR or by Southern blots in many other patients with Kallmann syndrome who do not have the associated X chromosomal abnormalities such as ichthyosis, a finding
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that would have suggested a contiguous gene syndrome caused by large chromosomal deletions. These findings raise the possibility that many patients may have point mutations or small deletions in the KAL gene, or that the disorder may also be caused by abnormalities at other genetic loci. Identification of the KAL gene as a neural adhesion molecule involved in the migration of GnRH-producing neurons provides a potential basis for explaining the variable penetrance of different clinical features of Kallmann syndrome. In some kindreds, affected individuals may have GnRH deficiency and anosmia, whereas other family members may have GnRH deficiency without anosmia, or anosmia alone. This clinical variability may reflect differences in the ability of the neurons to successfully migrate to and populate the olfactory system and hypothalamic structures. In its role as a neural adhesion molecule, mutations in the KAL gene may also explain some of the other clinical features that are occasionally seen in the syndrome. These include mirror movements, cerebellar dysfunction, oculomotor abnormalities, renal hypoplasia or aplasia, and abnormal bones. In this regard, it is notable that expression of the gene can be detected in a number of tissues other than the hypothalamus (26).
VII. Questions Remaining in the Human Model Despite these important advances, several questions remain to be addressed in the human model of GnRH deficiency. 1. What percentage of the patients with Kallmann syndrome will be explained by this Xp22.3 region deletion and/or other defects in the X-linked gene? Reasonable evidence exists for an autosomal recessive mode of inheritance in some of these patients (28, 29); male to male transmission has been documented in this disorder (30,31); there is also evidence for an autosomal dominant mode of inheritance (32), although this latter form of inheritance may bear some reexamination as possible sex-limited expression. There is also little information on the genetic basis for Kallmann syndrome in females. Are females homozygous for defects at the KAL locus, or can the disease be manifest in the hemizygous state, perhaps because of genomic imprinting with differential expression of genes inherited on paternal and maternal chromosomes (33)? These observations also raise the possibility that other as yet unidentified loci may contribute to the normal expression of the GnRH gene. 2. What percentage of spontaneous mutations also lie on the X chromosome? In the Massachusetts General Hospital series of nearly 200 individuals with IHH, at least half appear to harbor spontaneous mutations, without any evidence of prior familial defects. This high
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frequency of spontaneous mutations may reflect the fact that before the advent of hormonal treatment, the disease has precluded fertility, thereby limiting its transmission within kindreds. Because one cannot demonstrate Xlinkage in these cases, it will be of interest to determine whether patients with the spontaneous form of the disorder also have a defect in the KAL gene. 3. What is the link between mutations in Kallmann syndrome and the presence or absence of the other congenital anomalies, including anosmia, the renal abnormalities, and other neurological deficits (26, 27, 3442)? Can each of these apparently disparate abnormalities be linked to defects in the KAL gene product, resulting in abnormalities in embryonic migration? Are different types of mutations in the KAL gene associated with specific clinical phenotypes? 4. What is the nature of the defect in patients in whom GnRH deficiency occurs in the absence of anosmia? Presumably the normal sense of smell in more than half of these patients represents a normal olfactory bulb and tract (which also normally stains densely for GnRH). It is possible that these patients represent variable penetrance of the GnRH migratory defect. If so, one might expect there to be a group of patients with a partial defect in the migration of hypothalamic GnRH neurons and an enfeebled program of endogenous GnRH secretion. The latter phenotype has not yet been described. 5. Is there a human counterpart to the hypogonadal mouse? Since no apparent deletion or mutation within the GnRH gene itself has yet been found in patients with traditional Kallmann syndrome, perhaps other hypogonadotropic states associated abnormalities such as cerebellar ataxia, mental retardation, retinitis pigmentosa, etc. (37-42) have a defect on chromosome 8 that includes the GnRH gene along with other genetic loci? In this regard, several patients with hereditary spherocytosis have been documented to have a genomic deletion of ankyrin on the short arm of the eighth (8pll-p21.1) chromosome (43). Although the full extent of this deletion has not yet been charted in the region immediately adjacent to the GnRH gene, they would be strong candidates to harbor such a defect. It is also possible that a subset of patients with selective GnRH deficiency, without anosmia or other abnormalities suggestive of a defect in the KAL gene, may have mutations in the GnRH gene. 6. What lessons does this syndrome have for us regarding the general themes of development biology? One is reminded of a number of well-characterized mutations in Caenorhabditis elegans and in Drosophila that act by preventing a critical step during embryogenesis or development. In humans, there are a number of disorders in which a single defect can affect development in several different organ systems. One such example is Kartege-
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CLINICAL COUNTERPOINT: GnRH DEFICIENCY
ner's syndrome in which a defect in ciliary motility is echoed through several target organs including spermatozoa, cardiac development, and bronchial epithelium (44). Almost certainly there will be a number of lessons to be learned as this interesting story progresses. Elucidation of these issues will also provide some important clinical clues for physicians as to the genetic screening and counselling of these patients. Since gonadotropins are known to be elevated in utero and for a brief time during the neonatal period, is there any role for early treatment with pulsatile GnRH, perhaps to influence gonadal development?
VIII. Summary and Conclusions Advances in our understanding of the pathophysiology of Kallmann syndrome have come from an interdisciplinary approach involving developmental biology, clinical investigation, and molecular biology. It is equally clear that progress to date is but the first chapter of what will be a fascinating biological story. It now seems likely that the full expression of reproductive potential from the neuroendocrine perspective is likely to be as complicated as other aspects of reproduction, such as the mutigene control of external genital differentiation. An analogous story may well emerge for the neuroendocrine control of reproduction in which the GnRH gene is encoded on the eighth chromosome, the protein guiding the embryonic journey of the GnRH-producing neuron to the hypothalamus lies on the X chromosome, and many, as yet to be determined, other genetic loci collaborate in the full expression of reproductive potential. Such a detailed study is warranted not only because of the clinical and genetic implications for an individual patient with this disorder, but also from an organizational theme for the evolution of the species (and its potential regulation). Given the pressing nature of world population growth, obtaining such understanding and its applications to fertility and contraception is crucial. These advances will only come from enlightened interactions of clinical investigators, molecular geneticists, and developmental biologists in which interdisciplinary approaches should be fostered. This should be an exciting story to follow given the remarkable nature of the tools at hand to study these clinical conditions.
References 1. Kallmann FJ, Schoenfeld WA, Barrera SE 1944 The genetic aspects of primary eunuchoidism. Am J Ment Defic 48:203-236 2. de Morsier G 1954 Etudes sur les dysraphies cranio-encephaliques. Schweiz Arch Neurol Psychiatr 74:309-361 3. Spratt DI, Carr DB, Merriam GR, Scully RE, Rao PN, Crowley Jr WF 1987 The spectrum of abnormal patterns of gonadotropinreleasing hormone secretion in men and idiopathic hypogonadotropic hypogonadism: clinical and laboratory correlations. J Clin Endocrinol Metab 64:283-291
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4. Hashimoto T, Miyai K, Izumi K, Kumahara Y 1972 Isolated gonadotropin deficiency with response to luteinizing-hormonereleasing hormone. N Engl J Med 287:1059-1062 5. Naftolin F, Harris GW, Bobrow M 1971 Effect of purified luteinizing hormone releasing factor on normal and hypogonadotropic anosmic men. Nature 232:496-497 6. Hoffman AR, Crowley Jr WF 1982 Induction of puberty in men by long-term pulsatile administration of low-dose gonadotropin-releasing hormone. N Engl J Med 307:1237-1241 7. Crowley Jr WF, Whitcomb RW, Weiss J, Finkelstein JS, O'Dea LS 1991 The neuroendocrine control of human reproduction in the male. Recent Prog Horm Res 47:27-67 8. Finkelstein JS, Whitcomb RW, O'Dea LS, Longcope C, Schoenfeld DA, Crowley Jr WF 1991 Sex steroid control of gonadotropin secretion in the human male. I. Effects of testosterone administration in normal and GnRH-deficient men. J Clin Endocrinol Metab 73:609-620 9. Finkelstein JS, O'Dea LS, Whitcomb RW, Crowley Jr WF 1991 Sex steroid control of gonadotropin secretion in the human male. II. Effects of estradiol administration in normal and GnRH-deficient men. J Clin Endocrinol Metab 73:621-628 10. Crowley WF, Vale WW, Rivier J, McArthur JW 1981 LHRH in hypogonadotropic hypogonadism. In: Zatuchini GI, Shelton JD, Sciarra JJ (eds) LHRH Peptides as Female and Male Contraceptives. Program on Applied Research and Fertility Regulation Series on Fertility Regulation. Harper & Row, Philadelphia, pp 321-333 11. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E 1978 Hypophyseal response to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202:631633 12. Spratt DI, Finkelstein JS, Badger TM, Butler JP, Crowley Jr WF 1986 Bio- and immunoactive luteinizing hormone responses to low doses of gonadotropin-releasing hormone (GnRH): dose-response curves in GnRH-deficient men. J Clin Endocrinol Metab 63:143150 13. Spratt D, Finkelstein J, Butler JP, Badger TM, Crowley Jr WF 1987 Effects of increasing the frequency of low doses of gonadotropin-releasing hormone (GnRH) on gonadotropin secretion in GnRH-deficient men. J Clin Endocrinol Metab 64:1179-1186 14. Finkelstein JS, Badger TM, O'Dea LS, Spratt DI, Crowley WF 1988 Effects of decreasing the frequency of gonadotropin-releasing hormone stimulation on gonadotropin secretion in gonadotropinreleasing hormone-deficient men and perifused rat pituitary cells. J Clin Invest 81:1725-33 15. Butler JP, Spratt DI, O'Dea LS, Crowley Jr WF 1986 The interpulse interval sequence of luteinizing hormone in normal men essentially constitutes a renewal process. Am J Physiol 250:E338340 16. O'Dea L StL Finkelstein JS, Schoenfeld DA, Butler JP, Crowley Jr WF 1989 The interpulse interval of GnRH stimulation independently modulates LH secretion. Relation of interpulse interval to LH secretion. Am J Physiol 256:E510-515 17. Seeburg PH, Mason AJ, Stewart TA, Nikolics K 1987 The mammalian GnRH gene and its pivotal role in reproduction. Recent Prog Horm Res 43:69-98 18. Weiss J, Crowley Jr WF, Jameson JL 1989 Normal structure of the gonadotropin-releasing hormone (GnRH) gene in patients with GnRH deficiency and idiopathic hypogonadotropic hypogonadism. J Clin Endocrinol Metab 69:299-303 19. Nakayama Y, Wondisford FE, Lash RW, Bale AE, Weintraub BD, Cutler Jr GB, Radovick S 1990 Analysis of gonadotropin-releasing hormone gene structure in families with familial central precocious puberty adn idiopathic hypogonadotropic hypogonadism. J Clin Endocrinol Metab 70:1233-1238 20. Weiss J, Adams E, Whitcomb RW, Crowley Jr WF, Jameson JL 1991 Normal sequence of the gonadotropin-releasing hormone gene in patients with idiopathic hypogonadotropic hypogonadism. Biol Reprod 45:743-747 21. Schwanzel-Fukuda M, Bick D, Pfaff DW 1989 Luteinizing hormone- releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome. Mol Brain Res 6:311-326
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22. Ballabio A, Bardoni B, Carrozzo R, Andria G, Bick D, Campbell L, Hamel B, Ferguson-Smith MA, Gimelli G, Fraccaro M, Maraschio P, Zuffardi 0, Guioli S, Camerino G 1989 Contiguous gene syndromes due to deletions in the distal short arm of the human X-chromosome. Proc Natl Acad Sci USA 86:10001-10005 23. Schwanzel-Fukuda M, Pfaff DW 1989 Origin of luteinizing hormone-releasing hormone neurons. Nature 338:161-164 24. Wray S, Nieburgs A, Elkabes S 1989 Spatiotemporal cell expression of luteinizing hormone-releasing hormone in the prenatal mouse: evidence for an embryonic origin in the olfactory placode. Dev Brain Res 46:309-318 25. Gibson MJ, Krieger DT, Charlton HM, Zimmerman EA, Silverman AJ, Perlow MJ 1984 Mating and pregnancy can occur in genetically hypogonadal mice with preoptic area brain grafts. Science 225:949951 26. Franco B, Guioli S, Pragliola A, Incerti B, Bardoni B, Tonlorenzi R, Carrozzo R, Maestrini E, Pieretti M, Taillon-Miller P, Brown CJ, Willard HF, Lawrence C, Persico MG, Camerino G, Ballabio A 1991 A gene deleted in Kallmann syndrome shares homology with neural cell adhesion and axonal path-finding molecules. Nature 353:529-536 27. Legouis R, Hardelin JP, Levilliers J, Claverie JM, Compain S, Wunderle V, Millassean P, LePaslier D, Cohen D, Caterina D, Bougueleret L, Delemarre-Van de Waal H, Lutfalla G, Weissenbach J, Petit C 1991 The candidate gene for the X-linked Kallmann syndrome encodes a protein related to adhesion molecules. Cell 67:423-435 28. Ewer RW 1968 Familial monotropic pituitary gonadotropin insufficiency. J Clin Endocrinol 28:783-788 29. White BJ, Rogol AD, Brown KS, Lieblich JM, Rosen SW 1983 The syndrome of anosmia with hypogonadotropic hypogonadism: a genetic study of 18 new families and a review. Am J Med Genet 15:417-435 30. Merriam GR, Inese Z, Beitins MD, Bode HH 1977 Father-to-son transmission of hypogonadism with anosmia. Am J Dis Child 131:1216-1219 31. Rogol AD, Kamal KM, White BJ, McGinniss MH, Lieblich JM, Rosen SW 1980 HLA-compatible paternity in two "fertile eunuchs"
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with congenital hypogonadotropic hypogonadism and anosmia (the Kallmann syndrome). J Clin Endocrinol Metab 51:275-279 Santen RJ, Paulsen CA 1973 Hypogonadotropic eunuchoidism. I. Clinical study of the mode of inheritance. J Clin Endocrinol Metab 36:47-54 Hall JG 1992 Genomic imprinting and its clinical implications. N Engl J Med 326:827-829 Wegenke JD, Uehling DT, Wear JB, Gordon ES, Bargman JG, Deacon JSR, Herrmann JPR, Opitz JM 1975 Familial Kallmann syndrome with unilateral renal aplasia. Clin Genet 7:368-381 Swanson SL, Santen RJ, Smith DW 1971 Multiple lentigines syndrome: new findings of hypogonadotrophism, hyposmia, and unilateral renal agencies. J Pediat 78:1037-1039 Smals AGH, Kloppenborg PWC, Benraad THJ 1974 Hypogonadotropic hypogonadism and hyposmia—Kallmann's syndrome. Neth J Med 17:202-211 Berciano J, Amado JA, Freijanes J, Rebollo M, Vaquero A 1982 Familial cerebellar ataxia and hypogonadotropic hypogonadism: evidence for hypothalamic LHRH deficiency. J Neurol Neurosurg Psychiatr 45:747-751 Lowenthal A, Bekaert J, Van Dessel F, van Hauwaert J 1979 Familial cerebellar ataxia with hypogonadism. J Neurol 222:75-80 Neuhauser G, Opitz JM 1975 Autosomal recessive syndrome of cerebellar ataxia and hypogonadotropic hypogonadism. Clin Genet 7:426-434 Matthews WB, Rundle AT 1964 Familial cerebellar ataxia and hypogonadism. Brain 87:463-468 Boucher BJ, Gibberd FB 1969 Familial ataxia, hypogonadism and retinal degeneration. Acta Neurol Scand 45:507-510 Volpe R, Metzler WS, Johnston MW 1963 Familial hypogonadotrophic eunuchoidism with cerebellar ataxia. J Clin Endocrinol Metab 23:107-115 Lux SE, Tse WT, Menninger JC, John KM, Harris P, Shaler O, Chilcotte RR, Marchesi SL, Watkins PC, Bennett V, Mclntosh S, Collins FS, Francke U, Ward DC, Forget BG 1990 Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8. Nature 345:736-739 Eliasson R, Mossberg B, Camner P, Afzelius BA 1977 The immotile-cilio syndrome. A congenital ciliary abnormality as an etiologic factor in chronic airway infections and male sterility. N Engl J Med 297:1-6
Glycoprotein Hormones: Structure, Function and Clinical Implications Santa Barbara, California, March 11-14, 1993 This program will focus on current information relating to the production, structure, and function of the glycoprotein hormones. Topics to be addressed are: regulation of gene expression, receptor structure/function, hormonal protein structure/function, protein processing and posttranslational events, and clinical implications. Drs. Harold Papkoff, Robert Ryan and Darrell Ward will be honored for their pioneering and key contributions made to this field. For information regarding brochure, registration and poster abstract forms, please contact Bruce K. Burnett, Ph.D., Serono Symposia, USA, 100 Longwater Circle, Norwell, Massachusetts 02061. Telephone: (800) 283-8088, (617) 982-9000, or Fax: (617) 982-9481.
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CLINICAL COUNTERPOINT: Gonadotropin-Releasing Hormone Deficiency: Perspectives from Clinical Investigation* WILLIAM F. CROWLEY, JR., AND J. LARRY JAMESON Reproductive Endocrine (W.F.C.) and Thyroid (J.L.J.) Units of the Department of Medicine of the Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
I. Introduction II. Historical Overview of Kallmann Syndrome III. Use of Human GnRH-Deficient Human Models to Unravel the Physiology of Pulsatile GnRH IV. Genetic Basis for GnRH Deficiency in the Hypogonadal (hpg) Mouse V. Evidence for Developmental Abnormalities in Migration of GnRH-Producing Neurons VI. Identification of an X-Linked Gene Defect in Kallmann Syndrome VII. Questions Remaining in the Human Model VIII. Summary and Conclusions
I. Introduction
T
HESE are exciting times for clinicians and investigators interested in how the brain controls reproduction in the human. Patients with GnRH deficiency present to the physician with a failure to undergo puberty, low gonadotropin levels, and an absence of a discernible anatomical defect in their hypothalamus or anterior pituitary gland. Until the advent of GnRH as a therapeutic modality, the only treatments available to these patients were administration of sex steroids to provide secondary sexual characteristics or exogenous gonadotropins to induce fertility. However, several aspects of the central nervous system (CNS) control of reproduction have been elucidated by examining the pathophysiology of GnRH-deficient subjects and animal models, utilizing the tools of clinical investigation as well as more basic approaches of developmental and molecular biology (see accompanying review by SchwanzelFukuda, et al.). Taken together, these studies have not Address requests for reprints to: William F. Crowley, Jr., M.D., Reproductive Endocrine Unit, Bartlett Hall Extension 5, Massachusetts General Hospital, Boston, Massachusetts 02114. *Performed by the National Center for Infertility Research at the Massachusetts General Hospital as part of the NICHD's National Cooperative Program on Infertility Research as supported by Grant HD-29164.
only led to improved therapies for individual patients with these disorders, but also provided important insights into how the central mechanisms of GnRH secretion control reproduction. To put these recent advances into perspective, it is helpful to review the history of these disorders. II. Historical Overview of Kallmann Syndrome In 1943, Kallmann reported the initial descriptions of patients with hypogonadism and anosmia. Even in this early report, the first hints of a wider spectrum of clinical defects began to emerge (1). He made several important observations regarding this syndrome in his report including its: 1) occurrence in both men and women; 2) familial nature; and 3) association with other previously unrecognized defects including several midline congenital anomalies. Pathologists subsequently documented several neuroanatomical defects in these patients including agenesis of the olfactory bulb and tract as well as other midline neuroanatomical defects leading to the term olfactory-genital dysplasia (2). The syndrome occurring in anosmic patients with hypogonadism (who were later shown to be hypogonadotropic) has subsequently been referred to as Kallmann syndrome. What is often not widely appreciated is that many patients with hypogonadotropic hypogonadism lack several of these classical clinical features characteristic of Kallmann syndrome. For example, based upon the Massachusetts General Hospital series of nearly 200 individuals with hypogonadotropic hypogonadism, roughly 50-60% do not have a positive family history or the presence of anosmia (3). This nonanosmic group has been referred to as idiopathic hypogonadotropic hypogonadism (IHH). Until the discovery in 1971 of GnRH, the hypothalamic peptide responsible for gonadotropin secretion, it was not possible to determine whether the defect in patients with Kallmann syndrome resided in the hypothalamus or pituitary gland. Hence, several early reports 635
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referred to this disorder as isolated gonadotropin deficiency (4). Even after the discovery of GnRH, the location of the hormone deficiency was initially ambiguous as the majority of patients with hypogonadotropic hypogonadism failed to secrete gonadotropins in response to single bolus GnRH testing (5). It soon became clear, however, that the defect was hypothalamic in virtually all of these subjects. With repeated injections and eventually pulsatile administration of GnRH, the pituitarygonadal axis in these patients could be normalized by a physiologically designed replacement program of exogenous GnRH (6). Moreover, these paradigms eliminated a defect in the GnRH receptor or at a postreceptor step as the basis for gonadotropin deficiency.
III. Use of Human GnRH-Deficient Models to Unravel the Physiology of Pulsatile GnRH Once it became clear that patients with hypogonadotropic hypogonadism represented an isolated deficiency of GnRH, a series of clinical investigations using these patients as a model of normal reproduction contributed considerably to our understanding of the control of GnRH secretion. By varying the dose, frequency, timing, and interpulse interval of pulsatile exogenous GnRH administration to these subjects, each of these critical components of the GnRH secretory program from the hypothalamus could be isolated and examined experimentally (7). Hence, the hypothalamic or CNS input to human reproduction could be investigated in an isolated fashion not previously possible in normal men and women with an intact and free-running hypothalamicpituitary axis. Because input from GnRH could be controlled experimentally, responses in this 'hypothalamic' clamp model are mediated solely at the level of the pituitary-gonadal axis. Thus, by the tandem study of normal subjects, whose integrated responses incorporate both hypothalamic and pituitary adaptations, and GnRH-deficient subjects receiving physiologically fixed regimens of exogenous GnRH, the separate roles of the hypothalamus vs. pituitary feedback can be elucidated (8, 9). By using GnRH-deficient patients, it was determined that continuous occupancy of the GnRH receptor by long-acting GnRH agonists resulted in complete desensitization of gonadotropin secretion whereas only pulsatile administration could affect physiological levels and patterns of gonadotropin and gonadal sex steroid secretion (10). These results confirmed the earlier findings of Knobil's group utilizing continuous vs. pulsatile administration of natural sequence GnRH to castrated, GnRHdeficient (via anatomical lesions in the arcuate nucleus) Rhesus monkeys (11). Thus, the absolute requirement of the gonadotrope for an intermittent pattern of GnRH
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stimulation was first established utilizing GnRH-deficient primate and human models. Similarly, the frequency dependence of LH and FSH secretion was clarified using men with Kallmann syndrome whose GnRH dose could first be established to be physiological (12) and then varied in a systematic spectrum of increasing and decreasing frequencies (13, 14 ). While each of these previous experiments employed experimentally determinable doses and/or frequencies of exogenous GnRH administration to Kallmann or IHH men whose pituitary-gonadal axes were monitored, the ability to examine variabilities of GnRH interpulse intervals about a mean frequency of 2 h (15, 16) illustrates some of the more complicated neuroendocrine issues that can now be approached using this valuable human model. Whereas recreation of the normal spectrum of GnRH interpulse intervals was possible in GnRH-deficient men (15), the spectrum of resulting LH amplitudes varied from that encountered in normals. These findings inferred that the normal hypothalamus could vary the frequency as well as the dose of hypothalamic GnRH secretion, with the shorter interpulse intervals being associated with lower doses of GnRH secretion. On the other hand, the higher amplitude of LH pulses observed after the longer intervals between pulses of GnRH secretion does not appear to require higher doses of GnRH input; rather, merely lengthening the interpulse interval of GnRH secretion is sufficient to explain the higher LH amplitudes observed after long secretory pauses of hypothalamic GnRH secretion. Finally, the dissection of hypothalamic from pituitary sites of feedback effects of gonadal steroids has been addressed for the first time using this GnRH-deficient human model system (8, 9). This was previously an unapproachable issue in humans whose hypothalamicpituitary responses could only be monitored as an integrated response. Such studies have revealed a previously unappreciated pituitary site of androgen feedback of the pituitary level via aromatization. Thus, much of our updated understanding of the hypothalamic component of reproduction in the human has been considerably enhanced by clinical investigations of this most valuable model.
IV. Genetic Basis for GnRH Deficiency in the Hypogonadal (hpg) Mouse In a murine model of hypogonadotropic hypogonadism (the hpg mouse), GnRH deficiency was found to be caused by a deletion of the GnRH gene (17). Transgenic insertion of a normal GnRH gene restored reproductive function in the hpg mouse, confirming that the GnRH gene deletion was the cause of hypogonadism, and providing a rather dramatic example of gene replacement
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therapy (17). In contrast, humans with GnRH deficiency do not have analogous deletions of the GnRH gene (18, 19). Using Southern blot analyses, the GnRH gene, which is located on chromosome 8 in humans (8p21pll.2), was found to be intact in several subsets of IHH patients, including males and females and patients with and without anosmia (18,19). Moreover, point mutations or frame shifts in the coding sequence have been eliminated as a frequent genetic cause of IHH in humans by actual sequencing of the GnRH gene in several affected individuals (19, 20). V. Evidence for Developmental Abnormalities in Migration of GnRH-Producing Neurons The genetic basis of Kallmann syndrome remained unexplained until recently when two separate lines of evidence defined a locus on the X chromosome that appears responsible for at least some cases of the disorder (21, 22). The first line of evidence came from studies of the developmental biology of GnRH-secreting neurons (23, 24). These investigators found that the hypothalamic GnRH neurons are not of CNS origin embryonically. Rather, they migrate into the hypothalamus from an epithelial cluster of cells derived from the olfactory placode outside of the developing CNS. The GnRH neurons must migrate across and eventually populate the olfactory bulb and the olfactory tract before entering the hypothalamus, at which point a sharp turn is required to localize laterally in the mediobasal hypothalamus. This astounding trek requires transportation across several centimeters of the brain and its regulatory mechanisms remain poorly understood. These studies of GnRH neuronal migration fit very nicely with other innovative animal studies demonstrating that GnRH neurons from a normal fetal hypothalamus, when transplanted into the floor of the third ventricle of the GnRH-deficient (hpg) mice, eventually make their way to the hypothalamus, take up residence in their normal anatomical location in the arcuate nucleus, and send projections to the median eminence (25). Not only do the neurons from these fetal transplants migrate to the correct anatomical location, but they apparently function normally, secreting GnRH in a pulsatile fashion to restore fertility to these GnRH-deficient animals (25). VI. Identification of an X-Linked Gene Defect in Kallmann Syndrome The other observation of crucial importance to understanding the basis of GnRH deficiency was made by investigators examining the X chromosome for defects in a variety of genetic disorders including steroid sulfatase deficiency and ichthyosis (26, 27). These geneticists
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noted that a subset of patients with ichthyosis, known to be due to a steroid sulfatase gene deletion on the distal portion of the Xp region, also had Kallmann syndrome. Using the concept of contiguous gene syndromes in which progressively larger chromosomal deletions eliminate additional neighboring genes to expand the clinical phenotype, they were able to localize steroid sulfatase and Kallmann gene defects to adjacent regions of the X chromosome, near the tip of the short arm (Xp22.3). At this point, these two lines of investigation converged in the study of a male child born with agenesis of the olfactory tract, sexual infantilism, severe ichthyosis, and a variety of other congenital abnormalities that eventually proved fatal (21). Since this patient had a large and visible deletion of the distal tip of his X chromosome by karyotype, it was thought likely that his abnormalities could be attributed to the loss of several genes located on the distal X chromosome. When his mother subsequently conceived again, an amniocentesis demonstrated that the second child was similarly affected. Neuroanatomical studies of this aborted fetus revealed an arrest of the migration of the GnRH neurons at the cribriform plate of the ethmoid sinus, as compared to a normal fetus of a similar gestational age, who demonstrated that the GnRH neurons should have already completed their migration into the hypothalamus by this time of development. At this point, the hypothesis was enjoined that the failure of migration of the GnRH neurons in this family was linked to the X chromosome deletion and that some protein, coded for by a gene controlling the normal migration of GnRH neurons, was deleted in patients with Kallmann syndrome. This hypothesis has now been confirmed by two groups who have demonstrated that the deleted gene in the Xp22.3 region, referred to as KAL, encodes a protein that is homologous to members of the fibronectin gene family, known for having important functions in neural chemotaxis, cell adhesion, and now presumably in directing GnRH neuronal migration during embryogenesis (26, 27). Franco et al. (26) found that the KAL gene was deleted or interrupted in each of seven patients with X chromosomal deletions or translocations associated with Kallmann syndrome, whereas the gene was present in other cases with partial X chromosome deletions in which Kallmann syndrome was absent. Similarly, Legouis et al. (27) established that the chromosomal breakpoint in a Kallmann patient with an Xp22.3 to Yqll translocation occurred near the 3'-end of the coding region of the KAL gene, thereby interrupting the gene directly. Importantly, both groups have found that the KAL gene is present by PCR or by Southern blots in many other patients with Kallmann syndrome who do not have the associated X chromosomal abnormalities such as ichthyosis, a finding
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that would have suggested a contiguous gene syndrome caused by large chromosomal deletions. These findings raise the possibility that many patients may have point mutations or small deletions in the KAL gene, or that the disorder may also be caused by abnormalities at other genetic loci. Identification of the KAL gene as a neural adhesion molecule involved in the migration of GnRH-producing neurons provides a potential basis for explaining the variable penetrance of different clinical features of Kallmann syndrome. In some kindreds, affected individuals may have GnRH deficiency and anosmia, whereas other family members may have GnRH deficiency without anosmia, or anosmia alone. This clinical variability may reflect differences in the ability of the neurons to successfully migrate to and populate the olfactory system and hypothalamic structures. In its role as a neural adhesion molecule, mutations in the KAL gene may also explain some of the other clinical features that are occasionally seen in the syndrome. These include mirror movements, cerebellar dysfunction, oculomotor abnormalities, renal hypoplasia or aplasia, and abnormal bones. In this regard, it is notable that expression of the gene can be detected in a number of tissues other than the hypothalamus (26).
VII. Questions Remaining in the Human Model Despite these important advances, several questions remain to be addressed in the human model of GnRH deficiency. 1. What percentage of the patients with Kallmann syndrome will be explained by this Xp22.3 region deletion and/or other defects in the X-linked gene? Reasonable evidence exists for an autosomal recessive mode of inheritance in some of these patients (28, 29); male to male transmission has been documented in this disorder (30,31); there is also evidence for an autosomal dominant mode of inheritance (32), although this latter form of inheritance may bear some reexamination as possible sex-limited expression. There is also little information on the genetic basis for Kallmann syndrome in females. Are females homozygous for defects at the KAL locus, or can the disease be manifest in the hemizygous state, perhaps because of genomic imprinting with differential expression of genes inherited on paternal and maternal chromosomes (33)? These observations also raise the possibility that other as yet unidentified loci may contribute to the normal expression of the GnRH gene. 2. What percentage of spontaneous mutations also lie on the X chromosome? In the Massachusetts General Hospital series of nearly 200 individuals with IHH, at least half appear to harbor spontaneous mutations, without any evidence of prior familial defects. This high
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frequency of spontaneous mutations may reflect the fact that before the advent of hormonal treatment, the disease has precluded fertility, thereby limiting its transmission within kindreds. Because one cannot demonstrate Xlinkage in these cases, it will be of interest to determine whether patients with the spontaneous form of the disorder also have a defect in the KAL gene. 3. What is the link between mutations in Kallmann syndrome and the presence or absence of the other congenital anomalies, including anosmia, the renal abnormalities, and other neurological deficits (26, 27, 3442)? Can each of these apparently disparate abnormalities be linked to defects in the KAL gene product, resulting in abnormalities in embryonic migration? Are different types of mutations in the KAL gene associated with specific clinical phenotypes? 4. What is the nature of the defect in patients in whom GnRH deficiency occurs in the absence of anosmia? Presumably the normal sense of smell in more than half of these patients represents a normal olfactory bulb and tract (which also normally stains densely for GnRH). It is possible that these patients represent variable penetrance of the GnRH migratory defect. If so, one might expect there to be a group of patients with a partial defect in the migration of hypothalamic GnRH neurons and an enfeebled program of endogenous GnRH secretion. The latter phenotype has not yet been described. 5. Is there a human counterpart to the hypogonadal mouse? Since no apparent deletion or mutation within the GnRH gene itself has yet been found in patients with traditional Kallmann syndrome, perhaps other hypogonadotropic states associated abnormalities such as cerebellar ataxia, mental retardation, retinitis pigmentosa, etc. (37-42) have a defect on chromosome 8 that includes the GnRH gene along with other genetic loci? In this regard, several patients with hereditary spherocytosis have been documented to have a genomic deletion of ankyrin on the short arm of the eighth (8pll-p21.1) chromosome (43). Although the full extent of this deletion has not yet been charted in the region immediately adjacent to the GnRH gene, they would be strong candidates to harbor such a defect. It is also possible that a subset of patients with selective GnRH deficiency, without anosmia or other abnormalities suggestive of a defect in the KAL gene, may have mutations in the GnRH gene. 6. What lessons does this syndrome have for us regarding the general themes of development biology? One is reminded of a number of well-characterized mutations in Caenorhabditis elegans and in Drosophila that act by preventing a critical step during embryogenesis or development. In humans, there are a number of disorders in which a single defect can affect development in several different organ systems. One such example is Kartege-
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ner's syndrome in which a defect in ciliary motility is echoed through several target organs including spermatozoa, cardiac development, and bronchial epithelium (44). Almost certainly there will be a number of lessons to be learned as this interesting story progresses. Elucidation of these issues will also provide some important clinical clues for physicians as to the genetic screening and counselling of these patients. Since gonadotropins are known to be elevated in utero and for a brief time during the neonatal period, is there any role for early treatment with pulsatile GnRH, perhaps to influence gonadal development?
VIII. Summary and Conclusions Advances in our understanding of the pathophysiology of Kallmann syndrome have come from an interdisciplinary approach involving developmental biology, clinical investigation, and molecular biology. It is equally clear that progress to date is but the first chapter of what will be a fascinating biological story. It now seems likely that the full expression of reproductive potential from the neuroendocrine perspective is likely to be as complicated as other aspects of reproduction, such as the mutigene control of external genital differentiation. An analogous story may well emerge for the neuroendocrine control of reproduction in which the GnRH gene is encoded on the eighth chromosome, the protein guiding the embryonic journey of the GnRH-producing neuron to the hypothalamus lies on the X chromosome, and many, as yet to be determined, other genetic loci collaborate in the full expression of reproductive potential. Such a detailed study is warranted not only because of the clinical and genetic implications for an individual patient with this disorder, but also from an organizational theme for the evolution of the species (and its potential regulation). Given the pressing nature of world population growth, obtaining such understanding and its applications to fertility and contraception is crucial. These advances will only come from enlightened interactions of clinical investigators, molecular geneticists, and developmental biologists in which interdisciplinary approaches should be fostered. This should be an exciting story to follow given the remarkable nature of the tools at hand to study these clinical conditions.
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4. Hashimoto T, Miyai K, Izumi K, Kumahara Y 1972 Isolated gonadotropin deficiency with response to luteinizing-hormonereleasing hormone. N Engl J Med 287:1059-1062 5. Naftolin F, Harris GW, Bobrow M 1971 Effect of purified luteinizing hormone releasing factor on normal and hypogonadotropic anosmic men. Nature 232:496-497 6. Hoffman AR, Crowley Jr WF 1982 Induction of puberty in men by long-term pulsatile administration of low-dose gonadotropin-releasing hormone. N Engl J Med 307:1237-1241 7. Crowley Jr WF, Whitcomb RW, Weiss J, Finkelstein JS, O'Dea LS 1991 The neuroendocrine control of human reproduction in the male. Recent Prog Horm Res 47:27-67 8. Finkelstein JS, Whitcomb RW, O'Dea LS, Longcope C, Schoenfeld DA, Crowley Jr WF 1991 Sex steroid control of gonadotropin secretion in the human male. I. Effects of testosterone administration in normal and GnRH-deficient men. J Clin Endocrinol Metab 73:609-620 9. Finkelstein JS, O'Dea LS, Whitcomb RW, Crowley Jr WF 1991 Sex steroid control of gonadotropin secretion in the human male. II. Effects of estradiol administration in normal and GnRH-deficient men. J Clin Endocrinol Metab 73:621-628 10. Crowley WF, Vale WW, Rivier J, McArthur JW 1981 LHRH in hypogonadotropic hypogonadism. In: Zatuchini GI, Shelton JD, Sciarra JJ (eds) LHRH Peptides as Female and Male Contraceptives. Program on Applied Research and Fertility Regulation Series on Fertility Regulation. Harper & Row, Philadelphia, pp 321-333 11. Belchetz PE, Plant TM, Nakai Y, Keogh EJ, Knobil E 1978 Hypophyseal response to continuous and intermittent delivery of hypothalamic gonadotropin-releasing hormone. Science 202:631633 12. Spratt DI, Finkelstein JS, Badger TM, Butler JP, Crowley Jr WF 1986 Bio- and immunoactive luteinizing hormone responses to low doses of gonadotropin-releasing hormone (GnRH): dose-response curves in GnRH-deficient men. J Clin Endocrinol Metab 63:143150 13. Spratt D, Finkelstein J, Butler JP, Badger TM, Crowley Jr WF 1987 Effects of increasing the frequency of low doses of gonadotropin-releasing hormone (GnRH) on gonadotropin secretion in GnRH-deficient men. J Clin Endocrinol Metab 64:1179-1186 14. Finkelstein JS, Badger TM, O'Dea LS, Spratt DI, Crowley WF 1988 Effects of decreasing the frequency of gonadotropin-releasing hormone stimulation on gonadotropin secretion in gonadotropinreleasing hormone-deficient men and perifused rat pituitary cells. J Clin Invest 81:1725-33 15. Butler JP, Spratt DI, O'Dea LS, Crowley Jr WF 1986 The interpulse interval sequence of luteinizing hormone in normal men essentially constitutes a renewal process. Am J Physiol 250:E338340 16. O'Dea L StL Finkelstein JS, Schoenfeld DA, Butler JP, Crowley Jr WF 1989 The interpulse interval of GnRH stimulation independently modulates LH secretion. Relation of interpulse interval to LH secretion. Am J Physiol 256:E510-515 17. Seeburg PH, Mason AJ, Stewart TA, Nikolics K 1987 The mammalian GnRH gene and its pivotal role in reproduction. Recent Prog Horm Res 43:69-98 18. Weiss J, Crowley Jr WF, Jameson JL 1989 Normal structure of the gonadotropin-releasing hormone (GnRH) gene in patients with GnRH deficiency and idiopathic hypogonadotropic hypogonadism. J Clin Endocrinol Metab 69:299-303 19. Nakayama Y, Wondisford FE, Lash RW, Bale AE, Weintraub BD, Cutler Jr GB, Radovick S 1990 Analysis of gonadotropin-releasing hormone gene structure in families with familial central precocious puberty adn idiopathic hypogonadotropic hypogonadism. J Clin Endocrinol Metab 70:1233-1238 20. Weiss J, Adams E, Whitcomb RW, Crowley Jr WF, Jameson JL 1991 Normal sequence of the gonadotropin-releasing hormone gene in patients with idiopathic hypogonadotropic hypogonadism. Biol Reprod 45:743-747 21. Schwanzel-Fukuda M, Bick D, Pfaff DW 1989 Luteinizing hormone- releasing hormone (LHRH)-expressing cells do not migrate normally in an inherited hypogonadal (Kallmann) syndrome. Mol Brain Res 6:311-326
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22. Ballabio A, Bardoni B, Carrozzo R, Andria G, Bick D, Campbell L, Hamel B, Ferguson-Smith MA, Gimelli G, Fraccaro M, Maraschio P, Zuffardi 0, Guioli S, Camerino G 1989 Contiguous gene syndromes due to deletions in the distal short arm of the human X-chromosome. Proc Natl Acad Sci USA 86:10001-10005 23. Schwanzel-Fukuda M, Pfaff DW 1989 Origin of luteinizing hormone-releasing hormone neurons. Nature 338:161-164 24. Wray S, Nieburgs A, Elkabes S 1989 Spatiotemporal cell expression of luteinizing hormone-releasing hormone in the prenatal mouse: evidence for an embryonic origin in the olfactory placode. Dev Brain Res 46:309-318 25. Gibson MJ, Krieger DT, Charlton HM, Zimmerman EA, Silverman AJ, Perlow MJ 1984 Mating and pregnancy can occur in genetically hypogonadal mice with preoptic area brain grafts. Science 225:949951 26. Franco B, Guioli S, Pragliola A, Incerti B, Bardoni B, Tonlorenzi R, Carrozzo R, Maestrini E, Pieretti M, Taillon-Miller P, Brown CJ, Willard HF, Lawrence C, Persico MG, Camerino G, Ballabio A 1991 A gene deleted in Kallmann syndrome shares homology with neural cell adhesion and axonal path-finding molecules. Nature 353:529-536 27. Legouis R, Hardelin JP, Levilliers J, Claverie JM, Compain S, Wunderle V, Millassean P, LePaslier D, Cohen D, Caterina D, Bougueleret L, Delemarre-Van de Waal H, Lutfalla G, Weissenbach J, Petit C 1991 The candidate gene for the X-linked Kallmann syndrome encodes a protein related to adhesion molecules. Cell 67:423-435 28. Ewer RW 1968 Familial monotropic pituitary gonadotropin insufficiency. J Clin Endocrinol 28:783-788 29. White BJ, Rogol AD, Brown KS, Lieblich JM, Rosen SW 1983 The syndrome of anosmia with hypogonadotropic hypogonadism: a genetic study of 18 new families and a review. Am J Med Genet 15:417-435 30. Merriam GR, Inese Z, Beitins MD, Bode HH 1977 Father-to-son transmission of hypogonadism with anosmia. Am J Dis Child 131:1216-1219 31. Rogol AD, Kamal KM, White BJ, McGinniss MH, Lieblich JM, Rosen SW 1980 HLA-compatible paternity in two "fertile eunuchs"
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with congenital hypogonadotropic hypogonadism and anosmia (the Kallmann syndrome). J Clin Endocrinol Metab 51:275-279 Santen RJ, Paulsen CA 1973 Hypogonadotropic eunuchoidism. I. Clinical study of the mode of inheritance. J Clin Endocrinol Metab 36:47-54 Hall JG 1992 Genomic imprinting and its clinical implications. N Engl J Med 326:827-829 Wegenke JD, Uehling DT, Wear JB, Gordon ES, Bargman JG, Deacon JSR, Herrmann JPR, Opitz JM 1975 Familial Kallmann syndrome with unilateral renal aplasia. Clin Genet 7:368-381 Swanson SL, Santen RJ, Smith DW 1971 Multiple lentigines syndrome: new findings of hypogonadotrophism, hyposmia, and unilateral renal agencies. J Pediat 78:1037-1039 Smals AGH, Kloppenborg PWC, Benraad THJ 1974 Hypogonadotropic hypogonadism and hyposmia—Kallmann's syndrome. Neth J Med 17:202-211 Berciano J, Amado JA, Freijanes J, Rebollo M, Vaquero A 1982 Familial cerebellar ataxia and hypogonadotropic hypogonadism: evidence for hypothalamic LHRH deficiency. J Neurol Neurosurg Psychiatr 45:747-751 Lowenthal A, Bekaert J, Van Dessel F, van Hauwaert J 1979 Familial cerebellar ataxia with hypogonadism. J Neurol 222:75-80 Neuhauser G, Opitz JM 1975 Autosomal recessive syndrome of cerebellar ataxia and hypogonadotropic hypogonadism. Clin Genet 7:426-434 Matthews WB, Rundle AT 1964 Familial cerebellar ataxia and hypogonadism. Brain 87:463-468 Boucher BJ, Gibberd FB 1969 Familial ataxia, hypogonadism and retinal degeneration. Acta Neurol Scand 45:507-510 Volpe R, Metzler WS, Johnston MW 1963 Familial hypogonadotrophic eunuchoidism with cerebellar ataxia. J Clin Endocrinol Metab 23:107-115 Lux SE, Tse WT, Menninger JC, John KM, Harris P, Shaler O, Chilcotte RR, Marchesi SL, Watkins PC, Bennett V, Mclntosh S, Collins FS, Francke U, Ward DC, Forget BG 1990 Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8. Nature 345:736-739 Eliasson R, Mossberg B, Camner P, Afzelius BA 1977 The immotile-cilio syndrome. A congenital ciliary abnormality as an etiologic factor in chronic airway infections and male sterility. N Engl J Med 297:1-6
Glycoprotein Hormones: Structure, Function and Clinical Implications Santa Barbara, California, March 11-14, 1993 This program will focus on current information relating to the production, structure, and function of the glycoprotein hormones. Topics to be addressed are: regulation of gene expression, receptor structure/function, hormonal protein structure/function, protein processing and posttranslational events, and clinical implications. Drs. Harold Papkoff, Robert Ryan and Darrell Ward will be honored for their pioneering and key contributions made to this field. For information regarding brochure, registration and poster abstract forms, please contact Bruce K. Burnett, Ph.D., Serono Symposia, USA, 100 Longwater Circle, Norwell, Massachusetts 02061. Telephone: (800) 283-8088, (617) 982-9000, or Fax: (617) 982-9481.
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