domain. The LH receptor sequence of the parents was normal. The mutated receptor displayed an up ... domains of the glycoprotein hormone receptor proteins.
0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society
Vol. 83, No. 2 Printed in U.S.A.
A Mutation in the First Transmembrane Domain of the Lutropin Receptor Causes Male Precocious Puberty ¨ RG GROMOLL, CARL-JOACHIM PARTSCH, MANUELA SIMONI, JO VERENA NORDHOFF, WOLFGANG G. SIPPELL, EBERHARD NIESCHLAG, AND BRIJ B. SAXENA* Institute of Reproductive Medicine, University of Mu¨nster (J.G., M.S., V.N. E.N.), Mu¨nster; and the Department of Pediatrics, University of Kiel, (C.-J.P., W.S.), Kiel, Germany; and Cornell University Medical College (B.B.S.), New York, New York 10021 ABSTRACT We describe a patient with onset of puberty at the age of 5 yr, characterized by accelerated growth, enlargement of genitalia, pubarche, and serum hormone levels compatible with noncentral precocious puberty. Exon 11 of the LH receptor gene was amplified from genomic DNA by PCR and directly sequenced. We identified a heterozygous C to T base change at nucleotide position 1126, exchanging codon 373 from Ala to Val in the first transmembrane domain. The LH receptor sequence of the parents was normal. The mutated receptor displayed an up to 7.5-fold increase in basal cAMP production compared to that of the wild-type receptor in transiently
transfected COS-7 cells. Treatment of the patient with ketoconazole resulted in inconsistent suppression of serum testosterone levels. At the age of 9.1 yr, central activation of the hypothalamic-pituitarygonadal axis occurred. Additional treatment with a GnRH agonist led to complete suppression of testosterone secretion. This is the first description of constitutive activation of the LH receptor in the first transmembrane segment. It suggests the involvement of the first transmembrane helix in signal transduction and provides further insight into the structural organization of the seven transmembrane domains of the glycoprotein hormone receptor proteins. (J Clin Endocrinol Metab 83: 476 – 480, 1998)
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mutation in the LH receptor gene, resulting in continuous signal transduction and subsequent testosterone production by the Leydig cells, as the cause of FMPP. To date, nine inherited and five sporadic constitutively activating mutations in the LH receptor causing familial or sporadic malelimited precocious puberty have been reported (12–14). Such mutations have been found mostly in the sixth transmembrane domain and, with decreasing frequency, in the third intracellular loop and fifth transmembrane helix (12). We describe here a new heterozygous sporadic constitutively activating mutation in the LH receptor gene of a patient diagnosed with precocious puberty. This mutation causes a change of Ala to Val in codon 373, an amino acid in the first transmembrane domain that is highly conserved through phylogeny, and implies the participation of this domain in signal transduction.
UTATIONS of genes involved in reproductive hormone action result in abnormal gonadal function. Although only few mutations in the genes of gonadotropins have been described to date (1, 2), elucidation of the complementary DNA (cDNA) sequence and genomic organization of the LH and FSH receptor (3, 4) have made it possible to identify gonadotropin receptor mutations that can be directly linked to specific reproductive disorders. Gonadotropin receptor gene mutations can cause drastic effects on the protein structure-function relationship, posttranslational modification, and intracellular transport of the expressed protein (5, 6) and may lead to receptor activation in the absence of the hormone. To date, only one constitutively activating mutation of the FSH receptor gene has been reported that was capable of autonomously sustaining spermatogenesis in a hypophysectomized man (7). Familial male-limited precocious puberty (FMPP) has been known for more than 50 yr, but it was first characterized in 1981 (8). These patients have accelerated linear growth, development of the secondary sexual characteristics, increased secretion of testosterone, prepubertal levels of serum gonadotropins unresponsive to GnRH stimulation test, and lack of nocturnal pulsatile secretion of pituitary LH (9, 10). Shenker et al. (11) first identified a constitutively activating
Subjects and Methods Case report An 8.3-yr-old Caucasian male presented at the Department of Pediatrics, University of Kiel (Kiel, Germany), because of enlargement of genitalia and pubarche since the age of 5 yr. At the age of 8 yr he had been seen by his local pediatrician. Linear growth acceleration had started around the age of 6 yr, and signs of acne were apparent at the age of 6.5 yr. At age 8.3 yr, his height was 151.4 cm (.97th percentile, 13.6 sd). He showed Tanner stage 2–3 for pubic hair, pubertal testicular volumes (right, 6 mL; left, 8 mL), and beginning of axillary hair growth. His bone age was 13 yr (15, 16). Family history revealed no precocious puberty in either parent. Serum LH was undetectable, and the FSH level was 0.2 IU/L. Serum testosterone levels measured on two separate occasions were 8 and 9.7 nmol/L, respectively. LH was unresponsive to a standard exogenous GnRH stimulation test (100 mg). Serum FSH increased to 3.2 IU/L after GnRH stimulation. The clinical and hormonal data of the patient during
Received July 16, 1997. Revision received October 23, 1997. Accepted November 4, 1997. Address all correspondence and requests for reprints to: Prof. E. Nieschlag, Institute of Reproductive Medicine, University of Mu¨nster, Domagkstrabe 11, D-48129 Mu¨nster, Germany. E-mail: nieschl@ uni-muenster.de. * Guest Professor at the Institute of Reproductive Medicine, University of Mu¨nster.
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TABLE 1. Clinical and hormonal parameters of the patient Age (yr)
Pubertal stage (P/G)a
Testicular vol (mL), right/left
Ht (cm)
8 8.3 8.4 8.5 8.6 8.7 8.8 9 9.1 9.2 9.3 9.4
3– 4/5 2–3/5 2–3/5 2–3/5 2–3/5 2–3/5 4/5 4/4 4/4 4/4 4/4 3– 4/4
10/10 6/8 5/6 6/8 5/7 6/10 6/8 6/8 6/8 6/8
150.2 151.4 151.7 151.8 152.7 153.2 154.5 154.7 154.7 155.7
13.7 13.6 13.6 13.6 13.5 13.5 13.6 13.3 13.2 13.3
4/5
156.7
13.2
Ht
score CAb
SD
Growth rate (cm/yr)
Bone age (yr)
PAH (cm)c
LH (IU/L), basal/GnRH
FSH (IU/L), basal/GnRH
Testosterone (nmol/L)
10.8 (16.6 SD)
13.0
176.7
5.6 (10.3 SD)
14.0
170.7
,2 /,2 ,0.5 /5.0 1.6 1.5 2.5 1.8 1.3 2.5 2.5 /30.4
0.2 /3.2 ,0.5 /1.9 4.0 2.6 5.9 3.6 5.6 8.0 7.7 /18.5
3.4 (22.6 SD)
15.0
165.3
,0.5 ,0.5 /,0.5
,0.5 ,0.5 /,0.5
8.0 /9.7 6.9 2.0 0.2 2.6 0.5 4.1 4.8 5.9 8.0 0.5 1.1
a
P, Pubic hair stage; G, genital stage. score CA, SD score for chronological age. c Predicted adult height (Ref. 16). b
SD
the follow-up are summarized in Table 1. Treatment was started at the age of 8 yr with ketoconazole (600 mg daily) to suppress testosterone production. After 14 days of ketoconazole treatment, the GnRH stimulation test revealed a basal serum LH level less than 0.5 IU/L and a stimulated level of 5.0 IU/L. Serum FSH levels were less than 0.5 IU/L basally and 1.9 IU/L when stimulated. The serum testosterone level was 6.9 nmol/L. Subsequently the ketoconazole dose was changed several times by the patient’s mother, and testosterone suppression was variable. At the age of 9.1 yr, central activation of the hypothalamic-pituitary-gonadal axis was diagnosed by an elevated LH response to exogenous GnRH (Table 1) and spontaneous pulsatile LH secretion during the night (Fig. 1). The patient received additional treatment with the GnRH agonist triptorelin (Decapeptyl Depot, Ferring Arzneimittel, Kiel, Germany) at a dose of 3.75 mg every four weeks, sc. With this therapeutic regimen, LH secretion was completely suppressed, and serum testosterone levels returned to the prepubertal range (Table 1 and Fig. 1).
DNA isolation, PCR, and DNA sequencing DNA was isolated from the blood sample of the patient as well as from his parents and two normal male volunteers with normal hormonal levels and normal sperm count according to WHO criteria (17). Genomic DNA was purified by anion exchange chromatography (Qiagen, Dusseldorf, Germany). Exon 11 of the LH receptor gene (18) was amplified in two overlapping subfragments, indicated here as A and B. Fragment A was amplified using the forward primer 59-CCCTTACCTCAAGCCAATAA-39 and the reverse primer 59- TGAAGAAGGCCACCACATTG-39 from the intronic nucleotide position 231, 59 of exon 11, to nucleotide position 674. Fragment B was amplified using the forward primer 59-GATGTGGAAACCACTCTCTC-39 and the reverse primer 59ATGTTAAAATTACTGGTACAGG-39, corresponding to nucleotide positions 603-1231 in exon 11. Each PCR sample (50 mL) contained 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 0.01% gelatin, 2 mmol/L MgCl2, 0.2 mmol/L deoxy-NTPs, 2.5 U Taq polymerase (Promega, Heidelberg, Germany), and 100 nm/L primer. Denaturation at 94 C for 4 min was followed by 35 cycles at 94 C for 50 s, at 58 C for 35 s, at 72 C for 1 min and 30 s, and a final elongation step at 72 C for 10 min. The amplified fragments were directly sequenced with a PCR sequencing kit (U.S. Biochemical Corp., Braunschweig, Germany) using the same primers.
Mutagenesis The human LH receptor cDNA (18), cloned into the EcoRI restriction site of pSG5 (Stratagene, Heidelberg, Germany), was mutagenized at position 373 (Ala), which was converted into Val by oligonucleotidemediated site-directed mutagenesis using the Transformer site-directed mutagenesis kit (Clontech, ITC Biotechnology, Heidelberg, Germany). The selection primer (59-GAGTGCACCATGGGCGGTGTGAAAT-39) converted the unique NdeI restriction site of pSG5 into NcoI. The mutagenic primer was 59-GGCTGATTAATATTCTAGTCATCATGG-
FIG. 1. Nocturnal serum LH secretion pattern in a patient with testotoxicosis treated with ketoconazole (600 mg/daily for 1 yr) at the time of central activation of the hypothalamic-pituitary axis (closed circles) and after the addition of a GnRH agonist (open circles). Asterisks denote significant peaks.
GAAACATGAC-39. The mutation was confirmed by direct PCR sequencing. Plasmids were isolated and purified by anion exchange columns (Qiagen).
Transfection and cAMP assay COS-7 cells were grown in six-well plates in DMEM and 10% heatactivated FCS until the cells reached 50 –70% confluence. Transfection of wild-type and mutated LH receptor was performed separately, using the Lipofectamine reagent (Life Technologies, Eggenstein, Germany). Fortyeight hours after transfection, cells were washed and incubated for 2 h at 37 C in Dulbecco’s phosphate-buffered saline, 0.2% glucose, 0.1% BSA, and 0.1 mmol/L isobutylmethylxanthine in the absence and presence of increasing doses of hCG (Pregnesin, Organon, Oss, Holland). Media were collected for the RIA of cAMP as described previously (19), using an antiserum against cAMP obtained from the Institute of Hormone and Fertility Research (Hamburg, Germany). The transfection experiments were repeated at least twice.
Results
Upon sequencing exon 11 of the LH receptor, a heterozygous point mutation of nucleotide 1126 from C to T was detected, resulting in an amino acid transition from Ala (GCC) to Val (GTC) at position 373 (Fig. 2). This base exchange was not detected in the two normal control subjects or in the parents (data not shown), suggesting that the mutation is a de novo event in the patient. The Ala373Val mutation is located in the first transmembrane domain and causes the
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FIG. 2. Partial sequence of exon 11 of the mutated LH receptor of the patient with male-limited precocious puberty. The presence of the nucleotide T at the second position of codon 373 resulted in a heterozygous Ala to Val transition.
FIG. 3. Comparison of the cDNA deduced amino acid sequence around the first transmembrane domain of human LH, FSH, and TSH receptors and the corresponding amino acid sequence of the glycoprotein hormone receptors of Drosophila (31), mollusk (32), and sea anemone (33), indicating the highly conserved pattern of Ala373 throughout the animal kingdom. The box shows the limits of the transmembrane segment. The A at position 373 of the human LH receptor is perfectly conserved (bold letter).
substitution of an amino acid highly conserved in invertebrate, mammalian, and human glycoprotein hormone receptors (Fig. 3). COS-7 cells transiently transfected with increasing amounts of the mutant LH receptor cDNA consistently produced cAMP in the absence of hCG in a direct dose-response relationship, whereas increasing concentrations of the wildtype LH receptor did not stimulate cAMP production (Fig. 4). At the dose of 0.5 mg plasmid DNA, the mutant produced 7to 8-fold higher levels of cAMP than the wild-type receptor. This marked cAMP production by the mutant LH receptor in the absence of stimulation indicates that the receptor is constitutively active. When COS-7 cells were transfected with 2 mg/well wild-type and mutant LH receptor plasmid DNA, a dose-dependent increase in cAMP production was observed after stimulation with increasing amounts of hCG. However, the mutant receptor showed a 2- to 4-fold greater cAMP production than that by the wild-type receptor in the dose range 0 –1.5 mIU/mL, followed by a plateau at higher concentrations (Fig. 4). Discussion
Initiation of sexual development in boys is heralded by the surge of nocturnal gonadotropin secretion followed by an increase in serum testosterone, particularly in the early morning. These hormonal events result in the development of secondary sexual characteristics. FMPP is inherited with an autosomal dominant male-limited pattern in boys. The disorder usually becomes apparent between birth and the age of 4 yr (11, 12). On the basis of earlier reports, Shenker et al. (11) and Kremer et al. (20) predicted that FMPP may be the consequence of a mutant, constitutively activated LH
FIG. 4. Left panel, Basal and hCG-stimulated cAMP accumulation by COS-7 cells (105 cells/well) transiently transfected with 2 mg wild-type and mutated LH receptor constructs. Control, LH receptor cloned in the reverse orientation. Right panel, cAMP production of COS-7 cells (105 cells/well) transfected with increasing concentrations of LH receptor cDNA constructs. Results are expressed as the mean 6 SEM of an experiment performed in triplicate, considering cAMP production in mock-transfected cells as the control. Four other independently performed experiments gave similar results.
receptor. These researchers were the first to identify an A to G base change in the LH receptor gene that led to the substitution of Asp578 by Gly in the sixth transmembrane domain and was responsible for the autonomous Leydig cell activity. Idiopathic central precocious puberty is rare in boys. The absence of intracranial lesion and the suppressed gonadotropin levels in FMPP suggest that it is a gonadotropinindependent pathology, comparable to the McCune-Albright syndrome. Patients with male-limited precocious puberty due to sporadic mutation in the sixth transmembrane domain, as described by Laue et al. (13), and in the third intracellular loop, as described by Yano et al. (14), have a similar clinical history as FMPP patients. The clinical history of our patient with a sporadic mutation of Ala373Val in the first transmembrane domain was comparable to that of patients with FMPP. Such patients can be treated with either medroxyprogesterone acetate (9), ketoconazole (an inhibitor of testosterone biosynthesis) (21, 22), the antiandrogens cyproterone acetate and spironolactone (21, 22), the aromatase inhibitor testolactone (23), a GnRH agonist (23, 24), or a combination thereof (23). With advancing age, the advent of the physiological gonadotropin rise, and late pubertal development, FMPP patients tend to become normal fertile males (10, 25–27). However, the therapeutic management of inherited FMPP in early childhood is difficult. In our patient the treatment with ketoconazole was only partially successful. Growth potential was not preserved by ketoconazole treatment. Moreover, in association with the markedly accelerated bone age, which was 13 yr at first presentation, central precocious puberty had already occurred after 1 yr of treatment with ketoconazole. Combined treatment with ketoconazole and GnRH agonist then resulted in complete suppression of pituitary LH secretion and serum testosterone levels. This observation underscores earlier findings by Holland et al. (21), who demonstrated central activation of a GnRH pulse generator set at the adult level of sensitivity due to the marked acceleration of the maturational status (bone age, .11 yr). Central precocious puberty also has been re-
LH RECEPTOR-ACTIVATING MUTATION
ported as a complication after treatment of peripheral precocious puberty in cases of McCune-Albright syndrome, hormone-producing adrenal tumors, and congenital adrenal hyperplasia (28 –30). These cases demonstrate that the hypothalamic-pituitary-gonadal axis is functionally intact in such patients. To our knowledge, this is the first report of an activating mutation in the first membrane-spanning domain of the LH receptor. The recent identification of ancient forms of putative ancestors of the glycoprotein hormone receptors in Drosophila melanogaster (31), the mollusk Lymnaea stagnalis (32), and the sea anemone Anthopleura elegantissima (33) revealed a surprisingly high homology among the transmembrane domains and parts of the extracellular domain. The amino acid Ala, mutated in the patient described here, is conserved in all glycoprotein hormone receptors known throughout species ranging from flies to humans, indicating a crucial role of the first transmembrane domain in retaining the LH receptor in an inactive state. It would be interesting to see whether a mutation introduced into the corresponding position of the FSH and TSH receptor gene would cause a similar, high constitutive activation. Based on the high frequency of mutations, it has been postulated that the gonadotropin receptor region containing the fifth and sixth transmembrane segments and the third intracellular loop is a hot spot for point mutations; it may, therefore, be involved in G protein coupling and signal transduction (12). Mutation of Asp578Gly accounts for 82% of all reported FMPP families (11, 13). As described here, the mutation of the highly conserved Ala373 to Val in the first transmembrane domain also produced almost 7- to 8-fold greater accumulation of cAMP. The occurrence of activating mutations in the first and second transmembrane helixes indicates that these segments are important for signal transduction, suggesting their possible location in close proximity to the aforementioned hot spot region. The structural changes induced by these mutations are likely to create significant conformational modifications, such as retaining the receptor in an activated state. The current concepts of the arrangement of transmembrane helixes (34) may be further refined to accommodate the steric constraints imposed by new mutations emerging in the LH receptor gene. Activating LH receptor mutations do not impair the reproductive potential of the patients, although the clinical significance of these findings requires further studies. Gene mutations, however, present pathophysiologies challenging our understanding of the molecular basis of reproductive disease and providing the opportunity to develop improved and focussed therapeutic tools. Elucidation of the conformational changes involved in the transition from an inactive receptor to an autonomously active state and vice versa will lead to new approaches in the synthesis of agonistic and antagonistic compounds useful for the therapeutic manipulation of gonadal function. Acknowledgments We thank Dr. E. Milgrom, Unite´ de Recherches Hormones et Reproduction, Le Kremlin Bicetre, France, for the generous gift of the expression vector with the human LH receptor cDNA. We thank E. Pekel and
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B. Schuhmann for excellent technical assistance. We also thank PD Dr. J. Wiebel (Hamburg, Germany) for referring the patient.
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31. Hauser F, Nothacker HP, Grimmelikhuijzen JP. 1997 Molecular cloning, genomic organization and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals. J Biol Chem. 272:1002–1010. 32. Tensen CP, Van Kesteren ER, Planta RJ, et al. 1994 A G protein-coupled receptor with low density lipoprotein-binding motifs suggests a role for lipoproteins in G-linked signal transduction. Proc Natl Acad Sci USA. 91:4816 – 4820. 33. Nothacker HP, Grimmelikhuijzen CJ. 1993 Molecular cloning of a novel, putative G protein-coupled receptor from sea anemones structurally related to members of the FSH, TSH, LH/CG receptor family from mammals. Biochem Biophys Res Comun. 197:1062–1069. 34. Baldwin JM. 1993 The probable arrangement of the helices in G proteincoupled receptors. EMBO J. 12:1693–1703.
