| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Departments of Human Genetics (H.K., M.v.R., E.C.M.M., H.G.B.) and Pediatrics (B.J.O.), University Hospital, 6500 HB Nijmegen, The Netherlands; the Department of Endocrinology and Reproduction, Erasmus University (J.W.M.M., M.V.-P., A.P.N.T.), 3000 DR Rotterdam, The Netherlands; the Department of Pediatrics, University Hospital (J.M.W.), Leiden, The Netherlands; the Department of Pediatrics, Sophia Children’s Hospital (S.L.S.D.), 30156 Rotterdam, The Netherlands; the Department of Pediatrics, Free University (H.A.D.-v.d.W.), 1081 HV Amsterdam, The Netherlands; the Department of Pediatrics (M.P.-A.), Hospital General de Galacia 15705 Santiago de Compostella, Spain; the Department of Pediatrics, University Hospital (F.D.L.), 90123 Messina, Italy; Hospital Materna-Infantil (N.P.), 08035 Barcelona, Spain; Department of Pediatrics, University of Leeds (J.M.H.B.), Leeds, United Kingdom; Wilhelmina Children’s Hospital (M.J.), 3501 CA Utrecht, The Netherlands; the Department of Pediatrics, Emory University (J.S.P., H.A.L.), Atlanta, Georgia 30322; the Department of Pediatrics, University of Mississippi Medical Center (G.W.M.), Jackson, Mississippi 39216; the Department of Pediatrics, University of Dusseldorf (W.E.), 40225 Dusseldorf, Germany; and the Department of Pediatrics, University Hospital Pisa (G.S.), 56125 Pisa, Italy
Address all correspondence and requests for reprints to: Dr. H. G. Brunner, Department of Human Genetics, University Hospital Nijmegen, Postbox 9101, 6500 HB Nijmegen, The Netherlands. E-mail: h.brunner{at}antrg.azn.nl
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Patients (Table 1
) were recruited
into the study through contacts with individual pediatricians as well
as through a collaborative effort that was supported by the European
Society for Pediatric Endocrinology. For each patient the following
data were reviewed: family history, personal history, physical
examination at presentation (including penile length, pubic hair,
testicular volume, and axillary hair), growth curve, bone age,
testosterone, LH, FSH, results of GnRH test, androstenedione,
dehydroepiandrosterone, dehydroepiandrosterone sulfate,
17
-hydroxyprogesterone, and other investigations when applicable.
The results of treatment with ketoconazole or
testolactone/spironolactone were noted as well as subsequent data for
growth, testicular volume, and serum levels of testosterone, LH, and
FSH. Patients who were considered eligible for this study had signs of
precocious puberty before the age of 8 yr, accelerated growth velocity,
increased serum levels of testosterone, normal adrenal androgens, a
prepubertal response to GnRH, and the absence of clinically or
radiographically detectable tumor of the testis. Results for three of
these kindreds have been reported previously (7, 12). All but two of
the families were of European origin. In three patients, a feature was
present that is not usually seen in FMPP. These patients had unilateral
testicular enlargement without evidence of a tumor or of
McCune-Albright syndrome. As these patients had LH-independent
precocious puberty and no other specific diagnosis, they were included
in the sample as atypical FMPP.
|
Genomic DNA was isolated from peripheral blood as previously
described (13). Seven sets of primers were designed to perform single
strand conformation polymorphism (SSCP) analysis of overlapping
fragments of exon 11 (Table 2).
Amplification was performed in a 25-µL reaction mix containing 0.1
µg genomic DNA; 250 µmol/L deoxy (d)-ATP, dGTP, and dTTP; 0.125
µmol/L dCTP; 1 µCi [-32P]dCTP (3000 Ci/mmol); 10
mmol/L Tris-HCl (pH 8.5); 50 mmol/L KCl; 1.5 mmol/L MgCl2;
0.01% gelatin; 50 ng of each of the primers; and 1 U Taq
polymerase (Boehringer Mannheim, Mannheim, Germany). For amplification,
a first step of denaturation of 5 min at 94 C was followed by 35 cycles
of 1 min at 94 C, 2 min at a primer set-dependent temperature, and 1
min at 72 C. The amplification was followed by a 7-min extension at 72
C. The annealing temperatures were 56, 50, 50, 56, 58, 55, and 50 C for
primer sets 1–7, respectively (11).
|
-32P]dCTP was omitted. Amplified fragments were
recovered from 1.5% low melting point agarose using gelase (Epicentre,
Madison, WI) according to the manufacturer’s protocol. Purified
PCR fragments were sequenced using the ds-cycle sequencing system (BRL,
Gaithersburg, MD). If no mutation was found in exon 11, the portion of
the gene encoding the N-terminal extracellular domain (exons 1–10) was
also examined, using primers previously described (15). Functional analysis
In vitro expression of the wild-type and mutant human LH receptor and subsequent determination of CRE reporter activity was performed as described previously (16). Briefly, HEK293 cells were transfected with either a plasmid control, the wild-type LHR construct, or a mutant LHR (I575L) that was derived from the wild-type LHR by standard PCR mutagenesis. Three days after transfection, basal and maximal hCG-dependent CRE responses were measured by incubating the cells for 4 h with increasing concentrations of hCG. Subsequently, the cells were lysed, and luciferase activity was measured. Values were corrected for transfection efficiency after determination of the activity of a cotransfected ß-galactosidase construct (16).
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Genomic DNA of eight familial and nine sporadic patients with
LH-independent male precocious puberty was screened for the presence of
mutations of the LH receptor gene with SSCP analysis and direct
sequencing. Mutations were detected in all eight familial patients and
in four of six sporadic patients with a typical phenotype, but in none
of three patients with atypical clinical features (Table 1
).
Functional analysis
To assess the functional effects of the mutations, both mutated
and wild-type receptors were expressed in vitro in human
embryonic kidney cells (HEK293) and tested for stimulation of adenylyl
cyclase. This analysis was previously performed for the mutations
Met571Ile, Asp578Gly (7), and
Met398Thr, and results have been described in detail by
Kraaij et al. (12). The Ile575Leu mutation was
analyzed with similar methods, employing a cAMP-responsive element
luciferase reporter (16). Basal cAMP production was elevated 15- to
25-fold in the cells expressing the mutated receptors compared to that
in cells expressing the wild-type receptor after correction for
transfection efficiency (Fig. 1). This is
consistent with the findings of a previous report (17). In isolated
Leydig cells from rats, it has been shown that small increases in cAMP
production lead to maximal induction of steroid production (18).
Therefore, the elevated cAMP levels in response to the expression of
the mutant receptors explain the LH-dependent precocious puberty in the
patients. The activating effect of the remaining mutations has been
previously described (9).
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
We here present data on 17 patients with male LH-independent
precocious puberty who have been analyzed for the presence of mutations
of the LH receptor gene. To date, activating mutations in FMPP patients
have only been described for the transmembrane domains and the
interconnecting loops of the receptor (Table 3). In our sample of 17 FMPP patients (8
familial and 9 with a negative family history), we detected 7 different
mutations in 12 patients. Of these 7 mutations, 2 were detected more
than once. The Ile542Leu was present in 4 Dutch kindreds
(T3, T4, T6, and T17), suggesting a common ancestor as the cause
for this clustering, although no genealogical relationship could be
demonstrated. This mutation has also been reported in the literature in
3 families and in 1 sporadic case (de novo) from the U.S.
(9). The Met398Thr mutation was found in kindreds T7 and
T16 from Germany and in patient T10 from Sicily (Italy) in our sample
and has independently been found in a sporadic patient as well as in a
FMPP kindred from the United Kingdom (19) and an additional FMPP
patient from Japan (20). In contrast to previous reports (8, 9), the
Asp578Gly mutation was not frequent in our sample. In fact,
none of the 10 European kindreds with LH receptor mutations in the
present study had the Asp578Gly mutation, and the only
family with this mutation in our sample is from the U.S. (7). In this
family the mutation spans at least 9 generations (3). Thus, there is a
strong founder effect for this mutation in the U.S., where over 90% of
testotoxicosis families have the Asp578Gly mutation (8, 9).
Results similar to ours have been obtained in another study of 6 FMPP
kindreds from Europe, which also lacked the Asp578Gly
mutation (10, 11). Nevertheless, the presence of a founder effect for
Asp578Gly is not the only explanation for the frequent
occurrence of this specific mutation in American FMPP families, as the
Asp578Gly mutation has also been found to occur in 5
sporadic FMPP patients who had a de novo LH receptor gene
mutation (9, 21, 22, 23).
|
). The restricted
number of LH receptor mutations found in FMPP kindreds as well as in
sporadic cases strongly suggests that only mutations in specific areas
of the receptor, in particular the sixth transmembrane region, can
autonomously activate cAMP production. Activating mutations in the TSH
receptor, which has a very high similarity to the LH receptor, also
tend to cluster in the region of the sixth transmembrane domain and the
third intracellular loop (17, 24, 25, 26, 27, 28). Several of these activating TSHR
gene mutations involve the same residues as they do in the LH receptor
gene of FMPP patients. Taken together, the explanation for the
restricted number of mutations observed in FMPP patients from around
the world appears to be 2-fold. On the one hand, constitutive
activation is associated only with mutations of specific amino acids.
On the other hand, FMPP-associated LH receptor gene mutations may be
transmitted for many generations, allowing them to spread widely in
certain populations. Genotype-phenotype correlations
For most mutations in our study no obvious phenotypic differences were detected. Each was associated with onset of puberty between ages 2–6 yr, with as much variability in age at onset and growth velocity between cases with the same mutation as between cases with different mutations. An early age of onset (22 months) occurred in a patient (T13) from the United Kingdom who carries tyrosine for aspartic acid at codon 578. Very early onset of symptoms of testosterone excess has been noted previously for this mutation (9, 20, 23). This early onset is consistent with in vitro analysis of this mutation, which indicates a relatively high level of basal cAMP production compared to other LH receptor gene mutations (9).
Patients without LH receptor gene mutations
Of the three patients who had atypical features, none was found to carry a LH receptor mutation. Two cases (T11 and T15) had marked unilateral testicular enlargement, suggesting a contribution of both Leydig and Sertoli cell activation. In patient T11, testis size was 20 mL on the right and 4 mL on the left at age 2 yr, 8 months. Testis biopsy showed Leydig cell hyperplasia in the absence of neoplastic alterations. Patient T15 had a testicular volume of 20 mL on the right and 1 mL on the left at age 8 yr. Unilateral macroorchidism in this case was first noted at 8 months of age, at which time a testicular biopsy was performed, which was normal for age. Patient T5 had LH-independent precocious puberty at the age of 5.5 yr. A small difference in testis size was noted, the significance of which is unclear. The right testicular volume was 5 mL, and the left was 2–3 mL. In-depth investigations at age 8 yr revealed an elevated testosterone level (11 nmol/L) that failed to be suppressed with dexamethasone administration. hCG stimulation resulted in a prompt rise in plasma testosterone, suggesting a testicular instead of an adrenal origin of the excess androgens. Moreover, adrenal scintigraphy and selective phlebographic sampling of both adrenal veins failed to detect an adrenal origin of the excess androgens. A brain computed tomography scan was normal. Upon follow-up at the age of 22 yr, the right testicular volume was 13 mL, and the left was 9 mL. Serum LH and FSH were still suppressed [LH, <0.5 IU/L (normal, 2–15); FSH, 0.3 IU/L (normal, 3–16)]. The serum testosterone level was 21 nmol/L. Additional investigations in patients T11 and T15 (including ultrasound examination and magnetic resonance imaging of the testis) and follow-up have likewise failed to detect a tumor. Thus, hCG-secreting tumors are either absent or clinically undetectable in these patients. We hypothesize that these cases of LH-independent puberty could be due to a somatic mutation in one testis only, perhaps involving the stimulatory G protein gene, which has been shown to be somatically mutated in McCune-Albright syndrome (29, 30). However, molecular analysis of a testicular biopsy specimen from patient T11 excluded the gsp and gip mutations (data not shown), and additional signs of McCune-Albright syndrome (such as pigmentation or polyostotic fibrous dysplasia) were lacking in all three patients with unilateral testicular enlargement. Interestingly, an unusual G protein mutation has been reported in two unrelated boys with testotoxicosis and pseudohypoparathyroidism (31). This finding again underlines the propensity for G protein gene mutations to autonomously activate testicular cAMP production, thereby causing increased testosterone synthesis in the absence of LH receptor abnormalities. None of the present patients (T5, T11, or T15) had any signs of pseudo-hypoparathyroidism.
The final two patients with LH-independent precocious puberty without LH receptor mutation (T12 and T14) were phenotypically indistinguishable from the others in that both had LH-independent precocious puberty, virilization without distinct testicular growth at an early age, low gonadotropin values, increased testosterone levels exclusively from testicular origin, and rapid growth associated with an advanced bone age. After 6 yr of follow-up, one of these patients (case 12) developed central diabetes insipidus. A magnetic resonance imaging scan of the brain detected a lesion, which was subsequently found to be a dysgerminoma. The final patient with apparent FMPP is still clinically normal at age 12 yr. One might speculate that in this case the causative mutation involves a gene functioning downstream of the LH receptor. This could reside in the receptor kinase and arrestin genes, both of which function in the termination of receptor signaling. An example of a postreceptor defect interfering with inactivation of G protein-mediated signal transduction is provided by the finding that a form of night blindness in Japanese patients (Oguchi disease) is associated with a homozygous frameshift mutation in the retinal arrestin gene (32). In conclusion, based on our experience, almost all patients with clinically typical FMPP have an activating mutation of the LH receptor gene. Patients with marked unilateral testicular enlargement and LH-independent puberty do not have true FMPP, and no LH receptor gene mutations are found. We suggest that extensive clinical reevaluation of other causes should follow if no LH receptor mutation can be demonstrated in a male patient with LH-independent precocious puberty.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 These authors contributed equally to this work.
Received July 15, 1998.
Revised November 17, 1998.
Accepted November 19, 1998.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Tyr of the lutropin/choriogonadotropin receptor
gene. J Med Genet. 35:340–341. subunit of
the stimulatory G protein of adenylyl cyclase in McCune-Albright
syndrome. Proc Natl Acad Sci USA. 89:5152–5156. in patients with
gain and loss of endocrine function. Nature. 371:164–168.[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  S Gupta, T D Cheetham, H J Lambert, C Roberts, D Bourn, M G Coulthard, and S G Ball Thirst perception and arginine vasopressin production in a kindred with an activating mutation of the type 2 vasopressin receptor: the pathophysiology of nephrogenic syndrome of inappropriate antidiuresis Eur. J. Endocrinol., September 1, 2009; 161(3): 503 - 508. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ravindranathan, G. Joslyn, M. Robertson, M. A. Schuckit, J. L. Whistler, and R. L. White Functional characterization of human variants of the mu-opioid receptor gene PNAS, June 30, 2009; 106(26): 10811 - 10816. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Nurwakagari, A. Breit, C. Hess, H. Salman-Livny, D. Ben-Menahem, and T. Gudermann A conformational contribution of the luteinizing hormone-receptor ectodomain to receptor activation J. Mol. Endocrinol., February 1, 2007; 38(2): 259 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Richter-Unruh, M. Verhoef-Post, S. Malak, J. Homoki, B. P. Hauffa, and A. P. N. Themmen Leydig Cell Hypoplasia: Absent Luteinizing Hormone Receptor Cell Surface Expression Caused by a Novel Homozygous Mutation in the Extracellular Domain J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5161 - 5167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. M. Martens, S. Lumbroso, M. Verhoef-Post, V. Georget, A. Richter-Unruh, M. Szarras-Czapnik, T. E. Romer, H. G. Brunner, A. P. N. Themmen, and Ch. Sultan Mutant Luteinizing Hormone Receptors in a Compound Heterozygous Patient with Complete Leydig Cell Hypoplasia: Abnormal Processing Causes Signaling Deficiency J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2506 - 2513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Richter-Unruh, H. T. Wessels, U. Menken, M. Bergmann, K. Schmittmann-Ohters, J. Schaper, S. Tappeser, and B. P. Hauffa Male LH-Independent Sexual Precocity in a 3.5-Year-Old Boy Caused by a Somatic Activating Mutation of the LH Receptor in a Leydig Cell Tumor J. Clin. Endocrinol. Metab., March 1, 2002; 87(3): 1052 - 1056. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Latronico, H. Shinozaki, G. Guerra Jr., M. A. A. Pereira, S. H. V. Lemos Marini, M. T. M. Baptista, I. J. P. Arnhold, F. Fanelli, B. B. Mendonca, and D. L. Segaloff Gonadotropin-Independent Precocious Puberty Due to Luteinizing Hormone Receptor Mutations in Brazilian Boys: A Novel Constitutively Activating Mutation in the First Transmembrane Helix J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4799 - 4805. [Abstract] [Full Text]
|
|
|
|
 |
|
 A. P. N. Themmen and I. T. Huhtaniemi Mutations of Gonadotropins and Gonadotropin Receptors: Elucidating the Physiology and Pathophysiology of Pituitary-Gonadal Function Endocr. Rev., October 1, 2000; 21(5): 551 - 583. [Abstract] [Full Text]
|
|
|
|
 |
|
 H. G. Brunner and B. J. Otten Precocious Puberty in Boys N. Engl. J. Med., December 2, 1999; 341(23): 1763 - 1765. [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
