Familial gonadotropin-releasing hormone resistance and hypogonadotropic hypogonadism in a family with multiple affected individuals

Familial gonadotropin-releasing hormone resistance and hypogonadotropic hypogonadism in a family with multiple affected individuals

FERTILITY AND STERILITY威 VOL. 75, NO. 6, JUNE 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Print...

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FERTILITY AND STERILITY威 VOL. 75, NO. 6, JUNE 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

Familial gonadotropin-releasing hormone resistance and hypogonadotropic hypogonadism in a family with multiple affected individuals Lawrence C. Layman, M.D.,a,b Paul G. McDonough, M.D.,a David P. Cohen, M.D.,c Mary Maddox,a Sandra P. T. Tho, M.D.,a and Richard H. Reindollar, M.D.d The Medical College of Georgia, Augusta, Georgia

Received July 28, 2000; revised and accepted December 22, 2000. Supported by U.S. Public Health Service-National Institute of Child Health and Human Development grant HD33004 (Dr. Layman). Reprint requests: Lawrence C. Layman, M.D., Section of Reproductive Endocrinology, Infertility, and Genetics, Department of Obstetrics and Gynecology, The Medical College of Georgia, 1120 15th Street, Augusta, Georgia 30912-3360 (FAX: 706-721-6830; E-mail: [email protected]). a Section of Reproductive Endocrinology, Infertility, and Genetics, Department of Obstetrics and Gynecology. b Developmental Biology Program. c Section of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, The University of Chicago, Chicago, Illinois. d Section of Reproductive Endocrinology and Infertility, Beth Israel Deaconess Medical Center, Harvard University, Boston, Massachusetts. 0015-0282/01/$20.00 PII S0015-0282(01)01782-4

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Objective: To characterize the phenotype of idiopathic hypogonadotropic hypogonadism due to compound heterozygous GnRHR gene mutations (Arg262Gln/Tyr284Cys). Design: Retrospective review. Setting: Tertiary medical center. Patient(s): Family containing four siblings (three female and one male) with complete idiopathic hypogonadotropic hypogonadism. Intervention(s): Baseline and stimulated laboratory studies. One patient received GnRH treatment and one received human menopausal gonadotropins. Main Outcome Measure(s): Clinical phenotype vs. genotype is assessed by endocrine studies, karyotype, pedigree, and review of pathology slides of ovarian neoplasm. Result(s): With GnRH stimulation, two patients with idiopathic hypogonadotropic hypogonadism had maximum LH ⬍ 10 mIU/mL, and two others had peak LH ⬎ 10 mIU/mL. With repeated GnRH stimulation 24 hours later, gonadotropin levels in all patients were increased. Stimulation of thyroid-releasing hormone and tests for insulin-induced hypoglycemia were normal. One affected patient did not ovulate after GnRH treatment, but her sister ovulated with gonadotropin treatment. Another affected sibling had bilateral oophorectomy for seromucinous cystadenomas, and her hypogonadotropic state remained after castration. The man with idiopathic hypogonadotropic hypogonadism and his unaffected brother had a ring chromosome 21. Conclusion(s): All patients with complete idiopathic hypogonadotropic hypogonadism had the same GnRHR mutations, but clinical presentations and endocrinologic responses were heterogeneous. Gonadotropin levels remained low in patients with idiopathic hypogonadotropic hypogonadism after castration, and ring chromosome 21 was present, suggesting that sequences from this chromosome could affect the idiopathic hypogonadotropic hypogonadism phenotype. (Fertil Steril威 2001;75:1148 –55. ©2001 by American Society for Reproductive Medicine.) Key Words: Idiopathic hypogonadotropic hypogonadism, GnRH receptor, molecular genetics, ring chromosome 21

Genetic forms of human idiopathic hypogonadotropic hypogonadism have only recently been characterized (1–3). The first form of hypogonadotropic hypogonadism for which the genetic basis was elucidated was the Kallmann syndrome, an X-linked recessive disorder in which hypogonadotropic hypogonadism is accompanied by anosmia and occasionally by other anomalies, such as midfacial clefting, neurologic deficiencies, and unilateral renal agenesis (4).The KAL1 gene on Xp encodes a protein involved in embryologic migration of gonadotropin-releasing

hormone (GnRH) and olfactory neurons from the olfactory placode to the hypothalamus (5, 6). KAL1 gene mutations explain the phenotype of hypogonadotropic hypogonadism and anosmia (5–9), and KAL1 expression in midfacial mesenchyme, mesonephros and metanephros, cerebellum, and oculomotor nucleus parallels the phenotypic abnormalities in humans (4, 8, 9). Another X-linked recessive disease is adrenal hypoplasia congenita (AHC)/hypogonadotropic hypogonadism, which presents as adre-

nal failure after birth or during childhood (10 –15). Children who are treated and reach adolescence have delayed puberty secondary to hypogonadotropic hypogonadism. Mutations in the AHC gene encoding the dosage-sensitive sex reversalAHC critical region of the X-chromosome, gene 1 (DAX1) gene, an orphan receptor in the steroid receptor family, appear to cause both adrenal hypofunction and hypogonadotropic hypogonadism (10 –15). The defects in gonadotropin secretion appear to involve both the pituitary and hypothalamus (15). Until recently, KAL1 and DAX1 were the only causative genes involved in inherited human idiopathic hypogonadotropic hypogonadism, and they affected only men. The molecular basis for the first autosomal recessive form of idiopathic hypogonadotropic hypogonadism, affecting both women and men, was determined to be mutations in the GnRH receptor (16, 17). De Roux et al. (16) described compound heterozygous GnRH receptor mutations in two probands with partial idiopathic hypogonadotropic hypogonadism. On screening 46 unrelated idiopathic hypogonadotropic hypogonadism patients, we identified one family with four affected individuals who were compound heterozygotes for GnRH receptor gene mutations (17). The specific missense GnRH receptor gene mutations and their effects upon GnRH receptor function in vitro in our family with idiopathic hypogonadotropic hypogonadism have been described previously (17). Although the principal effect of both missense gene mutations was impaired signal transduction of GnRH, the clinical characteristics were not completely described (17). The purpose of the present study was to better define the clinical presentation, physical findings, and pedigree analysis and characterize completely the endocrinologic profiles in affected individuals.

MATERIALS AND METHODS The clinical features and pedigree of this family with idiopathic hypogonadotropic hypogonadism were reviewed retrospectively since their initial presentation in 1981 (Fig. 1). A complete history and physical was performed in all patients by faculty in the reproductive endocrine and genetics section of The Medical College of Georgia. Pubertal development was assessed by using Tanner staging, and a vaginal maturation index was applied in some cases. Laboratory studies for the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH), growth hormone (GH), morning cortisol, thyroxine (T4), tri-iodothyronine (T3), and thyroid-stimulating hormone (TSH) were performed by using radioimmunoassay (18). All patients with idiopathic hypogonadotropic hypogonadism were given an exogenous single bolus of 100 ␮g of GnRH subcutaneously. Serum FSH and LH were measured at 0, 15, 30, 45, 60, 90, 120, 150, and 180 minutes. Radiography of the sella turcica was also performed in each FERTILITY & STERILITY威

FIGURE 1 The pedigree of a family with idiopathic hypogonadotropic hypogonadism. Dark and light shading indicate heterozygosity for each of the GnRH receptor gene mutations. The hatched symbol (sibling II8) indicates delayed puberty of uncertain etiology, and the shaded quarter in this symbol indicates Down syndrome with a karyotype of 46,XX,21,⫹t(21q21q). The “railroad track” figure represents red– green color blindness. r21 ⫽ ring 21 chromosome.

Layman. Clinical effects of GnRHR gene mutations. Fertil Steril 2001.

patient to exclude a pituitary tumor. Two patients (II2 and II7) also had insulin-induced hypoglycemia (0.15 U insulin per kg of body weight intravenously to a blood glucose level that was 50% of the baseline value) and thyroid-releasing hormone (250 ␮g intravenously) stimulation tests with serial measurement of TSH, prolactin, GH, and cortisol. Both of these patients had a second GnRH stimulation test 24 hours after the first one to assess the priming effect of a repeated dose of GnRH. These provocative studies were performed to determine whether priming of the pituitary with GnRH increased pituitary responsiveness, whereas the other stimulation studies were performed to test pituitary reserve. Molecular analysis of the protein coding region of the GnRH receptor gene was performed previously and consisted of denaturing gradient gel electrophoresis of GCclamped polymerase chain reaction (PCR) products, followed by DNA sequencing (17, 19). Functional studies comparing the wild-type and mutant GnRH receptor were performed by using membrane fractions from COS-1 cells transiently transfected with either the wild type or a single mutant, as described elsewhere (17). The studies were approved by the institutional review boards at The Medical College of Georgia and the University of Chicago.

RESULTS Molecular Analysis Denaturing gradient gel electrophoresis of GG-clamped PCR products of exon 3 revealed two variant bands, which were then sequenced. One GnRH receptor point mutation resulted in the substitution of arginine with glutamine at codon 262 (Arg262Gln) in the third intracellular loop, 1149

whereas the other point mutation changed tyrosine to cysteine at codon 284 (Tyr284Cys) in the sixth transmembrane domain of the receptor (17). In vitro studies performed in membrane preparations from COS-1 cells transfected with wild-type or mutant receptors demonstrated that both missense mutations had no effect upon binding affinity of GnRH agonist to its receptor, but decreased receptor expression was observed (17). The major effect of these mutations in vitro was impaired production of second messenger inositol trisphosphate (IP3). The Arg262Gln substitution resulted in 10-fold reduced efficiency of IP3 production (median effective concentration, 57.7 ⫾ 4.97 nM vs. 5.8 ⫾ 0.71 nM) and a 35% decrease in total IP3 production. The Tyr284Cys substitution produced a more 20-fold reduction in the efficiency of IP3 production (median effective concentration, 127.0 ⫾ 17.6 nM vs. 5.8 ⫾ 0.71 nM) and a 75% reduction in total IP3 production (17).

FIGURE 2 Responses of LH to GnRH stimulation in four family members with idiopathic hypogonadotropic hypogonadism. Initial LH responses and those obtained on repeated testing 24 hours later (in parentheses) were as follows: proband II2: 0 minutes, 1 (1); 15 minutes, 3.7 (5.7); 30 minutes, 3.7 (6.8); 45 minutes, 3.6 (6.2); 60 minutes, 4.5 (5.2); 90 minutes, 3.1 (5.4); 120 minutes, 3 (4.7); 180 minutes, 2.5 (2.8). Sibling II7: 0 minutes, 2 (2.1); 15 minutes, 10.5 (16); 30 minutes, 11.5 (17); 45 minutes, 12.2 (17); 60 minutes, 8.8 (14.3); 90 minutes, 7.2 (10.8); 120 minutes, 7 (9.2); 180 minutes, 5.2 (7). All values expressed as mIU/mL.

The Proband (Patient II2) We studied a white family from Georgia that has four members with delayed sexual development due to hypogonadotropic hypogonadism who were evaluated in 1981. The proband (II2) was first seen at the Medical College of Georgia at age 30 (Fig. 1). She gave a history of vaginal spotting once at age 15 but reported no breast development or anosmia. No family member had anosmia, midline facial defects, or neurologic deficits. On physical examination, the proband had a height of 174 cm (68.5 inches), a weight of 50 kg, and a body mass index of 17.3 kg/m2. She had a normal result on thyroid examination, Tanner 1 breasts, Tanner 5 pubic hair, and a palpable infantile uterus without adnexal masses. As was true for all affected family members, results of neurologic examination were normal, which included normal cranial nerves, reflexes, strength, and sensation. On vaginal smear, the proband had 25% parabasal cells/ 75% intermediate cells/0 superficial cells, indicating hypoestrogenism. Radiography of the proband’s pelvis at age 30 revealed no ossification at the epiphyses, and skull radiography of the sella was normal. Her baseline laboratory data were as follows: LH, 2 mIU/mL; FSH, 1.8 mIU/mL; TSH, ⬍1.5 ␮U/mL; T4, 12.3 ␮g/dL, T3 resin uptake (RU), 34%; T3, 178 ng/dL; serum prolactin, 1.8 ng/mL; growth hormone (GH), 0.5 ng/mL; morning cortisol, 15.3 ␮g/dL; and evening cortisol, 7.3 ␮g/dL. Peripheral blood karyotype was 46,XX, counting 20 cells. On GnRH stimulation, the proband’s LH increased to a maximal level of 4.5 mIU/mL at 60 minutes, while her FSH level changed only minimally to 2.4 mIU/mL at 45 minutes (Fig. 2, 3). On repeated GnRH stimulation the following day, her maximal LH level increased to 6.8 mIU/mL at 30 minutes, and her FSH level increased to 5.0 mIU/mL at 150 minutes (Fig. 2, 3). After insulin-induced hypoglycemia (in which the glucose level decreased from 95 mg/dL at baseline to 26 mg/dL) and TRH stimulation, her levels of GH, pro1150 Layman et al.

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Layman. Clinical effects of GnRHR gene mutations. Fertil Steril 2001.

lactin, and TSH and cortisol responses were normal (Table 1). Idiopathic hypogonadotropic hypogonadism was diagnosed, which was thought to be due to GnRH deficiency. The proband was treated with conjugated equine estrogens to induce breast development followed by addition of medroxyprogesterone acetate once breast development was adequate. One year later, the proband was interested in pursuing pregnancy; she was therefore given pulsatile GnRH by subcutaneous pump up to a dose of 14 ␮g per pulse every 90 minutes (280 ng/kg/pulse). After 46 days of therapy, she remained hypoestrogenic and failed to ovulate, the pump was discontinued, and she did not desire further attempts at pregnancy.

Sibling II7 The proband’s sister (II7) also had delayed puberty since age 17 (Fig. 1). When first seen at the Medical College of Georgia, she was 21 years of age, with primary amenorrhea and no history of breast development. On physical examination, her height was 164.5 cm (64.8 inches), her weight was 46 kg, and her body mass index was 16.9 kg/m2. She had anisocoria, with the right pupil larger than the left; Tanner 1 breasts; Tanner 2–3 pubic hair; an atrophic vagina, a normal nulliparous cervix; and a palpable uterus. Vaginal smear revealed 17% parabasal cells/83% intermediate cells/0 Vol. 75, No. 6, June 2001

FIGURE 3 Responses of FSH to GnRH stimulation in four family members with idiopathic hypogonadotropic hypogonadism. Initial FSH responses and those obtained on repeated testing 24 hours later (in parentheses) were as follows: proband II2: 0 minutes, 1.7 (1.6); 15 minutes, 1.7 (3); 30 minutes, 2.3 (4); 45 minutes, 2.4 (4.5); 60 minutes, 2 (4.5); 90 minutes, 1.5 (4.9); 120 minutes, 2.2 (4.8); 180 minutes, 2.1 (4.8). Sibling II7: 0 minutes, 2.3 (3.1); 15 minutes, 3.3 (4.6); 30 minutes, 3.5 (5.8); 45 minutes, 3 (6); 60 minutes, 4.5 (6.5); 90 minutes, 4.7 (6.2); 120 minutes, 4.6 (5.7); 180 minutes, 4.5 (5.8).

was 12.2 mIU/mL at 45 minutes (Fig. 2, 3). Her FSH level increased to a maximum of 4.7 mIU/mL at 90 minutes. Stimulation with GnRH 24 hours after the first dose revealed an increased maximal LH response (17 mIU/mL) at 30 minutes and a slightly higher maximal FSH response (6.5 mIU/mL) at 60 minutes (Fig. 2, 3). Levels of GH, prolactin, and TSH and cortisol responses to provocative testing (baseline glucose decreased from 83 to 36 mg/dL) were normal and similar to those in her sister II3 (Table 2). Several years after puberty was initiated with exogenous estrogen and progesterone, sibling II7 desired conception. Hysterosalpingography revealed a normal cavity and bilaterally patent fallopian tubes. Treatment with hMG was started at a dosage of 150 U/d intramuscularly and increased up to a maximal dose of 300 U/d. After 12 days of hMG stimulation (total of 28 75-U ampules), sibling II7 received hCG, 10,000 IU intramuscularly, with an E2 level of 1741 pg/mL and two preovulatory follicles of 16 and 20 mm. During her second cycle of gonadotropins, she required 14 days of stimulation (49 ampules) and had one 20-mm follicle. In both cycles, ovulation occurred, but sibling II7 did not conceive.

Other Affected Siblings and Unaffected Family Members Layman. Clinical effects of GnRHR gene mutations. Fertil Steril 2001.

superficial cells. No hypothalamic–pituitary tumor was seen on skull radiography. Her laboratory values were as follows: FSH, 3.5 mIU/mL; LH, 3.8 mIU/mL; prolactin, 12.5 ng/mL; GH, 9.5 ng/mL; morning cortisol, 18.3 ␮g/dL; and evening cortisol, 12.5 ␮g/dL. Results of thyroid function tests were normal: T4, 11.1 ␮g/dL; T3RU, 32%; T3, 152 ng/dL, and TSH, ⬍1.5 ␮U/mL. Her karyotype was 46,XX, with 20 cells counted. In sibling II7, the maximal response to GnRH stimulation

The proband and sibling II7 reported that another sister (II6) had a history of delayed puberty and an unmarried brother (II3) had never shaved. Two other siblings were not seen at the Medical College of Georgia but had relevant history. Sister II8 had the phenotype of Down syndrome with a karyotype of 46,XX,⫺21,⫹t(21q21q) and delayed puberty but was not evaluated. Brother II5 had normal puberty, ring 21 chromosome, and red– green color blindness. One of the parents presumably also had ring chromosome 21 (Fig. 1). Sibling II6 At age 17, sibling II6 gave a history of absent breast development. She had previously had a laparoscopy, which

TABLE 1 Results of thyroid-releasing hormone and insulin-induced hypoglycemia in sibling proband II2. Time (min)

GH level (ng/mL)

Cortisol level (␮g/dL)

Prolactin level (ng/mL)

TSH level (␮U/mL)

0 15 30 45 60 90 120 150 180

5.9 4.8 6.6 9.2 10.4 7.5 2.9 2.2 1.3

14 13 10 27 30 25 17.5 15 9

12 26 26 35 33 27 21 17.5 16

⬍1.5 6.5 5.3 4.4 4.0 2.9 2.2 1.7 1.7

Note: GH ⫽ growth hormone; TSH ⫽ thyroid-stimulating hormone. Layman. Clinical effects of GnRHR gene mutations. Fertil Steril 2001.

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TABLE 2 Results of thyroid-releasing hormone and insulin-induced hypoglycemia in sibling proband II7. Time (min)

GH level (ng/mL)

Cortisol level (␮g/dL)

Prolactin level (ng/mL)

TSH level (␮U/mL)

0 15 30 45 60 90 120 150 180

3.2 4.4 7.2 9.6 8.7 5.5 5.2 6.3 4.6

15.5 14 23 26 26 22 14.5 15 11.5

12 39 37 33 28 22.5 18 17.5 16

⬍1.5 8.2 7.0 5.7 4.9 3.3 2.0 2.0 1.9

Note: GH ⫽ growth hormone; TSH ⫽ thyroid-stimulating hormone. Layman. Clinical effects of GnRHR gene mutations. Fertil Steril 2001.

revealed a small uterus and “streak” gonads. At age 22, she had a laparotomy and bilateral oophorectomy for bilateral ovarian seromucinous cystadenomas (a 10 ⫻ 7 cm right ovary and 8 ⫻ 4.5 cm left ovary). Some primordial follicles were seen on examination of pathologic specimens (Fig. 4). Imaging of the kidneys and urinary bladder revealed spina bifida occulta, and preoperative intravenous pyelography showed minimal right hydronephrosis, but kidneys were present. She also had a history of breast augmentation surgery. On physical examination at age 24, sibling II6 had a height of 164 cm (64.5 inches), weight of 54.5 kg, body mass index of 20.3 kg/m2, and anisocoria. Pelvic examination was normal. Her karyotype was 46,XX. Skull radiography did not reveal a tumor. Baseline laboratory data were as follows:

prolactin, 3 ng/mL; testosterone, ⬍30 ng/dL; TSH, 2.7 ␮U/ mL; T4, 10 ␮g/dL, T3RU, 28%; GH, 1.7 ng/mL; morning cortisol, 16 ␮g/dL; and evening cortisol, 10 ␮g/dL. GnRH stimulation test revealed a basal LH level of 3.5 mIU/mL, which increased maximally to 12.3 mIU/mL at 45 minutes, and a basal FSH level of 3.3 mIU/mL, which increased maximally at 120 minutes to 6 mIU/mL (Fig. 2, 3). Hormone replacement therapy was initiated. Approximately 8 years after hormone replacement therapy was begun, she was evaluated for anorgasmia of uncertain origin. Sibling II3 Brother II3 was 29 years of age at initial presentation and had never shaved. He had a history of red– green color

FIGURE 4 Ovarian tumors removed from a patient with idiopathic hypogonadotropic hypogonadism and compound heterozygous mutations for the GnRH receptor. Shown on the left is the serous portion of the neoplasm, demonstrating calcifications (dark-shaded regions) and cuboidal epithelium; in the middle, the mucinous portion of the tumor with columnar epithelium is shown; and on the right, primordial follicles are shown.

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blindness. His medical history was significant for the Guillain-Barre´ syndrome at age 14, which completely resolved. He had arm length of 78 cm, height of 176.5 cm (69.5 inches), weight of 89 kg, and body mass index of 28.7 kg/m2. He had no facial hair. He did not consent a genital examination but reported sparse pubic hair and no testicular or penile growth with puberty. Sibling II3’s testosterone level was 75 ng/dL. A GnRH stimulation test revealed a basal LH of 2.6 mIU/mL that increased to a maximal value of 7.5 mIU/mL at 45 minutes. His basal FSH level ⬍2 mIU/mL remained during the entire study. A karyotype revealed a 46,XY r(21). Results of laboratory testing were as follows: TSH, 1.9 ␮U/mL; prolactin, 3.5 ng/mL; morning cortisol, 11 ␮g/dL; and evening cortisol, 7.8 ␮g/dL. Thyroid function was reported to be normal, although the specific values were not located. Bone age was ⬎18 years, but both iliac epiphyses were only partly fused. A fracture of the right patella was noted, but osteoporosis was not observed. He was not interested in fertility because of his karyotype, and he was given intramuscular testosterone enanthate, 300 mg every 3 weeks.

DISCUSSION Kallmann syndrome is the most completely characterized form of human hypogonadotropic hypogonadism and is associated with anosmia and occasionally with other anomalies, such as neurologic deficits, midline facial defects, and unilateral renal agenesis (5– 8). Mutations in the KAL1 gene on chromosome Xp cause this X-linked recessive disorder in men (5– 8, 20). Of note, about half of men with KAL1 gene mutations have unilateral renal agenesis (8). The affected members of the family that we studied do not have anosmia, and the one patient who underwent intravenous pyelography (sibling II6) had bilateral normal kidneys. To date, no KAL1 gene mutations have been identified in women with the Kallmann syndrome. The observations that no adrenal disease was observed in any family members and that women were also affected make adrenal hypoplasia congenita/hypogonadotropic hypogonadism due to DAX1 gene mutations a very unlikely possibility. This family had delayed puberty due to hypogonadotropic hypogonadism. All patients clearly presented with irreversible delayed puberty (presenting at ages 21–30 years), low basal gonadotropin levels, and no evidence of a hypothalamic–pituitary tumor. The inheritance pattern was presumed to be autosomal recessive, which was confirmed by demonstration of compound heterozygous GnRH receptor gene mutations in all affected persons (17). Although the parents were deceased, they were presumed to be heterozygotes. An interesting finding in this family with idiopathic hypogonadotropic hypogonadism is the observation of a familial ring chromosome 21, which has not been previously reported. Ring chromosome 21 is rare, and familial forms in FERTILITY & STERILITY威

persons with normal intelligence (as in patient II3 with idiopathic hypogonadotropic hypogonadism and his unaffected brother II5) are even rarer (21, 22). Presumably, one of the parents had a ring chromosome 21. Even though the affected male (II3) had a ring 21 chromosome and his sister had Down syndrome, these conditions are probably not relevant to hypogonadotropic hypogonadism, since an unaffected brother (II5) also had a ring 21 chromosome and the GnRHR gene is localized to chromosome 4q (23). We cannot exclude the possibility that sequences on chromosome 21 modified the idiopathic hypogonadotropic hypogonadism phenotype in this family. Although the clinical presentation suggested GnRH deficiency, GnRH gene mutations were not identified (18, 24). Molecular analysis of this family revealed compound heterozygosity for two different missense mutations in exon 3 of the GnRH receptor gene (17), a G-protein coupled receptor with an extracellular amino terminus, a seven-transmembrane domain, three extracellular loops, and three intracellular loops but no carboxy-terminal tail (25–27). Compound heterozygous missense mutations (Arg262Gln in the third intracellular loop and Tyr284Cys in the sixth transmembrane domain of the receptor) were identified in all four patients with idiopathic hypogonadotropic hypogonadism (17). Our previous in vitro analysis demonstrated that both missense mutations had no effect on binding affinity of GnRH agonist to its receptor, but decreased receptor expression was seen (17). However, both the efficiency and total IP3 were decreased with both GnRH receptor mutations. These findings indicate that both mutations predominantly affect signal transduction rather than ligand binding. Clearly, these mutations had profound effects on normal GnRH receptor function in the pituitary and resulted in a state of GnRH resistance. Supportive clinical data include the inability to increase estradiol levels above baseline values with exogenous GnRH and induce ovulation in proband II2 at maximal pulsatile doses (280 ng/kg/pulse every 90 minutes) even at 46 days. Typically, doses of 50 –200 ng/ kg/pulse induce ovulation in hypogonadotropic women within a few weeks (28). However, we cannot exclude pituitary desensitization with higher doses of GnRH. In contrast, the proband’s sister (II7) responded with follicular growth and ovulation after administration of hMG, indicating normal gonadotropin receptor function. Although the findings in this family suggest that gonadotropins might be preferred over pulsatile GnRH for ovulation induction, increased doses might be able to induce ovulation in some patients with GnRHR mutations. This family with autosomal recessive GnRHR mutations has clinical features of complete hypogonadotropic hypogonadism. All three affected women lacked sexual development, manifested by a complete absence of breast development despite age older than 20 years. The man with hypogonadotropic hypogonadism (II3) did not permit a gen1153

ital examination, but he did not shave at age 29 years; had a serum testosterone level of 75 ng/dL; and had unfused iliac crests, which generally close after puberty. All of the available clinical evidence strongly supports a complete lack of steroid production. Our findings of complete idiopathic hypogonadotropic hypogonadism are in contrast to those of de Roux et al. (16), who described two affected siblings, one male and one female, with partial idiopathic hypogonadotropic hypogonadism due to GnRH receptor gene mutations. Both patients had some evidence of puberty and were compound heterozygotes for GnRH receptor gene mutations. The woman had breast development and only a single menses, and the man was hypogonadal but had evidence of spermatogenesis with a count of 39 million/mL (16). The Arg262Gln mutation was the same mutation as the one that we identified, which de Roux et al. also demonstrated to impair signal transduction. The other mutation was a Gln106Arg in the extracellular domain that interfered with binding of GnRH in vitro (16). On the basis of our findings, we would have expected these mutations to be more severe than ours and cause complete idiopathic hypogonadotropic hypogonadism. This variability could be due to DNA sequence differences in as yet unidentified genes or environmental factors. Even though all patients in the family that we studied had a severe form of idiopathic hypogonadotropic hypogonadism, the clinical presentation and endocrinologic findings in affected persons varied. Two siblings (II2 and II3) had a more attenuated response of LH to exogenous GnRH than their sisters (II6 and II7), who had maximal LH responses greater than 10 mIU/mL. Somewhat surprising is the observation that patients II2 and II7 demonstrated an increase in gonadotropin responses to exogenous GnRH in the second round of GnRH stimulation (Fig. 2, 3). This suggests that some priming of pituitary gonadotrophs occurs even with mutant GnRH receptors that cause complete idiopathic hypogonadotropic hypogonadism, indicating that the receptor must retain some function. Alternatively, it is possible that 100 ␮g of GnRH produces a pharmacologic rather than physiologic effect on the GnRH receptor. Similar findings were reported in a family of three siblings with complete idiopathic hypogonadotropic hypogonadism and GnRH receptor mutations (Ala129Asp/ Arg262Gln), in which pulsatile GnRH given every 90 minutes for 40 hours produced slight increases in gonadotropins but no change in steroid levels (29). More striking phenotypic variability was observed with compound heterozygous GnRHR mutations (Arg262Gln/Gln106Arg and Ser217Arg) in a family that included both complete and incomplete forms of idiopathic hypogonadotropic hypogonadism (30). Pituitary function is usually normal in patients with idiopathic hypogonadotropic hypogonadism (28). Decreased nocturnal release of prolactin (31) and GH (32) have been 1154 Layman et al.

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reported, but this is probably not clinically significant. Both of our patients who had the triple test with GnRH, TRH, and insulin-induced hypoglycemia had normal prolactin, cortisol, TSH, and GH responses. Although both tested patients (II2 and II7) had normal TSH responses to exogenous TRH over 3 hours, their responses were somewhat less than those in women with normal estrogen levels. However, these patients did not have clinical hypothyroidism. It has been shown that estrogen priming increases pituitary responses to GnRH and TRH stimulation (31). Several other interesting findings in this family with GnRH resistance were observed. Bilateral seromucinous cystadenomas occurred despite the presence of low serum gonadotropin levels, suggesting that factors other than gonadotropin stimulation are involved in the genesis of ovarian neoplasms. In addition, after bilateral oophorectomy, patient II6 gonadotropin levels remained low, suggesting that patients with idiopathic hypogonadotropic hypogonadism will probably have low gonadotropin levels at the expected age of menopause. This same patient also had a history of anorgasmia despite being treated with hormone replacement therapy. Although it is difficult to state with certainty on the basis of data from only one patient, GnRH is known to be involved in sexual function (33), and perhaps this sexual dysfunction is attributable to lack of normal GnRH function. Mutations of the GnRH receptor gene represent the first autosomal cause of human hypogonadotropic hypogonadism (16, 17), although leptin (34) and leptin receptor (35) gene mutations have been reported in some patients with autosomal idiopathic hypogonadotropic hypogonadism and obesity. The exact prevalence of GnRH receptor gene mutations is not known, but in screening 46 unrelated patients with idiopathic hypogonadotropic hypogonadism, we found a prevalence of 2.2% (17). When only probands with affected women are included, 7.1% had GnRH receptor gene mutations (17). Although all four affected persons in this family with hypogonadotropic hypogonadism possess the same GnRH receptor gene mutations, phenotypic and endocrinologic responses demonstrate pleiotropic effects. The finding of a ring chromosome 21 in this family is interesting, although its significance is currently unknown. It is possible that genes on chromosome 21 affect the phenotype of patients with idiopathic hypogonadotropic hypogonadism and produce the phenotypic differences exhibited in this family.

Acknowledgment: The authors thank Dr. Anthony C. Montag, Pathology Department, The University of Chicago, for assistance and interpretation of the ovarian neoplasm slides.

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