Clinical and hormonal features of selective folliclestimulating hormone (FSH) deficiency due to FSH beta-subunit gene mutations in both sexes

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1 Clinical and hormonal features of selective folliclestimulating hormone (FSH) deficiency due to FSH beta-subunit gene mutations in both sexes Karina Berger, M.D., a Haroldo Souza, M.D., b Vinicius Nahime Brito, M.D., a Catarina Brasil d Alva, M.D., a Berenice Bilharinho Mendonca, M.D., Ph.D., a and Ana Claudia Latronico, M.D., Ph.D. a a Endocrinology Development Unit, Hormone and Genetic Laboratory, LIM 42, Division of Endocrinology and Metabolism, Hospital das Clínicas, University of São Paulo School of Medicine, São Paulo, Brazil; and b Private Practice, São Paulo, Brazil Objective: To report the clinical, hormonal, and molecular features of a female adolescent with selective FSH deficiency. In addition, a complete review of previous cases is provided, focusing on hormonal aspects. Design: Clinical study. Setting: University hospital. Patient(s): A 16-year-old girl with primary amenorrhea and poor breast development due to isolated FSH deficiency. Intervention(s): Blood drawing before and after GnRH stimulation and pelvic ultrasound examination. Main Outcome Measure(s): Gonadotropin and E 2 measurements and sequencing of the FSH -subunit gene. Result(s): The patient was referred for primary amenorrhea and partial breast development (Tanner III). Her basal and GnRH-stimulated LH levels were elevated (31 IU/L and 98 IU/L, respectively), whereas her FSH levels were undetectable ( 1 IU/L) in both conditions. Estradiol levels were low ( 13 pg/ml). Automatic sequencing showed a nucleotide substitution of C for A in exon 3, resulting in a homozygous nonsense mutation in amino acid position 76 (Tyr76X) of the FSH -subunit. Conclusion(s): The Tyr76X mutation of the FSH -subunit was associated with a partial phenotype of FSH deficiency. To date, only four loss-of-function mutations of the FSH -subunit have been described in eight patients with undetectable serum FSH and high serum LH levels. Therefore, this unusual hormonal profile strongly suggests a defect in the FSH -subunit in both sexes. (Fertil Steril 2005;83: by American Society for Reproductive Medicine.) Key Words: Primary amenorrhea infertility, gonadotropins, FSH deficiency, FSH -subunit gene Human FSH is a heterodimeric pituitary glycoprotein that regulates gonadal function in both sexes. In females, FSH regulates the follicular development and sex steroid production, whereas in males, FSH stimulates Sertoli cell proliferation and maintenance of sperm quality. These functions are necessary for fertility in both sexes (1 3). Follicle-stimulating hormone is composed of an -subunit, which is common to other glycoproteins, and a specific -subunit. These subunits are encoded by separate genes (2, 3). No human -subunit mutation has been reported, although dysfunction of all glycoprotein hormones, including both gonadotropins, TSH, and hcg, would be expected on the basis of targeted disruption of this gene in mice (4). Because hcg plays an essential role in the maintenance of pregnancy, -subunit mutations might not be seen in humans (5). Received February 9, 2004; revised and accepted June 29, Supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico grants /96 (A.C.L.) and /95-5 (B.B.M). Reprint requests: Ana Claudia Latronico, M.D., Hospital das Clínicas, Prédio dos Ambulatórios, 2 andar, Bloco 6, Av. Dr. Enéas de Carvalho Aguiar, 155, São Paulo CEP , Brazil (FAX: ; anacl@usp.br). The FSH -subunit gene, located at the short arm of chromosome 11, is composed of three exons: exon 1 encodes a 5= untranslated sequence, exon 2 the 18-amino-acid propeptide and residues 1 35, and exon 3 encodes residues of the mature peptide (2). To date, four distinct FSH -subunit gene mutations have been described in four unrelated female patients with secondary hypogonadism due to selective deficiency of FSH (Table 1) (5 12). These women had delayed puberty, lack of or poor breast development, and primary amenorrhea. Normal primordial follicles were detected, and in two women follicular maturation, ovulation, and fertility were achieved after treatment with exogenous FSH (7 11). In the present study, we report the clinical and hormonal features of a new case, a girl with a loss-of-function FSH -subunit mutation. In addition, a complete review of the previous cases in female and male patients is provided, focusing on the hormonal aspects of this rare condition. CASE REPORT This work was approved by the institutional review board of Hospital das Clínicas, School of Medicine, São Paulo University, São Paulo, Brazil. 466 Fertility and Sterility Vol. 83, No. 2, February /05/$30.00 Copyright 2005 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 Fertility and Sterility TABLE 1 Clinical and hormonal data of five women and three men with selective FSH deficiency due to FSH -subunit gene mutations. Case CA (y) Sex/origin Clinical presentation LH (IU/L) FSH (IU/L) B P B P E 2 (pg/ml) T (ng/dl) Molecular defects Reference 1 27 Female/Italian Primary amenorrhea, no breast development, eunuchoidal proportions, infertility 2 15 Female/American Primary amenorrhea, no breast development 3 22 Female/Israeli Primary amenorrhea, partial breast development and eunuchoidal proportions 4 32 Female/Brazilian Primary amenorrhea, partial breast development, infertility 5 16 Female/Brazilian Primary amenorrhea, partial breast development, normal proportions 6 18 Male/Israeli Delayed puberty, small soft testes, azoospermia 7 28 Male/Serbian Infertility, small soft testes, azoospermia 8 30 Male/Brazilian Infertility, small testes, azoospermia Note: CA chronological age; B basal; P peak after GnRH (100 g, IV); Und undetectable. Berger. FSH -subunit mutations in both sexes. Fertil Steril Val61X homozygosis Val61X/Cys51Gly Val61X homozygosis 9,10, Tyr76X homozygosis Tyr76X homozygosis Present study Val61X homozygosis Und Und 246 Cys82Arg 19 homozygosis Tyr76X homozygosis

3 A 16-year-old girl was referred for evaluation of primary amenorrhea. She had breast and pubic hair development spontaneously at age 12, but her breast development was modest. Parents were consanguineous (second cousins), and there was no family history of menstrual disturbances. On physical examination, her height and weight were 158 cm ( 0.73 SD) and 48 kg, respectively, and arm span was 157 cm. Her target height was cm. Breast and pubic hair development were Tanner III. She had no eunuchoidal proportions and Turner syndrome stigmata. The karyotype was 46,XX. Basal and GnRH-stimulated LH levels were elevated (31 IU/L and 98 IU/L, respectively), whereas FSH levels were undetectable ( 1 IU/L) on both conditions. Estradiol levels were low ( 13 pg/ml). Thyroid function and prolactin were normal, and hcg levels were undetectable. Androgen levels were normal, including T (57 ng/dl), androstenedione (3 ng/ml), and DHEAS (3,262 ng/ml), as were 17 -hydroxyprogesterone levels (0.6 ng/ ml). Her bone age was 14 years. Ultrasound examination revealed a small uterus (volume, 7.0 cm 3 ) and ovaries of normal size. Computed tomography of the hypothalamic pituitary region was normal. Hormonal Assays Serum LH and FSH concentrations were determined by commercial, solid-phase, two-site fluoroimmunometric assays (AutoDELFIA hlh Spec and AutoDELFIA hfsh; Wallac Oy, Turku, Finland), according to the direct sandwich technique. Estradiol was measured by commercial solid-phase assay (AutoDELFIA Estradiol; Wallac Oy) with polyclonal antibodies and standards calibrated by gravimetric and spectrophotometric methods. Normal reference values were based on previously reported data (13). DNA Analysis After informed consent was obtained, genomic DNA of the patient and her mother were isolated from peripheral blood cells by conventional procedures. Exons 2 and 3 of the FSH -subunit gene were amplified by polymerase chain reaction (PCR) with flanking intronic oligonucleotide primers: exon 2 sense (5=-AGTTTCTAGTGGGCTTCATTGTTTG-3=) and antisense (5=-TGGCTAAAGGACTCATGGCTG-3=); exon 3 sense (5=-GCTAAATAGGAACTTCCAC-3=) and antisense (5=-TATGTGGCCTGAAATGTCC-3=). The amplification reaction for exon 2 consisted of 200 ng of genomic DNA, 30 pmol of each primer in a 100- L reaction volume containing 10 L 10 reaction buffer, 200 mol/l each of deoxyribonucleoside triphosphate (dntp) and 0.5 U Taq polymerase. Similar conditions were used for exon 3, except for an additional 50 mmol/l MgCl 2. Negative controls (without DNA) were always included in each amplification. After initial denaturation for 5 minutes at 95 C, samples were subjected to 30 cycles of 30 seconds at 95 C, 30 seconds at specific annealing temperatures (53 C for exon 2 and 55 C for exon 3) and 30 seconds at 72 C, followed by a final extension step of 10 minutes at 72 C. Exon 10 of the FSH receptor gene was also amplified by PCR with a specific oligonucleotide primer pair: sense 5=- CTGGATCTGAGATGTTGATTCTA-3= and antisense 5=- GTTTTGGGCTAAATGACTTAGAGG-3=. The amplification was performed in a 100- L reaction mixture containing 200 ng genomic DNA, 10 L 10 reaction buffer, 1.5 mmol/l MgCl 2, 200 mol/l each of dntp and 0.5 U Taq polymerase, and 30 pmol of each primer. After initial denaturation for 2 minutes at 94 C, samples were subjected to 35 cycles of 1 minute at 94 C, 1 minute at annealing temperature 60 C, and 2 minutes at 72 C, followed by a final extension step of 10 minutes at 72 C. All PCR products were purified with shrimp alkaline phosphatase and exonuclease I (PCR product presequencing kit; Amersham Life Sciences, Cleveland, OH) before sequencing with a commercial kit (ABI PRISM BigDye Terminator kit; Perkin-Elmer Applied Biosystems, Foster City, CA). The products were directly sequenced with a commercial analyzer (ABI PRISM 3100 Genetic Analyzer automatic DNA sequencer; Perkin-Elmer Applied Biosystems). Two additional inner primers were used for exon 10 of the FSH receptor gene sequencing (sense: 5=-TTCACTGACTTC CTCTGCAT-3= and antisense: 5=-ACACTGGCAGCATG GCG-3=). Results Automatic sequencing of the FSH -subunit gene revealed a nucleotide substitution of C for A in exon 3, resulting in a substitution of the amino acid tyrosine at codon 76 by a premature stop codon (Tyr76X) in the homozygous state in the patient (Fig. 1B). Her mother had the same mutation in the heterozygous state. A known silent polymorphism at codon 58 (TAT3TAC) was also identified in the patient and her mother, both in the homozygous state (14). The direct sequencing of exon 10 of the FSH receptor gene revealed no activating mutations. We found two known polymorphisms at codons 307 and 680 of the FSH receptor (15). DISCUSSION Normal pubertal development depends on normal secretory patterns of FSH and LH. In the ovary, FSH promotes steroidogenesis through interaction with its receptor on the surface of granulosa cells. The signal promotes the stimulation of the P450arom enzyme, which results in the aromatization of androstenedione and T to estrogens. Follicle-stimulating hormone also stimulates granulosa cell division and differentiation (1). Few mutations in the FSH -subunit gene have been described in humans (Fig. 1A, Table 1). The first female 468 Berger et al. FSH -subunit mutations in both sexes Vol. 83, No. 2, February 2005

4 FIGURE 1 (A) Schematic structure of the FSH -subunit gene with three exons and the mature protein. Four known inactivating mutations described in female and male patients with selective FSH deficiency are shown. (B) Automatic sequencing of the genomic DNA from the patient and her mother, showing a nucleotide substitution of C for A in exon 3 of the FSH -subunit gene. Berger. FSH -subunit mutations in both sexes. Fertil Steril patient with an FSH -subunit gene mutation was reported by Matthews et al. (7) in She had no breast development, primary amenorrhea, and infertility. A 2-base-pair (bp) nucleotide deletion (TG) gave rise to a completely altered amino acid sequence between codons 61 and 86 of the FSH chain, which was followed by a premature stop codon (Val61X) and lack of translation of amino acids 87 to 111 (7). The predicted truncated protein was unable to couple with the -subunit to form bioactive FSH (7). The second reported patient was an adolescent girl with delayed puberty (8). She presented with primary amenorrhea and absence of breast development. Two mutations in the FSH -subunit gene were found in this patient: one mutation was the same as the one described in the first reported case, and the other was a missense mutation that changed cysteine to glycine at codon 51 (Cys51Gly) (8). Another woman, initially reported as having FSH deficiency due to FSH circulating antibodies, was later re-evaluated and found to be homozygous for the same 2-bp deletion (Val61X) previously reported (9 11). More recently, Layman et al. (12) described a Brazilian woman with partial pubertal development, primary amenorrhea, and infertility due to a homozygous nonsense mutation (Tyr76X) in the -subunit of the FSH gene. All five women who presented with FSH -subunit mutations had undetectable FSH and high LH levels. Furthermore, they had no FSH response to GnRH stimulation (Table 1). Gonadotropin levels were measured by sensitive methods, such as immunofluorometric assay, in four of the five previously reported affected women, and basal LH levels ranged from 21 IU/L to 51 IU/L. Luteinizing hormone response to a GnRH stimulation test was elevated, ranging from 104 IU/L to 170 IU/L in these patients. In contrast, basal and stimulated FSH levels were lower than the detection limit of the methods in all cases. This unusual LH and FSH profile was also found in our patient, a Brazilian adolescent girl with primary amenorrhea and partial breast development, suggesting the clinical diagnosis of selective FSH -chain deficiency. Interestingly, eunuchoidal body habitus was not observed in our patient and was mentioned in two of the other female patients with selective FSH deficiency. These findings can suggest early diagnosis followed by steroid replacement in these patients with normal proportional bodies. Fertility and Sterility 469

5 The history of consanguinity in this family strongly supported a hypothesis of a recessive genetic defect. The DNA analysis in this patient confirmed the presence of an FSH -subunit abnormality, characterized by a homozygous nonsense mutation at codon 76 (Tyr76X). Her asymptomatic mother had the same mutation in the heterozygous state. Interestingly, this mutation was found in another Brazilian female patient with a similar FSH deficiency phenotype (breast development Tanner II III) (12). Both female patients were from the same region in Brazil, and it is possible that the Tyr76X mutation originated from a common ancestor. Layman et al. (12) demonstrated nonmeasurable immunoand bioactive FSH through the functional analysis of three mutations: Tyr76X, Val61X, and Cys51Gly. This finding is somewhat unexpected because both Brazilian female patients with the Tyr76X mutation had evidence of spontaneous breast development. The partial breast development in these patients can be secondary to ovarian E 2 production under an exclusive LH regimen or to ovarian androgen conversion to estrogen locally by breast aromatase or even by the presence of mutations in the FSH receptor gene (16 18). Regarding the latter hypothesis, three activating mutations within exon 10 of the FSH receptor gene, which encodes the transmembrane domain of the receptor, were recently described (16 18). The functional characterization of these mutations revealed promiscuous activation of the FSH receptor by hcg (16 18). However, the mutational analysis of the FSH receptor gene in our patient did not reveal any functional mutations. To date, three male patients with FSH -subunit mutations have been described (Table 1). Two of these patients, who harbored distinct mutations (Cys82Arg and Tyr76X), presented with small testes, azoospermia, and normal T (12, 19). In vitro analysis of the Cys82Arg mutation in Chinese hamster ovary cells also resulted in remarkable FSH deficiency (20). The third male patient, with the Val61X mutation, presented with absence of pubertal development, low T levels, small and soft testes, and azoospermia (21). The low serum T level despite high LH levels in this patient is a curious finding because Leydig cells do not have an FSH receptor. In conclusion, the Tyr76X mutation was associated with a partial phenotype of selective FSH deficiency in two apparently unrelated Brazilian female patients. Finally, the unusual hormonal profile characterized by undetectable serum FSH and high serum LH levels strongly suggests the presence of a molecular defect in the FSH -subunit chain in both sexes. Acknowledgments: The authors thank Dr. Ivo Jorge Prado Arnhold for his useful comments and suggestions. REFERENCES 1. Thorner MO, Vance ML, Laws ER, Horvath E, Kovacs K. The anterior pituitary. 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