CASE REPORT Primary amenorrhea in four adolescents revealed 5a-reductase deficiency confirmed by molecular analysis Laurent Maimoun, Ph.D.,a Pascal Philibert, Pharm.D., Ph.D.,a Philippe Bouchard, M.D., Ph.D.,b € G€ on€ ul Ocal, M.D.,c Bruno Leheup, M.D., Ph.D.,d Patrick Fenichel, M.D., Ph.D.,e Nad ege Servant, Ph.D.,a Françoise Paris, M.D., Ph.D.,a,f and Charles Sultan, M.D., Ph.D.a,f a Service d’Hormonologie, Hôpital Lapeyronie, CHU Montpellier, and UMI Montpellier, Montpellier, France; b Service d’Endocrinologie, Hôpital Saint Antoine, Paris, France; c Division of Pediatric Endocrinology, Department of Pediatrics, Ankara University Faculty of Medicine, Ankara, Turkey; d Médecine Infantile, Consultation Hôpital d’Enfants, CHU de Nancy, Vandoeuvre Les Nancy, France; e Service d’Endocrinologie, Hôpital L’archet, CHU Nice, Nice, France; and f Unité d’Endocrinologie and Gynécologie Pédiatrique, Hôpital Arnaud de Villeneuve, CHU Montpellier, and UMI Montpellier, Montpellier, France Objective: To determine the genetic cause of primary amenorrhea. Design: Case series. Setting: Pediatric endocrinology, endocrinology, and gynecology departments of academic hospitals. Patient(s): Three adolescents and one young woman 46, XY patients with srd5A2 gene mutations. Main Outcome Measure(s): Genetic analysis of srd5A2. Result(s): We report four srd5A2 gene mutations in three adolescents and one young woman with 46,XY primary amenorrhea. All presented clitoromegaly and two presented hypospadias; all had been reared as females. Virilization of the external genitalia was noted in the pubertal period in all four patients. Three were maintained in the female sex of rearing by personal choice, and the fourth switched gender. We identified the homozygous substitutions p.L55Q (exon 1), p.Q56R (exon 1), and p.N193S (exon 4), in patients 1, 2, and 3, respectively. Patient 4 had compound heterozygous mutations, a new c.34delG (exon 1) associated with p.R246W (exon 5). All patients had high plasma T levels (ranges, 16.2–23.2 nmol/L; normal female teenage range, 0.35–2 nmol/L). Conclusion(s): Our data clearly demonstrate that 5a-reductase deficiency should be considered in XY adolescents with primary amenorrhea and no breast development associated with virilization at puberty and high plasma T. Positive parental consanguinity should reinforce the diagnostic orientation. (Fertil Steril
2011;95:804.e1–e5. 2011 by American Society for Reproductive Medicine.) Key Words: Primary amenorrhea, 5 alpha-reductase deficiency, 46,XY adolescents The majority of XY females with primary amenorrhea have generally been assigned a diagnosis of complete androgen insensitivity syndrome (CAIS) (1). However, recent developments in molecular genetics have revealed other potential causes of XY primary amenorrhea. We recently reported the high prevalence of SF1 gene mutations in a group of 15 XY adolescent girls with primary amenorrhea (2). Investigations have also shown that SRY gene mutations and LH receptor defects are not exceptional (2–4). Other genetic causes, such as 17b-hydroxysteroid dehydrogenase or 5a-reductase type-2 Received May 12, 2010; revised and accepted August 4, 2010; published online September 20, 2010. L.M. has nothing to disclose. P.P. has nothing to disclose. P.B. has noth€ has nothing to disclose. B.L. has nothing to ing to disclose. G.O. disclose. P.F. has nothing to disclose. N.S. has nothing to disclose. F.P. has nothing to disclose. C.S. has nothing to disclose. Reprint requests: Charles Sultan, M.D., Ph.D., Unité d’Endocrinologie and Gynécologie Pédiatrique, Hôpital Arnaud de Villeneuve, CHU de Montpellier et UM1, 191 avenue Doyen Gaston Giraud, 34295 Montpellier, Cedex 5, France (TEL: 33-467-33-86-96; FAX: 33-467-33-83-27; E-mail: c-sultan@chu-montpellier.fr). 804.e1 deficiencies, may be implicated as well, although their prevalence is significantly lower (1). A 5a-reductase type-2 enzyme deficiency impairs the conversion of T to its more active metabolite dihydrotestosterone (DHT), which is required for the normal development of external genitalia, urethra, and prostate in the male fetus. T plays a major role in the virilization of Wolffian ducts into the seminal vesicles, vas deferens, and epididymis (5). Our group and others have described a wide spectrum of clinical phenotypes in patients with an alteration in the 5a-reductase enzyme (5–8). The spectrum ranges from minimal masculinization of the external genitalia presenting with isolated micropenis (9) or hypospadias to severe undervirilization appearing as normal female external genitalia with mild clitoral enlargement (5–8). Most of these patients are reared as females because of their phenotypic appearance at birth, although they are 46,XY individuals with bilateral testes and male Wolffian duct structures (5, 10, 11). When the diagnosis and its ramifications have not been fully considered in the neonatal period, the pubertal period with its expected changes may offer a second opportunity. Evidence of Fertility and Sterility Vol. 95, No. 2, February 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc. 0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.08.007 spontaneous virilization of both external genitalia and behavior at puberty along with a lack of breast development should indicate a 5a-reductase deficiency rather than CAIS. Moreover, the pubertal virilization is usually accompanied by a female-to-male gender and sex behavior switch (12–15). In this study, we report four young 46,XY women referred for primary amenorrhea associated with 5a-reductase deficiency confirmed by molecular analysis. PATIENTS Three adolescents and one young woman with primary amenorrhea and 46,XY karyotype were evaluated. Written informed consent for molecular analysis was obtained from the patients or their parents in the case of minors. tomy, followed by a deferred feminizing vaginoplasty associated with estrogen replacement therapy. Patient 4 Patient 4, a 15.7-year-old adolescent girl, was examined for primary amenorrhea and pubertal virilization. The patient was of French origin and no parental consanguinity was reported. Clinical examination showed clitoral hypertrophy associated with pubic hair (P4), but no breast development. Biological investigation revealed a high plasma T level (23.2 nmol/L) with elevated gonadotrophin levels (FSH, 11.1 UI/L; LH, 7.6 UI/L). Plasma DHT and the T/DHT ratio were 0.55 nmol/L and 42.1, respectively. No hCG stimulation test was performed. The diagnosis of partial androgen insensitivity syndrome was initially raised at birth, but the family was then lost to follow-up after the death of the father. Patient 1 Patient 1 was seen in the endocrinology clinic for disorders of sex development. The parents were of Turkish origin and were firstdegree cousins. Three other members of this family were reported to have similar symptoms. Clinical examination of this 18-year-old patient revealed pubertal virilization without breast development and clitoral hypertrophy (40 mm) with perineoscrotal hypospadias and bifid scrotum. Testes (6 mL) were in the inguinal position and pubic hair was P3. Pelvic ultrasonography showed Wolffian structures (prostate and epididymis) and confirmed the absence of M€ullerian ducts. Hormonal evaluation showed a high basal plasma T concentration (22.4 nmol/L), and plasma FSH and LH were elevated (22 mUI/mL and 9.3 mUI/mL, respectively). The adolescent maintained her female sex by her request. Patient 2 Patient 2, 24 years old, was admitted to the gynecology department for primary amenorrhea. The parents were of Tunisian origin and were first-degree cousins. Clinical examination showed clitoral hypertrophy with posterior hypospadias. Bilateral gonads were present in the scrotum. No breast development was observed, and pubic and axillary hair was P4. Pelvic ultrasonography did not show M€ullerian ducts. Basal plasma T values were high (16.2 nmol/L). DHT and the T/DHT ratio at basal level were 0.9 nmol/L and 18, respectively. Partial androgen insensitivity syndrome was first evoked, but analysis revealed that the entire androgen receptor gene was normal. This patient had clearly shown progressive male sex behavior and requested a change in sex identity. A few years later, he married but exhibited azoospermia. MATERIALS AND METHODS Molecular analysis of the srd5A2 gene was performed as reported previously (7). Genomic DNA was extracted from peripheral blood leukocytes following the manufacturer’s instructions (DNA QIAamp DNA blood Mini kit; Qiagen, Courtaboeuf, France). Exons 1–8 of the AR gene and exons 1–5 of the srd5A2 gene were amplified by PCR, and direct sequencing was performed using the BigDye Terminator v 1.1 Kit (Applied Biosystems, Courtaboeuf, France) and an ABI Prism 310 Genetic-Analyzer (Applied Biosystems). RESULTS The clinical, endocrine, and genetic results of the patients are summarized in Table 1. In these four young 46,XY patients with female phenotype and virilization at puberty, the high plasma T oriented our investigations toward molecular analysis of the srd5A2 gene, which revealed three homozygous and one compound heterozygous mutations (Fig. 1). Patients 1, 2, and 3 were found to be homozygous for the following srd5A2 gene mutations: p.L55Q in exon 1, p.Q56R in exon 1, and p.N193S in exon 4, respectively. For patient 1, the mutation was found in the heterozygous state in the father and the homozygous state in the mother. Patient 4 was found to have a compound heterozygous mutation, c.34delG, which induces a premature stop codon in position 40 in exon 1, associated with p.R246W in exon 5. The c.34delG is a new mutation that has never been reported (Fig. 2). DISCUSSION Patient 3 Patient 3, a 18.5-year-old adolescent, was admitted to the gynecology department for primary amenorrhea. The patient was of African origin, and no parental consanguinity was reported. Clinical examination showed an android morphotype without breast development and moderate pubic and axillary hair, corresponding to a female type. The patient exhibited clitoral hypertrophy with a single vaginal opening and partial fusion of the posterior labia. Magnetic resonance imaging of the abdominalpelvic region showed gonads at the inguinal orifice, two intrapelvic ullerian seminal vesicles with a draft prostate, and no evidence of M€ ducts. Basal plasma T (21.5 nmol/L) and DHT (8.6 nmol/L) were high, with elevated gonadotrophin levels (FSH, 11.6 UI/L; LH, 9.9 UI/L). Despite these hormonal data, this adolescent exhibited clear female sex behavior. In agreement with the patient, the medical team decided to perform a clitoridectomy with bilateral gonadecFertility and Sterility Primary amenorrhea is a frequent reason for consultation in pediatric or adult endocrinology and gynecology clinics. Management of primary amenorrhea should account for the clinical, biological, and molecular findings. The clinical examination reveals the degree of breast development, degree of pubic hair, evidence of hyperandrogenism, growth velocity, weight variation, and galactorrhea. The endocrine investigation should focus on the plasma FSH level and plasma T secretion. Last, karyotype analysis identifies the primary amenorrhea associated with the XX, X0, or XY karyotype. In this study, we report four young women with primary amenorrhea and 46,XY karyotype. The lack of pubertal breast development, subnormal pubic hair, and high plasma T were in favor of a 5a-reductase deficiency. We identified four known homozygous missense mutations of srd5A2 (p.L55Q, p.Q56R, p.N193S, and p.R246W) and one new deletion (c.34delG) (16). Although clitoral enlargement occurred at puberty, only patient 2, who exhibited clear male sex 804.e2 Maimoun. Primary amenorrhea and 5a-reductase deficiency. Fertil Steril 2011. French 15.7 4 African 18.5 3 Tunisian 24 2 Maimoun et al. Note: CM ¼ clitoromegaly; ND ¼ no data available. Negative F 23.2 0.55 42.1 Exon 4: p.N193S (homozygote) Exon 1: c.34delG; Exon 5: p.R246W (compound heterozygote) 2.5 8.6 21.5 F Negative Exon 1: p.Q56R (homozygote) 18 0.9 F to M Positive 16.2 Exon 1: p.L55Q (homozygote) ND ND F 18 1 Turkish CM þ perineoscrotal hypospadias þ no breast development CM þ penoscrotal hypospadias þ no breast development CM þ no breast development CM þ no breast development Positive 22.4 srd5A2 mutations Basal plasma T/DHT ratio Basal plasma DHT (nmol/L) Basal plasma T (nmol/L) Sex of rearing Parental consanguinity Phenotype Ethnic origin Age (y) Patient Main clinical, hormonal, and molecular data from four patients with primary amenorrhea and 5a-reductase deficiency. TABLE 1 804.e3 behavior, chose to change her sex of rearing at puberty. A female-tomale gender switch has been reported only twice before, once by Mendonca et al. (17) in four of four patients with compound heterozygous mutations (N193S/Q126R) and once by Hochberg et al. (18) in a 17-year-old patient with an L55Q homozygous mutation. In our experience in the clinical, biological, and genetic analysis of 55 children (L. Maı̈moun et al.), only five of these 55 patients raised as girls chose a sex reorientation. It is probable that social, cultural, and familial factors and the country of origin, play a crucial role in establishing gender identity (15). It is notable that two of our four patients presented a high degree of inbreeding, as generally reported (approximately 30%) (5). All patients presented high basal plasma T levels (mean 20.8  SD 3.2 nmol/l vs. 10.4–38.0 nmol/L, the reference range for males, and 0.35–2.00 nmol/L, the reference range for young women). Moreover, one patient did not show a high T/DHT ratio, although this is generally considered as a biological marker for the diagnosis of 5a-reductase deficiency. This finding supports our message that molecular analysis must be systematically performed. The homozygous substitution of leucine by glutamine at position 55 (p.L55Q), which was identified in patient 1, was originally reported in the context of a compound heterozygous mutation (6), but this mutation is most often identified in homozygous forms (18–20). Female phenotypes have been reported mainly for patients with p.L55Q, although a patient with male phenotype with micropenis and hypospadias, similar to patient 1 in this study, was reported (6, 18, 20). The predominantly female phenotype can be explained partly by the high degree of enzyme alteration. In vitro studies have demonstrated that this mutation leads to an almost complete loss of function (21). However, the lack of a systematic genotype–phenotype relationship for patients carrying the same mutation suggests that other genetic or hormonal factors are implicated in the variable clinical expression of the disorder (22). The clinical findings may also be dependent on the timing of the investigation—before or after puberty. The p.L55Q mutations have been found only in patients living in the Mediterranean basin, with Jordanian (23), southern Lebanese (18) or Turkish (18, 19) ancestry, and this was the case for our patient. These mutations are certainly derived from common ancestral mutations, which reinforces the founder-gene effect. To our knowledge, the substitution of glutamine by arginine at position 56 (p.Q56R) in the homozygous form, which was identified in patient 2, has never been reported. Only a heterozygous form (p.Q56R/p.L55Q) in a patient of Jordanian origin was identified (6). This patient, who was reared as female, exhibited clitoromegaly (3 cm) and hypospadias at 24 years old, in accordance with the phenotype described. The 5a-reductase enzyme activity in genital skin fibroblasts was lower than 0.2 pmol/h per mg protein, confirming the dramatic enzyme alteration (6). The substitution of asparagine by serine at position 193 (p.N193S) identified in patient 3 was reported in compound heterozygous form (p.Q126R/p.N193S) in three siblings of Brazilian origin (17). These patients exhibited micropenis and hypospadias and were reared as girls at birth, but they switched to male sex at puberty (13–14 years old), in contrast to our patient (17). p.N193S was also observed in a 16-year-old Polish patient who exhibited clitoral enlargement and virilization (7), and in a 17-year-old patient of Mexican-Mestizo origin with micropenis and perineoscrotal hypospadias without cryptorchidism (24). Patient 4 was found to carry compound heterozygous mutations (p.R246W/c.34delG). The substitution of arginine by tryptophan in position 246 has been reported mostly in the Primary amenorrhea and 5a-reductase deficiency Vol. 95, No. 2, February 2011 FIGURE 1 Partial sequences of the srd5A2 gene in the four patients. Maimoun. Primary amenorrhea and 5a-reductase deficiency. Fertil Steril 2011. homozygous form in patients of Dominican and Brazilian origin (6, 17, 25–27). It has also been reported in Spanish (28), African American, Austrian, Pakistani, and Egyptian patients (25). The identical p.R246W mutation found in individuals from widely divergent geographic and ethnic backgrounds suggests a mutational hot spot on exon 5 of the gene. This mutation was found to result in decreased affinity of the enzyme for its nicotinamide-adenine dinucleotide phosphate cofactor, a reduction in enzyme half-life, and residual enzyme activity of less than 5% of normal (21). This finding might explain the female phenotype of our patient. The previously unreported deletion identified in our study, c.34delG, induces a premature stop codon in position 40, which FIGURE 2 Comparison of the ethnic origin of the mutations described in this article with those reported in the literature. Maimoun. Primary amenorrhea and 5a-reductase deficiency. Fertil Steril 2011. 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