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Refining Hormonal Diagnosis of Type II 3ß-Hydroxysteroid Dehydrogenase Deficiency in Patients with Premature Pubarche and Hirsutism Based on HSD3B2 Genotyping

Departamento de Clinica Medica (L.M.M., A.C.M., M.d.C.) and Departamento de Fisiologia (L.L.K.E.), School of Medicine of Ribeirao Preto, University of São Paulo, Ribeirao Preto, São Paulo, Brazil 14049-900; and Unidade de Endocrinologia do Desenvolvimento, Laboratorio de Hormonios e Genetica Molecular (S.M., B.B.M.), School of Medicine of University of São Paulo, São Paulo, Brazil 01060-970

Address all correspondence and requests for reprints to: Margaret de Castro, M.D., Ph.D., Departamento Clinica Medica, Faculdade de Medicina de Ribeirao Preto, University of São Paulo, Avenida Bandeirantes, 14049-900, Ribeirao Preto, São Paulo, Brazil. E-mail: castrom{at}fmrp.usp.br.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Congenital adrenal hyperplasia due to 3ß-hydroxysteroid dehydrogenase/⁵-⁴-isomerase (3ßHSD), a rare autosomal recessive disorder that affects both sexes, has a heterogeneous clinical presentation ranging from the severe salt-wasting to the non-salt-wasting forms and results from mutations in the HSD3B2 gene. The hormonal criteria for diagnosing the mild variant of 3ßHSD deficiency have been controversial because the initial studies were not based on genetic evidence. We investigated the relationship between the hormonal phenotype and HSD3B2 genotype in 22 patients with clinical and/or biochemical features suggestive of 3ßHSD2 deficiency, including nine female children with premature pubarche, 12 hirsute females, and one boy with salt-wasting and ambiguous genitalia. Serum 17-hydroxypregnenolone (5-17P), cortisol (F), 17-hydroxyprogesterone, dehydroepiandrosterone, and androstenedione levels were determined by RIA and were compared with Tanner pubic hair stage-matched control groups. The genomic DNA was extracted, and the entire HSD3B2 gene was amplified by PCR followed by automatic sequencing. Besides two different mutations previously observed in three patients (T259M and G129R/P222Q mutations), we observed the P222Q mutation in the male patient with salt-wasting form of 3ßHSD2 deficiency. Basal and ACTH-stimulated 5-17P levels (nanomoles per liter) ranged from 4–41 (–0.2 to 14 SD) and 36–97 (3.5–15.5 SD), respectively, in patients without mutation in HSD3B2 and from 69–153 (25–57 SD) and 201–351 (36–65 SD), respectively, in patients with mutation in HSD3B2. Basal and ACTH-stimulated 5-17P to F ratios ranged from 11–159 (0.5–25 SD) and 42–122 (2.4–11.3 SD), respectively, in patients without mutation in HSD3B2 and from 181-1700 (29–282 SD) and 487-1523 (52–167 SD), respectively, in patients with mutation in HSD3B2. The hormone findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3ßHSD2 deficiency in children with premature pubarche: ACTH-stimulated 5-17P and 5-17P to F ratios at or greater than 201 and 487 nmol/liter, respectively, equivalent to or greater than 36 and 52 SD above matched control mean. Basal and ACTH-stimulated 5-17P and 5-17P to F ratios in all genotype-proven patients in childhood were unequivocally higher than the levels of either genotype-normal patients. All the other parameters overlapped between the patients with and without mutations in the HSD3B2 gene. In conclusion, genotyping more patients in the present study, we confirm that patients with mutations in the HSD3B2 gene have extremely elevated basal and ACTH-stimulated 5-17P levels and 5-17P to F ratios. Therefore, these data refine the hormonal criteria proposed to predict more accurately 3ßHSD2 deficiency.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
THE FUNCTIONAL MEMBRANE-BOUND nicotinamide adenine dinucleotide⁺-dependent enzyme 3ß-hydroxysteroid dehydrogenase/⁵-⁴-isomerase (3ßHSD) catalyzes the 3ß-hydroxysteroid dehydrogenation and 5 to 4-isomerization of the 5-steroid precursors pregnenolone, 17-hydroxypregnenolone (5-17P), dehydroepiandrosterone (DHEA), and androst-5-ene-3ß,17ß-diol into the respective 5-ketosteroids, namely progesterone, 17-hydroxyprogesterone (17-OHP), 4-androstenedione (4-A), and testosterone (1, 2). In human, there are two 3ßHSD isoenzymes, chronologically designated types I and II, which are 93.5% homologous and are encoded by two genes on chromosome 1p13.1 (1, 2, 3, 4, 5). The type I gene (HSD3B1) is almost exclusively expressed in the placenta and peripheral tissues including the mammary gland, prostate, and skin, whereas the type II gene (HSD3B2) is predominantly expressed in the human adrenal gland, ovary, and testis (2, 4, 6).

Congenital adrenal hyperplasia (CAH) due to 3ßHSD deficiency results from mutations in the HSD3B2 gene. This deficiency is a rare autosomal recessive disorder that affects both sexes and has a heterogeneous clinical presentation, with an impairment of both adrenal and gonad steroidogenesis (7, 8, 9, 10). Therefore, the clinical spectrum of inherited 3ßHSD deficiency ranges from the severe salt-wasting form, with or without ambiguous genitalia, to the non-salt-wasting form, with ambiguous genitalia and/or premature pubarche in young children and hirsutism and menstrual disorders in older females (11, 12, 13, 14, 15, 16).

In the past two decades, the hormonal criteria used to diagnose the less severe or non-salt-wasting form of 3ßHSD deficiency were ACTH-stimulated 5-17P and DHEA levels, and ratios of 5-17P to 17-OHP or DHEA to 4-A greater than 2 SD above the mean value for age- or pubertal stage-matched control or normal subjects (17, 18, 19, 20, 21, 22, 23). However, according to these hormonal criteria, an unusually large proportion of hirsute females and children with premature pubarche were diagnosed with the mild variant form of 3ßHSD, despite the fact that severe 3ßHSD deficiency is a rare disorder (16, 17, 18, 19, 20, 21, 22, 23). Finally, the hormonal criteria for diagnosing CAH due to the mild variant of 3ßHSD deficiency have been controversial because the initial studies were not based on genetic evidence (17, 18, 19, 20, 21, 22, 23).

In a recent report, Lutfallah et al. (24) defined new hormonal criteria to accurately predict 3ßHSD deficiency and proposed that it is necessary in a greater number of genotype-proven and genotype-normal patients to refine these hormonal findings. We therefore investigated the relationship between the hormonal findings and HSD3B2 genotype in patients presenting with clinical and/or hormonal abnormalities suggestive of 3ßHSD deficiency to establish hormonal criteria for accurate prediction of this disease.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

The present study was approved by the Research National Ethical Committee and was conducted after informed consent from the subjects or the subject’s legal guardians. We included patients with clinical and/or biochemical features suggestive of salt-wasting and non-salt-wasting forms of 3ßHSD2 deficiency. Our group comprised 22 patients (21 females and one male, from 9 months to 26 yr of age), including three previously reported children with 3ßHSD deficiency (25), six female children with premature pubarche, 12 hirsute females, and one boy with salt-wasting and ambiguous genitalia.

The nine female children presenting with premature sexual hair experienced onset of pubarche between the ages 3 and 7 yr (median age, 6 yr). All patients had Tanner stage II–III pubic hair. Three patients had facial acne, and two patients had mild clitoral enlargement. Five girls had advanced bone age. None of the patients had salt loss or siblings with male pseudohermaphroditism. Table 1 shows clinical and biochemical findings observed in children with premature pubarche.

Hormonal assays

All patients but one had a baseline hormone evaluation and an ACTH stimulation test without glucocorticoid therapy. The child with clinical findings suggestive of salt-wasting form of CAH started the substitutive glucocorticoid and mineralocorticoid therapy before biochemical evaluation for 3ßHSD2 deficiency. The family of this child did not agree to withdraw the therapy to allow further evaluation; however, they allowed the molecular analysis of the HSD3B2 gene.

A standard ACTH stimulation test was performed between 0800 and 1000 h. Blood samples were obtained before and 60 min after administering a synthetic ACTH (Tetracosactrin, Novartis Pharma S.A., Rueil Malmaison, France; 0.25 mg iv). Serum cortisol (F), 17-OHP, 11-deoxycortisol, DHEA, and 4-A levels were determined by RIA (25, 27). The ACTH test ruled out 21-hydroxylase and 11ß-hydroxylase deficiencies in all patients. The steroid assays were compared with Tanner pubic hair stage-matched control groups from previous reports (24, 27). Serum 5-17P was assayed by a homemade RIA after chromatography in celite columns, using tritiated steroid from NEN Life Science Products (Boston, MA) and antisera from ICN Biochemicals (Costa Mesa, CA) as previously described by Mendonca et al. (28).

PCR and sequencing of the HSD3B2 gene

All patients had HSD3B2 gene analysis. The genomic DNA from all subjects was extracted from peripheral white blood cells. PCR for the HSD3B2 gene was performed using primers previously described (29) with minor modifications. The four exons and the exon-intron boundaries of the HSD3B2 gene from all patients were amplified and sequenced. The PCR products were verified on an agarose gel for the predicted DNA size and treated by an enzymatic method (exonuclease I and shrimp alkaline phosphatase; Amersham Life Science, Buckinghamshire, UK). Direct sequencing of the PCR products was performed with a DNA sequencing kit (ABI Prism Big Dye Terminator Cycle Sequencing Read Reaction Kit; PE Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. After the sequencing reaction, the products were analyzed in an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems). The results obtained were compared with the normal HSD3B2 gene sequence (GenBank accession no. M77144).

Hormonal criteria

Hormonal differences between genotype-proven and -normal patients were calculated using a two-sided Mann-Whitney U test. P < 0.05 was considered to be significant (24). For the hormonal criteria for 3ßHSD2 deficiency in children and adult subjects, the lowest value of each hormonal parameter in the genotype-proven patients was used as a criterion if the values from the genotype-normal and genotype-proven patients did not overlap. To compare the hormonal criteria via genotypic proof and the past published hormonal criteria without genetic evidence (17, 18, 19, 20, 21, 22, 23), the lowest hormonal value in the genotype-proven patients was also expressed as SD from the mean value of age- or pubic hair stage-matched control/normal subjects.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
HSD3B2 genotype in patients with clinical and hormonal findings suggestive of 3ßHSD2 deficiency

Besides two different mutations previously published in three patients with premature pubarche (one had the homozygous T259M mutation that substitutes 8776 C>T, and two sisters had G129R and P222Q as a compound heterozygous mutation that substitutes 7985G>A and 8265C>A, respectively), we also found the homozygous P222Q mutation in the male patient with salt-wasting and ambiguous genitalia (Fig. 1) and the P222Q mutation in heterozygous state in his mother.

View larger version (31K): [in this window] [in a new window]   FIG. 1. Direct sequencing of a fragment of exon 4 of the HSD3B2 gene showing the mutations. A, G129R mutation. The arrow indicates the 7985G>A heterozygous substition. B, P222Q mutation. The arrow indicates the 8265C>A heterozygous substition. Compound heterozygous G129R/P222Q mutation was found in the siblings with permature pubarche. C, P222Q mutation. The arrow indicates the 8265C>A homozygous substitution in the male patient with salt wasting and ambiguous genitalia. D, T259M mutation. The arrow indicates the 8776C>A homozygous substitution found in the patient with premature pubarche and clitorimegaly.

Hormonal analysis

The absolute values and the range of SD above the mean values of the appropriate control subjects of 5-17P, F, 17-OHP, DHEA, and 4-A and the ratios 5-17P to 17-OHP, DHEA to 4-A, and 5-17P to F are shown in Table 1 for the premature pubarche group and in Table 2 for the hirsute adolescents and adult female groups.

In the premature pubarche group, the ACTH-stimulated 17-OHP levels and 11-deoxycortisol ranged from 2.1–24 and 1.8–14 nmol/liter, respectively, whereas in the hirsute female group, these values ranged from 2.4–22.3 and 2.5–7.5 nmol/liter, respectively, excluding the diagnosis of CAH due to 21-hydroxylase and 11ß-hydroxylase deficiencies in both groups.

Comparison of hormonal profiles between patients with normal genotype and patients with genotype-proven HSD3B2 gene mutations

Table 3 shows the hormonal criteria for 3ßHSD deficiency based on the lowest absolute hormonal value observed in genotype-proven 3ßHSD patients (Tanner II–III pubic hair) compared with appropriate control normal subjects in patients studied by Lutfallah et al. (24) and in patients studied in the present work.

5-17P levels.

Ratios of 5-17P to F. Baseline and ACTH-stimulated ratios of 5-17P to F in all genotype-proven patients were significantly higher than the ratios of either genotype-normal patients or age- or pubic stage-matched control subjects. This ratio was higher after ACTH stimulation due to the lower F response in the genotype-proven pubarche group. The lowest baseline ratio of 5-17P to F in the genotype-proven patients was 181 (29 SD above the control mean) in children with premature pubarche (Table 3). The highest baseline ratios of 5-17P to F in the genotype-normal patients were 159 (25 SD above the control mean) in children with premature pubarche. The lowest ACTH-stimulated ratio of 5-17P to F in the genotype-proven patients was 487 (52 SD above the control mean) in children with premature pubarche (Table 3). The highest ratio in the genotype-normal patients was 122 (11 SD above the control mean) in children with premature pubarche.

F and DHEA levels and ratios of 5-17P to 17-OHP and DHEA to 4-A. Both baseline and ACTH-stimulated ratios of all these parameters in the genotype-proven overlapped with the levels of genotype-normal patients (Tables 1 and 2). Baseline 5-17P to 17-OHP ratio and ACTH-stimulated F in the genotype-proven patients were statistically higher than genotype-normal patients. All other comparisons (baseline and ACTH-stimulated levels of DHEA and ratios of DHEA to 4-A, baseline F, and ACTH-stimulated 5-17P to 17-OHP ratio) in the genotype-proven patients were not significantly different from the levels of genotype-normal patients.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
In the present study, we evaluated the relationship between the hormonal phenotype and HSD3B2 genotype in 22 patients with suggestive clinical and/or hormonal abnormalities of salt-wasting and non-salt-wasting forms of 3ßHSD2 deficiency to establish hormonal criteria for accurate prediction of this disease. This is a very rare disease and difficult to be diagnosed in infants who do not show evidence of salt wasting. However, it should be suspected in female children presenting with premature pubarche and other progressive hyperandrogenism findings, such as facial acne, clitoral enlargement, and advanced bone age, whose diagnosis of CAH due to 21-hydroxylase and 11ß-hydroxylase deficiencies have been excluded by the ACTH stimulation test.

We previously published mutations in the HSD3B2 gene in three patients with the non-salt-wasting form of 3ßHSD2 deficiency (25). The homozygous T259M mutation was identified in a girl with premature pubarche and clitoral enlargement. The compound heterozygous G129R/P222Q was identified in two female sisters with premature pubarche. In the present study, we also identified the P222Q homozygous mutation in one boy with ambiguous genitalia and salt-wasting at birth. The residues threonine 259, glycine 129, and proline 222 are highly conserved in all vertebrates. Treonine 259 is located close to the YXXXK motif, which contains residues involved in the putative substrate-binding domain, suggesting an important role of this residue in the 3ßHSD2 activity (30). T259M and P222Q mutations have no enzyme activity in vitro; rather, the G129R mutation reduced the 3ßHSD2 activity to 2% compared with wild type (31).

The P222Q mutation, which has no enzyme activity in vitro (31), was associated with ambiguous genitalia and salt wasting in a male patient. This mutation has also been previously described in two Algerian siblings affected by the salt-wasting form of 3ßHSD2 deficiency (31). The G129R mutation also has been previously reported in two American siblings affected by the non-salt-wasting form of 3ßHSD2 deficiency (32). Consistent with a residual enzyme activity, we observed the compound heterozygous G129R/P222Q mutation in a patient with the non-salt-wasting form of 3ßHSD2 deficiency. T259M mutation was reported in a Taiwanese boy with ambiguous genitalia and the salt-wasting form of 3ßHSD2 deficiency (33) and also in the compound heterozygous state, associated with 867delG mutation, in two French siblings affected by the salt-wasting form of the disease (31). However, in the present study, the homozygous T259M mutation, which has no enzymatic activity in vitro and evidence of protein instability (31), was identified in a girl with the non-salt-wasting form. Despite the absence of salt wasting, this patient showed more severe hyperandrogenism findings, such as clitoral enlargement, more advanced bone age, and more precocious age of onset of pubic hair, and also very low basal and ACTH-stimulated F levels associated with high ACTH plasma level. In addition, this mutation had been previously identified in the homozygous state in a Brazilian woman who also presented a very severe virilization and hyperplastic ectopic adrenal tissue in ovaries (34). Our data, as well as data from the literature, show a very good genotype-phenotype correlation; however, the T259M mutation is an exception to the rule. In this case, the 3ßHSD1 activity in peripheral tissues might contribute to the phenotype variability.

There was no mutation in HSD3B2 gene in all other children with premature pubarche as well as in the 12 hirsute females. The absence of mutations in hirsute females confirms a previous study performed in another Brazilian population, which also found no mutation in 13 hirsute women (35). Pang’s group (24) also did not find mutations in HSD3B2 gene in 15 hirsute females in a previous published study. Taken together, these findings suggest that mutation in the HSD3B2 gene is not a frequent cause of this disorder. Indeed, a recent study demonstrated that the hormonal phenotype of compromised adrenal HSD3B activity, which has led to an incorrect diagnosis of the mild non-salt-wasting variant of 3ßHSD2 deficiency in hyperandrogenic females in the past, is associated with the insulin resistance and LH hypersecretion characteristic of polycystic ovary syndrome (36).

We previously described for the first time that young girls with premature pubarche with HSD3B2 gene mutations showed very high basal and ACTH-stimulated 5-17P and 5-17P to F ratios for diagnosing the non-salt-wasting form of 3ßHSD2 deficiency. These parameters did not overlap at all in genotype-proven and -normal patients (25). In agreement with the recent findings of Lutfallah et al. (24), in the present study we observed baseline and ACTH-stimulated 5-17P at or greater than 25 and 36 SD above the mean value, respectively. We also found that baseline and ACTH-stimulated 5-17P to F ratios were at or greater than 29 and 52 SD above the mean value, respectively. Therefore, baseline and ACTH-stimulated 5-17P and 5-17P to F ratio are much higher in genotype-proven patients, and they should be used as the hormonal diagnosis of 3ßHSD2 deficiency. On the other hand, baseline and ACTH-stimulated 4-A and DHEA levels, and ratios of 5-17P to 17-OHP or DHEA to 4-A showed an overlap in patients with and without HSD3B2 gene mutation and are useless for the diagnosis of this disease.

In conclusion, our findings, after genotyping more patients, confirm that mutation in the HSD3B2 gene is not a frequent cause of hirsutism in adult women. In addition, patients with mutations in the HSD3B2 gene have extremely elevated hormonal values compared with previous criteria for the diagnosis of 3ßHSD2 deficiency. Therefore, because 3ßHSD2 deficiency is a rare disease, our data contribute to the establishment of new hormonal parameters based on genotype proof. The compilation of the experience of different research groups will enable future meta-analysis studies, establishing the cutoff levels of hormonal criteria from infancy to adulthood to predict more accurately 3ßHSD2 deficiency.

	Acknowledgments

 
We thank Professor Marco Antonio Zago and Dr. Wilson A. Silva, Jr. (Fundação Hemocentro de Ribeirao Preto, Ribeirao Preto, Sãn Paulo, Brazil) for kindly allowing the use of the automated DNA sequencer and Ms. Natalia Torres and Mr. Fernando Amaral for technical assistance.

	Footnotes

 
This work was supported by a grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo (process no. 01/01148-4, to L.M.M.).

First Published Online December 7, 2004

Abbreviations: 4-A, Androstenedione; CAH, congenital adrenal hyperplasia; DHEA, dehydroepiandrosterone; F, cortisol; 3ßHSD, 3ß-hydroxysteroid dehydrogenase/⁵-⁴-isomerase; 17-OHP, 17-hydroxyprogesterone; 5-17P, 17-hydroxypregnenolone.

Received August 4, 2004.

Accepted November 3, 2004.

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Top Abstract Introduction Patients and Methods Results Discussion References

 

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