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Newly Proposed Hormonal Criteria via Genotypic Proof for Type II 3ß-Hydroxysteroid Dehydrogenase Deficiency

Department of Pediatrics (C.L., W.W., Y.T.C., S.P.), University of Illinois at Chicago, College of Medicine, Chicago, Illinois 60612; Department of Reproductive and Developmental Sciences (J.I.M.), University of Edinburgh, Edinburgh EH3 9YL, Scotland; Children’s Medical Center of Akron (A.H.), Akron, Ohio 44308; Department of Pediatrics (B.R.), University of Chicago, Chicago, Illinois 60637; Department of Pediatrics (M.C.-M.), Winthrop University Hospital, State University of New York, Long Island, New York 11501; Children’s Hospital of Oklahoma (K.C.C.), Oklahoma City, Oklahoma 73104; Department of Pediatrics (R.D.), New York University Medical Center, New York, New York 10016

Address all correspondence and requests for reprints to: Dr. Songya Pang, Department of Pediatrics (M/C 856), University of Illinois at Chicago, College of Medicine, 840 South Wood Street, Chicago, Illinois 60611. E-mail: . spang{at}tigger.cc.uic.edu

Abstract

To define the hormonal criteria via genotypic proof for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) deficiency in the adrenals and gonads, we investigated the type II 3ß-HSD genotype in 55 patients with clinical and/or hormonal presentation suggesting compromised adrenal with or without gonadal 3ß-HSD activity. Fourteen patients (11 males and 3 females) had ambiguous genitalia with or without salt wasting and with or without premature pubarche. One female neonate had salt wasting only. Twenty-five children (4 males and 21 females) had premature pubarche only. Fifteen adolescent and adult females had hirsutism with or without menstrual disorder. The type II 3ß-HSD gene, including the promoter region up to -1053 base, all exons I, II, III, IV, and exon and intron boundaries, was sequenced in all subjects. Eight patients had a proven or predictably deleterious mutation in both alleles of the type II 3ß-HSD gene, and 47 patients had no apparent mutation in the gene. ACTH-stimulated (1 h post iv bolus of 250 µg Cortrosyn) serum 17-hydroxypregnenolone (5–17P) levels and basal and ACTH-stimulated ratios of 5–17P to cortisol (F) in the genotypic proven patients were unequivocally higher than those of age-matched or pubic hair stage matched genotype-normal patients or control subjects (n = 7–30 for each group). All other baseline and ACTH-stimulated hormone parameters, including dehydroepiandrosterone (DHEA) levels, ratios of 5–17P to 17-OHP and DHEA to androstenedione in the genotype-proven patients, overlapped with the genotype-normal patients or control subjects. The hormonal findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3ß-HSD deficiency congenital adrenal hyperplasia (numeric and graphic reference standards from infancy to adulthood are provided): ACTH-stimulated 5–17P levels in 1) neonatal infants with ambiguous genitalia at or greater than 378 nmol/liter equivalent to or greater than 5.3 SD above the control mean level [95 ± 53 (SD) nmol/liter]; 2) Tanner I children with ambiguous genitalia at or greater than 165 nmol/liter equivalent to or greater than 35 SD above the control mean level [12 ± 4.3 (SD) nmol/liter]; 3) children with premature pubarche at or greater than 294 nmol/liter equivalent to or greater than 54 SD above Tanner II pubic hair stage matched control mean level [17 ± 5 (SD) nmol/liter]; and 4) adults with at or greater than 289 nmol/liter equivalent to or greater than 21 SD above the normal mean level [25 ± 12 (SD) nmol/liter]. ACTH-stimulated ratio of 5–17P to F in 1) neonatal infants at or greater than 434 equivalent to or greater than 6.4 SD above the control mean ratio [88 ± 54 (SD)]; 2) Tanner I children at or greater than 216 equivalent to or greater than 23 SD above the control mean ratio [12 ± 9 (SD)]; 3) children with premature pubarche at or greater than 363 equivalent to or greater than 38 SD above the control mean ratio [20 ± 9 (SD)]; and 4) adults at or greater than 4010 equivalent to or greater than 221 SD above the normal mean ratio [29 ± 18 (SD)]. Conversely, the hormonal data in the genotype-normal patients suggest the following hormonal criteria are not consistent with 3ß-HSD deficiency congenital adrenal hyperplasia: ACTH-stimulated 5–17P levels in children with premature pubarche up to 72 nmol/liter equivalent to up to 11 SD above the control mean level, and in hirsute females up to 150 nmol/liter equivalent to up to 12 SD above the normal female mean level [28 ± 10 (SD) nmol/liter]; and ACTH-stimulated 5–17P to F ratio in children with premature pubarche up to 67 equivalent to up to 5 SD above the control mean ratio, and in hirsute females up to 151 equivalent to up to 10 SD above the normal mean ratio [32 ± 12 (SD)]. These findings help define newly proposed hormonal criteria to accurately predict inherited 3ß-HSD deficiency.

3ß-HYDROXYSTEROID DEHYDROGENASE (3ß-HSD) is essential for the formation of progesterone, the precursor hormone for aldosterone, and 17-hydroxyprogesterone (17-OHP), the precursor hormone for cortisol (F) in the adrenal cortex (1, 2, 3). It is also essential for the formation of androstenedione (4-A), testosterone, and estrogen in the adrenals and gonads (1, 2, 3). In humans, the type II 3ß-HSD gene encodes for both adrenal and gonadal 3ß-HSD (3, 4). The clinical spectrum of inherited 3ß-HSD deficiency in the adrenals and gonads causing congenital adrenal hyperplasia ranges from the severe salt-wasting form, with or without ambiguous genitalia and hypogonadism, to the nonsalt-wasting form, with ambiguous genitalia and/or premature pubarche in young children and hirsutism and menstrual disorders in older females (5, 6, 7, 8, 9, 10, 11).

During the past two decades, a less severe or mild nonclassic variant of 3ß-HSD deficiency was reported to be a cause of premature sexual hair growth in many young children and a cause of hirsutism and menstrual disorders in a great number of adolescent and young women (12, 13, 14, 15, 16, 17, 18). The hormonal criteria used to diagnose the mild variant of 3ß-HSD deficiency in the past two decades were ACTH-stimulated 17-hydroxypregnenolone (5–17P) and dehydroepiandrosterone (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 (12, 13, 14, 15, 16, 17, 18). However, these hormonal criteria (12, 13, 14, 15, 16, 17, 18) have been controversial for diagnosing the mild variant of 3ß-HSD deficiency congenital adrenal hyperplasia because the precursor 5–17P abnormality was not as pronounced as the precursor 17-OHP abnormality reported in the mild variant of 21-hydroxylase deficiency congenital adrenal hyperplasia (19, 20, 21). Furthermore, despite the fact that severe 3ß-HSD deficiency congenital adrenal hyperplasia is a rare disorder, an unusually large proportion of hirsute females and children with premature pubarche were diagnosed with the mild variant form of 3ß-HSD deficiency congenital adrenal hyperplasia according to the previously reported hormonal criteria (11, 12, 13, 14, 15, 16, 17, 18). Finally, the validity of the past published hormonal criteria for diagnosing the mild variant of 3ß-HSD deficiency congenital adrenal hyperplasia was questionable because such diagnostic hormonal criteria were not based on any genetic evidence (12, 13, 14, 15, 16, 17, 18). Thus, it was essential to reevaluate the hormonal criteria from childhood to adulthood for the varying spectrum of inherited 3ß-HSD deficiency on the basis of genotypic proof. In humans, 3ß-HSD deficiency results from a deleterious mutation in the type II 3ß-HSD gene (11, 22, 23, 24, 25, 26, 27, 28, 29). We therefore investigated the relationship between the hormonal phenotype and type II 3ß-HSD genotype in the infants, children, adolescents, and adults presenting with clinical and/or hormonal abnormalities suggestive of altered adrenal or gonadal 3ß-HSD activity. We now report the hormonal phenotype in patients of all ages with proven or predictably deleterious mutations in the type II 3ß-HSD gene and in patients with normal type II 3ß-HSD genes. The hormonal findings in the genotype-proven 3ß-HSD deficiency congenital adrenal hyperplasia patients and in the genotype-normal patients helped develop newly proposed hormonal criteria for accurate prediction of inherited 3ß-HSD deficiency.

Patients and Methods

The study was approved by the Institutional Review Board at the University of Illinois at Chicago and was conducted after informed consent from the subjects or the subjects’ legal guardians.

Patient population

Fifty-five patients with clinical symptoms compatible with adrenal and/or gonadal 3ß-HSD deficiency were studied (Table 1). Fourteen patients, including 11 genetic males and 3 genetic females, had ambiguous genitalia. The male patients had various degrees of hypospadias and chordee with or without a bifid scrotum. The female patients had various degrees of clitoral enlargement with or without posterior labial fusion. Three of these patients had salt-wasting disorder from a neonatal age, and 11 had no evidence of salt wasting, including 2 males who developed premature sexual hair between the ages of 4 and 8 yr. One genetic female with normal genitalia presented with salt wasting only during the neonatal age. Twenty-five children with normal genitalia and no evidence of salt wasting (21 females and 4 males) presented with premature sexual hair between the ages of 1 month and 7 yr (median age, 4 yr). Fifteen hirsute females, ages ranging from 12–35 yr (median age, 19 yr), experienced onset of hirsutism between the ages 7 and 25 yr (median age, 15 yr). The hirsutism score of the patients was estimated to be greater than 8 in all subjects by the Ferriman and Gallwey method (30). Thirteen of these patients had irregular menses (every 2–3 month cycle) or secondary amenorrhea, or menometrorrhagia, one had regular menstrual cycle, and one was age appropriately premenarchal. All patients had type II 3ß-HSD gene analysis and had a baseline hormone evaluation without glucocorticoid therapy, except for one adult male pseudohermaphrodite whose glucocorticoid therapy was stopped for 7 d before the hormone evaluation. All but three patients had an ACTH stimulation test.

Control reference baseline and ACTH-stimulated hormonal data from the infants of three age groups were established and were in part previously reported (31): 7 neonates with ages ranging from 4–42 d, with a median age of 15 d, had an ACTH stimulation test for evaluation of hyponatremia, metabolic acidosis, or genital ambiguity and were ultimately proven to have normal adrenal function; 7 young infants with ages ranging from 2.5–6 months, median age 4.5 months, had an ACTH stimulation test for hypoglycemia or genital ambiguity and were ultimately proven to have normal adrenal function; 10 older infants with ages ranging from 7–20 months, median age 13 months, had an ACTH stimulation test for evaluation of a large adrenal gland noted on ultrasound, or hypoglycemia or genital ambiguity and were proven to have normal adrenal function. The control reference hormonal data obtained via ACTH stimulation test for 18 control prepubertal children and 7 control children with Tanner II–III pubic hair development and proven normal adrenal function were previously reported (31). The control reference hormonal data in 30 normal adult females and 10 normal adult males were also previously reported (32, 33).

ACTH stimulation test, hormonal assay, and hormonal indices to determine adrenal 3ß-HSD activity

An ACTH stimulation test was performed between 0830 and 1100 h. Blood samples were obtained before and 60 min after administering a synthetic ACTH 250 µg (Cortrosyn, Ben Venue Labs, Inc., Bedford, Ohio) iv by a bolus. Serum 5–17P, F, 17-OHP, DHEA, and 4-A levels were determined by a reported RIA following a celite chromatographic purification (31, 32, 33). The inter- and intra-assay variations of these assays were 10–15% and 5–10%, respectively. Hormonal indices suggesting compromised adrenal 3ß-HSD activity in the patients included a baseline or ACTH-stimulated 5–17P level and/or a DHEA level greater than 2 SD above age or pubertal stage matched control or normal subject’s mean value, with or without elevated ratio of 5–17P to F, 5–17P to 17-OHP ratio, or DHEA to 4-A ratio greater than 2 SD above control or normal subject’s mean value. Those with 21-hydroxylase deficiency and 11ß-hydroxylase deficiency were excluded from this study.

PCR and sequencing of the type II 3ß-HSD genes

The genomic DNA from all subjects was prepared from peripheral white blood cells. PCR for the type II 3ß-HSD gene was performed using primers as reported (28, 31). The type II 3ß-HSD gene region, including a putative promoter region up to 1053 bp, all exons I, II, III, IV, and exon and intron boundaries from all patients, was amplified and sequenced. The PCR products were verified on an agarose gel for the predicted DNA size. Direct sequencing or autosequencing of the PCR products was performed with a DNA sequencing kit (Promega Corp., Madison, WI) as previously described (28, 31). In addition, subcloning of PCR products in some cases was performed using TA cloning and then sequenced by dideoxynucleotide method using a kit (Invitrogen Inc., San Diego, CA) as previously described (23, 26).

Statistical analysis

Hormonal differences between type II 3ß-HSD genotype-proven patients and type II 3ß-HSD genotype-normal patients, and between genotype-proven patients and each group of control or normal subjects were analyzed using a two sample Wilcox and rank sum (Mann-Whitney) test. A P value less than 0.1 between the groups was considered to be significant.

Proposed new hormonal criteria

New hormonal criteria for the genotype-proven and genotype-normal patients were based on order statistics. For the new hormonal criteria for 3ß-HSD deficiency congenital adrenal hyperplasia, in infants, children, and adult subjects, the lowest value of each hormonal parameter in the genotype-proven patients became the new criterion if the values from the genotype-normal and genotype-proven patients did not overlap. The past published hormonal criteria for 3ß-HSD deficiency without any genotypic evidence (12, 13, 14, 15, 16, 17, 18) were based on hormonal parameters more than 2 SD above the control mean value. For the purpose of comparison between the new hormonal criteria via genotypic proof and the past published hormonal criteria without genetic evidence (12, 13, 14, 15, 16, 17, 18), the lowest hormonal value in the genotype-proven patients was also expressed as a certain SD from the mean value of age or pubic hair stage matched control/normal subjects. Conversely, the highest value of hormonal parameter among the genotype-normal patients became the hormonal criterion for exclusion of inherited 3ß-HSD deficiency if the values did not overlap between the genotype-normal and genotype-proven patients. The highest hormonal value in the genotype-normal patient was also expressed as a certain SD from the mean value of age or pubic hair stage matched control subjects.

Results

Type II 3ß-HSD genotype in the patients with clinical and hormonal findings suggestive of decreased adrenal with or without gonadal 3ß-HSD activity (Fig. 1 and Table 2)

Forty-seven of the 55 patients had no mutations in the putative promoter region, exons I, II, III, and IV, and all exon and intron boundaries on both alleles. Eight patients had a proven or predictably deleterious mutation in the gene on both alleles (Fig. 1 and genotype-proven patients in Table 2). The mutant type II 3ß-HSD genotypes of homozygous V248N-R249 Stop and homozygous T259M identified from two genetic males with salt-wasting disorder and ambiguous genitalia (Table 2, subjects 1 and 4) were previously proven or predicted to be seriously deleterious mutations for type II 3ß-HSD activity (23, 28). Compound heterozygous E142K and W171Stop mutant genotypes identified from a male with salt wasting and ambiguous genitalia (Table 2, subject 5) in this study were previously characterized by others (24) as seriously deleterious mutations. The homozygous P222T genotype identified from a genetic female with salt-wasting disorder (Table 2, subject 6) was proven to be a seriously deleterious mutation (29). The homozygous L6F mutant genotype identified from a genetic male with ambiguous genitalia (Table 2, subject 2) was proven to be less deleterious for the enzyme activity (28). The mutant Stop373C genotype identified from one allele of a genetic female with premature sexual hair growth (Table 2, subject 3) was characterized as less deleterious for the enzyme activity (29) whereas the E142K genotype identified from the second allele of the subject was previously reported by others (24) to be a seriously deleterious mutation. A mutant G129R identified from one allele each of a genetic male with premature pubarche and ambiguous genitalia and a genetic female with premature pubarche (Table 2, subjects 6 and 7) was previously proven to be a less deleterious mutation (25), whereas the N6651 allele identified on the second allele from these subjects was predicted to cause a splicing defect.

View larger version (22K): [in this window] [in a new window]   Figure 1. Mutant type II 3ß-HSD genotype identified in patients with salt-wasting patients (SW Pts) and nonsalt-wasting patients (NSW Pts) with 3ß-HSD deficiency congenital adrenal hyperplasia. A schematic of the type II 3ß-HSD gene map shows exon numbers in Roman numerals I, II, III, and IV, and the shaded region indicates translated coding region. Arabic numbers outside of boxes indicate codon numbers. Each mutant allele genotype of the patients appears in a square box, and lines connecting the genotype box to the gene map indicate the site of mutations in the exons or intron. A black circle indicates homozygous mutation, and a line connecting the square genotype boxes indicates compound heterozygous mutations in individual patients. Black boxes below the gene map indicate a schematic of putative or suggested functional domains.

5–17P levels (Fig. 2 and Table 2). Baseline 5–17P levels in all except one patient with the deleterious mutant type II 3ß-HSD genes (genotype-proven) were unequivocally higher than the age or pubic hair stage matched patients with normal type II 3ß-HSD genes (genotype-normal) and were unequivocally higher than the control subjects. Only one baseline 5–17P level in a genotype-proven (STOP373C/E142K) nonsalt-wasting female child with premature pubarche overlapped with the baseline 5–17P levels of children with premature pubarche and normal type II 3ß-HSD genes (Fig. 2 and Table 2). No mutation in the gene was identified in this study in the infants of age 2.5–20 months with either ambiguous genitalia or premature pubarche, and in young women with hirsutism with or without menstrual disorders. In the neonatal infants (age < 42 d) with salt wasting and/or ambiguous genitalia, or a positive newborn screening test for congenital adrenal hyperplasia, the lowest baseline 5–17P level in the genotype-proven patients was 84.3 nmol/liter, which was equivalent to 6.8 SD above the control neonatal infant mean value (Fig. 2 and Table 2). In the infants of ages 2.5–6 months, the highest baseline 5–17P level in the genotype-normal patients with ambiguous genitalia was 37 nmol/liter, which was 3.6 SD above the age-matched control mean value (Fig. 2 and Table 2). In the infants of ages 7–20 months, the highest baseline 5–17P level in the genotype-normal patients with either premature pubarche or ambiguous genitalia was 48 nmol/liter, which was equivalent to 14 SD above the age-matched control mean value. In children, the baseline 5–17P level in a genotype-proven prepubarchal patient with ambiguous genitalia was 26.4 nmol/liter, which was 12 SD above the control mean value of control children (Fig. 2 and Table 2). The lowest baseline 5–17P level in the genotype-proven patients with premature pubarche was 29 nmol/liter, which was equivalent to 10 SD above the normal mean value, whereas the highest baseline 5–17P level in the genotype-normal patients with premature pubarche was 35 nmol/liter, which was equivalent to 12 SD above the control mean value (Fig. 2 and Table 2). In adolescents and adults, the highest baseline 5–17P level in the genotype-normal hirsute females was 45 nmol/liter, which was equivalent to 16 SD above the mean value of normal females (Fig. 2 and Table 2), whereas a genotype-proven male pseudohermaphrodite had baseline levels at or greater than 159 nmol/liter, which was equivalent to 74 SD above the mean values of normal males (Fig. 2 and Table 2).

View larger version (30K): [in this window] [in a new window]   Figure 2. Comparison of baseline and ACTH-stimulated 5–17P levels between the patients with proven/predictably deleterious mutant type II 3ß-HSD genes (, Male; •, female; S, salt-wasting patients; NS, nonsalt-wasting patients) and the patients with normal type II 3ß-HSD genes (, Male; , female). indicates independent values of same male subject. AG, Ambiguous genitalia; SW, salt-wasting; + CAH test, a positive newborn screening 17-OHP level for congenital adrenal hyperplasia; PP, premature pubarche; HS, hirsutism; MPH, male pseudohermaphroditism. The shaded bars represent mean and range of hormonal levels in age or pubic hair stage matched control/normal subjects. Horizontal dotted line indicates the level at 2 SD above the control/normal mean value. Number followed by SD and dotted line indicates the level of SD values above the control/normal mean value in the patient. Please note the differences in the scale for the baseline and ACTH-stimulated 5–17P levels and the fact that two genotype-proven salt-wasting young infant patients did not have an ACTH-stimulation test.

Ratios of 5–17P to F (Fig. 3 and Table 2). Baseline and ACTH-stimulated ratios of 5–17P to F in all genotype-proven patients in neonatal infancy, childhood, and adulthood were unequivocally higher than the ratios of either genotype-normal patients or age or pubic stage matched control subjects (Fig. 3 and Table 2). The lowest baseline ratio of 5–17P to F in the genotype-proven patients was 461 (4.65 SD above the control mean) in the neonatal infants, 94 (15 SD above the control mean) in the preadrenarchal child, 103 (16 SD above the control mean) in children with premature pubarche, and 1943 (193 SD above the normal mean) in the adult (Fig. 3 and Table 2). The highest baseline ratios of 5–17P to F in the genotype-normal patient were 59 (8 SD above the control mean) in children with premature pubarche and 43 (5 SD above the normal mean) in hirsute females.

View larger version (42K): [in this window] [in a new window]   Figure 3. Comparison of baseline and ACTH-stimulated ratios of 5–17P to F between the patients with proven/predictably deleterious type II 3ß-HSD genes (, Male; •, female; S, salt-wasting patients; NS, nonsalt-wasting patients) and the patients with normal type II 3ß-HSD gene (, Male; , female). indicates two independent values of the same male patient. For abbreviations and other details, see Fig. 2 legend.

Ratios of 5–17P to 17-OHP (Fig. 4 and Table 2). The baseline and ACTH-stimulated ratios of 5–17P to 17-OHP in the genotype-proven neonatal patients were within the range of control infants, whereas the ratios in the genotype-proven children were close to or overlapped with the ratios of genotype-normal patients. Only the genotype-proven adult patient had unequivocally elevated ratios of baseline and ACTH-stimulated 5–17P to 17-OHP.

View larger version (41K): [in this window] [in a new window]   Figure 4. Comparison of baseline and ACTH-stimulated ratio of 5–17P to 17-OHP between the patients with proven/predictably deleterious type II 3ß-HSD gene (, Male; •, female; S, salt wasting patients; NS, nonsalt-wasting patients) and the patients with the normal type II 3ß-HSD gene (, Male; , female). indicates two independent values of the same male patient. For abbreviations and other details, see Fig. 2 legend.

DHEA levels (Fig. 5 and Table 2).

View larger version (38K): [in this window] [in a new window]   Figure 5. Comparison of baseline and ACTH-stimulated DHEA levels between the patients with proven/predictably deleterious mutant type II 3ß-HSD genes (, Male; •, females; S, salt-wasting patients; NS, nonsalt-wasting patients) and the patients with normal type II 3ß-HSD gene (, Male; , female). indicates two independent values of the same male patient. For abbreviations and other details, see Fig. 2 legend.

Ratios of DHEA to 4-A (Fig. 6 and Table 2). The baseline ratios of DHEA to 4-A in the genotype-proven neonates and in a genotype-proven preadrenarche child were in the range of the control subjects. ACTH-stimulated ratios in these infants and the preadrenarche patient were in the control range or higher than control subjects. Both baseline and ACTH-stimulated ratios of DHEA to 4-A in the genotype-proven patients with premature pubarche overlapped with or were higher than the ratios of genotype-normal patients. Baseline and ACTH-stimulated ratios in the genotype-proven adult patient were higher than the normal subjects or genotype-normal hirsute females.

View larger version (45K): [in this window] [in a new window]   Figure 6. Comparison of baseline and ACTH-stimulated ratios of DHEA to 4-A between the patients with proven/predictably deleterious mutant type II 3ß-HSD genes (, Male; •, female; S, salt-wasting patients; NS, nonsalt-wasting patients) and the patients with normal type II 3ß-HSD gene (, Male; , female). indicates two independent values of the same male patient. Please note that the scale above 30 differs between baseline and ACTH-stimulated ratios. For abbreviations and other details, see Fig. 2 legend.

Group comparison of hormonal profiles between the genotype-proven 3ß-HSD deficiency congenital adrenal hyperplasia patients and genotype-normal patients and between the genotype-proven patients and control subjects (Table 2).

Discussion

The past published hormonal criteria for diagnosing the nonclassic variant of 3ß-HSD deficiency congenital adrenal hyperplasia without any genotypic proof were based on ACTH-stimulated hormonal responses greater than 2 SD above the normal mean values for 5–17P and DHEA levels and ratios of 5–17P to F, 5–17P to 17-OHP, and DHEA to 4-A (12, 13, 14, 15, 16, 17, 18). Our updated hormonal and type II 3ß-HSD genotype findings from infancy to adulthood revealed that only ACTH-stimulated 5–17P levels and baseline and ACTH-stimulated ratios of 5–17P to F distinguished consistently and unequivocally the patients with genotype-proven 3ß-HSD deficiency from the patients with normal type II 3ß-HSD gene. ACTH-stimulated 5–17P levels and basal and ACTH-stimulated 5–17P to F ratios in the patients with genotype-proven 3ß-HSD deficiency were exceedingly higher than those in the patients with normal type II 3ß-HSD gene. This finding confirmed our preliminary observation reported earlier in young children with premature pubarche (31, 34), as well as the finding in a recent report by Mauri et al. (35). Baseline or random 5–17P levels in the genotype-proven 3ß-HSD deficiency patients were generally distinguishable from the genotype-normal patients except in one instance. Thus, ACTH-stimulated 5–17P levels and 5–17P to F ratios were the best indices to develop accurate hormonal criteria proven by genotypic study for 3ß-HSD deficiency congenital adrenal hyperplasia.

Although baseline and ACTH-stimulated ratios of 5–17P to 17-OHP in the genotype-proven 3ß-HSD deficiency patients as a group were statistically higher than the ratios of genotype-normal or control subjects, the ratios in the genotype-proven patients were indistinguishable or inconsistently distinguishable from the genotype-normal patients or control subjects. The DHEA level is expected to be elevated in patients with 3ß-HSD deficiency congenital adrenal hyperplasia. DHEA levels in the genotype-proven patients, however, were not consistently elevated from the levels of genotype-normal subjects or control subjects. Thus DHEA level in either baseline or ACTH-stimulated state from infancy to childhood was not a consistently useful index to define an accurate hormonal criterion for 3ß-HSD deficiency congenital adrenal hyperplasia. Likewise, the ratios of DHEA to 4-A in both the baseline and ACTH-stimulated state in the genotype-proven 3ß-HSD deficiency patients were not consistently distinguishable from either the genotype-normal patients or control subjects. Thus, the ratio of DHEA to 4-A was not useful to accurately and consistently detect 3ß-HSD deficiency congenital adrenal hyperplasia. The absence of elevation of 5–17P to 17-OHP and DHEA to 4-A ratios in the genotype-proven 3ß-HSD deficiency patients was predictably the result of an active peripheral conversion of 5 steroids by the efficient type 1 3ß-HSD activity in the extra-adrenal and extragonadal tissues (2, 3, 7, 8, 11).

The hormonal findings in the genotype-proven patients with 3ß-HSD deficiency helped define our newly proposed hormonal criteria for accurate prediction of 3ß-HSD deficiency congenital adrenal hyperplasia. Conversely, the hormonal findings in the patients with the normal type II 3ß-HSD genotype helped in defining the hormonal criteria for the exclusion of 3ß-HSD deficiency. These hormonal profiles in the genotype-normal patients have been incorrectly used to diagnose the mild, nonclassic variant of 3ß-HSD deficiency for the past two decades. We now propose new hormonal criteria using the consistently accurate hormonal indices, 5–17P levels and 5–17P to F ratios, proven by genotypic study, for inherited 3ß-HSD deficiency (Table 3); the criterion for ACTH-stimulated 5–17P level was at or greater than 378 nmol/liter in the neonates, at or greater than 165 nmol/liter in prepubarchal children, at or greater than 294 nmol/liter in children with premature pubarche, and at or greater than 289 nmol/liter in adults. The criterion of baseline 5–17P was at or greater than 84 nmol/liter in the neonatal infants and at or greater than 159 nmol/liter in adults; in children, greater than 29 nmol/liter was suggestive but not proven. The criterion of ACTH-stimulated ratio of 5–17P to F was at or greater than 434 in the neonatal infants, at or greater than 216 in preadrenarche children, at or greater than 363 in children with premature pubarche, and at or greater than 4010 in adults. The criterion of baseline ratio of 5–17P to F was at or greater than 461 in the neonatal infants, at or greater than 94 in Tanner I children, at or greater than 103 in children with premature pubarche, and at or greater than 1943 in adults (Table 3). These values are substantially higher than the past published hormonal criteria defined by greater than 2 SD above normal mean values (12, 13, 14, 15, 16, 17, 18).

In conclusion, substantial progress has been made in redefining hormonal criteria for accurate prediction of 3ß-HSD deficiency of varying spectrum by the study of hormonal phenotype and type II 3ß-HSD genotype in the patients discussed. The proposed new hormonal criteria predict more accurately 3ß-HSD deficiency than the previously published hormonal criteria (12, 13, 14, 15, 16, 17, 18) without genotypic proof. Our study also provides new hormonal criteria for genotype-normal children with premature pubarche and hirsute females for the exclusion of inherited 3ß-HSD deficiency. For patients whose hormonal profiles are between these proposed hormonal criteria for the genotype-proven 3ß-HSD deficiency and genotype-normal patients, it is necessary to investigate the type II 3ß-HSD genotype. Therefore, the proposed hormonal criteria may be further refined as a greater number of genotype-proven and genotype-normal patients from infancy to adulthood are identified.

Acknowledgments

We are grateful to Muhammad Jabar at the Hurley Medical Center (Flint, MI), Phyllis W. Speiser at Northshore University (Long Island, NY), Graeme Morgan at Prince of Wales Children’s Hospital (Randwick, Australia), David I. Schwartz at the University of Mississippi (Jackson, MS), and Robert P. Schwartz at the Wake Forest University School of Medicine (Winston-Salem, NC) for contributing to the patient samples for this study.

Footnotes

This work was supported by United States Public Health Service Grant R01 HD-36399 (to S.P.) and in part by a USPHS General Clinical Research Center grant to the University of Illinois at Chicago, College of Medicine.

Abbreviations: 4-A, Androstenedione; DHEA, dehydroepiandrosterone; F, cortisol; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; 17-OHP, 17-hydroxyprogesterone; 5–17P, 17-hydroxypregnenolone.

Received May 16, 2001.

Accepted March 1, 2002.

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