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Urine Steroid Hormone Profile Analysis in Cytochrome P450 Oxidoreductase Deficiency: Implication for the Backdoor Pathway to Dihydrotestosterone

Departments of Laboratory Medicine (K.H.) and Pediatrics (T.H.), Keio University School of Medicine, Tokyo 160-8582, Japan; Department of Pediatrics, Dokkyo University School of Medicine Koshigaya Hospital (T.N.), Koshigaya 343-8555, Japan; Division of Endocrinology and Metabolism, Kanagawa Children’s Medical Center (M.A.), Yokohama 232-8555, Japan; Division of Endocrinology and Metabolism, National Center for Child Health and Development (R.H.), Tokyo 157-8535, Japan; Department of Pediatrics, Tohoku University School of Medicine (I.F.), Sendai 980-0878, Japan; Department of Pediatrics, Hokkaido University School of Medicine (T.T.) Sapporo 060-8638, Japan; Department of Pediatrics, Shirakawa Kousei General Hospital (R.T.), Shirakawa 961-0907, Japan; and Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development (M.F., T.O.), Tokyo 157-8535, Japan

Address all correspondence and requests for reprints to: Dr. T. Ogata, Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, 2-10-1 Ohkura, Setagaya, Tokyo 157-8535, Japan. E-mail: tomogata{at}nch.go.jp.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: Although the "backdoor" pathway to dihydrotestosterone has been postulated in the fetal-to-early-infantile period of patients with cytochrome P450 oxidoreductase deficiency (PORD), clinical data in support of this pathway remain limited.

Objective: The objective of this study was to obtain clinical evidence for the presence of the backdoor pathway in PORD.

Setting: This was a collaboration study between laboratories and hospitals.

Subjects: Twenty-two Japanese patients with molecularly confirmed PORD and 1763 control subjects participated in this study.

Intervention: Urine steroid profile analysis was performed by gas chromatography/mass spectrometry. In five patients and 776 control subjects, urine samples were obtained before 12 months of age.

Main Outcome Measure: The main outcome measure was identification of a urine steroid(s) indicating the backdoor pathway.

Results: In the PORD patients, pregnanediol, pregnanetriolone, and pregnanetriol were obviously elevated, and the urine steroid ratios reflecting CYP17A1 and CYP21A2 activities were decreased throughout the examined ages. Furthermore, etiocholanolone and 11-hydroxyandrosterone, which should originate almost exclusively from androstenedione in the conventional "frontdoor" pathway, were grossly normal or somewhat decreased since early infancy, whereas androsterone, which can be derived not only from androstenedione and dihydrotestosterone in the conventional frontdoor pathway but also from 5-pregnane-3,17-diol-20-one in the backdoor pathway, was increased during early infancy and remained grossly normal thereafter. Thus, the androsterone to etiocholanolone ratio was increased during early infancy and remained grossly normal thereafter. 5-Pregnane-3,17-diol-20-one was elevated throughout the examined ages.

Conclusions: The increased androsterone excretion during early infancy, as compared with the etiocholanolone and 11-hydroxyandrosterone excretions in the same period, suggests the presence of the backdoor pathway in PORD.

	Introduction

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CYTOCHROME P450 OXIDOREDUCTASE (POR) deficiency (PORD) is an autosomal recessive disorder caused by mutations in the gene encoding a flavoprotein that serves as an electron donor to all microsomal P450 enzymes such as CYP51A1 (lanosterol 14-demethylase) involved in cholesterol biosynthesis and CYP17A1 (17-hydroxylase and 17,20 lyase), CYP21A2 (21-hydroxylase), and CYP19A1 (aromatase) involved in steroidogenesis (1, 2, 3). Clinical phenotypes of this condition are highly variable, and include abnormal skeletal development referred to as Antley-Bixler syndrome and insufficient glucocorticoid production with increased 17-hydroxyprogesterone (17-OHP) in patients of both sexes, undermasculinization during the fetal and pubertal periods in male patients, and virilization during the fetal life and poor pubertal development without worsening of virilization in female patients, together with maternal virilization during pregnancy (1, 2, 3, 4, 5, 6, 7). Because the complete absence of POR activity is assumed to be lethal (1), some residual POR activity should be present in the patients. Indeed, all the reported patients have at least one missense mutation that is likely to preserve some residual activity (2, 3, 4, 5, 6, 7).

Therefore, PORD is a unique disorder that can lead to impaired fetal sex development in both sexes. In particular, whereas masculinization can be preserved normally in a relatively large fraction of male patients, virilization is exhibited by nearly all female patients (2, 3, 4, 5, 6, 7) except for one adult woman who presented with primary amenorrhea (2). The defective sex development is primarily consistent with impaired gonadal CYP17A1 activity resulting in compromised testosterone (T) production in genetic males and defective placental CYP19A1 activity leading to accumulation of placental T as well as androstenedione (⁴A) that is transferred into the fetal and maternal circulations in both genetic male and female patients (2, 3, 4). In this situation, genetic male patients have nearly normal to underdeveloped external genitalia because of the combined effects of reduced gonadal androgens and accumulated placental androgens, whereas genetic female patients have virilized genitalia because of the effects of placenta-derived androgens. However, the virilization in genetic female patients appears to be inexplicable by defective placental CYP19A1 function alone: 1) in CYP19A1 deficiency, only female infants with nearly complete loss of enzyme activity (<1%) have virilization (8); and 2) in PORD, the supply of dehydroepiandrosterone (DHEA), the precursor of ⁴A and T, from the fetus to the placenta is reduced (4, 9).

Thus, the backdoor pathway to dihydrotestosterone (DHT) has been postulated in PORD (3, 10). The backdoor pathway, which has been demonstrated in the tammar wallaby pouch young testis (11) and in the immature mouse testis (12), is driven by the accumulation of 17-OHP, leading to the sequential conversion of 17-OHP into DHT via a T-independent route. Although the backdoor pathway is mediated by CYP17A1 as well as 5-reductase (presumably type 1) and other enzymes, the substrate for CYP17A1 in the backdoor pathway, 5-pregnane-3,17-diol-20-one, is known to have a much higher affinity for CYP17A1 than 17-OHP (13). Thus, the backdoor pathway would function better than the conventional frontdoor pathway in PORD. In this study, we report urine steroid profile data obtained in PORD patients that suggest the presence of the backdoor pathway in this condition.

	Patients and Methods

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Patients

Twenty-two Japanese patients with PORD (11 genetic male patients with 46,XY and 11 genetic female patients with 46,XX) were studied (Table 1). Fifteen of the 22 patients have been reported previously (2, 4, 6, 7). In all the patients, molecular analysis indicated the diagnosis of PORD, and the patients were divided into two groups in terms of the mutation type: 1) those with two missense mutations, mostly homozygotes for R457H with some residual activity (2, 3) (cases 1–3 and cases 12–17) (group 1); and 2) those with one missense mutation, mostly R457H, and one non-missense mutation that probably has no or very little residual activity (cases 4–11 and cases 18–22) (group 2). Skeletal phenotype was much milder in group 1 than in group 2, masculinization in genetic male patients was somewhat better preserved in group 1 than in group 2, and virilization in genetic female patients and maternal virilization during pregnancy appeared to be similar between groups 1 and 2; case 21 only exhibited normal female genitalia. This study was approved by the institutional review board committee of each investigator, and the mutation analysis was performed at the National Research Institute for Child Health and Development after obtaining written informed consent from each patient or the parents.

Urine steroid hormone profile was determined by gas chromatography/mass spectrometry (14), using 24-h urine samples or random spot urine samples. The age at the time of urine sampling in each case is shown in Table 1. Analyzed urine steroids included pregnanediol (PD), pregnenetriol (PT5), pregnanetriolone (Ptl), pregnanetriol (PT), tetrahydrocorticosterone (THB), tetrahydro-11-dehydrocorticosterone (THA), tetrahydrocortisol (THF), tetrahydrocortisone (THE), DHEA metabolites (Ms) (the sum of DHEA, androstenediol, 16-hydroxy-DHEA, 16ß-hydroxy-DHEA, 16-oxoandrostenediol, and androstenetriol), 11-hydroxyandrosterone (11-OHAn), etiocholanolone, androsterone, 5-pregnane-3,17-diol-20-one, and 5ß-pregnane-3,17-diol-20-one (Fig. 1) (the formal steroid names are described in Ref. 14). This analysis was performed after obtaining written permission from each patient or the parents.

View larger version (32K): [in this window] [in a new window]   FIG. 1. Simplified schematic representation indicating the steroid metabolism pathway. CYP51A1 (lanosterol 14-demethylase), CYP17A1 (17-hydroxylase and 17,20 lyase), CYP21A2 (21-hydroxylase), and CYP19A1 (aromatase) are POR-dependent enzymes. The conventional frontdoor pathway is shown by the solid lines with arrows, and the alternative backdoor pathway is indicated by the thick dotted lines with arrows. In the backdoor pathway, the conversion of 17-OH progesterone into 5-pregnane-3,17-diol-20-one is mediated by 5-R (5-reductase) and 3-HSD (R) (reductive 3-hydroxysteroid dehydrogenase), that of 5-pregnane-3,17-diol-20-one into androsterone by CYP17A1 (17,20 lyase), and that of androsterone into DHT by 17ß-HSD (R) (reductive 17ß-hydroxysteroid dehydrogenase) and 3-HSD (O) (oxidative 3-hydroxysteroid dehydrogenase). 17-OH progesterone is also converted into 5ß-pregnane-3,17-diol-20-one by 5ß-reductase and 3-HSD (R). Of the urine steroids analyzed in this study, those measurable since birth are surrounded by solid squares, and those measurable after 6 months of age are surrounded by broken squares. Note that androsterone can be derived from both the frontdoor and the backdoor pathways.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

Representative results for the assessment of the conventional frontdoor pathway are shown in Fig. 2A (the data are expressed using a logarithm scale). Throughout the examined ages, PD, Ptl, and PT were obviously elevated, and PT5 tended to be increased in the PORD patients, whereas THF and THE, as well as aldosterone-M, remained grossly normal, and THB and THA tended to be elevated (data not shown). Furthermore, the (THF + THE) to (THB + THA) ratio reflecting CYP17A1 (17-hydroxylase) activity was reduced, whereas the PT to PD ratio remained within the normal range (data not shown). Similarly, the THE to PT ratio reflecting CYP21A2 activity, the DHEA-Ms to PT5 ratio reflecting CYP17A1 (17,20 lyase) activity for the ⁵-steroid, and the 11-OHAn to PT ratio reflecting CYP17A1 (17,20 lyase) activity for the ⁴-steroid were all decreased in the PORD patients.

View larger version (32K): [in this window] [in a new window]   FIG. 2. Representative results of urine steroid profile analysis. A, The results for the assessment of the conventional frontdoor pathway. B, The results for the assessment of the alternative backdoor pathway. Cases 1–11 are genetic male patients, and cases 12–22 are genetic female patients. The triangles indicate group 1 patients with two missense mutations, and the circles represent group 2 patients with one missense and one non-missense mutation. Of the 22 cases, cases 1, 6, 12, 13, and 17 have been studied before 12 months of age, and the remaining 17 cases have been studied after 1 yr of age when the backdoor pathway is unlikely to exist. The light blue circles and the pink circles show the data obtained from 854 control males and 909 control females, respectively. Urine samples from the control subjects have been obtained before 12 months of age in 425 males and 351 females, and after 1 yr of age in the remaining 429 males and 558 females. Note that all the data are expressed using a logarithm scale except for the androsterone to etiocholanolone ratio that is expressed using a linear scale. (Figure continues on next page.)

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
The urine steroid profile analysis revealed the combined CYP17A1 and CYP21A2 deficiency characteristic of PORD, in the conventional frontdoor pathway. Although several data may be confounding, they would not be inconsistent with PORD. For example, the normal THF and THE excretions would be explained as the consequence of steroidogenic adjustment to normalize the cortisol production under residual CYP17A1 and CYP21A2 activities and mild adrenocorticotropic hormone excess (2, 3, 4, 5, 6, 7). Furthermore, because CYP21A2 is the sole POR-dependent enzyme involved in the mineralocorticoid biosynthesis, this would explain why THB and THA excretions tended to be increased in the presence of normal THF and THE excretions. In addition, because 17-OHP is markedly elevated, this would account for the normal PT to PD ratio and the normal to low-normal 11-OHAn and etiocholanolone excretions in the presence of residual CYP17A1 activity. Therefore, the results provide further support for the urine steroid profile analysis as a highly useful method for the diagnosis of PORD (4, 6, 7, 9, 16, 17).

Notably, urine androsterone excretion was elevated during early infancy in the PORD patients, despite the impaired CYP17A1 activity in the frontdoor pathway. By contrast, DHEA-Ms and 11-OHAn derived from the frontdoor pathway were at a low or low-normal range throughout the examined ages. Furthermore, etiocholanolone remained grossly normal throughout the examined ages including early infancy, and the androsterone to etiocholanolone ratio was elevated during early infancy. In this regard, of androsterone and etiocholanolone, which are considered to be net androgen Ms derived from both androgen precursors and active androgens, androsterone can be derived not only from the conventional frontdoor pathway via ⁴A as well as DHT but also from the backdoor pathway via 5-pregnane-3,17-diol-20-one, whereas etiocholanolone as well as DHEA-Ms and 11-OHAn should originate almost exclusively from the frontdoor pathway (9, 10, 11, 12, 13) (Fig. 1). Consistent with this, etiocholanolone remained grossly normal since birth, despite the elevation of 5ß-pregnane-3,17-diol-20-one in our study. This suggests that etiocholanolone production in the backdoor route, i.e. the conversion of 5ß-pregnane-3,17-diol-20-one into etiocholanolone by CYP17A1, remains scanty, if any.

Thus, the transient increase in androsterone secretion during the first several months of life implies the operation of the backdoor pathway during this time period (10). Consistent with our results, Shackleton et al. (9) have identified the backdoor pathway-derived steroids, including androsterone, in the urine of the mother of a fetus with PORD. In this context, because androsterone is a potent androgen and an efficient DHT precursor, even a small excess would have clinical effects. Thus, even if the backdoor pathway is a quantitatively minor source of 19-carbon steroids relative to the frontdoor pathway, it would be relevant to the relatively well-preserved masculinization in genetic male patients and to the virilization in genetic female patients and in mothers of affected fetuses.

The backdoor pathway would primarily exist in the steroidogenic tissue(s) that transiently expresses several key enzymes including CYP17A1 and 5-reductase to convert the accumulated 17-OHP into DHT (10) (Fig. 1). Thus, fetal to infantile gonads and/or adrenals would be the candidate tissue in the human (10). For the gonads, however, although steroidogenic cells are well developed in the fetal testis, they are barely present in the fetal to prepubertal ovary (18), yet the urine steroid profiles were similar between the male and female patients in this study. This may argue against the gonads being the major site for the backdoor pathway. For the adrenals, the fetal zone may be involved in the transient activity of the backdoor pathway, because it disappears shortly after birth (19). In this context, because 3ß-hydroxysteroid dehydrogenase activity is reduced in the fetal zone (19), it is unlikely that the fetal zone alone can produce a sufficient amount of 17-OHP to drive the backdoor pathway. However, because the fetal zone has abundant CYP17A1 activity (19), this may facilitate the conversion of the permanent zone derived 17-OHP into DHT via the backdoor pathway. In addition, other tissues/organs such as the fetal liver may also be relevant to the backdoor pathway. Therefore, this matter awaits further studies.

The urine steroid profile appeared grossly similar between groups 1 and 2. Thus, urine steroid profile analysis, although highly useful for the diagnosis of PORD, is unlikely to serve to differentiate between the two groups of PORD. This would be consistent with the extent of defective genital development of the patients and maternal virilization during pregnancy being similar between the two groups, although masculinization in genetic male patients tended to be better preserved in group 1 than in group 2 (Table 1). Indeed, the urine steroid profile and the total amount of androgens derived from the conventional frontdoor pathway, the backdoor pathway, and the placenta could be similar between the two groups, primarily because of the complexity of steroidogenesis in PORD (Fig. 1). For example, the amount of 17-OHP is determined by the balance between synthesis mediated by POR-dependent CYP17A1 and degradation mediated by POR-dependent CYP17A1 and CYP21A2, and such enzymatic reactions would also depend on the amount of substrates and products as well as the residual enzyme activity. In this regard, because cholesterol production relevant to the development of skeletal lesion should be carried out in a simple manner (Fig. 1), this would explain why skeletal development was clearly different between groups 1 and 2. However, other genetic and/or environmental factors would also be relevant to the genital development, because case 21 with normal female genitalia had the similar steroid profile pattern.

Two points should be made with regard to the present study. First, urine steroid profile analysis was performed for random spot urine samples in most of the patients and for different types of urine samples in the control subjects, although random spot urine samples were primarily examined before 12 months of age in both the patients and the control subjects. This may have reduced the accuracy of the data, especially the excretion dosage data even after the correction with creatinine, because of the circadian rhythms of steroidogenesis. Second, urine steroid profile analysis in the early infantile period was carried out only in five cases, with repeated examinations in case 6, and most patients received the examinations at later age. Thus, further studies in the fetal-to-early-infantile period are necessary to provide more evidence for the presence of the backdoor pathway.

In summary, the results suggest that the backdoor pathway to DHT exists in the fetal-to-early-infantile period of PORD patients. Further studies will clarify the relevance of backdoor pathway to abnormal genital development in PORD as well as to virilization in 21-hydroxylase deficiency and other pathological conditions such as polycystic ovary syndrome.

View larger version (34K): [in this window] [in a new window]   FIG. 2B. Continued

	Footnotes

The authors have nothing to declare.

First Published Online April 11, 2006

Abbreviations: ⁴A, Androstenedione; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; M, metabolite; 11-OHAn, 11-hydroxyandrosterone; 17-OHP, 17-hydroxyprogesterone; PD, pregnanediol; POR, cytochrome P450 oxidoreductase; PORD, POR deficiency; PT, pregnanetriol; PT5, pregnenetriol; Ptl, pregnanetriolone; T, testosterone; THA, tetrahydro-11-dehydrocorticosterone; THB, tetrahydrocorticosterone; THE, tetrahydrocortisone; THF, tetrahydrocortisol.

Received November 10, 2005.

Accepted April 3, 2006.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Homma, K. Articles by Ogata, T. Search for Related Content PubMed PubMed Citation Articles by Homma, K. Articles by Ogata, T. Related Collections Adrenal and Hypertension Pediatric Endocrinology Female Endocrinology Male Endocrinology