Metabolic Evidence for Impaired 17A-Hydroxylase Activity in A

European Journal of Endocrinology (2008) 158 385–392 ISSN 0804-4643

CLINICAL STUDY Metabolic evidence for impaired 17a-hydroxylase activity in a kindred bearing the E305G mutation for isolate 17,20-lyase activity Dov Tiosano1, Carlos Knopf2, Ilana Koren4, Nurit Levanon3, Michaela F Hartmann5, Ze’ev Hochberg1 and Stefan A Wudy5 1Pediatric Endocrinology, 2Metabolic Biochemistry Laboratory and 3Genetics Institute, Rambam Medical Center, Meyer Children’s Hospital, Technion- Israel Institute of Technology, 9602 Haifa, Israel, 4Endocrine Clinic, Clalit Health Services, 9602 Haifa, Israel and 5Steroid Research Unit, Center of Child and Adolescent Medicine, Justus Liebig University, Feulgenstr. 12, D-35392 Gießen, Germany (Correspondence should be addressed to D Tiosano; Email: [email protected])

Abstract Context: The CYP17A1 gene encodes many enzymatic reactions including 17a-hydroxylase and 17,20-lyase activities. Mutations that selectively ablate the 17,20-lyase activity, causing isolated 17,20-lyase deficiency, are exceedingly rare and may belong to the rarest of all disorders of steroidogenesis. We have previously reported an E305G mutation in the active site of CYP17A1 that apparently causes isolated 17,20-lyase deficiency. Expression studies suggested intact 17a-hydroxylase activity which was at odds with subnormal tetracosactrin stimulated cortisol in the patients. Objectives: To investigate the in vivo activity of the adrenal enzymes, we used the metabolomics approach with urinary steroid profiling by gas chromatography–mass spectrometry. Patients: Of the 11 subjects investigated, 6 patients in the kindred were found to be homozygous, 4 members were asymptomatic heterozygous, and 1 was homozygous for the wild-type allele. Results: In the homozygous patients for E305G, both serum and urinary steroids showed a severe lack of androgens (C19-steroids) pointing to the absence of 17,20-lyase activities. Furthermore, precursor/product ratios of urinary steroid metabolites characterizing 17a-hydroxylase activity showed variable decreases in 17a-hydroxylase activities. Conclusions: The results confirm the complete absence of 17,20-lyase activity in vivo, as in the in vitro expression studies. On the other hand, in vivo 17a-hydroxylase activity was partially impaired. Thus, the in vivo metabolic data seem to be more sensitive than the expression study and suggests that this mutation also impairs 17a-hydroxylase activity.

European Journal of Endocrinology 158 385–392

Introduction the residual mutant enzyme activity. Mutations in the steroid-binding domain mainly result in combined 17a- The CYP17 gene encodes the cytochrome P450 enzyme hydroxylase/lyase deficiency, whereas mutations in the that besides many other enzymatic reactions also redox partner domain have been found to result with catalyzes 17a-hydroxylase and 17,20-lyase activities. isolated lyase deficiency (5). Through its 17a-hydroxylase activity, the enzyme Clinically, males with isolated 17,20-lyase deficiency converts progesterone and pregnenolone to 17-hydroxy- have a female phenotype or ambiguous genitalia. progesterone and 17-hydroxypregnenolone with Biochemically, this enzyme defect is characterized by similar affinity. For this reaction, the enzyme requires low androgens and normal cortisol levels without an intact steroid-binding domain and P450 oxido- hypertension. reductase for electron transfer (1). For its lyase activity, In two members of this kindred, we have previously the human enzyme converts mainly 17-hydroxy- reported an E305G mutation in the active site of CYP17 pregnenolone to DHEA-S, whereas the conversion of that apparently causes isolated lyase deficiency (6) as 17-hydroxyprogesterone to androstenedione barely suggested by expression studies. However, this result is exists (2).Formaximallyaseactivity,theenzyme at odds with subnormal-stimulated cortisol during requires an interaction between the P450-oxido- tetracosactrin stimulation in some patients. We there- reductase in the redox partner-binding site and the fore used urinary steroid profiling by gas chromato- presence of cytochrome b5 (3, 4). The severity of the graphy–mass spectrometry (GC–MS) to characterize the phenotype and the clinical presentation are related to steroid metabolome of this unique kindred.

q 2008 Society of the European Journal of Endocrinology DOI: 10.1530/EJE-07-0712 Online version via www.eje-online.org

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Patients and methods had gynecomastia (Tanner stage B3) and he began shaving. Patients The third son of this nuclear family (IV12) was born after normal pregnancy and delivery with a birth weight The patients belonged to two families that are related by of 4500 g. He had micropenis (1!0.5 cm) perineal marital consanguinity; their pedigree is presented in hypospadias, chordeae, and a bifid scrotum with Fig. 1. The first family consisted of seven children, with testicles of 1 ml each. Treatment with three monthly three clinically affected boys and a girl (Table 1). injections of 25 mg i.m. testosterone depot doubled his The eldest son (IV6) was born after 38 weeks of penile length. He was operated for correction of normal pregnancy and delivery with a birth weight of hypospadias at the age of 11 months. 3700 g. On physical examination, he had micropenis ! All the daughters in this family have normal female of 1 0.5 cm with perineal hypospadias, chordeae, and genitalia. They were evaluated by tetracosactrin bifid scrotum, in which small testicles of 1 ml each were stimulation and urinary steroid profiling. The affected palpated. He was operated for hypospadias repair, until girl(IV10)wasfoundtohavelowtetracosactrin the age of nine when a pediatric endocrinologist saw stimulation-stimulated cortisol level. On physical him for the first time. At the age of ten, his height was examination at age ten, she had normal clitoris and 134.4 cm (50th percentile) and weight was 30.5 kg vaginal opening, she grew along the 25th percentile (75th percentile); the bone age was 9.5 years. He had a and her weight was on the 10th percentile. small penis (3 cm) with chordeae and scrotal testicles of The second family consisted of four boys and a girl. 4 ml each. At the age of 11, he developed gynecomastia One daughter died after delivery for an unknown cause. that reached Tanner stage B4 at the age of 14. At the Subject IV5 was referred during puberty for evaluation age of 15, treatment with i.m. testosterone depot was of micropenis and gynecomastia. On physical exami- begun and he started to shave at the age of 18. At the nation at the age of 14, he had gynecomastia (Tanner age of 19, his height was 179 cm (75th percentile), stage B4), normal testes (15 ml each), and a small weight was 66.5 kg (75th percentile), his genitalia (3.4 cm) phallus with sparse pubic hair. His history examination revealed a penis of length 7 cm and scrotal revealed an uncomplicated pregnancy and delivery. testicles of 20 ml. His endocrine evaluation is detailed in Severe hypospadias with chordeae, bifid scrotum, and Table 2. micropenis were noted at birth. The hypospadias was The second boy (IV7) was born after normal surgically repaired and the penile growth was treated pregnancy and delivery with birth weight of 3700 g. with three testosterone enanthate injections of He had micropenis without hypospadias or chordeae. A 25 mg/month during infancy. All his other brothers pediatric endocrinologist saw him first at the age of ten. had normal genitalia. His height was 133 cm (25th percentile) and weight In order to explore the suspected defect in his sister was 28.5 kg (50th percentile). Genitalia examination (IV2), a tetracosactrin stimulation was performed and revealed two mobile testicles of 2 ml each and a an abnormal response was detected at the age of 18 micropenis of 3 cm long. At the age of 14, he began (Table 2). Her history revealed normal pregnancy and treatment with i.m. testosterone depot. At the age of 16, delivery, normal growth, normal breast development, his height was 168 cm (25th percentile) and weight and irregular menses. On physical examination, normal was 66 kg (75th percentile), his scrotal testicles female genitalia with sparse pubic hair were observed. measured 20 ml and the penis length was 5 cm; he Ultrasonography showed a big cyst in each ovary of 6.5

Figure 1 The two nuclear families with affected children (S indicates that this family member’s DNA was analyzed). Subjects IV2, IV5, IV6, IV7, IV10, and IV12 were found to be homozygous and subjects IV8, IV9, III3, and III4 were found to be heterozygous whereas subject IV11 did not carry any mutant allele. Genotyping was not performed in IV1, IV3, and IV4. Subjects III1 and III2 are obligatory heterozygous. Patients IV2S* and IV 5S* were reported in Ref (6).

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Table 1 Clinical data of the children in the kindred.

Patient Year of birth Sex Birth weight (kg) Karyotype Genotype Clinical presentation at birth

IV 2 1985 F 46XX Homozygous Normal female genitalia IV 5 1984 M 46XY Homozygous Severe micropenis, perineal hypospadias, chordae, bifid scrotum, 1 ml testicles IV 6 1985 M 3.6 46XY Homozygous Severe micropenis, perineal hypospadias, chordae, bifid scrotum, 1 ml testicles IV 7 1988 M 3.7 46XY Homozygous Severe micropenis, perineal hypospadias, chordae, bifid scrotum, 1 ml testicles IV 8 1989 F 3.0 46XX Heterozygous Normal female genitalia IV 9 1991 F 3.6 46XX Heterozygous Normal female genitalia IV 10 1993 F 3.8 46XX Homozygous Normal female genitalia IV 11 1999 F 3.8 46XX WT Normal female genitalia IV 12 2000 M 4.5 46XY Homozygous Micropenis, without hypospadias, normal scrotum, with palpable testicles and 5.3 cm. None of the patients in the kindred had Serum steroids hypertension or hypokalemia. Serum cortisol, estradiol, and DHEA-S were measured by a solid phase chemiluminescent enzyme immunoassay Genotyping (CLEIA; DPC Los Angeles, CA, USA). The 11-deoxycortisol was measured by RIA Double Antibody (ICN Written informed consent was obtained from parents. Pharmaceuticals Inc., Costa Mesa, CA, USA). The Genomic DNA was extracted from peripheral leukocytes using standard protocol. Exon 5 of CYP17A1 was 17-hydroxyprogesterone was measured by RIA-coated amplified with Promega Go-Taq in a 25 ml reaction mix tubes (CIS Bio international, Bagnols/Ceze, France). according to the manufacturer’s protocol using Testosterone and progesterone were measured by CYP17A-F and CYP17A-R as primers (5-ATCTCTAG- competition principle electrochemiluminescent (ECLIA; TCAGGGACAGAAG-3 and 5-CAGGCCTAGTCTTCCTG- Elecsys 2010, F.Hoffmann-La Roche, Basel, Switzerland). CAC-3 respectively). The PCR parameters included 30 cycles of 30 s at 94 8C, 30 s at 60 8C, and extension 35 s Urinary steroids at 72 8C, giving a 410 bp PCR product. The presence of mutation E305G was sought by the elimination of an Urinary steroid profiles were determined from spot urine EcoR31I cleavage site, resulting in a single 410 bp specimens using quantitative data produced by GC–MS product, rather than the wild-type 240 and 170 bands analysis according to the method described elsewhere as shown in Fig. 2. (7). Free and conjugated steroids were extracted by solid

Table 2 Endocrine evaluation by tetracosactrin stimulation tests in ﬁve patients (basal and peak) and hCG test in two patients (peak).

60 min tetracosactrin- Normal tetracosactrin Basal Normal stimulation test stimulation test

Progesterone nmol/l 3.9–9.9 4.5–0.5 10.1–12.9 Corticosterone pmol/l 401 90–600 2022 1800–4500 Aldosterone pg/ml 57–111 40–300 Plasma renin activity ng/ml per h 0.4–1.8 !5 17 hydroxypregnenolone nmol/l 1.2–8 40–5 17 hydroxyprogesterone nmol/l 2.2–20.9 !15 1.9 –16 !30 11 Deoxycortisol nmol/l 8.1–8.4 !23 6.1–9.6 !23 Cortisol nmol/l 67–192 O90 200–383 O540 DHEA-S mmol/l 0.2–0.63 0.95 –11.3 Androstendione nmol/l !0.35 O0.35 !0.35 O0.35 Testosterone nmol/l !0.65 F 2.9 – 0.22 !0.65 M 0.98–39 Estrogen pmol/l 309 O73 F hCG test Age 11 years Age 12 years

Progesterone nmol/l 10.6 6.1 17-hydroxyprogesterone nmol/l 5.7 29.2 DHEA-S mmol/l 0.2 – Androstendione nmol/l !0.35 !0.35 Testosterone nmol/l 2.5 !0.5

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In each of these sites, one was homozygous for the wild type. The genotype/ mutations were directly responsible for a negligible phenotype relationship is summarized in Table 1. activity (!1–5%) of the 17,20-lyase in the in vitro expression studies. However, even in these cases where Serum steroids the mutation directly affects the17,20-lyase activity site, 17a-hydroxylase activity was affected with residual The adrenal steroids profile, summarized in Table 2, activities of 44.1–65, 13.6, and 65% respectively. demonstrated low C19-steroids. DHEA-S, androstene- The combination of low cortisol, testosterone, and dione, and testosterone were very low or undetectable in DHEA-S in our patients suggested a defect in CYP17, affected patients. The 17-hydroxysteroids were normal which is required for the conversion of C21-steroids to or high. The patients had low basal serum cortisol levels C19-steroids in both the adrenal glands and gonads, yet that ranged between 67 and 192 nmol/l, with a mean of 17-hydroxysteroids were normal, suggesting selective 130G45 nmol/l (normal O90 nmol/l). They also had impairment of lyase activity. insufficient response of cortisol to tetracosactrin Amplification and direct sequencing of all CYP17 stimulation, (250 mg Synacten, Novartis, France) with gene exons in our patients showed a GAG-to-GGG peak levels that ranged between 200 and 383 nmol/l, missense mutation at codon 305 in exon 5. This with a mean of 293G75 nmol/l (normal O540 nmol/l). mutation substitutes a glycine for the highly conserved Progesterone and estrogen levels were normal according glutamate at this position, which resides within the to their age. All patients had normal blood pressure, active site pocket. The Glu305 is found in a region of the normal aldosterone, and normal plasma renin activity. I-helix, corresponding to cytochrome P450 substrate Two patients underwent human chorionic gonado- recognition site (13); substitution of these residues may trophin (HCG) stimulation test (5000 units) at ages 11 uniquely influence the binding of various substrates. and 12: DHEA-S and androstenedione remained Kinetic studies that were conducted in yeast and undetectable; testosterone levels increased in one patient transfected human embryonic kidney (HEK)-293 cells from !0.69 to 2.5 nmol/l (normal O6.5 nmol/l) and expressing the mutant E305G demonstrated that in vitro did not respond in the other patient. lyase activity was seriously affected by this mutation; DHEA-S production was barely detectable, even when Urinary steroids cytochrome b5 was added to the incubation. However, the conversion of 17-hydroxyprogesterone to androste- A comparison between the urinary steroid metabolomes nedione was found to be slightly more rapid compared of a homozygote patient and a healthy subject as with wild-type CYP17. The conclusion from the assessed by GC–MS is given in Fig. 3. Our diagnostic expression studies was that the mutation E305G parameters for assessing enzymatic activity are based selectively impairs 17,20-lyase activity for DHEA-S on precursor/product ratios, as presented in Table 3. All synthesis while an increased capacity to form andros- affected subjects had highly elevated metabolic ratios of tenedione from 17-hydroxyprogesterone was found. corticosterone metabolites/C19-steroids (BKM/AnCEt) Assessment of the patients’ steroid metabolomes by reflecting impaired global activity of the enzymatic sys- urinary GC–MS steroid profiling allowed us to estimate tem 17a-hydroxylase/17,20-lyase. The highest values the relative activities of 17a-hydroxylase and 17,20- were found for the ratio of metabolites within the lyase. The 17,20-lyase activity was absent in the D5-pathway (P5T/(A5-3b,17b)) indicating no conver- homozygote patients as it has been found in the in vitro sion of 17-hydroxypregnenolone to DHEA-S. The 17a- expression studies. However, unlike the expression hydroxylase activity was impaired in all patients, studies, 17a-hydroxylase activity was found to be though not to the same extent as lyase activity. partially reduced. The discrepancy between the results Practically, all heterozygous relatives had pathological in the in vitro expression studies and those of the urine markers of 17,20-lyase activity, though not as expressed studies are a key finding of this study. Possible as in homozygote. Slightly impaired activity of the explanations remain speculative; they include different 17a-hydroxylase activity could only be found in two protein stability, different membrane phospholipid

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Figure 2 The presence of mutation E305G was sought by the elimination of an EcoR31I cleavage site, resulting in a single 410 bp product, rather than the wild-type 240 bp and 170 bp bands in nine subjects of the kindred. *represent non-speciﬁc bands.

Figure 3 GC–MS urinary steroid profiles (scan runs) in (A) a healthy male (20 years) and (B) a patient with isolated 17,20-lyase deficiency (23 years). In the healthy male’s profile, the presence of the cortisol metabolites (17-hydroxylated C21-steroids) tetrahydrocortisone (THE), tetrahydrocortisol (THF), and 5a-tetrahydrocortisol (aTHF) indicate normal 17a-hydroxylase activity. The presence of the androgen metabolites (C19-steroids) androsterone (An) and etiocholanolone (Et) reflects normal 17/20-lyase activity. In contrast, the patient’s profile is dominated by corticosterone metabolites such as tetrahydrocorticosterone (THB), 5a-tetrahydrocorticosterone (aTHB), and tetrahydro- 11-dehydrocorticosterone (THA) which, together with a reduction of cortisol metabolites, reflect impaired 17a-hydroxylase activity. The lack of 17-oxygenated C19-steroids (An and Et) proves the absence of 17,20-lyase activity. Internal standards are indicated by Roman numbers (I, 5a-androstane-3a,17a-diol; II, stigmasterol; and III, 5-cholestene-3b-ol-butyrate).

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Table 3 Urinary steroid metabolome of homozygous for E305G mutation and age and gender-matched controls (upper range limits in parentheses). Diagnostic ratios of precursor/product metabolites. Pathological values are in bold (22, 23).

Subject symbol IV 2 IV 5 IV 6 IV 7 IV 10 VI 12

Sex F M M M F M Age 19 years 23 years 18 years 15 years 10 years 3 years 10 months 11 months 10 months 5 months Global 17a-hydroxylase/lyase activity BKM/AnCEt 50.1 (0.3) 10.7 (0.5) 22.7 (0.3) 67.7 (0.6) 66.6 (1.7) 442.3 (2.6) 17a-hydroxylase activity D5-pathway P5D/P5T-17a 0.37 (0.29) 3.18 (0.11) 3.43 (0.29) 0.86 (0.46) 6.29 (1.21) 10.37 (0.93) D4-pathway PD/PT 0.54 (0.14) 0.83 (1.33) 0.64 (0.14) 0.53 (0.32) 19.13 (0.34) 1.25 (0.07) Global BKM/FKM 2.83 (0.17) 4.18 (0.30) 7.63 (0.17) 10.24 (0.29) 10.04 (0.43) 8.50 (0.34) 17,20-lyase activity D5-pathway P5T-17a/A5-3b,17b 262 (2.47) 65.47 (7.75) N (2.47) 182 (1.74) N (2.37) N (1.33) D4-pathway PT/AnCEt 5.43 (0.32) 0.64 (0.34) 1.62 (0.32) 2.5 (0.33) 2.0 (0.37) 4.9 (1.00) Global FKM/(AnCEt) 17.7 (1.88) 2.57 (1.85) 2.97 (1.88) 6.61 (2.36) 6.64 (4.04) 52.0 (7.75) 21-hydroxylase activity 11-O-PT/a-CL 0.02 (0.05) 0.01 (0.01) 0.05 (0.05) 0.02 (0.02) 0.09 (0.02) 0.04 (0.15)

B-M, sum of corticosterone metabolites; An, androsterone; Et, etiocolanolone; P5D, pregnenediol; P5T-17a, pregnenetriol; PD, pregnanediol; PT, pregnanetriol; F-M, cortisol-metabolites; A5-3b,17b, androstenediol, 11-O-PT, 11-oxopregnanetriol, a-CL, a-cortolone. composition and differences in P450 oxoreductase 21-hydroxylase deficiency are elevated in the urinary content. Urinary steroid profiles of heterozygotes for steroid profiles of patients with P450 oxidoreductase the E305G mutation inconsistently found slightly deficiency as well. However, our metabolic data show impaired 17a-hydroxylase and consistently impaired that 21-hydroxylation was not affected in our patients lyase activity. A clear differentiation between heterozy- and therefore 21-hydroxylase activity is not the reason gotes and unaffected relatives was not possible. for the blunted cortisol response to the tetracosactrin- The recently newly described form of congenital stimulation test. Other theoretical possibilities for the adrenal hyperplasia, P450 oxidoreductase deficiency blunted cortisol response are low 11b-HSD1 activity or (14), can be confused with 17-hydroxylase deficiency. low corticosteroid-binding globulin. In P450 oxidoreductase deficiency, the activity of not The results shed some light onto the role of the D5 only 17a-hydroxylase/lyase but also 21-hydroxylase steroid pathways in penile development. In these is impaired (15). Therefore, key metabolites of patients, the main production pathway of testosterone

Table 4 Urinary steroid metabolome of heterozygous for E305G mutation and one unaffected subject IV11, age and gender matched controls (upper range limits in parentheses). Diagnostic ratios of precursor/product metabolites. Pathological values are in bold. (22, 23)

Sex Mother Father F/IV 9 F/IV 8 F/IV 11

Age 12 years 3 months 11 years 1 month 4 years 7 months Global 17a-hydroxylase/lyase-activity B-M/AnCEt 2.66 (0.55) 0.99 (0.32) 0.66 (0.63) 0.27 (0.50) 4.28 (4.67) 17a-hydroxylase-activity D5-pathway P5D/P5T-17a 0.54 (0.11) 0.65 (0.29) 0.55 (0.56) 0.24 (1.02) 1.05 (1.36) D4-pathway PD/PT 0.61 (1.33) 0.18 (0.14) 0.36 (0.33) 0.11 (1.80) 0.26 (0.24) Global B-M/F-M 0.70 (0.30) 0.42 (0.17) 0.24 (0.21) 0.16 (0.24) 0.26 (0.20) 17,20-lyase-activity D5-pathway P5T-17a/A5-3b,17b 7.96 (7.75) 3.23 (2.47) 1.54 (5.40) 2.80 (1.71) 2.83 (1.14) D4-pathway PT/AnCEt 0.44 (0.34) 0.38 (0.32) 0.51 (0.24) 0.31 (0.14) 0.56 (0.92) Global F-M/(AnCEt) 3.79 (1.85) 2.36 (1.88) 2.81 (2.97) 1.70 (2.07) 16.78 (22.79) 21-hydroxase-activity 11-O-PT/a-CL 0.01 (0.01) 0.02 (0.05) 0.02 (0.02) 0.03 (0.02) 0.02 (0.13)

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The brisk metabolism of 5a-pregnan-3a,17a-diol-20-one to androsterone by CYP17 explains how, when 5a-reductases are expressed, Acknowledgements the testis generates C19-steroids androsterone and androstanediol from 17a-hydroxyprogesterone without The study was supported by a visiting scholarship from the intermediate of androstenedione and testosterone. It the European Society for Pediatric Endocrinology is possible that this pathway was also affected in our (ESPE). We thank Professor Richard Auchus for his patients in the same way that cortisol production was insight and helpful comments. D Tiosano and C Knopf blunted after tetracosactrin stimulation since both contributed equally and are to be regarded as first pathways’ origin is from 17a-hydroxyprogesterone. authors. The authors declare that there is no conflict of The ‘backdoor pathway’ is thought to be transient, interest that would prejudice its impartiality. being restricted to the fetus and the first months of life. All our subjects under investigation were older. In all our subjects investigated, the androsterone/etiocholanolone ratio was not elevated and thus not indicative of an active References back door pathway (16). The fact that our patients presented at birth with 1 Auchus RJ & Miller WL. Molecular modeling of human P450c17 micropenis and hypospadias and not with female genitalia (17a-hydroxylase/17,20-lyase): insights into reaction mechanisms and effects of mutations. Molecular Endocrinology indicates that they had sufficient testosterone and 1999 13 1169–1182. dihydrotestosterone levels during embryogenesis for the 2 Fluck CE, Miller WL & Auchus RJ. The 17, 20-lyase activity of development of a penis; however, the low levels of cytochrome p450c17 from human fetal testis favors the delta5 testosterone and dihydrotestosterone during the third steroidogenic pathway. Journal of Clinical Endocrinology and Metabolism 2003 88 3762–3766. trimester diminished penis growth and resulted in 3 Naffin-Olivos JL & Auchus RJ. Human cytochrome b5 requires micropenis with hypospadias. It turns out that androste- residues E48 and E49 to stimulate the 17,20-lyase activity of nedione formation from 17a-hydroxyprogesterone via the cytochrome P450c17. Biochemistry 2006 45 755–762. minor D4-steroid pathway and 5a-reductases is not 4 Auchus RJ, Lee TC & Miller WL. Cytochrome b5 augments the sufficient for complete formation of the male phenotype 17,20-lyase activity of human P450c17 without direct electron transfer. Journal of Biological Chemistry 1998 273 in humans. 3158–3165. One explanation for the testicles growth during 5 Geller DH, Auchus RJ & Miller WL. P450c17 mutations R347H puberty in the affected members IV5, IV6, and IV7 and R358Q selectively disrupt 17,20-lyase activity by disrupting can relate to their high follicle-stimulating hormone interactions with P450 oxidoreductase and cytochrome b5. Molecular Endocrinology 1999 13 167–175. (FSH) levels, stimulating the growth of testicular 6 Sherbet DP,Tiosano D, Kwist KM, Hochberg Z & Auchus RJ. CYP17 tubules (17). The gynecomastia in these patients can mutation E305G causes isolated 17,20-lyase deficiency by be due to the low levels of androgens that resolved selectively altering substrate binding. Journal of Biological Chemi- partially during testosterone replacement treatment. stry 2003 278 48563–48569. 7 Wudy SA & Hartmann MF. Gas chromatography–mass spec- Ultrasound studies revealed cystic ovaries in one trometry profiling of steroids in times of molecular biology. female. It confirms previous reports of the same in Hormone and Metabolic Research 2004 36 415–422. subjects with R347C mutation (18–21). Elevated FSH 8 Van Den Akker EL, Koper JW, Boehmer AL, Themmen AP, and luteinizing hormone levels, secondary to estrogen Verhoef-Post M, Timmerman MA, Otten BJ, Drop SL & De deficiency, might have caused ovarian stimulation. Jong FH. Differential inhibition of 17a-hydroxylase and 17,20- lyase activities by three novel missense CYP17 mutations The present study was initiated to understand the identified in patients with P450c17 deficiency. Journal of Clinical discrepancy between expression studies and the blunted Endocrinology and Metabolism 2002 87 5714–5721. serum cortisol response to tetracosactrin stimulation. To 9 Biason-Lauber A, Leiberman E & Zachmann M. A single amino study 17a-hydroxylase and lyase activities in vivo, acid substitution in the putative redox partner-binding site of P450c17 as cause of isolated 17,20-lyase deficiency. 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