Use of dissected paraffin block tissue as a source of mRNA for transcriptional profiling and biomarker identification: a review, with preliminary findings in adrenocortical carcinoma tissue.

Cancer tissue obtained by biopsy or at surgery is usually handled and stored as formalin-fixed, paraffin-embedded (FFPE) blocks. While primarily prepared for histological examination, this material represents a valuable resource that is stable and transportable, and which is ideal for examination in 3 dimensions. It has been used for DNA and protein analysis but the potential for transcriptional profiling has been little exploited: the quality of the extracted RNA is uncertain and there are few reports of its efficient use. We review the background and practicalities of this approach and describe a pilot study as a worked example in which RNA of good yield and quality could be consistently obtained from differentiated areas of adrenocortical carcinoma (ACC) tissue.

The broad phenotypic spectrum of 17α-hydroxylase/17,20-lyase (CYP17A1) deficiency: a case series.

CONTEXT: 17α-Hydroxylase/17,20-lyase deficiency (17OHD) caused by mutations in the CYP17A1 gene is a rare form of congenital adrenal hyperplasia typically characterised by cortisol deficiency, mineralocorticoid excess and sex steroid deficiency. OBJECTIVE: To examine the phenotypic spectrum of 17OHD by clinical and biochemical assessment and corresponding in silico and in vitro functional analysis. DESIGN: Case series. PATIENTS AND RESULTS: We assessed eight patients with 17OHD, including four with extreme 17OHD phenotypes: two siblings presented with failure to thrive in early infancy and two with isolated sex steroid deficiency and normal cortisol reserve. Diagnosis was established by mass spectrometry-based urinary steroid profiling and confirmed by genetic CYP17A1 analysis, revealing homozygous and compound heterozygous sequence variants. We found novel (p.Gly111Val, p.Ala398Glu, p.Ile371Thr) and previously described sequence variants (p.Pro409Leu, p.Arg347His, p.Gly436Arg, p.Phe53/54del, p.Tyr60IlefsLys88X). In vitro functional studies employing an overexpression system in HEK293 cells showed that 17,20-lyase activity was invariably decreased while mutant 17α-hydroxylase activity retained up to 14% of WT activity in the two patients with intact cortisol reserve. A ratio of urinary corticosterone over cortisol metabolites reflective of 17α-hydroxylase activity correlated well with clinical phenotype severity. CONCLUSION: Our findings illustrate the broad phenotypic spectrum of 17OHD. Isolated sex steroid deficiency with normal stimulated cortisol has not been reported before. Attenuation of 17α-hydroxylase activity is readily detected by urinary steroid profiling and predicts phenotype severity. SIGNIFICANCE STATEMENT: Here we report, supported by careful phenotyping, genotyping and functional analysis, a prismatic case series of patients with congenital adrenal hyperplasia due to 17α-hydroxylase (CYP17A1) deficiency (17OHD). These range in severity from the abolition of function, presenting in early infancy, and unusually mild with isolated sex steroid deficiency but normal ACTH-stimulated cortisol in adult patients. These findings will guide improved diagnostic detection of CYP17A1 deficiency.

The incidence of transient infantile pseudohypoaldosteronism in Ireland: A prospective study.

AIM: To review the clinical course, outcome and incidence of infantile salt wasting associated with urinary tract infection (UTI) and/or urinary tract malformation (UTM) over a two-year surveillance period on the island of Ireland. METHODS: A two-year (2013-14) prospective surveillance undertaken via the Irish and Ulster Paediatric Surveillance Units. Monthly prepaid postcards were circulated to consultant paediatricians (n = 260) at all paediatric units on the island of Ireland. Infants under one year of age presenting for the first time with hyponatraemia (Na < 130 mmol/L) and/or hyperkalaemia (K > 5.0 mmol/L) associated with urosepsis/UTM were reported. RESULTS: All 7 reported patients (6 male) had culture-proven UTI, and 5 (71%) also had an underlying UTM (one diagnosed antenatally). Four (57%) patients had a documented elevated serum aldosterone supporting secondary pseudohypoaldosteronism (PHA) as the underlying diagnosis. Data on aldosterone were not reported in the other 3 patients, but clinical features were suggestive of secondary PHA. The estimated incidence for the Irish population of transient PHA is 1 per 13,200 total live births per year. CONCLUSIONS: Salt wasting is a rare complication of UTI, especially if associated with underlying UTM. Boys appear to be at particular risk.

Posaconazole-Induced Hypertension Masquerading as Congenital Adrenal Hyperplasia in a Child with Cystic Fibrosis.

BACKGROUND: Deficiency of 11ß-hydroxylase is the second most common cause of congenital adrenal hyperplasia (CAH), presenting with hypertension, hypokalaemia, precocious puberty, and adrenal insufficiency. We report the case of a 6-year-old boy with cystic fibrosis (CF) found to have hypertension and cortisol insufficiency, which were initially suspected to be due to CAH, but were subsequently identified as being secondary to posaconazole therapy. Case Presentation. A 6-year-old boy with CF was noted to have developed hypertension after administration of two doses of Orkambi™ (ivacaftor/lumacaftor), which was subsequently discontinued, but the hypertension persisted. Further investigations, including echocardiogram, abdominal Doppler, thyroid function, and urinary catecholamine levels, were normal. A urine steroid profile analysis raised the possibility of CAH due to 11ß-hydroxylase deficiency, and a standard short synacthen test (SST) revealed suboptimal cortisol response. Clinically, there were no features of androgen excess. Detailed evaluation of the medical history revealed exposure to posaconazole for more than 2 months, and the hypertension had been noted to develop two weeks after the initiation of posaconazole. Hence, posaconazole was discontinued, following which the blood pressure, cortisol response to the SST, and urine steroid profile were normalized. CONCLUSION: Posaconazole can induce a clinical and biochemical picture similar to CAH due to 11ß-hydroxylase deficiency, which is reversible. It is prudent to monitor patients on posaconazole for cortisol insufficiency, hypertension, and electrolyte abnormalities.

Co-Existence of Congenital Adrenal Hyperplasia and Bartter Syndrome due to Maternal Uniparental Isodisomy of HSD3B2 and CLCNKB Mutations.

INTRODUCTION: We present a patient with co-existence of 3ß-hydroxysteroid dehydrogenase type 2 (HSD3B2) deficiency and Bartter syndrome, a unique dual combination of opposing pathologies that has not been reported previously in the literature. CASE: A female infant (46,XX) born at 34/40 weeks' gestation, weighing 2.67 kg (-1.54 standard deviation score) to non-consanguineous parents presented on day 4 of life with significant weight loss. Subsequent investigations revealed hyponatraemia, hypochloraemia, metabolic alkalosis, elevated 17-hydroxyprogesterone, ACTH, and renin. Urine steroid profile suggested HSD3B2 deficiency, which was confirmed by the identification of a homozygous HSD3B2 mutation. Due to the persistence of the hypochlo-raemic and hypokalemic alkalosis, an underlying renal tubulopathy was suspected. Sequence analysis of a targeted tubulopathy gene panel revealed a homozygous deletion in CLCNKB, consistent with Bartter syndrome type 3. The mother was found to be heterozygous for both mutations in -HSD3B2 and CLCNKB, and the father was negative for both. Single-nucleotide polymorphism microarray analysis confirmed 2 segments of homozygosity on chromosome 1 of maternal ancestry, encompassing both HSD3B2 and CLCKNB. DISCUSSION: Identification of a homozygous rare mutation in an offspring of non-consanguineous parents should raise suspicion of uniparental disomy, especially if the phenotype is unusual, potentially encompassing more than one disorder. The persistence of hypokalemic alkalosis, the biochemical fingerprint of hyperaldosteronism in a child with a form of CAH in which aldosterone production is severely impaired, challenges our current understanding of mineralocorticoid-mediated effects in the collecting duct.

Isolated 17, 20 Lyase Deficiency Secondary to a Novel CYB5A Variant: Comparison of Steroid Metabolomic Findings with Published Cases Provides Diagnostic Guidelines and Greater Insight into Its Biological Role.

OBJECTIVE: The objective of this study was to report CYB5A deficiency, to discuss the contribution of steroid metabolomics to diagnosis and interpretation, and to highlight the presence of testicular microlithiasis. METHODS: Two siblings with ambiguous genitalia at birth were later found to carry novel CYB5A variants, with resulting isolated 17, 20 lyase deficiency. We compared urine steroid data obtained between birth and adulthood with that from other cases. RESULTS: Neonatal urine steroid profiles show a relative increase of 16-hydroxylated pregnenolone metabolites. Thereafter, there are no distinguishing features until puberty, when sex steroid deficiency drives gonadotrophin production, resulting in marked increases of 17-hydroxyprogesterone metabolites derived from the gonads. This excess may be revealed pre-pubertally by gonadotrophin stimulation testing. Novel findings are first, a considerable capacity for DHEA synthesis in the neonatal period compared to childhood and adulthood, suggesting that DHEAS production is much less dependent on CYB5A at birth; second, no consistent change in "backdoor pathway" intermediates; third, side chain cleavage of cortisol is largely unaffected, supporting the existence of a different lyase not dependent on CYB5A; fourth, increased 17-hydroxyprogesterone metabolites and very low androgen metabolites are diagnostic post-pubertally. CONCLUSION: This is the fourth disease-causing variant in CYB5A in isolated 17, 20 lyase deficiency and the first associated with testicular microlithiasis. Establishing a biochemical diagnosis pre-pubertally should now be possible using urine steroid profiling, supported by synacthen and gonadotrophin stimulation testing. We recommend liquid chromatography-mass spectrometry/mass spectrometry rather than immunoassay for serum steroid analysis, early methaemoglobin measurement and surveillance should testicular microlithiasis be detected.

DIFFERENTIAL REGULATION OF 11ß-HYDROXYSTEROID DEHYDROGENASE TYPE 1 ACTIVITY IN PATIENTS WITH DIFFERING ETIOLOGIES OF HYPOPITUITARISM.

OBJECTIVE: Pituitary patients with different etiologies of hypopituitarism exhibit differing phenotypes, despite similar replacement therapy strategies. We hypothesized that differential regulation of the isoenzyme 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), which mediates the net autocrine conversion of cortisone to cortisol in adipose tissues and liver, may play a role. METHODS: We studied 11ß-HSD1 activity (using urine cortisol/cortisone metabolites ratio) in 36 hypopituitary patients with treated craniopharyngiomas, treated remitted Cushing disease, and treated nonfunctioning pituitary adenomas + prolactinomas on and off growth hormone (GH) replacement. RESULTS: 11ß-HSD1 activity was higher in subjects with craniopharyngioma both on and off GH, as evidenced by increased tetrahydrocortisol to tetrahydrocortisone metabolite ratios compared to other diagnostic groups, but there was no difference in body mass index, insulin levels, serum hormone measurements, or hydrocortisone dose between groups. CONCLUSION: Craniopharyngiomas are associated with enhanced 11ß-HSD1 activity compared to other diagnostic hypopituitary groups, and this may contribute to the adverse phenotypic and metabolic features seen in this condition. ABBREVIATIONS: BMI = body mass index; Em = cortisone metabolites; Fm = cortisol metabolites; GH = growth hormone; 11ß-HSD1 = 11ß-hydroxysteroid dehydrogenase type 1; IGF-1 = insulin-like growth factor 1; NFPA = nonfunctioning pituitary adenoma; THE = tetrahydrocortisone; THF = tetrahydrocortisol.

Modeling Congenital Adrenal Hyperplasia and Testing Interventions for Adrenal Insufficiency Using Donor-Specific Reprogrammed Cells.

Adrenal insufficiency is managed by hormone replacement therapy, which is far from optimal; the ability to generate functional steroidogenic cells would offer a unique opportunity for a curative approach to restoring the complex feedback regulation of the hypothalamic-pituitary-adrenal axis. Here, we generated human induced steroidogenic cells (hiSCs) from fibroblasts, blood-, and urine-derived cells through forced expression of steroidogenic factor-1 and activation of the PKA and LHRH pathways. hiSCs had ultrastructural features resembling steroid-secreting cells, expressed steroidogenic enzymes, and secreted steroid hormones in response to stimuli. hiSCs were viable when transplanted into the mouse kidney capsule and intra-adrenal. Importantly, the hypocortisolism of hiSCs derived from patients with adrenal insufficiency due to congenital adrenal hyperplasia was rescued by expressing the wild-type version of the defective disease-causing enzymes. Our study provides an effective tool with many potential applications for studying adrenal pathobiology in a personalized manner and opens venues for the development of precision therapies.

A 13-Steroid Serum Panel Based on LC-MS/MS: Use in Detection of Adrenocortical Carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare malignancy, with an annual incidence of 1 or 2 cases per million. Biochemical diagnosis is challenging because up to two-thirds of the carcinomas are biochemically silent, resulting from de facto enzyme deficiencies in steroid hormone biosynthesis. Urine steroid profiling by GC-MS is an effective diagnostic test for ACC because of its capacity to detect and quantify the increased metabolites of steroid pathway synthetic intermediates. Corresponding serum assays for most steroid pathway intermediates are usually unavailable because of low demand or lack of immunoassay specificity. Serum steroid analysis by LC-MS/MS is increasingly replacing immunoassay, in particular for steroids most subject to cross-reaction. METHODS: We developed an LC-MS/MS method for the measurement of serum androstenedione, corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone sulfate, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, and testosterone. Assay value in discriminating ACC from other adrenal lesions (phaeochromocytoma/paraganglioma, cortisol-producing adenoma, and lesions demonstrating no hormonal excess) was then investigated. RESULTS: In ACC cases, between 4 and 7 steroids were increased (median = 6), and in the non-ACC groups, up to 2 steroids were increased. 11-Deoxycortisol was markedly increased in all cases of ACC. All steroids except testosterone in males and corticosterone and cortisone in both sexes were of use in discriminating ACC from non-ACC adrenal lesions. CONCLUSIONS: Serum steroid paneling by LC-MS/MS is useful for diagnosing ACC by combining the measurement of steroid hormones and their precursors in a single analysis.

Kisspeptin Is a Novel Regulator of Human Fetal Adrenocortical Development and Function: A Finding With Important Implications for the Human Fetoplacental Unit.

Context: The human fetal adrenal (HFA) is an integral component of the fetoplacental unit and important for the maintenance of pregnancy. Low kisspeptin levels during pregnancy are associated with miscarriage, and kisspeptin and its receptor are expressed in the HFA. However, the role of kisspeptin in fetal adrenal function remains unknown. Objective: To determine the role of kisspeptin in the developing HFA. Design: Experiments using H295R and primary HFA cells as in vitro models of the fetal adrenal. Association of plasma kisspeptin levels with HFA size in a longitudinal clinical study. Setting: Academic research center and tertiary fetal medicine unit. Participants: Thirty-three healthy pregnant women were recruited at their 12-week routine antenatal ultrasound scan. Main Outcome Measures: The spatiotemporal expression of Kiss1R in the HFA. The production of dehydroepiandrosterone sulfate (DHEAS) from HFA cells after kisspeptin treatment, alone or in combination with adrenocorticotropic hormone or corticotropin-releasing hormone. Fetal adrenal volume (FAV) and kisspeptin levels at four antenatal visits (∼20, 28, 34, and 38 weeks' gestation). Results: Expression of Kiss1R was present in the HFA from 8 weeks after conception to term and was shown in the inner fetal zone. Kisspeptin significantly increased DHEAS production in H295R and second-trimester HFA cells. Serial measurements of kisspeptin confirmed a correlation with FAV growth in the second trimester, independent of sex or estimated fetal weight. Conclusions: Kisspeptin plays a key role in the regulation of the HFA and thus the fetoplacental unit, particularly in the second trimester of pregnancy.

Adrenocortical adenomas with regression and myelolipomatous changes: urinary steroid profiling supports a distinctive benign neoplasm.

BACKGROUND: Adrenocortical neoplasms are classically divided into adenomas (ACA) and carcinomas (ACC). Heterogeneous appearance and greater size are criteria to suggest malignancy, along with the urinary steroid profile (USP). The presence of regression and myelolipomatous changes in adenomas (ACA-RML) can contribute to confusion with ACC and its USP remains unknown. OBJECTIVE: To evaluate the features of ACA-RML in comparison with other adrenocortical neoplasms. METHODS: We selected consecutive ACA (11), ACA-RML (7) and ACC (13) cases for which USP analysis was performed before surgery and tissue was available for histological evaluation (King's College Hospital, 2005-2012). Cases were classified according to WHO and Armed Forces Institute of Pathology criteria. USPs were obtained by gas chromatography/mass spectrometry. Total excretion of individual steroids and indices (sums and ratios chosen to reflect steroid metabolic activity) were compared between ACA-RML, ACA and ACC. RESULTS: In comparison with ACA, tumours in ACA-RML were significantly larger (8·5 ± 2·4 vs 3·5 ± 1·0, P = 0·002), presented in older patients and showed relatively higher incidence in males. Mitotic figure counts were significantly lower (0·39 ± 0·04 vs 0·93 ± 0·11 in ACA, P = 0·001) and revealed higher frequency of apoptotic cells (100% vs 9% in ACA, P = 0·001). The USP of ACA-RML showed no diagnostic features of ACC. No differences from ACA were significant, but there was a tendency towards lower dehydroepiandrosterone DHA and DHA metabolites. CONCLUSIONS: ACA-RML reveals distinctive histological features and lack of USP markers of malignancy. More cases of this rare tumour may confirm differences from ACA in steroid excretion. It is important to recognize ACA-RML because its size and heterogeneous appearance raise the possibility of ACC.

Urinary steroid profiling.

Urinary steroid profiling is a long-established technique which has wide application in the study of disorders of human steroid biosynthesis and catabolism. The method divides into extraction of free and conjugated steroids, steroid conjugate hydrolysis, free steroid re-extraction, derivatization and analysis by GC or GC-MS. Several developments have led to better understanding of these processes and of ways to simplify and speed them up. The core method is described, together with some alternative options.

21-hydroxylase deficiency in the neonate - trends in steroid anabolism and catabolism during the first weeks of life.

Deficiency of 21-hydroxylase provides an in vivo model of intrauterine induction of enzymes participating in steroid anabolism and catabolism. Quantitative data for 93 steroid metabolites in urine from 111 patients and 7 controls (25 samples) were compared over the first six weeks of life. Net flux through the key anabolic enzymes was examined by comparison of the totals of steroids derived from the intermediates prior to and following each enzymatic step. Metabolic relationships were established on structural grounds and by Pearson correlation. The relative importance of each catabolic route was evaluated after summing metabolites classified according to their structure as fetal, neonatal, and classical (adult) type. Hierarchical cluster analysis identified the structure at C3-C5 as a key distinguishing feature of the major catabolic streams and demonstrated a split point in metabolic pattern in patients at 7 days. Changes with time in steroid metabolism, larger in patients than in controls, could be interpreted as reflecting increased cortisol demand post partum, the clinical onset of salt-wasting and a transition in catabolism from fetal to postnatal life. Faster involution of the fetal zone and pronounced enhancement of steroid production in zona fasciculata and zona glomerulosa were indicated in patients. Predominant at birth were 'planar' fetal-type 5α-reduced metabolites, adapted to placental excretion, which gave way to additionally hydroxylated neonatal-type metabolites, facilitating renal excretion. Classical metabolism made gains over the study period. Overproduction of steroids in utero in 21-hydroxylase deficiency would have induced fetal catabolic pathways dependent on 5α-reduction. A progressive increase of steroids likely to arise from 5α-reductase type 2 activity, again more distinct in disease, was observed. We demonstrate that the key intermediates in the hypothetical 'backdoor' pathway of androgen synthesis are part of a broader catabolic network and should not be examined in isolation.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 4. Characterization, using GC-MS and GC-MS/MS, of 11oxo-pregnanes and 11oxo-pregnenes.

In 21-hydroxylase deficiency, urinary metabolites of 21-deoxycortisol, mainly derived from its 11oxo form 21-deoxycortisone, are indicators of intra-adrenal overproduction of 17-hydroxyprogesterone. In affected neonates these metabolites are numerous and most have not been previously described. This work forms the concluding part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates. Cortisol metabolites found in untreated patients, similarly almost exclusively present in their 11oxo form in neonates, have been included for completeness. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of hitherto undescribed 11oxo-pregnane(enes). Few GC-MS features were associated with the presence of the non-derivatizeable 11oxo group in pregnane(ene)s. GC-MS/MS contributed only to the characterization of structures outside the C-ring, as described in the preceding parts of this study. Parallels were found between the metabolism of 21-deoxycortisone and cortisone. The major metabolic pathway was that of classical 3α,5ß-reduction with a prominent further hydroxylation, predominantly at C6. Oxidation of the 6-hydroxyl was also common. We conclude that further oxygenated metabolites of 21-deoxycortisone have potential as more reliable markers of 21-hydroxylase deficiency in the early neonatal period, because their levels are higher during that period of life than for the classical marker 11oxo-pregnanetriol.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 3. Characterization, using GC-MS and GC-MS/MS, of androstanes and androstenes.

Urine from neonates with 21-hydroxylase deficiency contains a large range of androstane(ene)s, many of which have not been previously described. We present their characterization as the third part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of hitherto undescribed androstane(ene)s. GC-MS/MS was pivotal for the structural characterization of 2-hydroxylated androstenediones but GC-MS was generally more informative for androstane(ene)s, in contrast to 17-hydroxylated pregnane(ene)s. Parallels were found between the GC-MS and GC-MS/MS characteristics of structurally similar androstenediones and progesterones without a substituent on the D-ring, but not with those of 17-hydroxylated progesterones. Assignment of 5α(ß) orientation, based on GC-MS characteristics, was possible for 11-oxo-androstanes. The major endogenous 3ß-hydroxy-5-enes in 21-hydroxylase deficiency did not differ from those in unaffected neonates. The key qualitative and quantitative differences encompassed 5α(ß)-androstanes and 3-oxo-androst-4-enes. Major positions of hydroxylation in these were C(2), C(6), C(11), C(16) and C(18). Additional oxo-groups were common at C(6), C(11) and C(16). We conclude that the presence of multiple further oxygenated metabolites of androstenedione in urine from neonates with 21-hydroxylase deficiency and their pattern indicate a predominance of the classical pathway of androgen synthesis and reflect an increased demand for clearance. The positions of oxygenation in androstane(ene)s are dependent on the configuration at C(3)-C(5).

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 2. Characterization, using GC-MS and GC-MS/MS, of pregnanes and pregnenes with an oxo- group on the A- or B-ring.

Urine from neonates with 21-hydroxylase deficiency contains a large range of metabolites of 17-hydroxyprogesterone, 21-deoxycortisol and androgens but few have been previously described. We present the second part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of the A- and B-rings containing an oxo group. Fragmentations indicating presence of 3-, 6-, and 7-oxo groups and also 1ß-, 2α-, 4ß-, and 6ß-hydroxyls are presented and discussed for the first time. Interpretation was aided by comparison with spectra of available relevant standards, of oxidation products of standards and urinary metabolites and of deuterated derivatives. Endogenous 1-enes and 2(3)-ene artifacts of non-hydrolyzed 3α-sulfates are also reported. D-ring and side chain structure was determined according to our previously published criteria. Likely metabolic relationships were also explored. We conclude that GC-MS combined with GC-MS/MS allows identification of the A- and B-ring structure of pregnane and pregnenes in the presence of an oxo group on one of these rings. Major oxygenations are 1ß, 15ß, 16α and 21-hydroxy and 6- and 7-oxo groups. Minor positions of hydroxylation are those at 2α, 4ß and 6ß. Three major metabolic streams exist in affected neonates in addition to the classical 3α-hydroxy-5ß-pregnane pathway, i.e. these of the 3-oxo-4-enes as well as 3α- and 3ß-hydroxy-5α-anes.

A missense mutation in the human cytochrome b5 gene causes 46,XY disorder of sex development due to true isolated 17,20 lyase deficiency.

CONTEXT: Isolated 17,20 lyase deficiency is commonly defined by apparently normal 17α-hydroxylase activity but severely reduced 17,20 lyase activity of the bifunctional enzyme cytochrome P450 (CYP) enzyme 17A1 (CYP17A1), resulting in sex steroid deficiency but normal glucocorticoid and mineralocorticoid reserve. Cytochrome b5 (CYB5A) is thought to selectively enhance 17,20 lyase activity by facilitating the allosteric interaction of CYP17A1 with its electron donor P450 oxidoreductase (POR). OBJECTIVE: We investigated a large consanguineous family including three siblings with 46,XY disorder of sex development (DSD) presenting with isolated 17,20 lyase deficiency. DESIGN: We investigated the clinical and biochemical phenotype, conducted genetic analyses, and functionally characterized the identified CYB5A mutation in cell-based CYP17A1 coexpression assays. RESULTS: All three siblings presented with 46,XY DSD, sex steroid deficiency, normal mineralocorticoids and glucocorticoids, and a urine steroid metabolome suggestive of isolated 17,20 lyase deficiency. CYP17A1 and POR sequences were normal, but we detected a homozygous CYB5A missense mutation (g.28,400A→T; p.H44L). Functional in vitro analysis revealed normal CYP17A1 17α-hydroxylase activity but severely impaired 17,20 lyase activity. In silico analysis suggested the disruption of CYB5A heme binding by p.H44L. CONCLUSION: We have identified the first human CYB5A missense mutation as the cause of isolated 17,20 lyase deficiency in three individuals with 46,XY DSD. Detailed review of previously reported cases with apparently isolated 17,20 lyase deficiency due to mutant CYP17A1 and POR reveals impaired 17α-hydroxylase activity as assessed by steroid metabolome analysis and short cosyntropin testing. This suggests that truly isolated 17,20 lyase deficiency is observed only in individuals with inactivating CYB5A mutations.