Long-term outcome of partial P450 side-chain cleavage enzyme deficiency in three brothers: the importance of early diagnosis.

OBJECTIVE: CYP11A1 mutations cause P450 side-chain cleavage (scc) deficiency, a rare form of congenital adrenal hyperplasia with a wide clinical spectrum. We detail the phenotype and evolution in a male sibship identified by HaloPlex targeted capture array. FAMILY STUDY: The youngest of three brothers from a non-consanguineous Scottish family presented with hyperpigmentation at 3.7 years. Investigation showed grossly impaired glucocorticoid function with ACTH elevation, moderately impaired mineralocorticoid function, and normal external genitalia. The older brothers were found to be pigmented also, with glucocorticoid impairment but normal electrolytes. Linkage studies in 2002 showed that all three brothers had inherited the same critical regions of the maternal X chromosome suggesting an X-linked disorder, but analysis of NR0B1 (DAX-1, adrenal hypoplasia) and ABCD1 (adrenoleukodystrophy) were negative. In 2016, next-generation sequencing revealed compound heterozygosity for the rs6161 variant in CYP11A1 (c.940G>A, p.Glu314Lys), together with a severely disruptive frameshift mutation (c.790_802del, K264Lfs*5). The brothers were stable on hydrocortisone and fludrocortisone replacement, testicular volumes (15-20 mL), and serum testosterone levels (24.7, 33.3, and 27.2 nmol/L) were normal, but FSH (41.2 µ/L) was elevated in the proband. The latter had undergone left orchidectomy for suspected malignancy at the age of 25 years and was attending a fertility clinic for oligospermia. Initial histology was reported as showing nodular Leydig cell hyperplasia. However, histological review using CD56 staining confirmed testicular adrenal rest cell tumour (TART). CONCLUSION: This kinship with partial P450scc deficiency demonstrates the importance of precise diagnosis in primary adrenal insufficiency to ensure appropriate counselling and management, particularly of TART.

Normal male external genitalia do not rule out CYP11A1 deficiency.

Defects in the initial steps of steroidogenesis usually present with female external genitalia in both 46,XX and 46,XY. Hence, they are not often considered in the differential diagnosis of primary adrenal insufficiency children with normal male external genitalia. Here, we report a boy with normal male external genitalia who presented with hyperpigmentation since the age of 2 years but diagnosis was delayed till 11 years of age. Evaluation revealed low-serum cortisol with elevated adrenocorticotropic hormone and direct renin level confirming primary adrenal insufficiency. Clinical exome sequencing analysis revealed a homozygous c.1351C>T (p.R451W) mutation in exon 8 of the CYP11A1 gene which was confirmed on Sanger sequencing. Both parents were heterozygous for the variation. To conclude, we report the first case of CYP11A1 deficiency from India. The report reiterates the existence of non-classic CYP11A1 deficiency characterised by primary adrenal insufficiency and normal male external genitalia in 46,XY.

Heterozygous mutations in the cholesterol side-chain cleavage enzyme gene (CYP11A1) can cause transient adrenal insufficiency and life-threatening failure to thrive.

The first and rate-limited step of steroidogenesis in all steroidogenic tissues is the conversion of cholesterol to pregnenolone, catalysed by P450scc side-chain cleavage enzyme (CYP11A1 gene-SCC). SCC deficiency has been characterised as an autosomal recessive disorder, although it may also be inherited as an autosomal dominant trait in humans. Here, we describe a family of three members carrying the same novel heterozygous CYP11A1 mutation, a c.235G > A missense variant in exon 1: pVal79Ile. A 46 XY boy (P1) was presented at the age of 3 months with early onset adrenal insufficiency and life-threatening failure to thrive, with low adrenal androgens but normal external genitalia. Five years later, the parents had twin girls, one of whom (P2) presented acute adrenal crisis a few hours after birth. The father (P3), born at term, was reported as having suffered from failure to thrive during the neonatal period, though not his only male sibling. This report of severe early adrenal insufficiency caused by a heterozygous mutation of the CYP11A1 gene clearly demonstrates that SCC deficiency may be inherited as an autosomal dominant trait in humans.

Aetiological bases of 46,XY disorders of sex development in the Hong Kong Chinese population.

OBJECTIVE: Disorders of sex development are due to congenital defects in chromosomal, gonadal, or anatomical sex development. The objective of this study was to determine the aetiology of this group of disorders in the Hong Kong Chinese population. SETTING: Five public hospitals in Hong Kong. PATIENTS: Patients with 46,XY disorders of sex development under the care of paediatric endocrinologists between July 2009 and June 2011. MAIN OUTCOME MEASURES: Measurement of serum gonadotropins, adrenal and testicular hormones, and urinary steroid profiling. Mutational analysis of genes involved in sexual differentiation by direct DNA sequencing and multiplex ligation-dependent probe amplification. RESULTS: Overall, 64 patients were recruited for the study. Their age at presentation ranged from birth to 17 years. The majority presented with ambiguous external genitalia including micropenis and severe hypospadias. A few presented with delayed puberty and primary amenorrhea. Baseline and post-human chorionic gonadotropin-stimulated testosterone and dihydrotestosterone levels were not discriminatory in patients with or without AR gene mutations. Of the patients, 22 had a confirmed genetic disease, with 11 having 5α-reductase 2 deficiency, seven with androgen insensitivity syndrome, one each with cholesterol side-chain cleavage enzyme deficiency, Frasier syndrome, NR5A1-related sex reversal, and persistent Müllerian duct syndrome. CONCLUSIONS: Our findings suggest that 5α-reductase 2 deficiency and androgen insensitivity syndrome are possibly the two most common causes of 46,XY disorders of sex development in the Hong Kong Chinese population. Since hormonal findings can be unreliable, mutational analysis of the SRD5A2 and AR genes should be considered the first-line tests for these patients.

Pregnenolone functions in centriole cohesion during mitosis.

Cell division is controlled by a multitude of protein enzymes, but little is known about roles of metabolites in this mechanism. Here, we show that pregnenolone (P5), a steroid that is produced from cholesterol by the steroidogenic enzyme Cyp11a1, has an essential role in centriole cohesion during mitosis. During prometa-metaphase, P5 is accumulated around the spindle poles. Depletion of P5 induces multipolar spindles that result from premature centriole disengagement, which are rescued by ectopic introduction of P5, but not its downstream metabolites, into the cells. Premature centriole disengagement, induced by loss of P5, is not a result of precocious activation of separase, a key factor for the centriole disengagement in anaphase. Rather, P5 directly binds to the N-terminal coiled-coil domain of short-form of shugoshin 1 (sSgo1), a protector for centriole cohesion and recruits it to spindle poles in mitosis. Our results thus reveal a steroid-mediated centriole protection mechanism.

Varied clinical presentations of seven patients with mutations in CYP11A1 encoding the cholesterol side-chain cleavage enzyme, P450scc.

CONTEXT: The cholesterol side-chain cleavage enzyme P450scc, encoded by CYP11A1, converts cholesterol to pregnenolone to initiate steroidogenesis. P450scc deficiency can disrupt adrenal and gonadal steroidogenesis, resembling congenital lipoid adrenal hyperplasia clinically and hormonally; only 12 such patients have been reported previously. OBJECTIVE: We sought to expand clinical and genetic experience with P450scc deficiency. PATIENTS AND METHODS: We sequenced candidate genes in 7 children with adrenal insufficiency who lacked disordered sexual development. P450scc missense mutations were recreated in the F2 vector, which expresses the fusion protein P450scc-Ferredoxin Reductase-Ferredoxin. COS-1 cells were transfected, production of pregnenolone was assayed, and apparent kinetic parameters were calculated. Previously described P450scc mutants were assayed in parallel. RESULTS: Four of five Bedouin children in one kindred were compound heterozygotes for mutations c.694C>T (Arg232Stop) and c.644T>C (Phe215Ser). Single-nucleotide polymorphism analysis confirmed segregation of these mutations. The fifth kindred member and another Bedouin patient presented in infancy and were homozygous for Arg232Stop. A patient from Fiji presenting in infancy was homozygous for c.358T>C (Arg120Stop). All mutations are novel. As assayed in the F2 fusion protein, P450scc Phe215Ser retained 2.5% of wild-type activity; previously described mutants Leu141Trp and Ala269Val had 2.6% and 12% of wild-type activity, respectively, and Val415Glu and c.835delA lacked detectable activity. CONCLUSIONS: Although P450scc is required to produce placental progesterone required to maintain pregnancy, severe mutations in P450scc are compatible with term gestation; milder P450scc mutations may present later without disordered sexual development. Enlarged adrenals usually distinguish steroidogenic acute regulatory protein deficiency from P450scc deficiency, but only DNA sequencing is definitive.

Distinguishing deficiencies in the steroidogenic acute regulatory protein and the cholesterol side chain cleavage enzyme causing neonatal adrenal failure.

OBJECTIVES: To determine the genetic basis of disordered steroidogenesis in Kuwaiti siblings. STUDY DESIGN: Two siblings (46,XX and 46,XY) had normal female external genitalia and severe glucocorticoid and mineralocorticoid deficiency presenting in the first month of life. Abdominal ultrasonography showed normal size adrenal glands, suggesting cholesterol side chain cleavage enzyme (P450scc) deficiency. The CYP11A1 gene encoding P450scc and the STAR gene encoding the steroidogenic acute regulatory protein (StAR) were directly sequenced from leukocyte DNA. RESULTS: All exons and intron/exon boundaries of the CYP11A1 gene were normal; the STAR gene was homozygous for a novel 14-base deletion/frameshift in exon 4 (g.4643_4656del), so that no functional protein could be produced. Both parents and an unaffected sibling were heterozygous; zygosity was confirmed with a BsmF1 restriction fragment length polymorphism. CONCLUSIONS: Unlike most patients with StAR deficiency, our patients did not have the massive adrenal hyperplasia typical of congenital lipoid adrenal hyperplasia. The distinction between StAR and P450scc deficiency may require gene sequencing.

Genetic defects in pregnenolone synthesis.

The pregnenolone synthesis is the first step in the biosynthesis of all steroid hormones. The disruption of this step is known to result in congenital lipoid adrenal hyperplasia (CLAH), a most severe form of congenital adrenal hyperplasia. CLAH was first demonstrated to be caused by mutations in the STAR gene encoding steroidogenic acute regulatory protein (StAR). Now genetic and phenotypic variations have been elucidated in pregnenolone synthesis defects; mutations in the CYP11A1 gene encoding cholesterol side-chain cleavage enzyme (SCC) also cause disordered pregnenolone synthesis, and STAR mutations do not necessarily results in typical CLAH. To define these conditions precisely, pathophysiological diagnoses, such as StAR deficiency and SCC deficiency, are more likely to be appropriate than the histopathological diagnosis like CLAH.

P450 side-chain cleavage deficiency--a rare cause of congenital adrenal hyperplasia.

Mitochondrial cytochrome P450 side-chain cleavage enzyme (P450scc) converts cholesterol to pregnenolone and is the initiating enzyme for steroidogenesis. It is encoded by a single-copy gene on chromosome 15. For a long time it was thought that deficiency of P450scc was not compatible with life due to lack of progesterone for maintenance of pregnancy. However, recently a total of 8 patients with missense or even nonsense mutations of CYP11A1 have been described. Depending on the severity of the enzyme dysfunction, patients present with mild to severe early-onset adrenal failure. In 46, XY patients also a disorder of sex development is prevalent with hypospadias to complete female phenotype. P450scc deficiency is the differential diagnosis of congenital lipoid adrenal hyperplasia caused by mutations in the steroidogenic acute regulatory protein, however, in contrast to the latter, these patients do not have adrenal hyperplasia but small adrenals and gonads.

Disorders of androgen synthesis--from cholesterol to dehydroepiandrosterone.

Androgens and estrogens are primarily made from dehydroepiandrosterone (DHEA), which is made from cholesterol via four steps. First, cholesterol enters the mitochondria with the assistance of the steroidogenic acute regulatory protein (StAR). Mutations in the StAR gene cause congenital lipoid adrenal hyperplasia (lipoid CAH), a potentially lethal disease in which virtually no steroids are made. Lipoid CAH is common among Palestinian Arabs and people from eastern Arabia, and among Korean and Japanese people. Second, within the mitochondria, cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc; disorder of this enzyme is very rare, probably due to embryonic lethality. Third, pregnenolone undergoes 17alpha-hydroxylation by microsomal P450c17. 17alpha-Hydroxylase deficiency, manifesting as female sexual infantilism and hypertension, is rare except in Brazil. Finally, 17-OH pregnenolone is converted to DHEA by the 17,20 lyase activity of P450c17. The ratio of the 17,20 lyase to 17alpha-hydroxylase activity of P450c17 determines the ratio of C21 to C19 steroids produced. This ratio is regulated posttranslationally by at least three factors: the abundance of the electron-donating protein P450 oxidoreductase (POR), the presence of cytochrome b5 and the serine phosphorylation of P450c17. Mutations of POR are a new, recently described disorder manifesting as the Antley-Bixler skeletal dysplasia syndrome, and a form of polycystic ovary syndrome.

Steroid deficiency syndromes in mice with targeted disruption of Cyp11a1.

Steroid deficiencies are diseases affecting salt levels, sugar levels, and sexual differentiation. To study steroid deficiency in more detail, we used a gene-targeting technique to insert a neo gene into the first exon to disrupt Cyp11a1, the first gene in steroid biosynthetic pathways. Cyp11a1 null mice do not synthesize steroids. They die shortly after birth, but can be rescued by steroid injection. Due to the lack of feedback inhibition by glucocorticoid, their circulating ACTH levels are exceedingly high; this results in ectopic Cyp21 gene expression in the testis. Male Cyp11a1 null mice are feminized with female external genitalia and underdeveloped male accessory sex organs. Their testis, epididymis, and vas deferens are present, but undersized. In addition, their adrenals and gonads accumulate excessive amounts of lipid. The lack of steroid production, abnormal gene expression, and aberrant reproductive organ development resemble various steroid deficiency syndromes, making these mice good models for studies of steroid function and regulation.

Diagnosis and management of congenital adrenal hyperplasia.

Congenital adrenal hyperplasia is a family of inborn errors of steroidogenesis, each characterized by a specific enzyme deficiency that impairs cortisol production by the adrenal cortex, and can lead to sexual ambiguity in both genetic males and females. The enzymes most often affected are 21-hydroxylase, 11 beta-hydroxylase, and 3 beta-hydroxysteroid dehydrogenase, and less often, 17 alpha-hydroxylase/17, 20-lyase and cholesterol desmolase. Decreased production of cortisol results in increased pituitary secretion of adrenocorticotropic hormone. The elevated adrenocorticotropic hormone stimulates both the accumulation of precursor steroids in the impeded pathways and excessive steroid synthesis in other adrenal biosynthetic pathways unaffected by the enzyme deficiency. Correct identification of the enzyme affected is achieved by the observation of clinical syndromes reflecting distinct hormonal patterns, and it is measured quantitatively as low levels of cortisol and other adrenal steroids, as well as increased levels of steroids proximal to the blocked step. Many of the corresponding genes for the described enzymes have been isolated and characterized, and specific mutations causing many cases of congenital adrenal hyperplasia have been identified. These advances have important implications for early prenatal diagnosis and prenatal treatment.

Familial male pseudohermaphroditism.

Familial male pseudohermaphroditism (MPH) due to 17,20-desmolase deficiency is rare. Here we present two siblings with MPH possibly due to 17,20-desmolase deficiency. The first patient presented with unambiguous female external genitalia and hypergonadotrophic hypogonadism. Chromosomal analysis revealed 46 XY. Ultrasound evaluation of pelvis revealed gonads in the inguinal canal, and no uterus. These findings were confirmed on laparotomy. Histology revealed the gonads to be testes. The second patient had ambiguous genitalia (perineoscrotal hypospadias, bifid scrotum with palpable gonads) with a 46 XY chromosomal pattern. Both patients had high plasma 17-hydroxy progestrone (17 OHP), low normal dehydro epiandrosterone sulphate (DHEAS) and low plasma testosterone. Plasma testosterone and DHEAS showed no response to ACTH or HCG. These features are compatible with the diagnosis of 17,20-desmolase deficiency.

P450scc deficiency (congenital lipoid adrenal hyperplasia): first reported case in Thailand and literature review.

A male infant presented with hyponatremia, hyperkalemia, generalized skin hyperpigmentation, and female type external genitalia. These clinical findings were compatible with mineralocorticoid, glucocorticoid and androgen insufficiency. Serum cortisol, progesterone and testosterone levels were extremely low after ACTH stimulation test, suggestive of defect in all of the adrenal steroidogenesis. Computed tomography demonstrated enlarged adrenal glands. The diagnosis of P450scc deficiency or lipoid congenital adrenal hyperplasia was based on all these characteristics. Physiologic replacement therapy with hydrocortisone and 9 alpha-fluorocortisol were effective and the patient achieved normal growth. The clinical characteristics, differential diagnoses, and prenatal diagnosis are discussed and reviewed.

Defects in steroidogenic enzymes. Discrepancies between clinical steroid research and molecular biology results.

Molecular biology has clarified the understanding of steroidogenic enzyme genetics. Nevertheless, there are discrepancies between fundamental and clinical experience. (1) Why do patients with "pure" 17 alpha-hydroxylase or 17,20-desmolase deficiency exist, when one cytochrome regulates both steps? A case of interest is discussed, who had "pure" 17,20-desmolase deficiency until adolescence, but additional 17 alpha-hydroxylase deficiency thereafter. (2) In 11 beta-hydroxylase deficiency, it was puzzling to find 18-hydroxylated compounds, and, in isolated hypoaldosteronism, normal cortisol, since 11 beta- and 18-hydroxylation were thought to be regulated together. This has now been explained by differences in the fasciculata and glomerulosa. The occurrence of 11 beta-hydroxylase deficiency of 17-hydroxylated steroids only, however, remains enigmatic. (3) 3 beta-Hydroxysteroid dehydrogenase deficiency does not only seem to exist in classic (mutations of type II gene), but also in late-onset cases. In them, no molecular basis could be found. (4) Also, in cholesterol side-chain cleavage, there is an inequity: while evidently one cytochrome regulates 20- and 22-hydroxylation, pregnenolone is formed when 20 alpha OH-cholesterol, but not when cholesterol, is added to adrenal tissue of deficient patients. Other factors (promoters, fusion proteins, adrenodoxin, cAMP-dependent expression of genes, and/or proteases), or hormonal replacement in patients may be responsible for these discrepancies.

Congenital lipoid adrenal hyperplasia--genes for P450scc, side chain cleavage enzyme, are normal.

In the most severe form of congenital adrenal hyperplasia (CAH), termed lipoid CAH, both the adrenals and gonads fail to convert cholesterol to pregnenolone, so that no steroid hormones are made. Newborns have female external genitalia irrespective of karyotype, and suffer a severe salt-losing form of CAH. Previous studies have shown that adrenal or gonadal mitochondria from these patients also fail to convert cholesterol to pregnenolone in vitro, implicating a lesion in the single gene for P450scc, which is the sole enzyme converting cholesterol to pregnenolone. Two patients with XY karyotypes had female genitalia and unmeasurable steroids after stimulation with ACTH and hCG. ACTH stimulation tests of parents, obligate heterozygotes, showed normal stimulation of all precursor steroids. Southern blotting patterns of the P450scc gene were normal. Oligonucleotide-initiated enzymatic amplification (PCR) of all P450scc exons showed normal sequences on multiple amplifications and sequencing reactions, indicating normal P450scc genes. Northern blots of testicular RNA from a 6-month-old patient and from a control fetus showed normal P450scc mRNA, indicating a normal P450scc promoter. Reprobing of the blot with our cloned human cDNAs for adrenodoxin reductase and adrenodoxin showed that these electron transport cofactors used by P450scc were also normal. Similarly, probing with cDNAs for all three known factors involved in cholesterol transport to the mitochondria-sterol carrier protein 2, endozepine, and steroidogenesis activator peptide were also normal. These results suggest that the lesion in lipoid CAH is not in the P450scc system or in any known step upstream from P450scc.