13.5.2 Congenital adrenal hyperplasia 2360 Nils P.

13.5.2 Congenital adrenal hyperplasia 2360 Nils P. Krone and Ieuan A. Hughes

SECTION 13  Endocrine disorders 2360 Isidori AM, et  al. (2006). The ectopic adrenocorticotropin syn- drome:  clinical features, diagnosis, management, and long-​term follow-​up. J Clin Endocrinol Metab, 91, 371–​7. Monaghan PJ, et al. (2011). Comparison of serum cortisol measure- ment by immunoassay and liquid chromatography-​tandem mass spectrometry in patients receiving the 11β-​hydroxylase inhibitor metyrapone. Ann Clin Biochem, 48(Pt 5), 441–​6. Nieman LK, et al. (2008). The diagnosis of Cushing’s syndrome: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 93, 1526–​40. Nieman LK, et al.; Endocrine Society (2015). Treatment of Cushing’s syndrome: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab, 100, 2807–​31. Sbiera S, et al. (2015). The new molecular landscape of Cushing’s dis- ease. Trends Endocrinol Metab, 26, 573–​83. Stratakis CA (2008). Cushing syndrome caused by adrenocortical tumors and hyperplasias (corticotropin-​independent Cushing syndrome). In: Flück CE, Miller WL (eds) Disorders of the human adrenal cortex. Endocr Dev. Basel, Karger, vol 13, pp. 117–​32. Theodoropoulou M, et al. (2015). Decoding the genetic basis of Cushing’s disease: USP8 in the spotlight. Eur J Endocrinol, 173, M73–​83. Tritos NA, Biller BMK (2019). Current management of Cushing’s dis- ease. J Intern Med, doi: 10.1111/joim.12975. Vaidya A, et al. (2019). The evaluation of incidentally discovered adrenal masses. Endocr Pract, 25, 178–92. Adrenal insufficiency Agha A, et al. (2006). The long-​term predictive accuracy of the short synacthen (corticotropin) stimulation test for assessment of the hypothalamic-​pituitary-​adrenal axis. J Clin Endocrinol Metab, 91, 43–​7. Arlt W, Allolio B (2003). Adrenal insufficiency. Lancet, 361, 1881–​93. Arlt W, et al. (1999). Dehydroepiandrosterone replacement in women with adrenal insufficiency. N Engl J Med, 341, 1013–​20. Boonen E, Bornstein SR, Van den Berghe G (2015). New insights into the controversy of adrenal function during critical illness. Lancet Diabetes Endocrinol, 3, 805–​15. Boonen E, Van den Berghe G (2016). Mechanisms in endocrin- ology: new concepts to further unravel adrenal insufficiency during critical illness. Eur J Endocrinol, 175, R1–​9. Bornstein SR, et al. (2016). Diagnosis and treatment of primary ad- renal insufficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab, 101, 364–​89. Erturk E, Jaffe CA, Barkan AL (1998). Evaluation of the integrity of the hypothalamo-​pituitary adrenal axis by insulin hypoglycaemia test.
J Clin Endocrinol Metab, 83, 2350–​4. Johannsson G, et al. (2012). Improved cortisol exposure-​time profile and outcome in patients with adrenal insufficiency: a prospective randomized trial of a novel hydrocortisone dual-​release formula- tion. J Clin Endocrinol Metab, 97, 473–​81. Marik PE, et  al. (2008). Recommendations for the diagnosis and management of corticosteroid insufficiency in critically ill adult patients: consensus statements from an international task force by the American College of Critical Care Medicine. Crit Care Med, 36, 1937–​49. Oelkers W (1996). Adrenal insufficiency. N Engl J Med, 335, 1206–​12. Rushworth RL, Torpy DJ, Falhammar H (2019). Adrenal crisis. N Engl J Med, 381, 852–61. Stewart PM, et  al. (1988). A rational approach for assessing the hypothalamo-​pituitary adrenal axis. Lancet, 1, 1208–​10. Teblick A, et al. (2019). Adrenal function and dysfunction in critically ill patients. Nat Rev Endocrinol, 15, 417–27. Woods CP, et al. (2015). Adrenal suppression in patients taking in- haled glucocorticoids is highly prevalent and management can be guided by morning cortisol. Eur J Endocrinol, 173, 633–​42. Mineralocorticoids Burton TJ, et al. (2012). Evaluation of the sensitivity and specificity of (11)C‐metomidate positron emission tomography (PET)-​CT for lateralizing aldosterone secretion by Conn’s adenomas. J Clin Endocrinol Metab, 97, 100–​9. Funder JW, et  al. (2016). The management of primary aldoster- onism:  case detection, diagnosis, and treatment:  an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab, 101, 1889–​916. Hundemer GL, et al. (2018). Cardiometabolic outcomes and mortality in medically treated primary aldosteronism: a retrospective cohort study. Lancet Diabetes Endocrinol, 6, 51–​9. Monticone S, et  al. (2018). Cardiovascular events and target organ damage in primary aldosteronism compared with essential hyper- tension:  a systematic review and meta-​analysis. Lancet Diabetes Endocrinol, 6, 41–​50. Monticone S, et al. (2018). Genetics in endocrinology: the expanding genetic horizon of primary aldosteronism. Eur J Endocrinol, 178, R101–​11. Rossi GP (2011). A comprehensive review of the clinical aspects of pri- mary aldosteronism. Nat Rev Endocrinol, 7, 485–​95. Rossi GP, et al. (2014). An expert consensus statement on use of ad- renal vein sampling for the subtyping of primary aldosteronism. Hypertension, 63, 151–​60. Adrenal tumours Chortis V, et  al. (2013). Mitotane therapy in adrenocortical cancer induces CYP3A4 and inhibits 5α-​reductase, explaining the need for personalized glucocorticoid and androgen replacement. J Clin Endocrinol Metab, 98, 161–​71. Fassnacht M, et al. (2016). Management of adrenal incidentalomas: European Society of Endocrinology clinical practice guideline in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol, 175, G1–​G34. Fassnacht M, Kroiss M, Allolio B (2013). Update in adrenocortical carcinoma. J Clin Endocrinol Metab, 98, 4551–​64. 13.5.2  Congenital adrenal hyperplasia Nils P. Krone and Ieuan A. Hughes ESSENTIALS Congenital adrenal hyperplasia results from enzymatic defects in the pathways of adrenal steroidogenesis, with over 90% of cases being due to 21-​hydroxylase deficiency caused by autosomal recessive mutations in the CYP21A2 gene.

13.5.2  Congenital adrenal hyperplasia 2361 Classical presentation—​this is in the neonatal period with virilized external genitalia of a female infant, with the phenotype tradition- ally subdivided according to the presence (75%) or absence of salt wasting, which in affected males is the sole manifestation, and can, if unrecognized, be life-​threatening. Delayed presentations can occur, manifesting in women as hirsutism, oligomenorrhoea, and infertility, and in men as infertility or testicular adrenal rest tumours. Biochemical diagnosis—​in the newborn this is made on the basis of an elevated plasma concentration of 17-​OH-​progesterone; the diagnosis of non​classic congenital adrenal hyperplasia often re- quires a Synacthen (ACTH1-​24) stimulation test, with confirmation by sequencing of the CYP21A2 gene for specific mutations. Management—​this requires glucocorticoid and mineralocorticoid replacement sufficient to replenish salt balance and control ACTH hyperstimulation without incurring steroid side effects. In the ado- lescent and young adult attention is focused on continuing optimal steroid replacement, with clinical endpoints being potential repro- ductive function rather than linear growth. Fertility in women is compromised by scarring effects of surgery following genitoplasty in childhood, inadequate adrenal suppression that leads to anovulation, and an overall reduced maternal desire in women with congenital adrenal hyperplasia. Men with congenital adrenal hyperplasia should be screened for testicular adrenal rest tumours after puberty, and semen preservation should be considered in young adulthood. Genetic testing of the index case, their partner, and fetus allows pre- vention of major congenital malformation in an affected female in- fant by maternal treatment with dexamethasone during pregnancy. Introduction Congenital adrenal hyperplasia (CAH) comprises a family of in- herited autosomal recessive disorders of adrenal steroidogenesis (Fig. 13.5.2.1), characterized by deficiency of cortisol and an accu- mulation of substrate precursors. A pathophysiological consequence of inadequate cortisol production is ACTH hypersecretion associ- ated with hyperplastic adrenal glands. Genital anomalies are not a universal feature of all forms of CAH, and the original adrenogenital syndrome nomenclature is now seldom used. The rate-​limiting step is the delivery of cholesterol from the outer to the inner mitochondrial membrane to act as substrate for P450 side-​chain cleavage enzyme (CYP11A1), a mixed-​function oxidase side-​chain cleavage enzyme. The intracellular transport of cholesterol is controlled by several proteins, including steroidogenic acute regu- latory protein (StAR). The synthesis of cortisol is predominantly con- trolled by ACTH, acting via a G-​protein-​coupled receptor activation of cAMP. Table 13.5.2.1 is a summary of the types of enzymes involved in adrenal steroidogenesis and the location of the genes that encode each enzyme. Deficiency of 21-​hydroxylase activity is the cause of CAH in more than 90% of cases; it occupies the bulk of this chapter. CAH resulting from 21-​hydroxylase deficiency Clinical presentation Deficiency of 21-​hydroxylase presents with a wide range of clinical manifestation representing a disease continuum that can manifest from birth to adult life (Table 13.5.2.2). The classical form pre- sents in infancy, with ambiguous genitalia of the female newborn. An affected female fetus becomes virilized in utero as a result of the effect of excess adrenal androgens, converted peripherally to testos- terone, masculinizing the external genital anlagen. CAH is the com- monest cause of ambiguous genitalia of the newborn, now classified as 46,XX DSD (disorder of sex development; see Chapter 13.7.3). Milder forms of virilization manifest either as isolated clitoromegaly or as isolated labial fusion. Clinically relevant impairment of aldos- terone biosynthesis can be found in at least 75% of cases; in affected males, salt loss is initially the sole manifestation, as the onset of vir- ilization in males is delayed beyond infancy. Left unrecognized, this can lead to a life-​threatening salt-​losing crisis. The male without clinically apparent salt loss may not manifest until the second year of life or beyond, with signs of precocious sexual development, rapid growth, and tall stature with acceleration of the bone age. The testes remain prepubertal in size (<4 ml in volume), which is a useful distinguishing feature from central precocious puberty associated with increased gonadotropin secretion due to activation of the hypothalamic-​pituitary-​gonadal axis. Biochemistry In the absence of 21-​hydroxylase, accumulation of 17OH-​ progesterone (17OHP) occurs due to reduced cortisol pro- duction, initiating a compensatory ACTH-​induced increased steroidogenesis. 17OHP is generally an inert steroid, apart from displaying some competitive inhibition of aldosterone binding to the mineralocorticoid receptor when produced in excessive amounts. However, 17OHP serves as the substrate for increased androgen production that leads to the profound virilization characteristics of the newborn female with CAH. The pathways to excess androgen production are threefold. First, some 17OHP is converted to andro- stenedione and thence to testosterone in the liver and most likely the adrenal cortex. Similarly, increased dehydroepiandrosterone (DHEA) production leads via androstenedione conversion to tes- tosterone synthesis by action of enzymes with 17-​β hydroxysteroid dehydrogenase activity. Finally, another pathway has been pro- posed as a source of the increased androgen production in CAH that involves 5α and 3α reduction of 17OH-​progesterone to 17OH-​ allopregnanolone, which—​via androsterone and androstanediol intermediary steps—​leads to production of DHT (dihydrotes- tosterone), the most potent androgen. This has been dubbed the ‘backdoor pathway’ of adrenal steroidogenesis and is referred to later in the section on P450 oxidoreductase (POR) deficiency. It has first been suggested that this alternative pathway to androgens in humans is only present during fetal life and up to 1 year after birth; however, recent evidence suggests that this pathway might serve as source of androgens in untreated patients with CAH beyond the first year of life. Non​classical forms of 21-​hydroxylase deficiency are also recog- nized, and have an incidence as high as 1 in 500 to 1 in 1000 among white populations. The non​classical form in females may present with early onset of pubic hair growth, or after puberty with signs of hirsutism and symptoms of menstrual dysfunction. It is important to exclude an adrenal tumour as the cause of late-​onset signs of viril- ization. In adult females the symptoms and signs are similar to those associated with polycystic ovary syndrome. Male infertility has also been ascribed to 21-​hydroxylase deficiency. Tumours arising from

SECTION 13  Endocrine disorders 2362 the testicular adrenal rests may also be a presenting feature (see ‘Reproductive function’, next). The characteristic biochemical hallmark is an elevated plasma con- centration of 17OHP, generally greater than 300 nmol/​litre (normal <10 nmol/​litre). In most cases, 17OHP concentrations in patients with SW-​CAH are higher than in non-​salt-​losing patients. A more specific marker for 21-​hydroxylase deficiency is the metabolite 21-​ deoxycortisol, which is generated by 11-​hydroxylation of 17OHP (Fig. 13.5.2.1). The analysis of a spot urine steroid profile is diag- nostic with increased metabolites of 17OHP and 21-​deoxycortisol. A  24-​h-​urine collection is generally not required to achieve the diagnosis of inborn errors of steroidogenesis. Plasma testosterone can reach adult male concentrations. The patient with salt loss has hyponatraemia, hyperkalaemia, and elevated plasma renin levels. The newborn with salt-​losing CAH may also have hypoglycaemia. The diagnosis of non​classic CAH often requires a Synacthen (ACTH1-​24) stimulation test. This might be necessary to distinguish it from premature adrenarche, which is characteristically accom- panied by elevated dehydroepiandrosterone sulphate (DHEAS) and androstenedione concentrations. It is also useful to test if pa- tients with non​classic CAH are glucocorticoid replete and re- quire glucocorticoid stress cover. Studies of women with signs of hyperandrogenism show only about 5% of hormone profiles consistent with non​classic CAH. Polycystic ovary syndrome is a Cholesterol Cholesterol outer mitochondrial membrane inner ADR/Adx POR POR POR POR POR H6PDH H6PDH POR PAPSS2 b5 b5 ADR/Adx ADR/Adx ADR/Adx ADR/Adx ADR/Adx ADR/Adx ADR/Adx StAR CYP11A1 CYP17A1 CYP17A1 CYP11B1 CYP11B1 CORTISOL TESTOSTERONE ALDOSTERONE CYP21A2 HSD3B2 17OH-Pregnenolone 17OH-Progesterone 11-deoxycortisol 21-deoxycortisol CYP17A1 Dehydroepi androsterone Androste- nedione 11OH-Andro- stenedione 11keto-Andro stenedione CYP11B1 CYP11B1 CYP17A1 DHEA-S SULT2A1 HSD3B2 HSD11B1 HSD11B1 HSD11B2 HSD11B2 11keto-Testosterone 11OH-Testosterone Androstenediol HSD17B HSD17B HSD17B HSD3B2 HSD17B1 HSD3B2 CYP21A2 CYP11B2 CYP11B2 CYP11B2 18OH-corticosterone Corticosterone 11-deoxycorticosterone Pregnenolone Pregnenediol Pregnenediol THDOC THA, THB 5α THA, 5α THB 18OH-THA THALDO THF, 5α THF THS Pregnanetriolone 11O-Etiocholanolone 11O-Androsterone 11OH-Etiocholanolone Androsterone Etiocholanolone Androsterone Etiocholanolone DHEA, 16αOH-DHEA 11OH-Androsterone Pregnanetriol 17OH-pregnanolone Pregnanetriol Progesterone Fig. 13.5.2.1  Pathways of adrenal steroid biosynthesis. Enzymes are marked with boxes. Mitochondrial P450 cytochrome (CYP) type I enzymes requiring electron transfer via adrenodoxin reductase (ADR) and adrenodoxin (Adx) CYP11A1, CYP11B1, CYP11B2, are marked with a labelled box ADR/​Adx. Microsomal CYP II enzymes receive electrons from P450 oxidoreductase, CYP17A1, CYP21A2, are marked by circled POR. The 17,20-​lyase reaction catalysed by CYP17A1 requires in addition to POR also cytochrome b5 indicated by a circled b5. Hexose-​6-​ phosphate dehydrogenase (H6PDH) is the cofactor to HSD11B1 and given as ellipse. Recent evidence suggests that androstenedione and testosterone can be further metabolized by the adrenal. Urinary steroid hormone metabolites are given in italics below the plasma hormones. The asterisk (*) indicates the pathognomonic 11-​hydroxylation of 17OHP to 21-​deoxycortisol in 21-​hydroxylase deficiency. The conversion of androstenedione to testosterone is catalysed by HSD17B3 in the gonad and aldo-​keto reductase (AKR) 1C3 (HSD17B5) in the adrenal. StAR, steroidogenic acute regulatory protein; CYP11A1, P450 side-​chain cleavage enzyme; HSD3B2, 3β-​hydroxysteroid dehydrogenase type 2; CYP17A1, 17α-​hydroxylase; CYP21A2, 21-​hydroxylase; CYP11B1, 11β-​hydroxylase; CYP11B2, aldosterone synthase; HSD17B, 17β-​ hydroxysteroid dehydrogenase facilitated by AKR1C3 and/​ or HSD17B3; SULT2A1, sulfotransferase 2A1; PAPPS2, 3’-​phosphoadenosine 5’-​ phosphosulfate synthase 2; PAPPS2, 3’-​phosphoadenosine 5’-​phosphosulfate synthase 2.

13.5.2  Congenital adrenal hyperplasia 2363 well-​recognized and more frequent cause of hirsutism and infer- tility, although ultrasonographic evidence of polycystic ovaries is common in CAH. The definitive diagnosis of idiopathic hirsutism as being the result of 21-​hydroxylase deficiency can be confirmed by sequencing the CYP21A2 gene for one of the mutations associated with the non​classic form of CAH. Management in infancy and childhood Medical For the infant in salt-​losing crisis, treatment with intravenous saline and hydrocortisone is required. Blood glucose levels need moni- toring for hypoglycaemia. Otherwise, the well infant with CAH re- quires glucocorticoid replacement with oral hydrocortisone. Since most affected infants are losing salt, mineralocorticoid replacement is recommended in all patients with classic CAH at least during infancy. The average daily hydrocortisone replacement dose is aligned to the daily physiological cortisol production, which is approximately 6–​8 mg/​m2 per day and enterohepatic cortisol circulation, together yielding an average hydrocortisone replacement dose of 8–​10 mg/​m2 per day. In CAH, where suppression of the HPA-​axis is required to control androgen excess, a higher dose of up to 15 mg/​m2 per day is recommended. Importantly, doses of more than 17 mg/​m2 per day during puberty have a significant deleterious effect on growth vel- ocity and final height. In infancy there is evidence to suggest that an- drogen excess is not associated with increased height velocity and that lower hydrocortisone replacement doses can therefore be used (8–​10 mg/​m2 per day). Due to the lack of circadian rhythm of cortisol secretion in the first months of life, the dose can be split into ideally four even doses. However, after about 6 months of age half to two-​ thirds of the daily hydrocortisone dose should be replaced as early as possible in the morning. The total daily dose is divided into three or four daily doses in view of the short half-​life of hydrocortisone. In all forms of CAH a degree and spectrum of aldosterone de- ficiency and salt loss is present. During infancy there is relative aldosterone resistance with the immature kidney tubular system being unable to adequately respond to aldosterone action to regu- late water and sodium homeostasis. Thus, in the neonatal period and in early infancy a higher dose of fludrocortisone is required. Fludrocortisone doses during the first year of life are commonly 150 µg/​m2 per day. Sodium supplementation is also required to replace the sodium losses. If hyponatraemia persists on a standard dose of Table 13.5.2.1  Genes and proteins involved in adrenal steroidogenesis Activity Protein and synonyms Site of protein Gene/​chromosome Cholesterol transport Steroidogenic acute regulatory protein StAR Mitochondrial surface STAR/​8p11.2 Cholesterol side-​chain cleavage Cytochrome P450, subfamily XIA, polypeptide 1 P450scc Mitochondrion CYP11A/​15q23-​q24 3β-​hydroxysteroid dehydrogenase/​ isomerase 3βHSD Endoplasmic reticulum HSD3B1, HSD3B2/​1p11–​p13 17α-​hydroxylase and 17,20-​lyase Cytochrome P450, family 17, subfamily A, polypeptide 1 P450c17 Endoplasmic reticulum CYP17A1/​10q24–​q25 Oxidoreductase Cytochrome P450, oxidoreductase P450 Endoplasmic reticulum (membrane bound) POR/​7q11.2 21-​hydroxylase Cytochrome P450, subfamily XXIA, polypeptide 2 P450c21 Endoplasmic reticulum CYP21A2, CYP21P/​6p21.3 11β-​hydroxylase Cytochrome P450, subfamily XIB, polypeptide 1 P450c11 Mitochondrion CYP11B1/​8q21–​q22 Aldosterone synthase Cytochrome P450, subfamily XIB, polypeptide 2 Mitochondrion CYP11B2/​8q21–​q22 Table 13.5.2.2  Clinical manifestations of 21-​hydroxylase deficiency from birth to adulthood Type Female Male Age Clinical signs Age Clinical signs Classic Neonatal Ambiguous genitalia Occasional male phenotype Salt loss in 75% Late neonatal Occasional pigmented scrotum Salt loss in 75% Unexpected death Early childhood Penile growth Pubic hair Rapid linear growth Increased musculature Adult Testicular adrenal rest tumour Oligospermia Non​classic Late infancy Clitoromegaly Late infancy Occasional delayed salt loss Childhood Pubic hair Rapid growth Childhood Pubic hair Tall stature Adolescence Abnormal menses Hirsutism Acne Adolescence Not known Adult Hirsutism Oligomenorrhoea Adult Infertility

SECTION 13  Endocrine disorders 2364 fludrocortisone (150 µg/​m2 per day), the dose of fludrocortisone should only be increased further after 10–​12 mmol/​kg/​day of so- dium supplements have failed to normalize serum sodium. If higher fludrocortisone doses are necessary, close monitoring of blood pressure and renin is indicated to avoid iatrogenic hypertension. Commonly, sodium supplementation can safely be discontinued from usually 6 to 8 months of age when salt intake is sufficient via food. This should be approached on an individualized basis taking growth, development, and compliance into account. Commonly, the fludrocortisone requirement decreases with age. The principle of longer-​term medical treatment for CAH in child- hood is to provide sufficient glucocorticoid and, if necessary, min- eralocorticoid replacement for adequate homeostasis, but not at the expense of steroid side effects such as growth suppression, obesity, arterial hypertension, and other metabolic effects. There is a ten- dency to overtreat during infancy in particular when hydrocorti- sone doses above 10 mg/​m2 per day are given. This is compounded by the need to increase the hydrocortisone dose during episodes of intercurrent infection with fever, which often occur at this time. This may result in the suppression of the infantile growth spurt as well as the impairment of later growth and the development of obesity, a problem more common in adolescent girls with CAH. Serial measurements of growth are thus the clinical mainstay of monitoring treatment for CAH in childhood. This is supplemented by calculating the bone age at intervals of about 1 to 2 years. The assessment is particularly valuable as an index of undertreatment, where the resulting increase in adrenal androgens leads to an ad- vanced bone age. If left unchecked, this will eventually lead to a sig- nificant reduction in adult height. The child with non​classic CAH presenting in mid childhood often already has a marked increase in bone age (often >3–​4 years in advance of chronological age). In those cases, final height will be considerably reduced. Furthermore, the advanced bone age is asso- ciated with an earlier puberty, thus further shortening the period for statural growth. Linear growth ceases when bony epiphyses fuse at the ends of the long bones. This is mediated by oestrogens in both sexes, based on studies of a rare male with a disrupted oestrogen receptor, and several reported males with aromatase deficiency. These individuals were excessively tall because of continued growth in young adulthood resulting from lack of closure of the growth plate. Oestrogen treatment was effective in fusing the epiphyses in aromatase-​deficient patients, but not in the man with an oestrogen-​ receptor defect. There is a wide age range for the onset of puberty in normal chil- dren, but in girls with CAH the onset of menarche at a normal age (12–​13 years) and subsequent regular menses is a reasonable index of adequate control. Hydrocortisone is the predominant glucocorticoid used in infancy and childhood and remains the glucocorticoid of first choice during later life. However, when growth is mostly complete, a longer-​acting glucocorticoid such as prednisolone is occasionally substituted when therapy adherence is an issue. Dexamethasone is in current practice rarely used. It has a potency 80-​ to 100-​fold greater than hydrocortisone in suppressing ACTH-​induced steroidogenesis and its use is reserved for specific treatments. For example, it can usefully restore regular menses in adolescent girls with poorly controlled CAH or shrink testicular adrenal rest tumours in males. However, the dose must be carefully titrated to avoid side effects such as weight gain, striae, metabolic effects, and hypertension. Effective doses can be as small as 0.1 to 0.3 mg daily; its long half-​life enables a single daily dose to be employed. CAH requires biochemical monitoring to complement clinical indices of control, not dissimilar to the use of serial blood glu- cose and glycosylated haemoglobin measurements in diabetes. Figure 13.5.2.1 indicates that the equivalent analytes in CAH are 17OHP, androstenedione, and testosterone. Several monitoring strategies have been proposed; however, no data on validating a su- perior marker regarding long-​term outcomes exists. 17OHP has a marked diurnal rhythm and shows stress-​induced increases in con- centrations. Consequently, random single measurements can be misleading. Some have suggested a daily profile made up of samples collected in the early morning, at midday, in the late afternoon, and at bedtime might be more appropriate and informative. Capillary blood spot and saliva assays of 17OHP enable families to undertake home sampling. However, 17OHP concentrations can be used to monitor overtreatment with glucocorticoids indicated by normal- ization to suppression of 17OHP concentrations. Plasma andro- stenedione and testosterone are both useful longer-​term markers of control, and an age-​ and sex-​related increase usually reflects prolonged undertreatment, which in due course leads to excessive linear growth and an advancing bone age. Testosterone measure- ments are not reflective of CAH control in males from puberty on- wards because of the predominance of testicular testosterone at this age. Recent reports suggest that the use of additional plasma ster- oids such as 11-​keto-​testosterone, specific for adrenal testosterone production, might be a specific biomarker of control in adult males. Urinary steroid analysis by specific chromatographic techniques is primarily for diagnosis, but is also used in some centres to monitor treatment. The adequacy of mineralocorticoid replacement is best assessed by renin measurement. Renin values are normally higher in infants and young children than in adults and should be kept in the upper normal range to slightly elevated concentrations to avoid overexposure to mineralocorticoids. Surgical The degree of virilization of the external genitalia in female infants born with CAH can vary from mild clitoromegaly and some labial fusion, to marked clitoromegaly, complete labial fusion resembling a scrotal sac without palpable gonads, and a urethral opening on the tip of the phallus. In this circumstance the infant may initially be wrongly designated male. However, the absence of palpable gonads in the ‘scrotal sac’ on routine newborn examination should alert to the need for further investigation. A Prader scoring system, as shown in Fig. 13.5.2.2, is used to denote the degree of clitoromegaly and the site of insertion of the vagina into the common urogenital sinus. The surgery that might be required is a reduction clitoroplasty and a vaginoplasty to enable separate urethral and vaginal open- ings to be exposed on the perineum. There has been a change in policy regarding the threshold for deciding that the clitoris is too large and needs reducing in size. The current practice is not to op- erate on a clitoris of Prader stage less than III. Some centres have adopted the practice delaying surgery until the patient can provide full informed consent. Decisions about early surgery have been in- fluenced by the results of studies in women with CAH who report dissatisfaction with sexual function, purported to be the result of clitoral surgery undertaken when they were infants. In the presence

13.5.2  Congenital adrenal hyperplasia 2365 of marked clitoromegaly (Prader stages III–​V), parents generally want surgery performed early to make the appearance consonant with the female sex of rearing, even though they understand this may have consequences for their daughter in adulthood. The number of clitoroplasties can be significantly reduced by assessing the situation after 12–​18 months of age under adequate hormone re- placement therapy, when the clitoris is often partly or fully covered by the labia majora. Technical details of clitoroplasty can be found in surgical textbooks, but it is vitally important to preserve as much of the highly innervated neurovascular bundle surrounding the clitoris as possible. It is questionable whether vaginoplasty is needed before puberty, but many surgeons also undertake this procedure early to take advantage of favourable tissue healing at a young age. A fur- ther examination under anaesthetic is usually required at puberty to assess the vaginal anatomy and the need for any revision surgery or the use of vaginal dilators. For those infants where a decision has been taken not to perform a clitoroplasty, medical treatment must be adequate to avoid further clitoral enlargement generated by elevated testosterone levels. Genetics of 21-​hydroxylase deficiency CAH due to 21-​hydroxylase deficiency is an autosomal recessive condition. The CYP21A2 gene (also known as CYP21) is closely linked to the highly polymorphic major HLA histocompatibility complex on chromosome 6p21.3. It is 98% homologous with a pseudogene, CYP21AP1 (also known as CYP21P), which has ac- cumulated several mutations that render it functionally inactive. The genes are in tandem repeat with neighbouring genes such as tenascin TNXA/​B, complement C4A/​B, and the serine/​threonine nuclear protein kinase RP. The CYP21A2 gene comprises 10 exons. Misalignment and unequal crossing over between sister chroma- tids during meiosis leads to large gene deletions and chimeric genes (previously called large gene conversions). These are, when homo- zygous, always associated with the severe, salt-​losing form of CAH. The allele frequency of large gene deletions and chimeric genes as a cause of 21-​hydroxylase deficiency is about 25–​30% and is highest in northern European populations. The majority of CYP21A2-​inactivating mutations have occurred by gene-​conversion events between CYP21A2 and its pseudo- gene (CYP21AP1) that are small-​scale in nature. These include the eight most common point mutations and an 8bp deletion in exon 3. In most populations, pseudogene derived mutations can be de- tected in similar frequencies. Novel or rare mutations account for about 3–​5% of detected mutations in large cohorts. To date, over 90 additional rare pseudogene-​independent mutations have been described (http://​www.hgmd.cf.ac.uk; http://​www.cypalleles.ki.se/​ cyp21.htm). Linked microsatellites are useful for prenatal diagnosis when the family genotype has previously been ascertained. About 65–​75% of patients with 21-​hydroxylase deficiency are compound heterozygous (e.g. they are affected, but carry different mutations on each chromosome). The clinical phenotype of CAH correlates well with the less severely mutated allele, and conse- quently with the allele encoding for the higher residual activity of 21-​hydroxylase. The mutations that cause more than 90% of cases of 21-​hydroxylase deficiency are shown in Fig. 13.5.2.3 in relation to the expected phenotype. A common mutation in classic 21-​hydroxylase deficiency affects mRNA splicing and results from a nucleotide base change in the second intron. A stretch of nucleotides that is normally spliced out is retained, so that the translational reading frame is altered and an inactive protein synthesized. Most patients with this muta- tion have the salt-​losing form of CAH, but some patients who are homozygous for this mutation are salt replete. Presumably, enough normally spliced mRNA is generated to produce some enzyme activity (sometimes referred to as leaky transcription). Other ex- amples leading to salt loss are shown in Fig. 13.5.2.3. In vitro func- tional assays of wild-​type and mutant CYP21A2 enzymes using progesterone and 17-​hydroxyprogesterone as substrates show total absence of enzyme activity for mutations leading to salt loss. A specific mutation not associated with salt wasting occurs in exon 4, changing isoleucine to asparagine (Ile172Asn). This mutation results in an enzyme with about 1 to 2% of normal activity, suffi- cient for adequate aldosterone production. The non​classic form of 21-​hydroxylase deficiency is associated with a mutant enzyme that displays 20 to 50% of normal activity in functional in vitro assays. An example is Val281Leu in exon 7; this single mutation accounts for most non​classical cases of CAH. Other common examples of non​classic alleles include Pro30Leu and Pro453Ser. The definitive diagnosis of non​classic CAH as a cause of premature adrenarche in a child or idiopathic hirsutism in a woman may not even be Normal I II III IV V Normal Fig. 13.5.2.2  Prader genital scores (reproduced from Helv Paediatrc Acta). The upper panel denotes the stages of virilization, resulting in a penile urethra and high insertion of the vagina to a common urogenital sinus by stage V. The lower panel depicts the degree of clitoral hypertrophy with stage V resembling a penis.

SECTION 13  Endocrine disorders 2366 confirmed by an ACTH1-​24 stimulation test, but only secured by CYP21A2 analysis. Prenatal diagnosis and treatment Prenatal treatment is effective to prevent severe genital virilization in 46,XX individuals with 21-​hydroxylase deficiency. However, it remains controversial as its safety, including metabolic and psycho-​ intellectual consequences, remains to be fully defined. Counselling regarding prenatal dexamethasone therapy should be carried out well before conception, involving an endocrinologist, fetal medicine specialist, and clinical geneticist. Chorionic villus sampling and molecular analysis of the CYP21A2 gene has enabled an earlier and more reliable diagnosis to be made. A recent report suggests that the fetal CAH status can be correctly determined by targeted massively parallel sequencing of DNA in maternal plasma, as early as 5 weeks 6 days of gestation. Dexamethasone is the chosen glucocorticoid as it crosses the pla- centa unmetabolized by the placental 11β-​hydroxysteroid enzyme and is not protein-​bound. Maternal dexamethasone treatment needs to start once pregnancy is confirmed and ideally before the sixth and no later than the eighth week of gestation to achieve significant benefit in preventing 46,XX DSD, as fetal adrenal steroidogenesis is established by 7 to 8 weeks of gestation. CYP21A2 genotyping of the index case, parents, and unaffected siblings should have been performed previously. DNA analysis is then more reliable and can be coupled with using additional linked microsatellite markers. The conventional starting dose is 20–​25 μg/​kg per day based on prepregnancy body weight, administered in three divided doses (total maximum dose 1.5 mg/​day). In the traditional protocols, treatment was only continued to term in the case of an affected female fetus. Thus, seven out of eight fetuses will be exposed unnecessarily to dexamethasone for about 6 weeks during early gestation. However, analysis of free fetal DNA in the maternal circulation enables Y chromosome material to be detected by specific probes (e.g. for SRY, the male sex-​determining gene) as early as 7 weeks of gestation if the fetus is male. Thus, dexametha- sone exposure would be avoided in male fetuses. Both approaches have raised significant ethical concerns due to the high number of unaffected fetuses unnecessarily treated. With the most recent de- velopment determining the CAH status from maternal plasma, it appears possible that only affected fetuses will be treated overcoming previous ethical problems. gene del/conversion ∆8bp E6 cluster p.Leu307PhefsX6 p.Gin318X p.Arg356Trp intron splice p.lle172Asn p.Pro30Leu p.Val281Leu p.Pro453Ser Nonclassic Simple virilizing Salt wasting Mutation group Phenotypic severity 21-hydroxylase deficiency CAH form Speiser et al., 1992 96% Positive predictive values Prader genital stages 100% 97% 91% 100% 85% 90% 96% 92% 84% 73% 74% 53% 85% 87% 63% 65% 100% 98% 100% Krone et al., 2000 Stikkelbroeck et al., 2003 Finkielstain et al., 2011 Marino et al., 2011 Speiser et al., 1992 II–IV (IV) III–V (IV) II–IV III–V (IV) III–IV II–V II–V (IV) II–V (III) II–V I–IV I–IV (III) 0–IV (0) 0–IV (0) 0 0–V Wedell et al., 1994 Jääskeläinen et al., 1997 Krone et al., 2000 Null A B C 0–1% 1–5% 20–30% 30–50% 0% Fig. 13.5.2.3  Genotype–​phenotype correlation for most common mutations causing 21-​hydroxylase deficiency. The genotype–​phenotype correlation in CAH due to 21-​hydroxylase deficiency is based on in vitro CYP21A2 activity. Mutation groups Null and A are associated with the salt wasting (SW) form of 21-​hydroxylase deficiency (21OHD), group B with the simple virilizing (SV) form, and group C with the non​classic (NC) form. Positive predictive values are calculated from the cited publications. The variability in the degree of virilization of the female external genitalia in the different mutation groups (grading according to Prader genital stages) is shown in the lower panel. Modal values are provided in brackets where possible. Intron 2 splice, refers to the c.293–​13A/​C>G mutation (other names: I2G, IVS2-​13A/​C>G), Δ8bp refers to the p.Gly110ValfsX21 mutation, E6 cluster to the p.Ile236Asn, p.Val237Glu, and p.Met239Leu mutation cluster at exon 6; and the p.Leu307PhefsX6 mutation is also described as insT exon 7.

13.5.2  Congenital adrenal hyperplasia 2367 Fetal adrenal suppression is monitored by serial measurement of maternal plasma or urinary oestriol concentrations. This steroid metabolite is formed as a result of placental aromatization of weak androgen substrates uniquely produced by the fetal adrenal gland. This monitoring also enables the dexamethasone dose to be lowered in later pregnancy. More direct evidence of adrenal suppression can be obtained by collecting amniotic fluid for measurement of 17OHP and testosterone. The outcome of prenatal treatment is satisfactory in most cases when treatment is started early and continues uninterrupted to term. Thus the external genitalia in affected females are completely normal, or so mildly affected that surgery is not required. There have been isolated reports of other abnormalities in dexamethasone-​exposed infants, but no cluster of anomalies that appear to be teratogenically specific to glucocorticoids. In animal studies, exposure to steroids has resulted in growth restriction, cleft palate, thymic hypoplasia, and features of metabolic syndrome, such as hypertension and im- paired glucose tolerance. The hippocampus was also smaller in some species. However, the sensitivity of rodents to prenatal dexametha- sone is significantly higher than in human and studies in primates used significantly higher prenatal dexamethasone doses than being employed in human prenatal dexamethasone treatment. Thus, a direct translation of results from animal experiments into the human context has to be done with some caution. Studies of cognitive function and verbal and visuospatial working memory, in a controlled study of children aged 7 to 17 years who had been prenatally exposed to dexamethasone, generally gave normal results, with perhaps poorer verbal working memory in the treated group. There is some evidence that gender role behaviour may be affected in males who were exposed to prenatal dexamethasone. A more recent report analysing behavioural and emotional problems as well as social anxiety did not reveal differences between prenatally dexamethasone-​exposed unaffected individuals and controls. Maternal side effects occur in 10% of treated pregnancies, com- prising excess weight gain, striae, and hypertension in some. It is clear that virilization of the external genitalia in a female fetus with CAH can largely be prevented if appropriate doses of dexamethasone are started soon enough in pregnancy. However, a number of fetuses will be exposed unnecessarily to such treatment, and the uncertain- ties about effects on behavioural development during childhood and metabolic effects in adulthood mandates that prenatal treatment of CAH should be considered experimental and conducted in the con- text of clinical trials. Neonatal screening for CAH It is possible to screen newborn infants for CAH by measurement of 17OHP in dried blood spots collected on the Guthrie card cur- rently used for other conditions such as phenylketonuria and con- genital hypothyroidism. Most centres use standard immunoassays, but improved positive predictive values can be achieved with the use of techniques such as tandem mass spectrometry. False-​positive results may occur from sampling on the day of birth in low birth weight and sick preterm infants, and because of assay interference by cross-​reacting steroids. It is essential that laboratories establish cut-​off values of 17OHP that are specific for birth weight and gesta- tional age. The classic form of CAH has an incidence of 1 in 10 000 to 1 in 15 000 live births, based on newborn screening. Non​classical CAH is much more common (at least 1 in 1000), but is often not detected by newborn 17OHP measurement. A false-​negative result can occasionally occur with the simple virilizing form of CAH, or if the mother has been treated with glucocorticoids during pregnancy. CAH forms other than 21-​hydroxylase deficiency can be detected if tandem mass spectrometry is used as a second-​tier method. The main benefit of newborn screening for CAH is the detection of affected males early enough to prevent a life-​threatening salt-​ losing adrenal crisis and to avoid the risk of delayed diagnosis pre- senting with precocious pseudopuberty. Retrospective case studies have shown a preponderance of females over males with CAH, suggesting an increased male mortality when screening is not em- ployed. Other benefits include the avoidance of incorrect sex assign- ment (the Prader V virilized female thought to be a boy at birth) and earlier treatment, which may improve later growth and pubertal de- velopment. Not all countries, including the United Kingdom, have yet incorporated CAH in the panoply of conditions included in the newborn blood-​spot screening programme. Longer-​term outcome in CAH CAH is a condition that extends across the lifespan, with manage- ment issues that vary according to development and maturation in adulthood (Fig. 13.5.2.4). It is during adolescence and young adult- hood that the longer-​term outcomes of treatment instigated during infancy, early childhood, and even before birth become manifest. Adult stature and medical management Management of CAH in childhood has primarily focused on growth, but increasing awareness exists regarding the prevention of long-​term comorbidities. Growth velocity is a dynamic biomarker of control, and is sensitive to any deviation in age-​appropriate gluco- corticoid replacement doses. Closure of the growth plate at a bone-​ age of around 16 to 17 years of age signals the end of linear growth, and final height adjustment. Most adults with CAH are shorter than predicted from mean parental height, but are generally within the normal population range for adult height. Two meta-​analyses from 2001 and 2010 showed similar impairment of final height in patients with 21-​hydroxylase deficiencies with mean final height standard deviation scores (SDS) of –​1.37 and –​1.38, which, when corrected for target height in a subgroup, resulted in a mean final height SDS of –​1.21 and –​1.03, respectively. Interestingly, the more recent meta-​analysis did not find a significant association with age at diagnosis, gender, type and dose of steroid, and age of onset of pu- berty; however, patients treated with mineralocorticoids had better height outcomes compared to non​users. Final height outcome was better in a single large clinic population based in Munich, Germany, where final height SDS corrected for target height was –​0.6 for fe- males and –​0.9 for males with the salt-​losing form of CAH. The infantile growth spurt during the first two years of life deserves spe- cial attention as this growth phase is characterized by the highest postnatal growth velocity. An impaired growth velocity during this time significantly impacts on final height outcome. Thus, the lowest optimal dose for glucocorticoid replacement and normalization of sex hormone needs to be found as early in life as possible. An add- itional decrement in final height is attributed to a reduction in total pubertal growth and the use of longer-​acting glucocorticoids such as prednisolone. There is also a relationship between total cumula- tive glucocorticoid dose and a reduction in bone mineral density, the impact being more pronounced during puberty. Paradoxically,

SECTION 13  Endocrine disorders 2368 some studies show that the milder form of CAH that presents in later childhood might have a worse outcome for final height because of the combination of advanced bone age and earlier onset of puberty. In such a situation, the addition of a long-​acting gonadotropin-​ releasing hormone analogue to delay puberty, and supplementation with growth hormone, can have a beneficial effect on final height. Glucocorticoid replacement is preferably provided as hydrocor- tisone in adulthood, but longer-​acting glucocorticoid preparations, such as prednisolone and dexamethasone, are more often used. There is no fixed dose; the amount has to be calculated according to general well-​being, the absence of steroid side effects, and biochem- ical monitoring using indices such as serum 17OHP, androstene- dione, and testosterone. No current oral glucocorticoid preparation can replicate the normal diurnal cortisol rhythm, characterized by a rise in early morning levels. Modified-​release formulations of hydro- cortisone that better mimic circadian cortisol profiles are becoming available and are likely to optimize future glucocorticoid replace- ment in CAH. The requirement for mineralocorticoid replacement is lower in adulthood, so lower relative doses of fludrocortisone are used. Plasma renin activity or renin (upper normal range) and blood pressure measurements are needed to adjust the dose to avoid hypertension. There is a tendency for a higher body mass index in CAH, which is related to glucocorticoid dose. This can be associated with frank obesity, particularly occurring in the female during adolescence. At this time, control of CAH (as indicated by regularity of menses and measurements of 17OHP and testosterone) may be inad- equate, yet increasing the glucocorticoid dose merely compounds the weight problem. This is also associated with insulin resistance, which perpetuates the irregular menses and anovulation. A problem of compliance may be the explanation, but medical manipulation with longer-​acting steroids, the combined oral contraceptive pill, gonadotropin-​releasing hormone analogues, or antiandrogens can all be to no avail in restoring control towards regular menses, ovu- lation, and reduced hyperandrogenism. Bilateral adrenalectomy is sometimes undertaken in these circumstances to good effect redu- cing the adrenal hyperandrogenism; however, this procedure leaves the patient completely adrenal insufficient increasing the risk of ad- renal crisis. In the last decade, several studies explored the health status in adults with CAH. Prevalent features were obesity, hypercholester- olaemia, insulin resistance, osteopenia, and reduced quality of life and more recently the finding of increased cardiovascular and meta- bolic morbidity. Some studies identified a significant shortfall in the expected numbers of patients who should be attending specialized adult services for CAH, in stark contrast to facilities provided for children with this condition. Furthermore, individuals with 21-​ hydroxylase deficiency might be at increased risk to suffer from psychiatric disorders emphasizing the requirement for a multidis- ciplinary approach for all age groups. Prenatal

• Prevention of virilization balancing potential side effects
• Early diagnosis
• Gender assignment
• Surgical management
• Growth and development
• Prevention of
• hypoglycaemia
• adrenal crisis Psychology parents Psychology patients Primary/ secondary prevention of comorbidities - When/ if to monitor? Biochemical monitoring Surgery when, how, and in whom? Dexamethansone
• Hydrocortisone
• Hydrocortisone
• Hydrocortisone
• Long-acting glucocorticoids
• NaCI
• GnRH agonists
• Fludrocortisone
• Fludrocortisone
• Fludrocortisone
• Oral contraceptives with/or without antiandrogens
• Fertility drugs
• Antiandrogens
• Growth hormone
• Aromatase inhibitors YES/NO? Outcome?
• Growth and development -Androgen excess -Adrenal rest tumours -Fertility (female/male)
• Psychosexual issues
• Obesity
• Insulin resistance
• Cardiovascular morbidity
• Osteoporosis
• Quality of life
• Puberty
• Psychosexual issues
• Forerunners of metabolic risk Birth to childhood Childhood - 16 years

16 years to adulthood Fig. 13.5.2.4  CAH is a disorder that extends across the lifespan, with management issues that vary according to the development and maturation in adulthood. It is during adolescence and young adulthood that the longer-​term outcomes of treatment instigated during infancy, early childhood, and even before birth become manifest. It is now widely accepted that psychological support plays an important role, but is often not provided due to lack of resources. Due to a current lack of evidence, variable strategies to screen for comorbidities and monitor biochemical parameters exist. Prenatal dexamethasone treatment, surgery, and the use of additional drugs is controversially discussed.

13.5.2  Congenital adrenal hyperplasia 2369 Reproductive function Female The overall fertility rate is reduced in women with CAH. However, pregnancy rates for women with classical CAH trying to conceive appear to be normal. A number of factors appear to contribute to impaired fertility; these include vaginal stenosis and unsatisfactory sexual intercourse, ovulatory dysfunction from inadequate adrenal suppression, elevated progesterone levels acting like a contracep- tive mini-​pill, and a lower desire of women with CAH to become mothers. Studies of clitoral sensation and measures of sexual func- tion in adult women with CAH show impaired genital sensitivity and difficulties in sexual function (vaginal penetration and inter- course frequency) in those who had feminizing genitoplasty, com- pared with the minority of CAH women who did not have surgery and with non-​CAH controls. Elevated progesterone levels during the follicular phase of the cycle are associated either with anovula- tory cycles or a thin endometrium that is not receptive to blastocyst implantation. It is important to maintain androgen concentrations within the age-​related range for females throughout childhood and adolescence, as permanent effects such as voice lowering can be a feature in adulthood. Studies of psychosexual issues in women with CAH indicate overall satisfaction with their gender assignment, irrespective of the degree of prenatal masculinization. However, the rates of bisexual and homosexual orientation are increased, even in the milder forms of CAH. Women with CAH are less likely to have partners, are de- layed in their sexual debut, and have decreased frequency of sexual intercourse. These features are more evident with higher Prader vir- ilization scores. Even so, pregnancy rates in CAH have improved with better hor- monal control, and rates in non-​salt-​losers are similar to those in women without CAH. Indeed, the pregnancy rate does not differ between the types of CAH or even the normal population when the rate is expressed as the proportion of women who are actively wanting to become pregnant. Glucocorticoid replacement for the mother should not be dexamethasone, as this steroid will readily cross the placenta unmetabolized. Prepregnancy doses of hydrocortisone or prednis- olone can be used without the need to oversuppress slightly elevated levels of 17OHP and testosterone. The latter is efficiently converted to oestrogens by placental aromatase, thus protecting a female fetus from being virilized. Fludrocortisone requirements may sometimes increase due to the antimineralocorticoid properties of progesterone. The mineralocorticoid dose is adjusted according to postural blood pressure response, and serum sodium and potassium concentrations. Renin is physiologically increased during pregnancy and thus cannot serve as a monitoring tool. Parenteral hydrocortisone is provided during labour, although most women are likely to need a caesarean section because of previous genital surgery. Women may have an in- creased risk of gestational diabetes, but the pregnancies are other- wise normal. The offspring have normal birthweight, no increased frequency of malformations, and normal development. Intriguingly, there is a 2:1 ratio of girls to boys born to mothers with CAH. Male The adult male with CAH is not devoid of problems of a repro- ductive nature. Non​adherence with treatment is prevalent, which can result in elevated adrenal androgen secretion and suppression of pituitary luteinizing hormone secretion following aromatization to oestrogens. Some men have not gone through true puberty due to suppression of the hypothalamic-​pituitary-​gonadal axis if CAH control has already slipped during adolescence. Hypogonadotropic hypogonadism with small soft testes may ensue, with consequent oligospermia. This can be potentially rectified by reinstituting gluco- corticoid replacement. Adult males with CAH may develop benign testicular adrenal rest tumours that are the result of hyperstimulation of adrenal rest cells by ACTH. The origin of such cells is the common site of adrenal and gonadal development at the urogenital ridge. The prevalence of testicular adrenal rest tumours is reported to be up to 90% in males with CAH. They are evident on routine testicular ultrasound examination while often not palpable on clinical examination. Histological examination shows sheets of polygonal cells separated by dense fibrous tissue with focal lymphocytic infiltrates. There is a decrease in diameter of the tubules and reduced spermatogen- esis. Infertility is probably the result of long-​standing obstruction of the seminiferous tubules. Testis-​sparing surgery has not been suc- cessful in restoring normal pituitary–​gonadal function. Ultrasound examination shows that nearly one-​quarter of prepubertal boys with CAH have testicular adrenal rest tumours. It is now recommended that routine ultrasound screening should start at puberty. Early op- timization of treatment to reduce these tumours appears to be key to improve long-​term fertility outcomes. Semen preservation in young adulthood is an option to consider. Adrenal myelolipomas are also frequently found in adults with CAH. Other forms of CAH Steroid acute regulatory protein (StAR)
deficiency—​lipoid adrenal hyperplasia The first key initial step to enable cholesterol to be utilized as a sub- strate for steroidogenesis is the intracellular cholesterol transport from the outer to the inner mitochondrial membrane, mediated by the StAR (Fig. 13.5.2.1). StAR-​independent cholesterol transport only occurs at a low rate. Therefore, a defect in StAR leads to almost no substrate provision for P450 side-​chain cleavage enzyme (CYP11A1) and the production of all steroid hormones from adrenal and gonad is severely reduced. StAR is not necessary for placental progesterone production and does therefore not affect placental progesterone syn- thesis. In contrast to other CAH forms, the adrenals show an ac- cumulation of lipids, predominantly cholesterol esters resulting in large adrenals of yellow appearance. The most severe form presents with 46,XY DSD and combined adrenal insufficiency. Salt wasting typically develops in the neonatal period or after few weeks of life, but later onset also occurs. Females can show spontaneous pubertal development. The pathophysiology of StAR deficiency is explained by a ‘two-​hit’ hypothesis, whereby there is reduced transport of chol- esterol into the mitochondria which, under the continued stimula- tion of ACTH, leads to engorgement of steroid cells from cholesterol accumulation that ultimately severely disrupts cell function. Girls with StAR deficiency may start puberty spontaneously, but might later develop hypergonadotropic hypogonadism. Since fetal infant ovarian follicles are quiescent until puberty these are undamaged

SECTION 13  Endocrine disorders 2370 by the cholesterol accumulation and the length of the ovulatory cycle is too short to impair follicular hormone production. It is only after puberty, with elevated gonadotropins, that the ovarian cells become engorged and any cycles that occur are likely to be an- ovulatory. Polycystic changes may also ensue. A milder non​classical form of StAR deficiency is now recognized. Non​classic Lipoid CAH manifests later in life with adrenal insufficiency resembling non-​ autoimmune Addison’s disease and mild 46,XY DSD or normal male genital development. Adult phenotypes suggest that fertility may also be compromised in the non​classic form. In some patients, hypocortisolism is the only clinical manifestation. In such cases, non​classic CAH might be misdiagnosed as familial glucocorticoid deficiency. Treatment consists of glucocorticoid and mineralocor- ticoid replacement, and substitution of sex hormones in later life tailored to the individual requirements. P450 Side-​chain cleavage enzyme (CYP11A1) deficiency Cholesterol is converted to pregnenolone via three enzymatic re- actions (20α-​hydroxylation, 22-​hydroxylation, and cleavage of the cholesterol side chain) via the mitochondrial enzyme, CYP11A1, also known as P450scc. These initial steps in steroidogenesis are rate limiting and ACTH dependent. In contrast to StAR, CYP11A1 is generally required to maintain sufficient progesterone synthesis in pregnancy. However, a few cases of P450scc deficiency as a re- sult of mutations in the CYP11A1 gene have now been reported. The clinical presentation varies from prematurity and early onset of salt-​losing adrenal failure, to adrenal failure presenting in later childhood. Generally affected XY male patients have female external genitalia, but sometimes with clitoromegaly. However, some milder mutations seem to allow for normal masculinization of the external genitalia in 46,XY individuals clinically presenting with ‘isolated’ adrenal insufficiency. By contrast with StAR deficiency, the adrenals are small rather than grossly enlarged. 3β-​Hydroxysteroid dehydrogenase deficiency 3β-​Hydroxysteroid dehydrogenase/​isomerase (3βHSD) is a non-​ P450 membrane-​bound enzyme which converts Δ5 to Δ4 steroids in the adrenals and gonads. Hence it is needed for the synthesis of glucocorticoids, mineralocorticoids, progesterone, androgens, and oestrogens. HSD3B2 on chromosome 1p13.1 expresses the type II enzyme in the adrenals and gonads. The type I enzyme is expressed predominantly in the placenta and peripheral tissues, and is there- fore essential for maintaining high levels of progesterone in preg- nancy. Consequently, deficiency of 3βHSD activity is only associated with mutations in HSD3B2. Genital abnormalities occur, mainly in males, from inadequate masculinization resulting from the produc- tion of weak androgens by the testis and peripheral tissues. Affected females may be mildly virilized. Salt loss generally occurs. Diagnosis is confirmed by an elevated ratio of Δ5 (17-​hydroxypregnenolone, DHEA) to Δ4 (17OHP, androstenedione) steroids and analysis of urinary steroid metabolites. Molecular studies show that most patients have missense mu- tations in the HSD3B2 gene, and many are compound heterozy- gotes. There is close concordance between genotype and phenotype with respect to salt-​wasting forms. Significant conversion of Δ5 to Δ4 steroids occurs in peripheral tissues through the action of type I  3βHSD. Consequently, some patients have elevated concentra- tions of Δ4 steroids (17OHP, androstenedione), which has led to a mistaken diagnosis of 21-​hydroxylase deficiency. Spontaneous onset of puberty and menarche may occur in females, and gynaecomastia in males, presumably the result of conversion of Δ5 to Δ4 steroids by the type I enzyme. Based on HSD3B2 genotyping, the hormonal criteria have been refined for the diagnosis of HSD3B2 deficiency. 17OH-​pregnenolone concentrations and 17OH-​pregnenolone to cortisol ratios at baseline and after ACTH stimulations are of the highest discriminatory value in differentiating between patients af- fected by HSD3B2 deficiency and patients with milder biochemical abnormalities, who are negative for HSD3B2 mutations 17α-​Hydroxylase deficiency A single P450c17 (CYP17A1) microsomal enzyme catalyses 17α-​hydroxylase and 17,20-​lyase reactions. Both are required for the synthesis of sex hormones (C19 steroids), whereas only 17α-​hydroxylase activity is required for the synthesis of cortisol (C21, 17-​hydroxysteroids). The conversion of pregnenolone to 17-​ hydroxypregnenolone, and progesterone to 17-​hydroxyprogesterone, is 17α-​hydroxylase dependent, whereas the 17,20-​lyase reaction converts 17-​hydroxypregnenolone to DHEA and with by far lesser efficiency 17OHP to androstenedione. Mineralocorticoid biosyn- thesis is not dependent on the presence of the CYP17A1 enzyme, thus ACTH-​stimulated, low-​renin hypertension is a typical feature of the combined 17α-​hydroxylase/​17,20-​lyase deficiency from ex- cess production of C21,17-​deoxysteroids such as deoxycortico- sterone (DOC). There is an accompanying hypokalaemic metabolic alkalosis. Inadequate androgens in affected males cause a phenotype ranging from female genitalia to an ambiguous appearance or fea- tures of a hypospadic male. Female patients lack breast development and have primary amenorrhea. Patients with 46,XY karyotype may only present at adolescence because of pubertal failure. The external genitalia are female in appearance, with a blind-​ending vagina, testes that may be abdominal or inguinal in location, and absent pubic and axillary hair. The presentation is not unlike complete androgen in- sensitivity syndrome, apart from the lack of breast development. The excess production of corticosterone, a weak glucocorticoid, gener- ally prevents adrenal crisis in patients with CYP17A1 deficiency. Increased corticosterone, deoxycorticosterone, and progesterone and decreased concentrations of testosterone, oestradiol, and renin characterize this enzyme defect. Measurements of steroid metabol- ites delineate patterns indicative of 17α-​hydroxylase or 17,20-​lyase deficiency alone or combined. A rare variant of CYP17A1 deficiency has been described, as isolated 17,20 lyase deficiency, with largely preserved 17α-​hydroxylase activity. This manifests clinically with impaired sex steroid biosynthesis only, without apparent clinical evidence of mineralocorticoid excess or glucocorticoid deficiency. Affected boys have genital anomalies, whereas in girls the presenta- tion is one of delayed puberty. The 6.5 kb human CYP17A1 gene on chromosome 10q24.3 has eight exons. Over 100 different mutations have been described, without evidence of a hot spot apart from mutations in specific population. A frequent mutation is a 4 bp duplication in exon 8, which, as a result of altering the reading frame, leads to a short- ened C-​terminal sequence. This mutation is shared by Mennonites and other individuals in the Friesland region of the Netherlands, suggesting a founder effect. There are other geographic clusters in Southeast Asia (in-​frame deletion of residues 487–​489) and Brazilians of Portuguese and Spanish ancestry (Arg362Cys and

13.5.2  Congenital adrenal hyperplasia 2371 Trp406Arg, respectively). CYP17A1 mutations underlying the iso- lated 17,20 lyase deficiency variant are located within the area of the CYP17A1 molecule that is thought to interact with the cofactor cytochrome b5, thereby disrupting the electron transfer from P450 oxidoreductase to CYP17A1 specifically disrupting the conversion of 17OH-​pregnenolone to DHEA. Further biochemical work-​up has revealed that subclinical impairment of 17-​hydroxylation is present in these patients. Interestingly mutations in cytochrome b5 (CYB5A), a key redox cofactor for the 17,20-​lyase enzyme have been shown to be the cause for ‘true’ isolated 17,20-​lyase deficiency. P450 oxidoreductase (POR) deficiency Apparent combined deficiencies of the 17α-​hydroxylase (CYP17A1) and 21-​hydroxylase (CYP21A2) enzymes were reported in patients with ambiguous genitalia, but in whom analysis of the CYP17A1 and CYP21A2 genes revealed no mutations. The phenotypes com- prised mild degrees of virilization of affected female infants (and in some cases their mothers during pregnancy), which was self-​ limiting after birth, and some undermasculinization in affected males. This diagnostic conundrum was resolved when mutations were found in P450 oxidoreductase (POR), which is a membrane-​ bound flavoprotein that functions to transfer electrons from NADPH to all microsomal cytochrome P450 enzymes, including CYP17A1 and CYP21A2. Most patients with POR deficiency have skeletal malformations similar to malformations observed in Antley-​Bixler syndrome (OMIM 207410), adrenal and gonadal insufficiency, and disorder of sex development in both sexes (46,XX, DSD, and 46,XY DSD). However, normal development of the external genitalia has also been observed in either sex. Classic Antley–​Bixler syndrome is caused by autosomal dominant mutations in the fibroblast growth factor receptor 2 (FGFR2) gene, and manifests neither with abnor- malities of steroid metabolism nor ambiguous genitalia. Impairment of sterol biosynthesis, specifically of POR-​dependent 14α-​lanosterol demethylase (CYP51A1), may be causative. This is supported by the finding that children born to mothers treated during pregnancy with the CYP51A1 inhibitor fluconazole show evidence of Antley–​Bixler-​ like skeletal malformations. Skeletal abnormalities associated with POR deficiency include craniosynostosis, radiohumeral synostosis, choanal atresia, femoral bowing, and joint contractures. The dual deleterious effects of virilizing an affected girl and causing undermasculinization in an affected boy have two explan- ations. First, placental aromatase is also POR-​dependent and thus requires this cofactor for adequate aromatization of fetal adrenal androgen. Mutations in POR would thus explain virilization of an affected female at birth, as well as the mother, and the self-​limiting nature of the disorder. A second, more speculative, suggestion for the virilizing effect is the presence of a fetus-​specific ‘back door’ pathway of dihydrotestosterone production from 17OHP, which does not utilize androstenedione and testosterone as intermedi- aries. Such a scheme has been documented in the tammar wallaby (Macropus eugenii), and there is evidence that a similar pathway may be functional in the human fetus. The affected XY male is probably undermasculinized because of partial disturbance of 17,20-​lyase activity. POR deficiency can also manifest in the form of delayed and disordered puberty (ovarian cysts in girls). Milder de- fects in POR can manifest as polycystic ovary syndrome in women or as gonadal dysfunction in men. The POR gene is located on chromosome 7q11.2. More than 80 different mutations have been described, which are distrib- uted throughout all four functional domains of the POR protein. Most missense mutations are located within the central electron-​ transfer domain; Arg287Pro is prevalent in patients of European ancestry, whereas Arg457His is common in Japan. Baseline gluco- corticoid secretion is often sufficient, but the cortisol response to stress is often impaired, whereas mineralocorticoid deficiency is not common. Some patients have increased excretion of mineralo- corticoid metabolites and mild hypertension. Since most clinically used drugs are metabolized by POR-​dependent hepatic P450 en- zymes, POR mutations and polymorphisms can impact on variant drug metabolism. 11β-​Hydroxylase deficiency Deficient 11β-​hydroxylase activity accounts for 5 to 8% of cases of CAH, with an incidence of about 1 in 100 000 in non​consanguineous populations. The enzyme is required for the terminal conversion of 11-​deoxycortisol to cortisol, and DOC to corticosterone, but is lacking noteworthy 18-​hydroxylase and 18-​oxidase activity. The impaired cortisol production leads to increased ACTH stimulation and as a consequence to salt and water retention, low-​renin hyper- tension, and virilization, which appear to be more profound than in 21-​hydroxylase deficiency. Hypertension is commonly identi- fied in late childhood or adolescence, the severity not necessarily correlating with plasma concentrations of DOC. Complications of long-​standing hypertension include cardiomyopathy, retinal vein occlusion, blindness, and stroke. There is typically hypernatraemia, hypokalaemia, and sup- pressed renin concentrations. The diagnosis is confirmed by ele- vated concentrations of 11-​deoxycortisol and DOC in plasma, and their tetrahydro metabolites in urine. Plasma concentrations of androstenedione and testosterone are increased. Moderately elevated levels of 17OHP may lead to an erroneous diagnosis of 21-​hydroxylase deficiency. Of note 17OHP concentrations are commonly magnitudes lower than in 21-​hydroxylase deficiency and often paired with excessively increased androstenedione con- centrations. Treatment requires only glucocorticoid replacement, although transient salt wasting may follow an initial fall in levels of the mineralocorticoid, DOC. Treatment can be monitored by measuring 11-​deoxycortisol, androstenedione, and testosterone as well as normalization of renin and aldosterone concentrations. Antihypertensive treatment may be necessary if hypertension has been long-​standing and should be initiated with a mineralo- corticoid receptor blocker. Milder or non​classic deficiency also occurs, and manifests similar features to the non​classic form of 21-​hydroxylase deficiency. Steroid 11β-​hydroxylase activity is a function of the CYP11B1 gene located on chromosome 8q21–​22 in tandem with CYP11B2, which encodes aldosterone synthase, the enzyme involved in mineralocorticoid synthesis. CYP11B1 is expressed in the zona fasciculata, whereas CYP11B2 is exclusively expressed in the zona glomerulosa, where it not only catalyses the 11β-​hydroxylation of DOC to corticosterone, but also catalyses the terminal steps of al- dosterone synthesis. More than 90 mutations causing 11β-​hydroxylase deficiency are distributed throughout the CYP11B1 gene. CYP11B1-​inactivating mutations have been shown to be distributed over the entire

SECTION 13  Endocrine disorders 2372 coding region consisting of 9 exons. Although a cluster is reported in exons 2, 6, 7, and 8, real hot spots such as in 21OHD do not exist. A  higher incidence of this form of CAH occurs in some populations such as in Moroccan Jews, and is associated with an Arg448His mutation. Prenatal treatment with dexamethasone has been used successfully in this form of CAH to prevent virilization of an affected female fetus. A non​classic form of 11β-​hydroxylase deficiency is described that can manifest as premature adrenarche, or hirsutism and infertility in late childhood, adolescence, or adulthood. Aldosterone synthase, encoded by CYP11B2, catalyses the ter- minal steps of aldosterone synthesis: the 11β-​hydroxylation of DOC, 18-​hydroxylation to 18-​hydroxycorticosterone, and 18-​oxidation to aldosterone. Patients with CYP11B2 deficiency generally respond well to fludrocortisone (start dose 150 µg/​m2 per day in neonates and infancy) and can also benefit from salt supplementation. Patients who presented with failure to thrive generally show a good catch-​up growth after initiation of treatment. The need for mineralocorticoid treatment lessens in later life. A chimaeric form of the two CYP11B genes, under the control of the CYP11B1 promoter, leads to an autosomal dominant form of hyper- tension, termed glucocorticoid remediable hyperaldosteronism. This condition is suppressible with dexamethasone because of its ACTH dependence. FURTHER READING Arlt W, et al. (2010). Health status of adults with congenital adrenal hyperplasia:  a cohort study of 2013 patients. J Clin Endocrinol Metab, 95, 5110–​21. Bidet M, et  al. (2010). Fertility in women with nonclassical con- genital adrenal hyperplasia due to 21-​hydroxylase deficiency. J Clin Endocrinol Metab, 95, 1182–​90. Carroll AE, Downs SM (2006). Comprehensive cost-​utility analysis of newborn screening strategies. Pediatrics, 117, 5287–​95. Casteras A, et al. (2009). Reassessing fecundity in women with classical congenital adrenal hyperplasia (CAH): normal pregnancy rate but reduced fertility rate. Clin Endocrinol, 70, 833–​7. Crouch NS, et al. (2008). Sexual function and genital sensitivity fol- lowing feminizing genitoplasty for congenital adrenal hyperplasia. J Urol, 179, 634–​8. Dhir V, et  al. (2009). Steroid 17alpha-​hydroxylase deficiency: functional characterization of four mutations (A174E, V178D, R440C, L465P) in the CYP17A1 gene. J Clin Endocrinol Metab, 94, 3058–​64. Falhammer H, et al. (2014). Increased mortality in patients with con- genital adrenal hyperplasia due to 21-​hydroxylase deficiency. J Clin Endocrinol Metab, 99, E2715–​21. Falhammar H, et al. (2015). Increased cardiovascular and metabolic morbidity in patients with 21-​hydroxylase deficiency:  a Swedish population-​based national cohort study. J Clin Endocrinol Metab, 100, 3520–​8. Finkielstain GP, et al. (2012). Clinical characteristics of a cohort of 244 patients with congenital adrenal hyperplasia. J Clin Endocrinol Metab, 97, 4429–​38. Hagenfeldt K, et al. (2008). Fertility and pregnancy outcome in women with congenital adrenal hyperplasia due to 21-​hydroxylase defi- ciency. Hum Reprod, 23, 1607–​13. Hirvikoski T, et al. (2007). Cognitive functions in children at risk for congenital adrenal hyperplasia treated prenatally with dexametha- sone. J Clin Endocrinol Metab, 92, 542–​8. Hirvikoski T, et  al. (2011). Gender role behaviour in prenatally dexamethasone-​treated children at risk for congenital adrenal hyperplasia—​a pilot study. Acta Paediatr, 100, e112–​9. Hughes IA (2006). Prenatal treatment of congenital adrenal hyper- plasia: do we have enough evidence? Treat Endocrinol, 5, 1–​6. Hughes IA (2007). Congenital adrenal hyperplasia: a lifelong disorder. Horm Res, 68, Suppl 5, 84–​9. Kamrath C, et  al. (2012). Increased activation of the alternative ‘backdoor’ pathway in patients with 21-​hydroxylase deficiency: evi- dence from urinary steroid hormone analysis. J Clin Endocrinol Metab, 97, E367–​75. Khattab A, Marshall I (2019). Management of congenital adrenal hyperplasia: beyond conventional glucocorticoid therapy. Curr Opin Pediatr, 31, 550–4. Kim CJ, et al. (2008). Severe combined adrenal and gonadal deficiency caused by novel mutations in the cholesterol side chain cleavage en- zyme, P450ssc. J Clin Endocrinol Metab, 93, 696–​702. Krone N, Arlt W (2009). Genetics of congenital adrenal hyperplasia. Best Pract Res Clin Endocrinol Metab, 23, 181–​92. Lajic S, et al. (2011). Long-​term outcome of prenatal dexametha- sone treatment of 21-​hydroxylase deficiency. Endocr Dev, 20, 96–​105. Mallappa A, et al. (2015). A phase 2 study of chronocort, a modified-​ release formulation of hydrocortisone, in the treatment of adults with classic congenital adrenal hyperplasia. J Clin Endocrinol Metab, 100, 1137–​45. Meyer-​Bahlburg HFL, et  al. (2008). Sexual orientation in women with classical or non-​classical congenital adrenal hyperplasia as a function of degree of prenatal androgen excess. Arch Sex Behav, 37, 85–​99. Miller WL, Auchus RJ (2011). The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocr Rev, 32, 81–​151. Nermoen I, et al. (2011). High frequency of adrenal myelolipomas and testicular adrenal rest tumours in adult Norwegian patients with classical congenital adrenal hyperplasia due to 21-​hydroxylase de- ficiency. Clin Endocrinol, 75, 753–​9. New MI, et al. (2014). Noninvasive prenatal diagnosis of congenital adrenal hyperplasia using cell-​free fetal DNA in maternal plasma. J Clin Endocrinol Metab, 99, E1022–​30. Nimkaru S, New MI (2008). Steroid 11β-​hydroxylase deficiency con- genital adrenal hyperplasia. Trends Endocrinol Metab, 19, 96–​9. Nordenskjöld A, et al. (2007). Type of mutation and surgical procedure effect long-​term quality of life for women with congenital adrenal hyperplasia. J Clin Endocrinol Metab, 93, 380–​6. Nordenstrom A (2011). Adult women with 21-​hydroxylase deficient congenital adrenal hyperplasia, surgical and psychological aspects. Curr Opin Pediatr, 23, 436–​42. Ogilvie CM, et al. (2006). Congenital adrenal hyperplasia in adults: a review of medical, surgical and psychological issues. Clin Endocrinol, 64, 2–​11. Parajes S, et al. (2009). Functional consequences of seven novel mu- tations in the CYP11B1 gene:  four mutations associated with nonclassic and three mutations causing classic II beta-​hydroxylase deficiency. J Clin Endocrinol Metab, 95, 779–​88. Prader A, Gurtner HP (1955). The syndrome of male pseudoherm- aphroditism in congenital adrenocortical hyperplasia without

13.5.2  Congenital adrenal hyperplasia 2373 overproduction of androgens (adrenal male pseudohermaphrodism). Helv Paediatr Acta, 10, 397–​412. Reisch N (2019). Pregnancy in congenital adrenal hyperplasia. Endocrinol Metab Clin North Am, 48, 619–41. Reisch N (2019). Review of health problems in adult patients with classic congenital adrenal hyperplasia due to 21-hydroxylase defi- ciency. Exp Clin Endocrinol Diabetes, 127, 171–7. Scott RR, Willer WL (2008). Genetic and clinical features of P450 oxidoreductase deficiency. Horm Res, 69, 266–​75. Speiser PW (2009). Nonclassic adrenal hyperplasia. Rev Endocr Metab Disord, 10, 77–​82. Speiser PW, et al. (2010). Congenital adrenal hyperplasia due to steroid 21-​hydroxylase deficiency: an Endocrine Society Clinical Practice guideline. J Clin Endocrinol Metab, 95, 4133–​60. Turcu AF, et  al. (2016). Adrenal-​derived 11-​oxygenated 19-​carbon steroids are the dominant androgens in classic 21-​hydroxylase defi- ciency. Eur J Endocrinol, 174, 601–​9. Wallensteen L, el al. (2016). Evaluation of behavioral problems after prenatal dexamethasone treatment in Swedish adolescents at risk of CAH. Horm Behav, 85, 5–​11. White PC (2009). Neonatal screening for congenital adrenal hyper- plasia. Nat Rev Endocrinol, 5, 490–​8.