Congenital Adrenal Hyperplasia Owing to 21-Hydroxylase Deficiency

C H A P T E R

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Congenital Adrenal Hyperplasia Owing to 21-Hydroxylase Deficiency Maria I. New, Oksana Lekarev, Denesy Mancenido, Alan Parsa, Tony Yuen Department of Pediatrics, Mount Sinai School of Medicine, New York, NY, USA

INTRODUCTION

stimulates production and pulsatile release of ACTH [1–3]. The synthesis of cortisol is regulated directly by ACTH. ACTH, a peptide with a relatively short plasma half-life, is cleaved from its high molecular weight ­precursor, pro-opiomelanocortin (POMC), and is stored in the corticotroph cells of the anterior pituitary, from which ACTH is released. CRH controls the pulsatile release of ACTH from these cells. The hypothalamic–pituitary–adrenal axis forms a regulated system (Fig. 3A.1). Negative feedback control is exerted by cortisol, and the hypothalamus then determines the setpoint for the expected cortisol level. ACTH release is required by the body at basal, diurnal, and stress-induced levels. When the plasma cortisol level is lower than required, ACTH rises. Therefore, with adrenal enzyme deficiencies that cause impaired synthesis and decreased secretion of cortisol, there is chronic elevation of ACTH and subsequent overstimulation and hyperplasia of the adrenal cortex. The primary regulation of aldosterone synthesis is via the renin–angiotensin system (RAS), the activity of which is regulated by electrolyte balance and plasma volume. Renin, an enzyme produced by the renal juxtaglomerular apparatus, cleaves angiotensinogen into the decapeptide angiotensin I. Angiotensin I is then converted to the octapeptide angiotensin II in the lungs. Angiotensin II is a potent vasoconstrictor that directly stimulates aldosterone secretion from the ZG. In addition, high serum K+ concentration and low serum Na+ concentration, which increase plasma renin activity (PRA), stimulate aldosterone secretion. Aldosterone is also under ACTH control as the ZG is sensitive to ACTH [4], especially at the latter stages of aldosterone synthesis when there is chronic angiotensin II stimulation or electrolyte imbalance (hyperkalemia and hyponatremia) [5]. In addition,

Congenital adrenal hyperplasia (CAH) refers to a group of genetic disorders that arise from defective ­steroidogenesis. In a physiologic state, the production of cortisol in the zona fasciculata of the adrenal cortex occurs in five major enzyme-mediated steps. When one of the enzymes is deficient, cortisol synthesis is impaired, leading to elevation of adrenocorticotropic hormone (ACTH) via the negative feedback system. As a result, the adrenal cortex is overstimulated and becomes hyperplastic. Each deficient enzymatic step produces a unique combination of elevated precursors and deficient product hormones. There are five forms of CAH. The most common form is 21-hydroxylase deficiency, which accounts for more than 90% of all cases of CAH.

ENDOCRINE FUNCTION OF THE ADRENAL CORTEX The adrenal cortex is divided into three histological regions: the outer zona glomerulosa (ZG), the middle zona fasciculata (ZF), and the inner zona reticularis (ZR). Synthesis of the mineralocorticoid aldosterone is dependent on enzymatic activity in the ZG, while p ­ roduction of cortisol and androgens requires enzymes found in the ZF and ZR, respectively. The different zones are regulated as if they are separate glands (see below). Adrenal steroid synthesis depends predominately on the secretion of ACTH from the anterior pituitary. ACTH exhibits acute and chronic effects on adrenocortical cell processes, which produce up to 10-fold amplification of the rate of steroidogenesis. Corticotropin-releasing hormone (CRH), which is produced by the hypothalamus,

Genetic Steroid Disorders. http://dx.doi.org/10.1016/B978-0-12-416006-4.00003-X

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3A.  CONGENITAL ADRENAL HYPERPLASIA OWING TO 21-HYDROXYLASE DEFICIENCY

the fetal zone and the definitive (adult) zone [8]. The fetal zone is much larger than the definitive zone and is responsible for the majority of fetal steroidogenesis. While ACTH is the primary trophic regulator of the fetal adrenal cortex, growth, transcription, and placental factors also contribute to the development of the cortex. In the neonatal period the fetal zone atrophies and the three zones of the adult adrenal cortex develop [9]. The zonal theory of the adrenal cortex demonstrates that the ZG and the ZF are histologically distinct. New and Seaman [10] indicated that the adrenal gland behaves hormonally and biochemically as two separate glands with separate regulation and hormonal secretion. Steroidogenesis in the fasciculata is regulated primarily by ACTH, which stimulates secretion of cortisol, deoxycorticosterone, corticosterone, and androgens. Steroidogenesis in the glomerulosa is regulated primarily by angiotensin II and potassium, and aldosterone secretion via ACTH stimulation exerts only a secondary influence in this zone [10].

PATHOGENESIS OF 21-HYDROXYLASE DEFICIENCY FIGURE 3A.1  The hypothalamic–pituitary–adrenal (HPA) axis.  Neural stimuli from the central nervous system (CNS) stimulate the production of corticotropin-releasing hormone (CRH) in the hypothalamus. CRH, together with vasopressin, stimulate the release of adrenocorticotropic hormone (ACTH) from the anterior lobe of the ­pituitary gland. In the adrenal cortex, ACTH stimulates the production of cortisol along with a small amount of androgens. Cortisol inhibits the higher CNS center, the hypothalamus, and the anterior pituitary through a negative feedback mechanism. This decreases CRH secretion and reduces pro-opiomelanocortin (POMC) cleavage into ACTH and β-endorphins.

cis-regulatory elements also seem to influence transcription of aldosterone synthase (CYP11B2), the enzyme that catalyzes the final steps in aldosterone synthesis, but further studies are necessary to define these elements [6]. Therefore, in summary, the four major factors affecting aldosterone synthesis by the ZG are angiotensin II, K+, Na+, and ACTH.

THE ADRENAL CORTEX AS TWO GLANDS Normal maturation, growth, and development of a fetus, as well as perinatal survival, are largely dependent on proper development of the fetal adrenal glands [7]. The adrenal medulla cells are derived from the neuroderm, the adrenal cortex cells are derived from the mesoderm. The primitive adrenal cortex cells can be identified in the fourth week of gestation. In the following weeks, the migrating cells segregate into two separate zones:

The production of cortisol in the ZF of the adrenal cortex occurs in five major enzyme-mediated steps (Fig. 3A.2). CAH arises from reduced or absent enzymatic activity at one of the steps of steroid ­ ­synthesis, and each deficiency produces characteristically abnormal adrenal hormones and precursor hormone l­ evels. In 2­ 1-hydroxylase deficiency, the function of the 21-hydroxylating cytochrome 450 is inadequate, which creates a block in the cortisol production pathway. The 21-hydroxylase defect leads to an accumulation of 17-hydroxyprogesterone (17-OHP), a hormone precursor to the 21-hydroxylation step. Excess 17-OHP is then shunted into the intact androgen pathway, where the 17,20-lyase enzyme converts 17-OHP to Δ4-androstenedione, which is then converted into androgens. The most severe form of 2­1-hydroxylase deficiency, the salt-wasting form, also consists of ­mineralocorticoid (aldosterone) deficiency, leading to renal sodium wasting. The 21-hydroxylase enzyme defect in the mild non-classical form of the disease is only partial and salt-wasting does not occur. Salt wasting also does not occur in the simple virilizing form of CAH.

CLASSICAL CAH Salt-Wasting Form By definition, classical CAH is characterized by the ambiguity (or masculinization) of the genitalia at birth in affected female patients. Approximately

Classical CAH

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FIGURE 3A.2  Adrenal steroidogenesis.  In the first step of a­ drenal steroidogenesis, cholesterol enters the mitochondria via a transport protein called steroidogenic acute regulatory protein (StAR). ACTH stimulates cholesterol cleavage, the rate-limiting step of adrenal steroidogenesis. The five enzymes required for cortisol production are cholesterol side chain cleavage enzyme, 17α-hydroxylase, 3β-hydroxysteroid dehydrogenase, 21-hydroxylase, and 11β-hydroxylase.

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t­hree-quarters of patients with classical 21-hydroxylase deficiency (21OHD) have salt-wasting disease [11]. When the loss of 21-hydroxylase function is severe, adrenal aldosterone secretion is not sufficient for sodium reabsorption by the distal renal tubules, leading to salt-wasting [12,13]. In vitro expression studies show that as little as 1% of 21-hydroxylase activity is necessary to generate enough aldosterone to prevent significant salt-wasting [14]. In addition, hormonal precursors of the 2­ 1-hydroxylase enzyme, which accumulate and become elevated, may act as antagonists to mineralocorticoid action in the sodium-conserving mechanism of the immature newborn renal tubule [15]. The patient develops the hallmark findings of hyponatremia and hyperkalemia, disproportionately high urinary sodium, low serum and urinary aldosterone, and elevated PRA. Infants with renal salt-wasting have poor feeding, weight loss, failure to thrive, vomiting, dehydration, hypotension, hyponatremia, and hyperkalemic metabolic acidosis progressing to adrenal crisis (azotemia, vascular collapse, shock, and death). Adrenal salt-wasting crisis can occur as early as 1–4 weeks [12]. More patients with salt-wasting disease are being identified with the advent of the newborn screening, leading to lower morbidity and mortality rates. It has been demonstrated that salt-wasting evident in infancy may improve with age [16]. Speiser et al. reported a spontaneous partial recovery of aldosterone biosynthesis in an adult patient with a homozygous deletion of the CYP21A2 gene who had documented severe salt-wasting in infancy. Therefore, it is recommended that sodium and mineralocorticoid requirements be carefully followed by measuring PRA and electrolytes in patients who have been diagnosed as salt-wasters in the neonatal period [17]. Although correlation of the severity of salt-wasting to the severity of genital masculinization has been suggested [18], it is widely accepted that the degree of genital masculinization does not correlate with salt-wasting. Even mildly virilized females with 21-hydroxylase deficiency may be salt-wasters and should be observed carefully in the first weeks of life. Conversely, patients without salt-wasting can have severely masculinized genitalia [19,20,88]. While generally there are some exceptions, the presence of salt-wasting is seen consistently within families [16,21]. However, discordance for salt-wasting has been found in several families among siblings with identical mutations on the CYP21A2 gene [19,22,23].

Simple-Virilizing Form The hallmark features of classical simple-virilizing 21OHD are prenatal masculinization and progressive postnatal virilization with rapid somatic growth and advanced epiphyseal maturation owing to elevated

androgens and subsequent aromatization to estrogens. This frequently leads to early epiphyseal fusion and short stature in adulthood. There is no evidence of mineralocorticoid deficiency in this disorder. At birth, the diagnosis of simple-virilizing CAH in a female is easily made because of the genital ambiguity. Since the external genitalia are not affected in newborn males, hyperpigmentation may be the only clue suggesting increased ACTH secretion and cortisol deficiency. Diagnosis at birth in males is largely dependent on prenatal or newborn screening. If a female is not treated with glucocorticoid replacement therapy postnatally, her genitalia may continue to virilize because of continued excess adrenal androgens, and pseudoprecocious puberty can occur. As in patients with salt-wasting 21OHD, signs of hyperandrogenism in children affected with CAH include early onset of sexual hair, adult body odor, acne, and rapid somatic growth and bone age advancement [24].

NON-CLASSICAL CAH A mild, late-onset form of adrenal hyperplasia, now known as the non-classical form, was first suspected during the early 1950s by gynecologists in clinical practice who used glucocorticoids on an empirical basis to treat women with signs and symptoms of hyperandrogenism, including infertility. The first biochemical report of 21OHD was by Baulieu and colleagues in 1957 [25]. Diagnosis of 21OHD on the basis of serum steroid measurements became feasible with the development of a specific radioimmunoassay for 17-OHP [26]. Dupont and colleagues [27] reported the first genetic linkage of CAH (21OHD) with human leukocyte antigen (HLA); this report was followed by numerous family studies of classical 21OHD [28–30]. It is now known that mutations in the gene for 21-hydroxylase associated with the ­non-classical defect are distinct from those found in the classical forms and often differ by ethnicity [19,31]. The phenotype of non-classical 21OHD is variable and may present at any age. Children may present with premature development of pubic hair. To our knowledge, the youngest patient was noted to have pubic hair at 6 months of age [32]. Elevated adrenal androgens promote the early fusion of epiphyseal growth plates; children with this disorder commonly have an advanced bone age [32] and are among the tallest in their class in early childhood [33], though ultimately they have early growth arrest and short stature as adults [34]. Severe cystic acne refractory to oral antibiotics and retinoic acid has been observed in non-classical 21OHD [35]. In one study comparing the responses of 11 female

Diagnosis (Hormonal and Genetic)

patients with acne and eight (female) control subjects to a 24-hour infusion of ACTH, elevated urinary excretion of pregnanetriol suggestive of a partial 21OHD was found in six patients [36]. Conversely, in another study of 31 young female patients with acne and/or hirsutism tested with low-dose ACTH stimulation after overnight dexamethasone suppression, no cases of 21OHD were found [37]. Male-pattern baldness has been noted in other cases as the sole presenting symptom in young women with non-classical 21OHD. Menarche in females may be normal or delayed. Secondary amenorrhea occurs frequently [32], and is often the presenting sign of non-classical CAH. Polycystic ovarian syndrome (PCOS) can be a complication of nonclassical 21OHD [38,39] because the elevated adrenal sex steroids disrupt the usual cyclicity of gonadotropin release and have direct effects on the ovaries. This disruption may ultimately lead to production of ovarian cysts, which produce excess androgens. The prevalence of non-classical 21OHD as an etiology of these endocrine complaints in women in other published series ranges from 1.2% to 30% [32,38,40–43]. Because the disease frequency is ethnicity-dependent, the large variability of frequencies in these reports may be because of ethnic variability. In boys, early facial hair development, acne, and growth spurt may be detected. In men, signs of androgen excess are difficult to appreciate. The manifestations of adrenal androgen excess may be limited to short stature, oligospermia, and diminished fertility. These manifestations are owed to adrenal sex steroid-induced gonadal suppression; they are reversible with glucocorticoid treatment [44–46]. Occasionally, a patient will be diagnosed with 21OHD via genetic analysis as part of a family study, yet he or she will have no clinical symptoms of disease while demonstrating biochemical abnormalities compatible with CAH. Longitudinal follow-up evaluation of these cases often shows signs of hyperandrogenism that wax and wane with time. The reason has not yet been elucidated.

DIAGNOSIS (HORMONAL AND GENETIC) Hormonal Diagnosis The diagnosis of CAH must be suspected in infants born with ambiguous genitalia and should be made as early as possible so that therapy can be initiated. The genetic sex often determines the sex assignment. However, the hormonal determination of the specific deficient enzyme, and an assessment of the patient's potential for future sexual activity and fertility should be considered along with the karyotype during sex

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assignment. Physicians are urged to recognize the physical characteristics of CAH in newborns (e.g. ambiguous genitalia) and to refer such patients to appropriate clinics for full endocrinologic evaluation. In the past, laboratories measured urinary excretion of adrenal hormones or their urinary metabolites (e.g. 17-ketosteroids). However, collection of 24-hour urine excretion is difficult, particularly in neonates [47]. Therefore, simple and reliable radioimmunoassays were utilized in the past for measuring circulating serum levels of adrenal steroids [48]. Recently, tandem mass spectrometry determination has provided precise results for serum concentrations. Alternatively, a non-invasive random urine collection in the first days of life for steroid hormone metabolites and precursor:product ratio assessments can be measured simultaneously. The ratio may be used independently or in conjunction with serum steroid assays to increase accuracy and confidence in making the diagnosis and distinguishing the separate enzymatic forms of the disorder [49,50]. Diagnosis of 21OHD CAH is supported biochemically by a hormonal evaluation. A very high concentration of 17-OHP, the precursor of the defective enzyme, is diagnostic of classical 21OHD. Such testing is the basis of the newborn screening program developed to identify classically affected patients who are at risk for salt-wasting crisis [51,52]. Only 20 μl blood, obtained by heel prick and blotted on microfilter paper, is used for the test to provide a reliable diagnostic measurement of 17OHP. The simplicity of the test and the ease of transporting microfilter paper specimens by mail has facilitated the implementation of CAH newborn screening programs worldwide. As of 2009, all 50 states in the USA screen for CAH. False positive results are common in premature infants [53]; therefore, appropriate references based on weight and gestational age are in place in many screening programs [54]. The majority of screening programs use a single screening test without retesting of questionable 17-OHP concentrations. To improve efficacy, a small number of programs perform a second screening test of the initial sample to re-evaluate borderline cases identified by the first screening. Current immunoassay methods used in newborn screening programs yield a high false positive rate. To decrease this high false positive rate, liquid chromatography–tandem mass spectrometry measuring different hormones (17OHP, Δ4-androstenedione, and cortisol) has been suggested as a second-tier method of analyzing positive results [55]. False negative rates of CAH diagnosis on newborn screening are not well documented, but one publication reported 15 missed cases out of more than 800 000 neonates tested from 1999 to 2010 [56]. Thus some have advocated genetic testing as the preferred mode of newborn screening.

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Hormonal diagnosis is determined by performing a corticotropin stimulation test (250 μg cosyntropin intravenously), measuring levels of 17-OHP and Δ4androstenedione at baseline and at 60 minutes. These values, which correlate with the severity of the disease, can then be plotted in the published nomogram (Fig. 3A.3) [57]. The corticotropin stimulation test should not be performed during the initial 24 hours of life as samples from this period are typically elevated in all infants and may yield false positive results. The corticotropin stimulation test is particularly important in establishing hormonal diagnosis of the non-classical form of the disease since e­ arly-morning values of 17-OHP may not be sufficiently elevated to allow accurate diagnosis.

Molecular Genetics of 21OHD The steroid 21-hydroxylase belongs to the cytochrome P450 superfamily, a diverse group of heme proteins. The

gene is encoded by CYP21A2, which is located on the short arm of chromosome 6 (6p21.3) at the human major histocompatibility complex (MHC) between the structurally, genetically, and evolutionarily closely related and highly polymorphic HLA class I and class II genes (Fig. 3A.4). The region between MHC class I and class II is sometimes referred to as MHC class III, although it has no functional significance and contains genes unrelated to HLA. The RCCX Module Within this 730-kb stretch of DNA in the MHC class III region lies the CYP21A2 gene, which is surrounded by three other genes: RP1, C4, and TNXB. The 11-kb RP1 gene encodes a nuclear serine/threonine protein kinase [58] that is ubiquitously expressed [59–61]. The primarily liver-expressed C4 gene encodes the fourth component of the complement system [62–64]. There are two C4 variants, an acidic C4A isoform and a basic

FIGURE 3A.3  Nomogram relating baseline to ACTH-stimulated serum concentrations of 17-hydroxyprogesterone.  The scales are logarithmic. A regression line for all data points is shown. Data points cluster, as shown, into three non-overlapping groups: classic and non-classic forms of 21OHD are readily distinguished from each other and from those that are heterozygotes and unaffected. Distinguishing unaffected from heterozygotes is difficult.

Diagnosis (Hormonal and Genetic)

C4B isoform that differ from each other by only a few amino acids. Whereas the C4A gene is almost always 20.5 kb long, the C4B gene can either be 14.2 kb or 20.5 kb in size. The size difference is due to a 6.3-kb insertion of a human endogenous retrovirus HERV-K(C4) in intron 9 of the C4 gene [65,66]. Both long and short C4 variant forms are functional. The 3.4-kb CYP21A2

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gene encodes steroid 21-hydroxylase and is expressed primarily in the adrenal cortex [67–71]. The 68.2-kb TNXB gene encodes tenascin X, an extracellular matrix protein expressed mainly in skin, tendons, and blood vessels [72–75]. This RP–C4–CYP21–TNX (RCCX) module is usually tandemly duplicated in humans, and 70–80% of all copies of chromosome 6 are bimodular,

FIGURE 3A.4  Chromosomal location of CYP21A2.  Steroid 21-hydroxylase is encoded by the CYP21A2 gene located at 6p21.3 on the short arm of chromosome 6. The gene is situated in the MHC class III region which is flanked by MHC class I and class II genes. While many genes and pseudogenes exist in each of these regions, for illustration purposes, only a few HLA genes are shown. Here, the most common bimodular arrangement of the RCCX region is displayed, although monomodular, trimodular, and quadrimodular arrangements are also observed in humans. The RCCX module can exist in long and short forms. The long module features a 6.3-kb insertion in intron 9 of the C4 gene. Whereas the telomeric RCCX module is almost always a long one, the centromeric module can exist in either form. A long telomeric RCCX module and a short centromeric RCCX module is illustrated here.

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3A.  CONGENITAL ADRENAL HYPERPLASIA OWING TO 21-HYDROXYLASE DEFICIENCY

containing two RCCX modules. Not all genes in the RP1–C4A–CYP21A1P–TNXA/RP2–C4B–CYP21A2– TNXB modules are active. RP2 and TNXA are severely truncated and do not encode any functional proteins. While CYP21A1P shares approximately 98% sequence homology with CYP21A2, several mutations in the CYP21A1P gene render it inactive. Steroid 21-hydroxylase is 494/495 amino acids long, where the size difference is attributed to a polymorphism in the hydrophobic N-terminal domain that serves as membrane anchorage. The longer variant has an additional leucine residue, highlighted in bold in the following N-terminal sequence (MLLLGLLLLLPLLAGA…). Both variants have identical enzymatic activity. In this article, sequence nomenclature for the short form is used. The enzyme contains various functional domains, which include membrane anchorage, interaction with cytochrome P450 oxidoreductase (POR), heme binding, substrate binding, and oxygen/water binding. Recently, the crystal structure for the bovine 2­ 1-hydroxylase has been solved [76]. One intriguing feature of the bovine 21-hydroxylase is that there are two substrate-binding sites in the enzyme. Using this crystal structure as a template, a humanized model of 21-hydroxylase has been constructed and utilized to correlate disease severity, with over 100 known missense mutations [77]. Mutations affecting critical enzyme functions, such as membrane anchoring, heme binding, substrate binding, or enzyme stability, result in a complete loss of function, leading to salt-wasting CAH. Mutations affecting the transmembrane region or conserved hydrophobic patches result in up to 98% reduction in enzyme activity, leading to simple-virilizing CAH. Mild non-classical disease arises from interference in cytochrome POR interactions, salt-bridge and hydrogen bonding networks, and non-conserved hydrophobic clusters [77].

MOLECULAR MECHANISMS CREATING CYP21A2 GENETIC DEFECTS Gene Conversion Because of the high degree of sequence homology between the tandemly repeated RCCX modules, misalignment of sister chromatids during mitosis can result in gene conversion, where a small part of one sister ­chromatid is copied to the other (Fig. 3A.5A). A sequence of the CYP21A1P gene may be copied to the CYP21A2 gene (Fig. 3A.5Aii), and vice versa (Fig. 3A.5Aiii). Gene ­conversion can take place with transfer of short sequences (50–200 bp), sometimes called microconversion, or with longer sequences in the case of multi-exon conversion. When a pathogenic mutation from the CYP21A1P pseudogene is copied to the CYP21A2 gene, partial or complete

inactivation of enzymatic activity will result. A list of pathogenic pseudogene mutations is shown in Figure 3A.5B. There are four promoter mutations, two ­frameshift mutations (exons 3 and 7), one intronic mutation (intron 2), and eight single-base missense mutations (exons 1, 4, 6, 7, and 8) in the CYP21A1P pseudogene (Fig. 3A.5B).

Large-Scale Gene Deletion via Unequal Crossover Misalignment of homologous chromosomes during meiosis can also lead to unequal crossover resulting in large-scale gene deletions. Crossover can take place at the C4, CYP21, or TNX genes (Fig. 3A.6). Meiotic unequal crossover results in the transfer of one RCCX module from one chromosome to another, and the location of the crossover point determines the composition of the resultant hybrid. As a result of the unequal crossover, one bimodular chromosome loses one RCCX module and becomes monomodular, whereas the other chromosome gains one RCCX module and becomes trimodular (Fig. 3A.6). Depending on the site of crossover, there are three scenarios regarding inheritance of the active CYP21A2 gene. It should be noted that it is often difficult to pinpoint the exact crossover point because of the high sequence similarity between the RCCX modules.

  

1. C  rossover at C4 results in C4A/C4B and C4B/C4A chimeras, both of which are functional (Fig. 3A.6A). With respect to CYP21, one chromosome loses the CYP21A1P pseudogene but retains the CYP21A2 active gene (Fig. 3A.6Ai), whereas the other chromosome inherits two copies of the CYP21A1P pseudogene and one copy of the CYP21A2 active gene (Fig. 3A.6Aii). Since each of the chromosomes carries one copy of the CYP21A2 gene, crossover at C4 does not result in 21-hydroxylase deficiency. 2. Crossover at CYP21 genes, on the other hand, results in one chromosome bearing a CYP21A1P/ CYP21A2 chimera (Fig. 3A.6Bi) and the other chromosome bearing a CYP21A2/CYP21A1P chimera and a functional CYP21A2 gene (Fig. 3A.6Bii). Progeny inheriting the CYP21A1P/ CYP21A2 chimera (Fig. 3A.6Bi) will be at risk for 21OHD, as the chimera carries disease-causing mutations that affect the function of 21-hydroxylase. Based on the location of the crossover, nine CYP21A1P/CYP21A2 chimeras have been classified (CH1–CH9) [67,78–84] (Fig. 3A.7). CH-4 and CH-9 carry the pseudogene promoter and the P30L mutation. Since these two mutations are rather mild, CH-4 and CH-9 chimera types are usually associated with moderate simple-virilizing CAH. The other seven chimera types are associated with severe salt-wasting CAH.

Molecular Mechanisms Creating Cyp21a2 Genetic Defects

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FIGURE 3A.5  Pseudogene-derived CYP21A2 mutation via gene conversion.  A. The RCCX modules display high sequence similarity. Misalignment of a telomeric module (gray boxes) and a centromeric module (open boxes) sometimes occurs between sister chromatids during mitosis (i). DNA sequences can be copied from the CYP21A1P pseudogene to the CYP21A2 active gene, thereby bringing disease-causing mutations to the active gene (ii). DNA sequences can also be copied from the CYP21A2 gene to the CYP21A1P pseudogene, replacing disease-causing mutations with wild-type normal sequences from the active gene (iii). However, owing to the extent of the mutations present in the CYP21A1P pseudogene, small-scale microconversion will not revert the pseudogene into an active gene.  B. The 3.4-kb CYP21A2 gene consists of 10 exons. There are 15 common mutations derived from CYP21A1P microconversion that render the CYP21A2 gene inactive. For the four promoter and one intronic mutations, nucleotide changes in the genomic sequence are displayed. For the 10 mutations in the coding region, nucleotide changes in the genomic and cDNA sequence, along with the amino acid residue changes in the protein sequence, are shown. The coding sequences are shown in open boxes, whereas the untranslated regions are in gray.

FIGURE 3A.6  Large-scale gene deletion via unequal crossover between two bimodular chromosomes.  Misalignment of a telomeric RCCX module (gray boxes) and a centromeric RCCX module (open boxes) can occur between homologous chromosomes during meiosis. Crossover can take place in the C4, CYP21, or TNX genes. The products of crossover between two bimodular chromosomes are one monomodular chromosome with one RCCX module deleted and one trimodular chromosome with RCCX module duplicated, resulting in an unequal crossover.  A. Crossover at the C4 genes results in CYP21A1P deletion in one chromosome (Ai) and CYP21A1P duplication in the other chromosome (Aii). Since each of the two chromosomes still carries one copy of the CYP21A2 active gene, unequal crossover at C4 does not result in CAH.  B. Crossover at the CYP21 genes results in a monomodular chromosome carrying a CYP21A1P/CYP21A2 chimera (Bi) and a trimodular chromosome carrying CYP21A1P, CYP21A2 genes, and an additional CYP21A2/CYP21A1P chimera (Bii). Offspring inheriting the monomodular chromosome (Bi) will be at risk for CAH. Offspring inheriting the trimodular chromosome (Bii) will not be affected with CAH because a copy of the CYP21A2 gene is present.  C. Crossover at the TNX genes results in CYP21A2 deletion in one chromosome (Ci) and CYP21A2 duplication in another chromosome (Cii). Offspring inherited the monomodular chromosome (Ci) will be at risk for CAH.

Molecular Mechanisms Creating Cyp21a2 Genetic Defects

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FIGURE 3A.7  CYP21A1P/CYP21A2 chimera types.  To date, nine types of CYP21A1P/CYP21A2 chimera (CH1–CH9) with different junction sites have been identified. These chimeras are ordered here according to the location of the junction sites with respect to the 5' end of the gene. The first two chimeras, CH-4 and CH-9, contain mild promoter and P30L mutations and are associated with moderate simple-virilizing CAH. The other seven chimeras contain severe mutations and are associated with salt-wasting CAH.

While the CYP21A2/CYP21A1P chimera (Fig. 3A.6Bii) is likely to be non-functional, there is a functional CYP21A2 copy on the same chromosome and so progeny inheriting this chromosome will not be affected with 21OHD. 3. Crossover at TNX genes results in a TNXA/TNXB chimera and a complete deletion of the CYP21A2 gene on one chromosome (Fig. 3A.6Ci), and a TNXB/TNXA chimera and a duplication of CYP21A2 on the other chromosome (Fig. 3A.6Cii). Progeny inheriting the chromosome lacking the CYP21A2 gene (Fig. 3A.6Ci) will be at risk for 21OHD.   

Whereas unequal crossover between two bimodular chromosomes generates monomodular and trimodular chromosomes, other configurations are also possible. For example, crossover between a bimodular chromosome and a trimodular chromosome can generate a monomodular and a quadrimodular product.

Non-Pseudogene-Derived and De Novo Mutations Over 90% of all 21OHD cases are caused by pseudogene-derived conversion or deletion. Non-pseudogenederived mutations are relatively rare and are observed

in less than 10% of 21OHD cases. De novo mutations are even rarer, and can be caused by:   

1. A  new meiotic mutation in one of the parents. 2. A new somatic mutation in the germline of one of the parents. 3. Uniparental isodisomy, where the offspring inherits two identical copies of mutated CYP21A2 from a parent who is a 21OHD carrier.   

In some cases, non-paternity may explain why a patient carries a mutation that is not present in the parents.

Genotype–Phenotype Association CAH caused by 21OHD is inherited in an autosomal recessive manner. The patient’s phenotype is usually determined by the milder affected allele. Thus, the presence of two severely affected alleles results in classical 21OHD, whereas the presence of at least one mildly affected allele usually results in non-classical 21OHD. There is generally a good correlation between the severity of the clinical disease and the discrete mutations observed. On the other hand, several studies have demonstrated that this correlation is less than perfect

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3A.  CONGENITAL ADRENAL HYPERPLASIA OWING TO 21-HYDROXYLASE DEFICIENCY

6. 7. 8. 9.

 igase detection of amplified fragments reaction [105]. L Cleavase fragment length polymorphism analysis [106]. Reverse dot-blot hybridization [107]. Minisequencing [108].

  

CLINICAL FEATURES FIGURE 3A.8  Genotype–phenotype association in CAH owing to 21OHD.  There is a high genotype–phenotype concordance in the V281L, del, G110fs, E6 cluster, L307fs, Q318X, and R356W mutations. In contrast, P30L, I172N, and I2G mutations show considerable genotype–phenotype variability.

[20,85–88]. In the largest 21OHD genotype–phenotype study to date, about half of the genotypes are associated with a definitive phenotype [88]. However, certain mutations, such as P30L, I2G, and I172N, show variable CAH phenotypes. While the P30L mutation is frequently associated with non-classical CAH, ∼30% of patients carrying the P30L mutation display classical CAH phenotypes. Similarly, the I2G mutation is mostly associated with salt-wasting CAH but is also observed in 20% of simple-virilizing CAH patients. Likewise, although the I172N mutation is usually associated with ­simple-virilizing CAH, it is also seen in ∼25% of saltwasting CAH patients (Fig. 3A.8). Nonetheless, there is a high genotype–­phenotype concordance in the V281L, large gene deletion, G110fs, E6 cluster, L307fs, Q318X, and R356W mutations.

Molecular Diagnosis of CAH Owing to 21OHD Before cloning of the CYP21A2 gene, genetic linkage analysis through HLA haplotyping was used to determine inheritance of the mutant allele [27,89,90]. Characterization of the CYP21A2 gene [70,71] provides the genetic basis for other diagnostic methods that are more accurate and robust. These methods include:   

1. A  llele-specific polymerase chain reaction (PCR) with oligonucleotide primers that have a single-base mismatch at the 3’ end [91–93]. 2. Hybridization of allele-specific oligonucleotides to digested genomic DNA on Southern blots [94] or in dried agarose gels [95–97]. 3. Restriction fragment length polymorphism analysis of either genomic DNA or PCR products with restriction enzymes (e.g. TaqI) chosen to recognize sequence differences [98]. 4. Amplification-generated restriction sites, where an enzyme restriction site is created by part of an oligonucleotide and part of either the normal or the mutated sequence [99–102]. 5. Direct sequencing of PCR products [103,104].

External Genitalia Females with classical 21OHD generally present at birth with masculinization of the genitalia. Adrenocortical function begins around the seventh week of gestation [109]; thus, a female fetus with classical CAH is exposed to adrenal androgens at the critical time of sexual differentiation (approximately 9–15 weeks gestational age). Androgens interact with the receptors on genital skin and induce changes in the developing external female genitalia. This leads to clitoral enlargement, fusion, and scrotalization of the labial folds, and rostral migration of the urethral/vaginal perineal orifice, placing the phallus in the male position. The degree of genital virilization may range from mild clitoral enlargement alone to, in rare cases, a penile urethra. Degrees of genital v ­ irilization are classified into five Prader stages [110] (Fig. 3A.9).

Internal Genitalia In contrast to the masculinization of the external genitalia, internal female genitalia, such as the uterus, Fallopian tubes, and ovaries, develop normally. Females with CAH do not have testicular tissue and do not produce anti-Müllerian hormone (AMH), which is produced by the testicular Sertoli cells. These internal female structures are Müllerian derivatives. Therefore, the affected female who is born with masculinized external genitalia will have a normal uterus, normal Fallopian tubes, and normal ovaries. 46, XX females with classical CAH have the internal genitalia potential for fertility.

Puberty In the majority of patients treated in recommended doses from early life, the onset of puberty in both girls and boys with classical 21OHD occurs at the expected chronological age. However, a recent study showed that the mean ages at onset of puberty in both males and females were somewhat younger than the general population, but did not differ significantly among the three forms of 21OHD [111]. In those who are inadequately treated, advanced epiphyseal development results, which may lead to central precocious puberty. In those with advanced

Clinical Features

41

FIGURE 3A.9  Different degrees of virilization according to the scale developed by Prader.  Stage I: clitoromegaly without labial fusion. Stage II: clitoromegaly and posterior labial fusion. Stage III: greater degree of clitoromegaly, single perineal urogenital orifice, and almost complete labial fusion. Stage IV: increasingly phallic clitoris, urethra-like urogenital sinus at base of clitoris, and complete labial fusion. Stage V: penile clitoris, urethral meatus at tip of phallus, and scrotum-like labia (appear like males without palpable gonads).

epiphyseal maturation at the initial presentation owing to exposure to elevated androgens, the sudden decreased androgen levels after initiation of glucocorticoid treatment may cause an early activation of the hypothalamic–pituitary–gonadal axis. Studies suggest that excess adrenal androgens (aromatized to estrogens) inhibit the pubertal pattern of gonadotropin secretion by the hypothalamic–pituitary axis. This inhibition, which probably takes place via a negative feedback effect, can be reversed by glucocorticoid treatment [111,112]. Following the onset of puberty, in a majority of successfully treated patients, the milestones of further development of secondary sex characteristics in general appear to be normal [111,112]. In adolescents and adults signs of hyperandrogenism may include malepattern alopecia (temporal balding) and acne. Female patients may develop hirsutism and menstrual irregularities. Although the expected age of menarche may be delayed in females with classical CAH [113], when adequately treated many have regular menses after menarche [114]. Menstrual irregularity and secondary amenorrhea with or without hirsutism occur in a subset of postmenarchal females, especially those in poor hormonal control. Primary amenorrhea or delayed menarche can occur if a woman with classical CAH is untreated, inadequately treated, or overtreated with glucocorticoids [115]. In addition, women with CAH may develop PCOS [116].

Growth Lack of appropriate postnatal treatment in boys and girls results in continued exposure to excessive androgens, causing progressive penile or clitoral

enlargement, the development of premature pubic hair (pubarche), axillary hair, and acne. Advanced somatic and epiphyseal development occurs with exaggerated growth during childhood. This rapid linear growth is usually accompanied by premature epiphyseal maturation and closure, resulting in a final adult height that is typically below that expected from parental heights (on average –1.1 to –1.5 SD below the midparental target height) [117,118]. This is, on average, 10 cm below the mid-parental height [34,119]. On the other hand, poor growth can occur in patients with 21OHD as a result of excess glucocorticoid treatment. Short stature occurs even in patients with good hormonal adrenal control. A study of growth hormone therapy alone or in combination with a gonadotropin-releasing hormone (GnRH) analog in CAH patients with compromised height prediction showed improvement in short- and long-term growth to reduce the height deficit [34,119,120].

Fertility In a retrospective survey of fertility rates in a large group of females with classical CAH, simple-virilizers were shown to be more likely to become pregnant and carry the pregnancy to term. Adequate ­glucocorticoid therapy is probably an important variable with respect to fertility outcome. The development of PCOS in CAH patients is not uncommon and may be related to both prenatal and postnatal excess androgen exposure, which can affect the hypothalamic–pituitary–gonadal axis. An inadequate vaginal introitus can affect up to a third of classical CAH adult females. Since vaginal dilatation is needed to maintain good patency, vaginoplasty is delayed until sexual intercourse is regular or

42

3A.  CONGENITAL ADRENAL HYPERPLASIA OWING TO 21-HYDROXYLASE DEFICIENCY

when the patient can assume responsibility for vaginal ­dilatation [121]. Males with CAH, particularly if poorly treated, may have reduced sperm counts and low testosterone as a result of small testes owing to suppression of gonadotropins. Intratesticular adrenal rests may contribute to impaired fertility [34,46]. All of these complications may result in impaired fertility. In male patients with classical CAH, several long-term studies indicate that those who have been adequately treated undergo normal pubertal development, have normal testicular function, and normal spermatogenesis and fertility [122,123]. However, small testes and aspermia may occur in patients as a result of inadequately controlled disease [124,125]. Testicular adrenal rest tumor may lead to end-stage damage of testicular parenchyma as a result of longstanding obstruction of the seminiferous tubules [126]. In contrast, some investigators have reported normal testicular maturation as well as normal spermatogenesis and fertility in patients who had never received glucocorticoid treatment [127]. Studies demonstrate that postpubertal males with inadequately treated CAH are at a very high risk of developing hyperplastic nodular testes. In one study, almost all these patients were found to have adenomatous adrenal rests within the testicular tissue, as indicated by the presence of specific 11β-hydroxylated steroids in the blood from gonadal veins [128]. These tumors have been reported to be ACTH-dependent and to regress following adequate steroid therapy [129–132]. These testicular adrenal rests are more frequent in males with salt-wasting CAH and are associated with an increased risk of infertility [46,133]. Regular testicular examination and periodic testicular ultrasonography are recommended for early detection of testicular lesions.

Gender Role Behavior and Cognition Please refer to Chapter 10, “Psychoendocrinology of Congenital Adrenal Hyperplasia.”

TREATMENT Hormone Replacement The goal of therapy in CAH is both to correct the deficiency in cortisol secretion and to suppress ACTH overproduction. Proper treatment with glucocorticoid reduces stimulation of the androgen pathway, thus preventing further virilization and allowing normal growth and development. The usual requirement of hydrocortisone (or its equivalent) for the treatment of the classical 21OHD form of CAH is about 10–15 mg/m2/day

divided into two or three doses per day. Dosage requirements for patients with non-classical 21OHD CAH are typically less. Adults may be treated with the longer ­acting dexamethasone, alone or in combination with hydrocortisone. A small dose of dexamethasone at bedtime (0.25– 0.5 mg) is usually adequate for androgen suppression in ­non-classical patients. Anti-androgen treatment may be useful as adjunctive therapy in adult women who continue to have hyperandrogenic signs despite good adrenal suppression. Females with concomitant PCOS may benefit from an oral contraceptive. Treatment of adult males with non-classical 21OHD may not be necessary, although our group has found that it is helpful in preventing adrenal rest tumors and preserving fertility. Optimal corticosteroid therapy is determined by adequate suppression of adrenal hormones balanced against normal physiological parameters. The goal of corticosteroid therapy is to give the lowest dose required for optimal control. Adequate biochemical control is assessed by measuring serum levels of 17-OHP and androstenedione; serum testosterone can be used in females and prepubertal males (but not in newborn males). We recommend that hormone levels are measured at a consistent time in relation to medication dosing, usually 2 hours after the morning corticosteroid. Titration of the dose should be aimed at maintaining androgen levels at age- and sex-appropriate levels and 17-OHP levels of <1000 ng/dL, and less than 200ng/dL for androstenedione. Overtreatment should be avoided because it can lead to Cushing’s syndrome and growth suppression. Depending on the degree of stress, stress dose coverage may require doses of up to 50–100 mg/m2/day. Patients with salt-wasting CAH have elevated plasma renin in response to the sodium-deficient state, and they require treatment with the salt-retaining 9α-fludrocortisone acetate. The average dose is 0.1 mg daily, ranging from 0.05–0.2 mg daily. Infants should also be started on salt supplementation, as sodium chloride, at 1–2 g daily, divided into several feedings [53]. Although patients with the simple-virilizing and nonclassical forms of CAH can make adequate aldosterone, the aldosterone-to-renin ratio (ARR) has been found to be lower than normal, though not to the degree seen in the salt-wasting form [134]. It has not been customary to supplement conventional glucocorticoid replacement therapy with the administration of salt-retaining steroids in the simple-virilizing and non-classical forms of CAH, though there are reports that adding fludrocortisone to patients with elevated PRA may improve hormonal control of the disease [135]. The requirement for fludrocortisone appears to diminish with age, and oversuppression of the PRA should be avoided to prevent complications from hypertension and excessive mineralocorticoid activity. Measurements of plasma renin and aldosterone are used to monitor the efficacy of mineralocorticoid

Prenatal Diagnosis and Treatment

therapy in all patients with the salt-wasting form of the disease. Because patients with CAH are at risk for short stature as adults, other adjunct therapies are being utilized. Studies have demonstrated significant improvement in growth velocity, final adult height prediction [35], and final adult height [34,119] with the use of growth hormone in conjunction with a GnRH analog. Please see Chapter 3B for a detailed description of treatments aimed to improve stature in patients with CAH. In non-life-threatening periods of illness or physiologic stress, the corticosteroid dose should be increased to two or three times the maintenance dose for the duration of that period. Each family should be given injection kits of hydrocortisone for emergency use (25 mg for infants, 50 mg for children who weigh less than 40 kg, and 100 mg for children who weigh more than 40 kg, and for adults). In the event of a surgical procedure, a total of five to ten times the daily maintenance dose may be required during the first 24–48 hours, which can then be tapered over the following days to the normal preoperative schedule. Stress doses of dexamethasone should not be given because of the delayed onset of action. It is not necessary for increased mineralocorticoid doses during these periods of stress. It is imperative for all patients who are receiving corticosteroid replacement therapy, including patients with CAH, to wear a Medic-Alert bracelet or medallion that will enable correct and appropriate therapy in case of emergencies. Additionally, all responsible family members should be trained in the intramuscular administration of hydrocortisone.

Surgery Please also refer to Chapter 9. The aim of surgical repair in females with ambiguous genitalia caused by CAH, when the decision is made by parents or patients themselves, is generally to remove the redundant erectile tissue, preserve the sexually sensitive glans clitoris, and provide a normal vaginal orifice that functions adequately for menstruation, intromission, and delivery. A medical indication for early surgery other than for sex assignment is recurrent urinary tract infections as a result of pooling of urine in the vagina or urogenital sinus. In the past, it was routine to recommend early corrective surgery for neonates born with ambiguous genitalia. However, in recent years, the implementation of early corrective surgery has become increasingly controversial owing to lack of data on long-term functional outcome. Recent data show that genital sensitivity is impaired in areas where feminizing genital surgery had been done, leading to difficulties in sexual function [136]. Another recent study showed that patients with more severe mutations in the CYP21A2 gene, i.e. those with

43

the null genotype and thus those more severely virilized, had more surgical complications than those less severely virilized and were less satisfied with their sexual life [137]. Because of the scarcity of this data, the role of the parents in sex assignment becomes crucial in all aspects of the decision-making process, and should include full discussion of the controversy and all possible therapeutic options for the intersex child, particularly early versus delayed surgery. Further, a multidisciplinary case-by-case approach, involving pediatric endocrinology, urology, genetics, and psychoendrocrinology, is imperative when considering sex assignment and possible surgical repair.

Other Treatment Strategies Glucocorticoid replacement has been an effective treatment for CAH for over 50 years and remains its primary therapy; however, the management of these patients presents a challenge because inadequate treatment, as well as oversuppression, may cause complications. Bilateral adrenalectomy is a radical but effective measure in some cases; however, it should be conserved for patients whose genotypes consist of null mutations. A few patients who were extremely difficult to control with medical therapy alone showed improvement in their symptoms after bilateral adrenalectomy [138,139]. Because this approach renders the patient completely adrenal-insufficient, however, it should be reserved for extreme cases and is not a good treatment option for patients who have a history of poor compliance with medication.

PRENATAL DIAGNOSIS AND TREATMENT The earliest accounts of prenatal diagnosis are surprisingly crude, consisting principally of a physician palpating a pregnant woman’s abdomen to assess fetal growth. It was not until the 1950s, with the development of amniocentesis and the optimization of techniques to culture fetal cells from amniotic fluid, that modern day prenatal diagnosis began [140]. Building on these advances, the first prenatal diagnosis of CAH was based on hormonal measurements of amniotic fluid obtained by amniocentesis. In 1965, Jeffcoate et al. reported the first successful prenatal diagnosis of 21OHD, based on elevated levels of 17-ketosteroids and pregnanetriol in the amniotic fluid [141]. A decade later, Frasier et al. improved the method by establishing the presence of elevated levels of testosterone [142] and 17α-hydroxyprogesterone (17OHP) [143] in the amniotic fluid of CAH-affected fetuses obtained as early as the second trimester. Subsequently, this assay, which measures elevated levels of 17OHP in the amniotic fluid, was widely adopted as diagnostic for fetal CAH.

44

3A.  CONGENITAL ADRENAL HYPERPLASIA OWING TO 21-HYDROXYLASE DEFICIENCY

However, advances in genotyping of the CYP21A2 gene have made molecular genetic studies of fetal DNA the ­preferred method to diagnose 21OHD. Chorionic villus sampling (CVS), rather than amniocentesis, is the preferred diagnostic procedure for obtaining tissue for molecular genetic analysis, as CVS can be performed at the weeks 9–11 of gestation, while amniocentesis is usually performed in the second trimester. Early prenatal diagnosis is particularly important since dexamethasone treatment must be initiated during the first trimester to prevent masculinization of the female genitalia. Because only affected females should be treated until term and only ¼ of the fetuses will be affected and ½ will be males, seven out of eight fetuses do not require treatment. Amniocentesis, which is performed later in gestation than CVS, results in treatment of unaffected fetuses for a longer period of time than CVS. Nonetheless, pitfalls do occur in a small percentage of the patients undergoing prenatal diagnosis utilizing genetic diagnosis, such as undetectable mutations [144], allele dropouts [145], or maternal DNA contamination. Determination of satellite markers may increase the accuracy of molecular genetic analysis [146]. In 21OHD, prenatal treatment with dexamethasone was introduced in France in 1978 [147] and in the USA in 1986 [148]. Institution of therapy before the ninth week of gestation, prior to the onset of adrenal androgen secretion, effectively suppresses excessive adrenal androgen production and prevents masculinization of external female genitalia in the female fetus affected with CAH. Dexamethasone is used because it binds minimally to cortisol binding globulin (CBG) in the maternal blood and, unlike hydrocortisone, it escapes inactivation by the placental ­ 11-dehydrogenase enzyme. Thus, dexamethasone crosses the placenta from the mother to the fetus and suppresses ACTH secretion with longer halflife compared to other synthetic steroids [148]. When dexamethasone administration begins as early as the eighth week of gestation, the treatment is blind to the disease status and sex of the fetus. If the fetus is later determined to be a male upon karyotype or an unaffected female upon DNA analysis, treatment is discontinued. Otherwise, treatment is continued to term. A simplified algorithm of management of potentially affected pregnancies is shown in Figure 3A.10. The optimal dosage is 20 μg/kg/day of dexamethasone per maternal pre-pregnancy body weight, in three divided daily doses, up to a maximum dose of 1.5 mg per day [149]. It is recommended to start the treatment as soon as pregnancy is confirmed, and no later than 9 weeks after the last menstrual period [150]. The mother’s blood pressure, weight, glycosuria, HbA1C, symptoms of edema, striae, and other possible adverse effects of dexamethasone treatment should be carefully observed throughout pregnancy [186].

Outcome of Prenatal Treatment of 21OHD Prenatal treatment of 21OHD has proven to be successful in significantly reducing genital masculinization in affected females. Our group has performed prenatal diagnosis in over 700 pregnancies at risk for 21OHD, and 63 affected female fetuses have been treated to term [151]. Treatment was highly effective in preventing genital ambiguity when the mother was compliant until term. In all the female fetuses treated to term, the degree of masculinization was on average 1.69, as measured using the Prader scoring system (Fig. 3A.9). Late treatment as well as no treatment resulted in much greater masculinization, and the average Prader score was 3.73 for those female fetuses not treated [148,151]. Another group corroborated these findings and reported decreased masculinization of the external genitalia in an affected female fetus prenatally treated with dexamethasone as compared to a similarly affected female sibling (index case) which was not treated [152]. Not only does prenatal treatment effectively minimize the degree of female genital masculinization in patients, it also decreases the high-level androgen exposure of the brain during differentiation. Elevated levels of androgens in the developing fetal brain are thought to cause a higher tendency to gender ambiguity in some females with CAH [153,154]. Genital masculinization in female newborns with classical 21OHD CAH has potential adverse psychosocial implications that may be alleviated by prenatal treatment [149]. Prenatal dexamethasone treatment has recently been a subject of controversy and heated debate [155–160]. Some uncertainties and concerns have been expressed about the long-term safety of prenatal diagnosis and treatment [161,162]. Concerns have been raised in regard to the glucocorticoid effects on the fetal brain, which arise from studies of other conditions rather than direct studies on prenatal treatment of 21OHD CAH. These include studies whereby much higher doses of dexamethasone were given to human subjects later in pregnancy [163] or to animals [164,165], and therefore hold little relevance to using dexamethasone prenatally in CAH. In a small-sample study of children prenatally treated with dexamethasone, Lajic and her colleagues found no effects on intelligence, handedness, memory encoding, or long-term memory, but short-term-treated CAH-unaffected children had significantly poorer performance than controls on a test of verbal working memory. These patients also had lower questionnaire scores in self-perceived scholastic competence and social anxiety [166]. However, parents described these children as more sociable than controls, without significant difference in psychopathology, school performance, adaptive functioning, or behavioral problems [167]. A larger study led by our group indicated no adverse cognitive effects

Prenatal Diagnosis and Treatment

45

FIGURE 3A.10  Algorithm with recommendations for prenatal management of pregnancies where the fetus is at risk for 21OHD. Dexa-

methasone is given orally to the mother at 20 μg/kg pregnancy weight per day (divided into two or three doses) beginning before the ninth week of gestation. Prenatal diagnosis will be performed using fetal DNA samples obtained either by chorionic villus sampling (CVS) at gestational week 9–11 or amniocentesis at gestational week 15–18. Dexamethasone treatment will be stopped if the fetus is a male or an unaffected female. If the fetus is an affected female, the mother will be treated with dexamethasone to term. For all treated and untreated cases, the following neonatal confirmation and postnatal follow-up will be performed: the birthweight, birth length, head circumference, developmental assessment, and other physical findings will be recorded. 17-Hydroxyprogesterone levels from cord blood and post-72-hour blood sample, either from dried blood spot on filter paper or from whole blood (serum), will be measured. For affected infants, electrolytes and plasma renin activity (PRA) will be determined. A peripheral blood sample will also be obtained for DNA analysis.

of short-term prenatal dexamethasone exposure, including no adverse influence on verbal working memory. By contrast, long-term prenatal dexamethasone exposure in a small sample of girls affected with CAH showed lower

scores on several neuropsychological tests, which contribute to concerns about potentially detrimental cognitive effects of prenatal dexamethasone treatment [168]. However, given the variability of cognitive findings

46

3A.  CONGENITAL ADRENAL HYPERPLASIA OWING TO 21-HYDROXYLASE DEFICIENCY

in dexamethasone-unexposed CAH-affected patients, retrospective study design, and low statistical power owing to a small sample size, this result cannot be linked to dexamethasone with certainty. This indicates that further studies are needed. Compelling data from large cohorts of pregnancies with prenatal diagnosis and treatment of 21OHD CAH prove its efficacy and safety [148]. In our studies all the mothers who received prenatal treatment (partial or fullterm) stated that they would take dexamethasone again for a future pregnancy [169]. Rare adverse events have been reported in treated children, but no harmful effects have been documented that can be clearly attributed to the treatment [170]. Another long-term follow-up study in Scandinavia showed that 44 children who were variably treated prenatally demonstrated normal prenatal and postnatal growth compared to matched controls. Further, there was no observed increase in fetal abnormalities or fetal death [152]. Although some abnormalities in postnatal growth and behavior were observed among dexamethasone-exposed offspring, none could logically be explained by the present knowledge of teratogenic effects of glucocorticoids. Nonetheless, the group has halted further recruitment of patients for an ongoing prospective study of prenatal dexamethasone treatment of CAH in Sweden [171]. No significant or enduring side effects were noted in either the mothers or the fetuses. Greater weight gain in treated versus untreated mothers did occur, as well as the presence of striae and edema. Excessive weight gain was lost after birth. No differences were found regarding gestational diabetes or hypertension [150]. No cases have been reported of cleft palate, placental degeneration, or fetal death, which have been observed in the rodent model of in utero exposure to high-dose glucocorticoids [172]. One explanation for the safety of human versus rodent is that glucocorticoid receptor–ligand systems in humans differ from those of rodents [173]. Most recently, we conducted a comprehensive, long-term outcome study looking at 149 male and female patients 12 years of age and older, affected and unaffected with CAH, who were treated with dexamethasone partially or to term. To date, this is the largest study evaluating the long-term effects of dexamethasone. We found no adverse effects, such as increased risk for cognitive defects, disorders of gender identity and behavior, sexual function in adulthood, hypertension, diabetes, and osteopenia [174]. The general consensus for prenatal dexamethasone treatment is inconclusive; however, what is agreed upon is the need for continuing long-term prospective and retrospective studies. Assessment methods should include a battery of tests that will allow measurement of both behavioral and somatic outcomes, in order to arrive at a definitive conclusion about the efficacy and safety of prenatal dexamethasone treatment.

Preimplantation Genetics Please see Chapter 13C. Preimplantation genetic diagnosis (PGD) identifies genetic abnormalities in preimplantation embryos prior to embryo transfer, so only unaffected embryos established from in vitro fertilization (IVF) are transferred. The procedure has been utilized in many monogenic recessive disorders such as cystic fibrosis, hemoglobinopathies, spinal muscular atrophy, and Tay–Sach’s disease. PGD is being used for a growing number of genetic diseases [175]. There is only one report of PGD utilized in a family in whom offspring were at risk for CAH [176]; however, we know from experience that families are seeking PGD with greater frequency. It would be desirable to have further studies of preimplantation diagnosis in CAH families. The future may hold promise for prenatal diagnosis of CAH. Circulating cell-free fetal DNA has been detected in maternal plasma from the seventh week of gestation [177,178] (before dexamethasone treatment must be initiated for CAH). It has been used for several monogenic disorders [179] but only once for CAH [180]. The first non-invasive prenatal diagnosis application translated into clinical practice is fetal sex determination, which can be reliably performed from the seventh week of gestation to detect sequences from the Y chromosome of male fetuses in maternal plasma [181]. This test stratifies the need for antenatal dexamethasone therapy for CAH and excludes males from prenatal treatment [182]. In addition, the advent of next-generation sequencing technology has enabled whole genome sequencing of the fetus [183–185]. The development of novel methods for non-invasive prenatal diagnosis for genetic disorders deserves careful investigation.

References [1]  Ganong WF. Neurotransmitters and pituitary function: regulation of ACTH secretion. Fed Proc 1980;39(11):2923–30. [2]  Rivier CL, Plotsky PM. Mediation by corticotropin releasing factor (CRF) of adenohypophysial hormone secretion. Annu Rev Physiol 1986;48:475–94. [3]  Scott LV, Dinan TG. Vasopressin and the regulation of hypothalamic-pituitary-adrenal axis function: implications for the pathophysiology of depression. Life Sci 1998;62(22):1985–98. [4]  Lifton RP, Gharavi AG, Geller DS. Molecular mechanisms of human hypertension. Cell 2001;104(4):545–56. [5]  Ganong W. Cortisol and aldosterone secretion. In: Martini L, Gordan G, Sciarra F, editors. Steroid Modulation of Neuroendocrine Function, Sterols, Steroids, and Bone Metabolism. New York: Elsevier; 1984. p. 111. [6]  Rainey WE. Adrenal zonation: clues from 11beta-hydroxylase and aldosterone synthase. Mol Cell Endocrinol 1999;151(1–2):151–60. [7]  Liggins G. Endocrinology of Parturition. In: Novy MJ, Resco JA, editors. Fetal Endocrinology. New York: Academic Press; 1981. p. 211. [8]  Hornsby P. The regulation of adrenocortical function by control of growth and structure. In: Anderson DC, Winter JSD, editors. Adrenal Cortex. London: Butterworths; 1985. p. 1.

References

[9]  Ishimoto H, Jaffe RB. Development and function of the human fetal adrenal cortex: a key component in the feto-placental unit. Endocr Rev 2011;32(3):317–55. [10]  New MI, Seaman MP. Secretion rates of cortisol and aldosterone precursors in various forms of congenital adrenal hyperplasia. J Clin Endocrinol Metab 1970;30(3):361–71. [11]  New MI, Wilson RC. Steroid disorders in children: congenital adrenal hyperplasia and apparent mineralocorticoid excess. Proc Natl Acad Sci U S A 1999;96(22):12790–7. [12]  Klein R. Evidence for and against the existence of a salt-losing hormone. J Pediatr 1960;57:452–60. [13]  Kowarski A, Finkelstein JW, Spaulding JS, Holman GH, Migeon CJ. Aldosterone Secretion Rate in Congenital Adrenal Hyperplasia. A Discussion of the Theories on the Pathogenesis of the SaltLosing Form of the Syndrome. J Clin Invest 1965;44:1505–13. [14]  Tusie-Luna MT, Traktman P, White PC. Determination of functional effects of mutations in the steroid 21-hydroxylase gene (CYP21) using recombinant vaccinia virus. J Biol Chem 1990;265(34):20916–22. [15]  Kuhnle U, Land M, Ulick S. Evidence for the secretion of an antimineralocorticoid in congenital adrenal hyperplasia. J Clin Endocrinol Metab 1986;62(5):934–40. [16]  Stoner E, Dimartino-Nardi J, Kuhnle U, Levine LS, Oberfield SE, New MI. Is salt-wasting in congenital adrenal hyperplasia due to the same gene as the fasciculata defect? Clin Endocrinol (Oxf) 1986;24(1):9–20. [17]  Speiser PW, Agdere L, Ueshiba H, White PC, New MI. Aldosterone synthesis in salt-wasting congenital adrenal hyperplasia with complete absence of adrenal 21-hydroxylase. N Engl J Med 1991;324(3):145–9. [18]  Verkauf BS, Jones Jr HW. Masculinization of the female genitalia in congenital adrenal hyperplasia: relationship to the salt losing variety of the disease. South Med J 1970;63(6):634–8. [19]  Wilson RC, Mercado AB, Cheng KC, New MI. Steroid 21-hydroxylase deficiency: genotype may not predict phenotype. J Clin Endocrinol Metab 1995;80(8):2322–9. [20]  Krone N, Braun A, Roscher AA, Knorr D, Schwarz HP. Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany. J Clin Endocrinol Metab 2000;85(3):1059–65. [21]  Rosenbloom AL, Smith DW. Varying expression for salt losing in related patients with congenital adrenal hyperplasia. Pediatrics 1966;38(2):215–9. [22]  Bormann M, Kochhan L, Knorr D, Bidlingmaier F, Olek K. Clinical heterogeneity of 21-hydroxylase deficiency of sibs with identical 21-hydroxylase genes. Acta Endocrinol (Copenh) 1992;126(1):7–9. [23]  Chemaitilly W, Betensky BP, Marshall I, Wei JQ, Wilson RC, New MI. The natural history and genotype-phenotype nonconcordance of HLA identical siblings with the same mutations of the 21-hydroxylase gene. J Pediatr Endocrinol Metab 2005;18(2):143–53. [24]  White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000;21(3):245–91. [25]  Decourt J, Jayle MF, Baulieu E. Clinically late virilism with excretion of pregnanetriol and insufficiency of cortisol production. Ann Endocrinol (Paris) 1957;18(3):416–22. [26]  Abraham GE, Odell WD, Swerdloff RS, Hopper K. Simultaneous radioimmunoassay of plasma FSH, LH, progesterone, 17-hydroxyprogesterone, and estradiol-17 beta during the menstrual cycle. J Clin Endocrinol Metab 1972;34(2):312–8. [27]  Dupont B, Oberfield SE, Smithwick EM, Lee TD, Levine LS. Close genetic linkage between HLA and congenital adrenal hyperplasia (21-hydroxylase deficiency). Lancet 1977;2(8052–8053): 1309–12.

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[28]  Laron Z, Pollack MS, Zamir R, Roitman A, Dickerman Z, Levine LS, et al. Late onset 21-hydroxylase deficiency and HLA in the Ashkenazi population: a new allele at the 21-hydroxylase locus. Hum Immunol 1980;1(1):55–66. [29]  Pollack MS, Levine LS, O'Neill GJ, Pang S, Lorenzen F, Kohn B, et al. HLA linkage and B14, DR1, BfS haplotype association with the genes for late onset and cryptic 21-hydroxylase deficiency. Am J Hum Genet 1981;33(4):540–50. [30]  Levine LS, Dupont B, Lorenzen F, Pang S, Pollack M, Oberfield SE, et al. Genetic and hormonal characterization of cryptic 21-hydroxylase deficiency. J Clin Endocrinol Metab 1981;53(6):1193–8. [31]  New MI. Extensive Clinical Experience: Nonclassical 21-Hydroxylase Deficiency. J Clin Endo Metab 2006;91:4222–4231. [32]  Kohn B, Levine LS, Pollack MS, Pang S, Lorenzen F, Levy D, et al. Late-onset steroid 21-hydroxylase deficiency: a variant of classical congenital adrenal hyperplasia. J Clin Endocrinol Metab 1982;55(5):817–27. [33]  Jaaskelainen J, Voutilainen R. Growth of patients with 21-hydroxylase deficiency: an analysis of the factors influencing adult height. Pediatr Res 1997;41(1):30–3. [34]  Lin-Su K, Harbison MD, Lekarev O, Vogiatzi MG, New MI. Final adult height in children with congenital adrenal hyperplasia treated with growth hormone. J Clin Endocrinol Metab 2011;96(6):1710–7. [35]  Marynick SP, Chakmakjian ZH, McCaffree DL, Herndon Jr JH. Androgen excess in cystic acne. N Engl J Med 1983;308(17):981–6. [36]  Rose LI, Newmark SR, Strauss JS, Pochi PE. Adrenocortical hydroxylase deficiencies in acne vulgaris. J Invest Dermatol 1976;66(5):324–6. [37]  Lucky AW, Rosenfield RL, McGuire J, Rudy S, Helke J. ­Adrenal androgen hyperresponsiveness to a­ drenocorticotropin in women with acne and/or hirsutism: adrenal enzyme defects and exaggerated adrenarche. J Clin Endocrinol Metab 1986;62(5):840–8. [38]  Lobo RA, Goebelsmann U. Adult manifestation of congenital adrenal hyperplasia due to incomplete 21-hydroxylase deficiency mimicking polycystic ovarian disease. Am J Obstet Gynecol 1980;138(6):720–6. [39]  Gibson M, Lackritz R, Schiff I, Tulchinsky D. Abnormal adrenal responses to adrenocorticotropic hormone in hyperandrogenic women. Fertil Steril 1980;33(1):43–8. [40]  Chetkowski RJ, DeFazio J, Shamonki I, Judd HL, Chang RJ. The incidence of late-onset congenital adrenal hyperplasia due to 21-hydroxylase deficiency among hirsute women. J Clin Endocrinol Metab 1984;58(4):595–8. [41]  Child DF, Bu'lock DE, Anderson DC. Adrenal steroidogenesis in hirsute women. Clin Endocrinol (Oxf) 1980;12(6):595–601. [42]  Chrousos GP, Loriaux DL, Mann D, Cutler Jr GB. Late-onset 21-hydroxylase deficiency is an allelic variant of congenital adrenal hyperplasia characterized by attenuated clinical expression and different HLA haplotype associations. Horm Res 1982;16(4):193–200. [43]  Kuttenn F, Couillin P, Girard F, Billaud L, Vincens M, Boucekkine C, et al. Late-onset adrenal hyperplasia in hirsutism. N Engl J Med 1985;313(4):224–31. [44]  Augarten A, Weissenberg R, Pariente C, Sack J. Reversible male infertility in late onset congenital adrenal hyperplasia. J Endocrinol Invest 1991;14(3):237–40. [45]  Bonaccorsi AC, Adler I, Figueiredo JG. Male infertility due to congenital adrenal hyperplasia: testicular biopsy findings, hormonal evaluation, and therapeutic results in three patients. Fertil Steril 1987;47(4):664–70. [46]  Cabrera MS, Vogiatzi MG, New MI. Long term outcome in adult males with classic congenital adrenal hyperplasia. J Clin Endocrinol Metab 2001;86(7):3070–8.

48

3A.  CONGENITAL ADRENAL HYPERPLASIA OWING TO 21-HYDROXYLASE DEFICIENCY

[47]  Pang S, Levine LS, Chow DM, Faiman C, New MI. Serum androgen concentrations in neonates and young infants with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Clin Endocrinol (Oxf) 1979;11(6):575–84. [48]  Korth-Schutz S, Virdis R, Saenger P, Chow DM, Levine LS, New MI. Serum androgens as a continuing index of adequacy of treatment of congenital adrenal hyperplasia. J Clin Endocrinol Metab 1978;46(3):452–8. [49]  Shackleton CH. Profiling steroid hormones and urinary steroids. J Chromatogr 1986;379:91–156. [50]  Caulfield MP, Lynn T, Gottschalk ME, Jones KL, Taylor NF, Malunowicz EM, et al. The diagnosis of congenital adrenal hyperplasia in the newborn by gas chromatography/mass spectrometry analysis of random urine specimens. J Clin Endocrinol Metab 2002;87(8):3682–90. [51]  Pang S, Hotchkiss J, Drash AL, Levine LS, New MI. Microfilter paper method for 17 alpha-hydroxyprogesterone radioimmunoassay: its application for rapid screening for congenital adrenal hyperplasia. J Clin Endocrinol Metab 1977;45(5):1003–8. [52]  Therrell Jr BL, Berenbaum SA, Manter-Kapanke V, Simmank J, Korman K, Prentice L, et al. Results of screening 1.9 million Texas newborns for 21-hydroxylase-deficient congenital adrenal hyperplasia. Pediatrics 1998;101(4 Pt 1):583–90. [53]  Speiser PW, Azziz R, Baskin LS, Ghizzoni L, Hensle TW, Merke DP, et al. A Summary of the Endocrine Society Clinical Practice Guidelines on Congenital Adrenal Hyperplasia due to Steroid 21-Hydroxylase Deficiency. Int J Pediatr Endocrinol 2010;2010:494173. [54]  Gruneiro-Papendieck L, Prieto L, Chiesa A, Bengolea S, Bossi G, Bergada C. Neonatal screening program for congenital adrenal hyperplasia: adjustments to the recall protocol. Horm Res 2001;55(6):271–7. [55]  Janzen N, Peter M, Sander S, Steuerwald U, Terhardt M, Holtkamp U, et al. Newborn screening for congenital adrenal hyperplasia: additional steroid profile using liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab 2007;92(7):2581–9. [56]  Sarafoglou K, Banks K, Kyllo J, Pittock S, Thomas W. Cases of congenital adrenal hyperplasia missed by newborn screening in Minnesota. JAMA 2012;307(22):2371–4. [57]  New MI, Lorenzen F, Lerner AJ, Kohn B, Oberfield SE, ­Pollack MS, et al. Genotyping steroid 21-hydroxylase deficiency: hormonal reference data. J Clin Endocrinol Metab 1983;57(2):320–6. [58]  Gomez-Escobar N, Chou CF, Lin WW, Hsieh SL, Campbell RD. The G11 gene located in the major histocompatibility complex encodes a novel nuclear serine/threonine protein kinase. J Biol Chem 1998;273(47):30954–60. [59]  Sargent CA, Anderson MJ, Hsieh SL, Kendall E, Gomez-Escobar N, Campbell RD. Characterisation of the novel gene G11 lying adjacent to the complement C4A gene in the human major histocompatibility complex. Hum Mol Genet 1994;3(3):481–8. [60]  Shen L, Wu LC, Sanlioglu S, Chen R, Mendoza AR, Dangel AW, et al. Structure and genetics of the partially duplicated gene RP located immediately upstream of the complement C4A and the C4B genes in the HLA class III region. Molecular cloning, exonintron structure, composite retroposon, and breakpoint of gene duplication. J Biol Chem 1994;269(11):8466–76. [61]  Yang Z, Shen L, Dangel AW, Wu LC, Yu CY. Four ubiquitously expressed genes, RD (D6S45)-SKI2W (SKIV2L)-DOM3Z-RP1 (D6S60E), are present between complement component genes factor B and C4 in the class III region of the HLA. Genomics 1998;53(3):338–47. [62]  Carroll MC, Porter RR. Cloning of a human complement component C4 gene. Proc Natl Acad Sci U S A 1983;80(1):264–7. [63]  Belt KT, Carroll MC, Porter RR. The structural basis of the multiple forms of human complement component C4. Cell 1984;36(4):907–14.

[64]  Carroll MC, Belt T, Palsdottir A, Porter RR. Structure and organization of the C4 genes. Philos Trans R Soc Lond B Biol Sci 1984;306(1129):379–88. [65]  Yu CY. The complete exon-intron structure of a human complement component C4A gene. DNA sequences, polymorphism, and linkage to the 21-hydroxylase gene. J Immunol 1991;146(3):1057–66. [66]  Dangel AW, Mendoza AR, Baker BJ, Daniel CM, Carroll MC, Wu LC, et al. The dichotomous size variation of human complement C4 genes is mediated by a novel family of endogenous retroviruses, which also establishes species-specific genomic patterns among Old World primates. Immunogenetics 1994;40(6):425–36. [67]  White PC, New MI, Dupont B. HLA-linked congenital adrenal hyperplasia results from a defective gene encoding a cytochrome P-450 specific for steroid 21-hydroxylation. Proc Natl Acad Sci U S A 1984;81(23):7505–9. [68]  White PC, Grossberger D, Onufer BJ, Chaplin DD, New MI, Dupont B, et al. Two genes encoding steroid 21-hydroxylase are located near the genes encoding the fourth component of complement in man. Proc Natl Acad Sci U S A 1985;82(4):1089–93. [69]  Carroll MC, Campbell RD, Porter RR. Mapping of steroid 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major histocompatibility complex in man. Proc Natl Acad Sci U S A 1985;82(2):521–5. [70]  White PC, New MI, Dupont B. Structure of human steroid 21-hydroxylase genes. Proc Natl Acad Sci U S A 1986;83(14): 5111–5. [71]  Higashi Y, Yoshioka H, Yamane M, Gotoh O, Fujii-Kuriyama Y. Complete nucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome: a pseudogene and a genuine gene. Proc Natl Acad Sci U S A 1986;83(9):2841–5. [72]  Morel Y, Bristow J, Gitelman SE, Miller WL. Transcript encoded on the opposite strand of the human steroid 21-hydroxylase/ complement component C4 gene locus. Proc Natl Acad Sci U S A 1989;86(17):6582–6. [73]  Gitelman SE, Bristow J, Miller WL. Mechanism and consequences of the duplication of the human C4/P450c21/gene X locus. Mol Cell Biol 1992;12(5):2124–34. [74]  Matsumoto K, Arai M, Ishihara N, Ando A, Inoko H, Ikemura T. Cluster of fibronectin type III repeats found in the human major histocompatibility complex class III region shows the highest homology with the repeats in an extracellular matrix protein, tenascin. Genomics 1992;12(3):485–91. [75]  Bristow J, Tee MK, Gitelman SE, Mellon SH, Miller WL. TenascinX: a novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B. J Cell Biol 1993;122(1):265–78. [76]  Zhao B, Lei L, Kagawa N, Sundaramoorthy M, Banerjee S, Nagy LD, et al. Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J Biol Chem 2012;287(13):10613–22. [77]  Haider S, Islam B, D'Atri V, Sgobba M, Poojari C, Sun L, et al. Structure-phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia. Proc Natl Acad Sci U S A 2013;110(7):2605–10. [78]  Lee HH. The chimeric CYP21P/CYP21 gene and 21-hydroxylase deficiency. J Hum Genet 2004;49(2):65–72. [79]  Lee HH, Lee YJ, Chan P, Lin CY. Use of PCR-based amplification analysis as a substitute for the southern blot method for CYP21 deletion detection in congenital adrenal hyperplasia. Clin Chem 2004;50(6):1074–6. [80]  Lee HH, Chang SF, Lee YJ, Raskin S, Lin SJ, Chao MC, et al. Deletion of the C4-CYP21 repeat module leading to the formation of a chimeric CYP21P/CYP21 gene in a 9.3-kb fragment as a cause of steroid 21-hydroxylase deficiency. Clin Chem 2003;49(2):319–22.

References

[81]  L'Allemand D, Tardy V, Gruters A, Schnabel D, Krude H, Morel Y. How a patient homozygous for a 30-kb deletion of the C4-CYP 21 genomic region can have a nonclassic form of 21-hydroxylase deficiency. J Clin Endocrinol Metab 2000;85(12):4562–7. [82]  Concolino P, Mello E, Minucci A, Giardina E, Zuppi C, Toscano V, et al. A new CYP21A1P/CYP21A2 chimeric gene identified in an Italian woman suffering from classical congenital adrenal hyperplasia form. BMC Med Genet 2009;10:72. [83]  Vrzalova Z, Hruba Z, Hrabincova ES, Vrabelova S, Votava F, Kolouskova S, et al. Chimeric CYP21A1P/CYP21A2 genes identified in Czech patients with congenital adrenal hyperplasia. Eur J Med Genet 2011;54(2):112–7. [84]  Chen W, Xu Z, Sullivan A, Finkielstain GP, Van Ryzin C, Merke DP, et al. Junction site analysis of chimeric CYP21A1P/CYP21A2 genes in 21-hydroxylase deficiency. Clin Chem 2012;58(2): 421–30. [85]  Wedell A. Molecular genetics of congenital adrenal hyperplasia (21-hydroxylase deficiency): implications for diagnosis, prognosis and treatment. Acta Paediatr 1998;87(2):159–64. [86]  Marino R, Ramirez P, Galeano J, Perez Garrido N, Rocco C, ­Ciaccio M, et al. Steroid 21-hydroxylase gene mutational spectrum in 454 Argentinean patients: genotype-phenotype correlation in a large cohort of patients with congenital adrenal hyperplasia. Clin Endocrinol (Oxf) 2011;75(4):427–35. [87]  Stikkelbroeck NM, Hoefsloot LH, de Wijs IJ, Otten BJ, ­Hermus AR, Sistermans EA. CYP21 gene mutation analysis in 198 patients with 21-hydroxylase deficiency in The Netherlands: six novel mutations and a specific cluster of four mutations. J Clin Endocrinol Metab 2003;88(8):3852–9. [88]  New MI, Abraham M, Gonzalez B, Dumic M, ­Razzaghy-Azar M, Chitayat D, et al. Genotype-phenotype correlation in 1,507 f­amilies with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Proc Natl Acad Sci U S A 2013; ­ 110(7):2611–6. [89]  Levine LS, Zachmann M, New MI, Prader A, Pollack MS, O'Neill GJ, et al. Genetic mapping of the ­21-hydroxylase-deficiency gene within the HLA linkage group. N Engl J Med 1978;299(17):911–5. [90]  Levine LS. HLA-B14 and nonclassical 21-hydroxylase deficiency in a heterogeneous New York population. Ann N Y Acad Sci 1985;458:65–70. [91]  Wedell A, Luthman H. Steroid 21-hydroxylase deficiency: two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations. Hum Mol Genet 1993;2(5):499–504. [92]  Wilson RC, Wei JQ, Cheng KC, Mercado AB, New MI. Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene. J Clin Endocrinol Metab 1995;80(5):1635–40. [93]  Carrera P, Barbieri AM, Ferrari M, Righetti PG, Perego M, Gelfi C. Rapid detection of 21-hydroxylase deficiency mutations by allele-specific in vitro amplification and capillary zone electrophoresis. Clin Chem 1997;43(11):2121–7. [94]  Higashi Y, Tanae A, Inoue H, Fujii-Kuriyama Y. Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450(C21) gene: implications for steroid 21-hydroxylase deficiency. Am J Hum Genet 1988;42(1):17–25. [95]  Amor M, Parker KL, Globerman H, New MI, White PC. Mutation in the CYP21B gene (Ile-172–--Asn) causes steroid 21-hydroxylase deficiency. Proc Natl Acad Sci U S A 1988;85(5):1600–4. [96]  Globerman H, Amor M, Parker KL, New MI, White PC. Nonsense mutation causing steroid 21-hydroxylase deficiency. J Clin Invest 1988;82(1):139–44. [97]  Speiser PW, New MI, White PC. Molecular genetic analysis of nonclassic steroid 21-hydroxylase deficiency associated with HLA-B14, DR1. N Engl J Med 1988;319(1):19–23.

49

[98]  Urabe K, Kimura A, Harada F, Iwanaga T, Sasazuki T. Gene conversion in steroid 21-hydroxylase genes. Am J Hum Genet 1990;46(6):1178–86. [99]  Lee HH, Chao HT, Ng HT, Choo KB. Direct molecular diagnosis of CYP21 mutations in congenital adrenal hyperplasia. J Med Genet 1996;33(5):371–5. [100] Oriola J, Plensa I, Machuca I, Pavia C, Rivera-Fillat F. Rapid screening method for detecting mutations in the 21-hydroxylase gene. Clin Chem 1997;43(4):557–61. [101] Tajima T, Fujieda K, Nakae J, Toyoura T, Shimozawa K, Kusuda S, et al. Molecular basis of nonclassical steroid 21-hydroxylase deficiency detected by neonatal mass screening in Japan. J Clin Endocrinol Metab 1997;82(7):2350–6. [102] Ko TM, Kao CH, Ho HN, Tseng LH, Hwa HL, Hsu PM, et al. Congenital adrenal hyperplasia. Molecular characterization. J Reprod Med 1998;43(4):379–86. [103] Rumsby G, Skinner C, Honour JW. Genetic analysis of the steroid 21-hydroxylase gene following in vitro amplification of genomic DNA. J Steroid Biochem Mol Biol 1992;41(3-8):827–9. [104] Chin KK, Chang SF. The -104G nucleotide of the human CYP21 gene is important for CYP21 transcription activity and protein interaction. Nucleic Acids Res 1998;26(8):1959–64. [105] Day DJ, Speiser PW, White PC, Barany F. Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction. Genomics 1995;29(1):152–62. [106] Wei WL, Killeen AA. Analysis of Four Common SaltWasting Mutations in CYP21 (Steroid 21-Hydroxylase) by Cleavase Fragment Length Polymorphism Analysis and Characterization of a Frequent Polymorphism in Intron 6. Mol Diagn 1998;3(3):171–7. [107] Yang YP, Corley N, Garcia-Heras J. Reverse dot-blot hybridization as an improved tool for the molecular diagnosis of point mutations in congenital adrenal hyperplasia caused by 21-hydroxylase deficiency. Mol Diagn 2001;6(3):193–9. [108] Ohlsson G, Schwartz M. Mutations in the gene encoding 21-hydroxylase detected by solid-phase minisequencing. Hum Genet 1997;99(1):98–102. [109] Goto M, Piper Hanley K, Marcos J, Wood PJ, Wright S, Postle AD, et al. In humans, early cortisol biosynthesis provides a mechanism to safeguard female sexual development. J Clin Invest 2006;116(4):953–60. [110] Prader A. Der genitalbefund beim pseudohermaphroditimus feminus der kengenitalen adrenogenitalen sydroms. Helv Paediatr Acta 1954;9(3):231–48. [111] Trinh L, Nimkarn S, New MI, Lin-Su K. Growth and pubertal characteristics in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Pediatr Endocrinol Metab 2007;20(8):883–91. [112] Klingensmith GJ, Garcia SC, Jones HW, Migeon CJ, Blizzard RM. Glucocorticoid treatment of girls with congenital adrenal hyperplasia: effects on height, sexual maturation, and fertility. J Pediatr 1977;90(6):996–1004. [113] Helleday J, Siwers B, Ritzen EM, Carlstrom K. Subnormal androgen and elevated progesterone levels in women treated for congenital virilizing 21-hydroxylase deficiency. J Clin Endocrinol Metab 1993;76(4):933–6. [114] Premawardhana LD, Hughes IA, Read GF, Scanlon MF. Longer term outcome in females with congenital adrenal hyperplasia (CAH): the Cardiff experience. Clin Endocrinol (Oxf) 1997;46(3):327–32. [115] Richards GE, Grumbach MM, Kaplan SL, Conte FA. The effect of long acting glucocorticoids on menstrual abnormalities in patients with virilizing congenital adrenal hyperplasia. J Clin Endocrinol Metab 1978;47(6):1208–15.

50

3A.  CONGENITAL ADRENAL HYPERPLASIA OWING TO 21-HYDROXYLASE DEFICIENCY

[116] Barnes RB, Rosenfield RL, Ehrmann DA, Cara JF, Cuttler L, Levitsky LL, et al. Ovarian hyperandrogynism as a result of congenital adrenal virilizing disorders: evidence for perinatal masculinization of neuroendocrine function in women. J Clin Endocrinol Metab 1994;79(5):1328–33. [117] DiMartino-Nardi J, Stoner E, O'Connell A, New MI. The effect of treatment of final height in classical congenital adrenal hyperplasia (CAH). Acta Endocrinol Suppl (Copenh) 1986;279: 305–14. [118] New MI, Gertner JM, Speiser PW, Del Balzo P. Growth and final height in classical and nonclassical 21-hydroxylase deficiency. J Endocrinol Invest 1989;12(8 Suppl. 3):91–5. [119] Lin-Su K, Vogiatzi MG, Marshall I, Harbison MD, Macapagal MC, Betensky B, et al. Treatment with growth hormone and luteinizing hormone releasing hormone analog improves final adult height in children with congenital adrenal hyperplasia. J Clin Endocrinol Metab 2005;90(6):3318–25. [120] Quintos JB, Vogiatzi MG, Harbison MD, New MI. Growth hormone therapy alone or in combination with gonadotropinreleasing hormone analog therapy to improve the height deficit in children with congenital adrenal hyperplasia. J Clin Endocrinol Metab 2001;86(4):1511–7. [121] Mulaikal RM, Migeon CJ, Rock JA. Fertility rates in female patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. N Engl J Med 1987;316(4):178–82. [122] Prader A. Perfect male external genital development and saltloss syndrome in girls with congenital adrenogenital syndrome. Helv Paediatr Acta 1958;13(1):5–14. [123] Urban MD, Lee PA, Migeon CJ. Adult height and fertility in men with congenital virilizing adrenal hyperplasia. N Engl J Med 1978;299(25):1392–6. [124] Claahsen-van der Grinten HL, Sweep FC, Blickman JG, ­Hermus AR, Otten BJ. Prevalence of testicular adrenal rest tumours in male children with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Eur J Endocrinol 2007;157(3): 339–44. [125] Molitor JT, Chertow BS, Fariss BL. Long-term follow-up of a patient with congenital adrenal hyperplasia and failure of testicular development. Fertil Steril 1973;24(4):319–23. [126] Claahsen-van der Grinten HL, Otten BJ, Hermus AR, Sweep FC, Hulsbergen-van de Kaa CA. Testicular adrenal rest tumors in patients with congenital adrenal hyperplasia can cause severe testicular damage. Fertil Steril 2008;89(3):597–601. [127] Wilkins L, Crigler Jr JF, Silverman SH, Gardner LI, Migeon CJ. Further studies on the treatment of congenital adrenal hyperplasia with cortisone. III. The control of hypertension with cortisone, with a discussion of variations in the type of congenital adrenal hyperplasia and report of a case with probable defect of carbohydrate-regulating hormones. J Clin Endocrinol Metab 1952;12(8):1015–30. [128] Blumberg-Tick J, Boudou P, Nahoul K, Schaison G. Testicular tumors in congenital adrenal hyperplasia: steroid measurements from adrenal and spermatic veins. J Clin Endocrinol Metab 1991;73(5):1129–33. [129] Schoen EJ, Di Raimondo V, Dominguez OV. Bilateral testicular tumors complicating congenital adrenocortical hyperplasia. J Clin Endocrinol Metab 1961;21:518–32. [130] Miller Jr EC, Murray HL. Congenital adrenocortical hyperplasia: case previously reported as "bilateral interstitial cell tumor of the testicle". J Clin Endocrinol Metab 1962;22:655–7. [131] Rutgers JL, Young RH, Scully RE. The testicular "tumor" of the adrenogenital syndrome. A report of six cases and review of the literature on testicular masses in patients with adrenocortical disorders. Am J Surg Pathol 1988;12(7):503–13. [132] Glenn JF, Boyce WH. Adrenogenitalism with testicular adrenal rests simulating interstitial cell tumor. J Urol 1963;89:457–63.

[133] Sugino Y, Usui T, Okubo K, Nagahama K, Takahashi T, Okuno H, et al. Genotyping of congenital adrenal hyperplasia due to 21-hydroxylase deficiency presenting as male infertility: case report and literature review. J Assist Reprod Genet 2006;23(9-10):377–80. [134] Nimkarn S, Lin-Su K, Berglind N, Wilson RC, New MI. Aldosterone-to-renin ratio as a marker for disease severity in 21-hydroxylase deficiency congenital adrenal hyperplasia. J Clin Endocrinol Metab 2007;92(1):137–42. [135] Rosler A, Levine LS, Schneider B, Novogroder M, New MI. The interrelationship of sodium balance, plasma renin activity and ACTH in congenital adrenal hyperplasia. J Clin Endocrinol Metab 1977;45(3):500–12. [136] Crouch NS, Liao LM, Woodhouse CR, Conway GS, Creighton SM. Sexual function and genital sensitivity following feminizing genitoplasty for congenital adrenal hyperplasia. J Urol 2008;179(2):634–8. [137] Nordenstrom A, Frisen L, Falhammar H, Filipsson H, ­Holmdahl G, Janson PO, et al. Sexual function and surgical outcome in women with congenital adrenal hyperplasia due to CYP21A2 deficiency: clinical perspective and the patients' perception. J Clin Endocrinol Metab 2010;95(8):3633–40. [138] Van Wyk JJ, Gunther DF, Ritzen EM, Wedell A, Cutler Jr GB, Migeon CJ, et al. The use of adrenalectomy as a treatment for congenital adrenal hyperplasia. J Clin Endocrinol Metab 1996;81(9):3180–90. [139] Gmyrek GA, New MI, Sosa RE, Poppas DP. Bilateral laparoscopic adrenalectomy as a treatment for classic congenital adrenal hyperplasia attributable to 21-hydroxylase deficiency. Pediatrics 2002;109(2):E28. [140] Parrish HM, Lock FR, Rountree ME. Lack of congenital malformations in normal human pregnancies after transabdominal amniocentesis. Science 1957;126(3263):77. [141] Jeffcoate TN, Fliegner JR, Russell SH, Davis JC, Wade AP. Diagnosis of the adrenogenital syndrome before birth. Lancet 1965;2(7412):553–5. [142] Frasier SD, Weiss BA, Horton R. Amniotic fluid testosterone: implications for the prenatal diagnosis of congenital adrenal hyperplasia. J Pediatr 1974;84(5):738–41. [143] Frasier SD, Thorneycroft IH, Weiss BA, Horton R. Letter: Elevated amniotic fluid concentration of 17 alpha-hydroxyprogesterone in congenital adrenal hyperplasia. J Pediatr 1975;86(2):310–2. [144] Mao R, Nelson L, Kates R, Miller CE, Donaldson DL, Tang W, et al. Prenatal diagnosis of 21-hydroxylase deficiency caused by gene conversion and rearrangements: pitfalls and molecular diagnostic solutions. Prenat Diagn 2002;22(13):1171–6. [145] Day DJ, Speiser PW, Schulze E, Bettendorf M, Fitness J, Barany F, et al. Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees. Hum Mol Genet 1996;5(12):2039–48. [146] Technical report: congenital adrenal hyperplasia. Section on Endocrinology and Committee on Genetics. Pediatrics 2000; 106(6):1511–8. [147] David M, Forest MG. Prenatal treatment of congenital adrenal hyperplasia resulting from 21-hydroxylase deficiency. J Pediatr 1984;105(5):799–803. [148] New MI, Carlson A, Obeid J, Marshall I, Cabrera MS, Goseco A, et al. Prenatal diagnosis for congenital adrenal hyperplasia in 532 pregnancies. J Clin Endocrinol Metab 2001;86(12):5651–7. [149] Forest MG, David M, Morel Y. Prenatal diagnosis and treatment of 21-hydroxylase deficiency. J Steroid Biochem Mol Biol 1993;45(1–3):75–82. [150] Mercado AB, Wilson RC, Cheng KC, Wei JQ, New MI. Prenatal treatment and diagnosis of congenital adrenal hyperplasia owing to steroid 21-hydroxylase deficiency. J Clin Endocrinol Metab 1995;80(7):2014–20.

References

[151] New MI, Abraham M, Yuen T, Lekarev O. An update on prenatal diagnosis and treatment of congenital adrenal hyperplasia. Semin Reprod Med 2012;30(5):396–9. [152] Lajic S, Wedell A, Bui TH, Ritzen EM, Holst M. Long-term somatic follow-up of prenatally treated children with congenital adrenal hyperplasia. J Clin Endocrinol Metab 1998;83(11):3872–80. [153] Saenger P. Abnormal sex differentiation. J Pediatr 1984;104(1):1–7. [154] Meyer-Bahlburg HF, Dolezal C, Baker SW, Ehrhardt AA, New MI. Gender development in women with congenital adrenal hyperplasia as a function of disorder severity. Arch Sex Behav 2006;35(6):667–84. [155] Dreger A, Feder EK, Lindemann H. Still concerned. Am J Bioeth 2010;10(9):46–8. [156] New MI. Vindication of prenatal diagnosis and treatment of ­congenital adrenal hyperplasia with low-dose dexamethasone. Am J Bioeth 2010;10(12):67–8. [157] New M. Description and defense of prenatal diagnosis and treatment with low-dose dexamethasone for congenital adrenal hyperplasia. Am J Bioeth 2010;10(9):48–51. [158] McCullough LB, Chervenak FA, Brent RL, Hippen B. A case study in unethical transgressive bioethics: "Letter of concern from bioethicists" about the prenatal administration of dexamethasone. Am J Bioeth 2010;10(9):35–45. [159] McCullough LB, Chervenak FA, Brent RL, Hippen B. The intellectual and moral integrity of bioethics: response to commentaries on "A case study in unethical transgressive bioethics: 'Letter of concern from bioethicists' about the prenatal administration of dexamethasone". Am J Bioeth 2010;10(9):W3–5. [160] Dreger A, Feder EK, Tamar-Mattis A. Prenatal Dexamethasone for Congenital Adrenal Hyperplasia: An Ethics Canary in the Modern Medical Mine. J Bioeth Inq 2012;9(3):277–94. [161] Seckl JR. Prenatal glucocorticoids and long-term programming. Eur J Endocrinol 2004;151(Suppl. 3):U49–62. [162] Seckl JR, Miller WL. How safe is long-term prenatal glucocorticoid treatment? JAMA 1997;277(13):1077–9. [163] Yeh TF, Lin YJ, Lin HC, Huang CC, Hsieh WS, Lin CH, et al. Outcomes at school age after postnatal dexamethasone therapy for lung disease of prematurity. N Engl J Med 2004;350(13):1304–13. [164] Slotkin TA, Zhang J, McCook EC, Seidler FJ. Glucocorticoid administration alters nuclear transcription factors in fetal rat brain: implications for the use of antenatal steroids. Brain Res Dev Brain Res 1998;111(1):11–24. [165] Uno H, Lohmiller L, Thieme C, Kemnitz JW, Engle MJ, Roecker EB, et al. Brain damage induced by prenatal exposure to dexamethasone in fetal rhesus macaques. I. Hippocampus. Brain Res Dev Brain Res 1990;53(2):157–67. [166] Hirvikoski T, Nordenstrom A, Lindholm T, Lindblad F, Ritzen EM, Wedell A, et al. Cognitive functions in children at risk for congenital adrenal hyperplasia treated prenatally with dexamethasone. J Clin Endocrinol Metab 2007;92(2):542–8. [167] Hirvikoski T, Nordenstrom A, Lindholm T, Lindblad F, Ritzen EM, Lajic S. Long-term follow-up of prenatally treated children at risk for congenital adrenal hyperplasia: does dexamethasone cause behavioural problems? Eur J Endocrinol 2008;159(3):309–16. [168] Meyer-Bahlburg HF, Dolezal C, Haggerty R, Silverman M, New MI. Cognitive outcome of offspring from dexamethasone-treated pregnancies at risk for congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Eur J Endocrinol 2012;167(1):103–10.

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[169] Trautman PD, Meyer-Bahlburg HF, Postelnek J, New MI. Mothers' reactions to prenatal diagnostic procedures and dexamethasone treatment of congenital adrenal hyperplasia. J Psychosom Obstet Gynaecol 1996;17(3):175–81. [170] Lajic S, Nordenstrom A, Ritzen EM, Wedell A. Prenatal treatment of congenital adrenal hyperplasia. Eur J Endocrinol 2004;151(Suppl. 3):U63–9. [171] Hirvikoski T, Nordenstrom A, Wedell A, Ritzen M, Lajic S. Prenatal dexamethasone treatment of children at risk for congenital adrenal hyperplasia: the Swedish experience and standpoint. J Clin Endocrinol Metab 2012;97(6):1881–3. [172] Goldman AS, Sharpior BH, Katsumata M. Human foetal palatal corticoid receptors and teratogens for cleft palate. Nature 1978;272(5652):464–6. [173] Nandi J, Bern HA, Biglieri EG, Pieprzyk JK. In vitro steroidogenesis by the adrenal glands of mice. Endocrinology 1967;80(4):576–82. [174] New MI, Parsa AA. Long range outcome of prenatal treatment. Adv Exp Med Biol 2011;707:33–5. [175] Simpson JL. Preimplantation genetic diagnosis at 20 years. Prenat Diagn 2010;30(7):682–95. [176] Fiorentino F, Biricik A, Nuccitelli A, De Palma R, Kahraman S, Iacobelli M, et al. Strategies and clinical outcome of 250 cycles of Preimplantation Genetic Diagnosis for single gene disorders. Hum Reprod 2006;21(3):670–84. [177] Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350(9076):485–7. [178] Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62(4):768–75. [179] Hill M, Barrett AN, White H, Chitty LS. Uses of cell free fetal DNA in maternal circulation. Best Pract Res Clin Obstet Gynaecol 2012;26(5):639–54. [180] Chiu RW, Lau TK, Cheung PT, Gong ZQ, Leung TN, Lo YM. Noninvasive prenatal exclusion of congenital adrenal hyperplasia by maternal plasma analysis: a feasibility study. Clin Chem 2002;48(5):778–80. [181] Devaney SA, Palomaki GE, Scott JA, Bianchi DW. Noninvasive fetal sex determination using cell-free fetal DNA: a systematic review and meta-analysis. JAMA 2011;306(6):627–36. [182] Rijnders RJ, van der Schoot CE, Bossers B, de Vroede MA, Christiaens GC. Fetal sex determination from maternal plasma in pregnancies at risk for congenital adrenal hyperplasia. Obstet Gynecol 2001;98(3):374–8. [183] Lo YM, Chan KC, Sun H, Chen EZ, Jiang P, Lun FM, et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med 2010;2(61); 61ra91. [184] Fan HC, Gu W, Wang J, Blumenfeld YJ, El-Sayed YY, Quake SR. Non-invasive prenatal measurement of the fetal genome. Nature 2012;487(7407):320–4. [185] Kitzman JO, Snyder MW, Ventura M, Lewis AP, Qiu R, Simmons LE, et al. Noninvasive whole-genome sequencing of a human fetus. Sci Transl Med 2012;4(137); 137ra76. [186] New MI, Parsa A. “Long-range Outcome of Prenatal Treatment for CAH.” Hormonal and Genetic Basis of Sexual Differentiation Disorders and Hot Topic in Endocrinology: Proceedings of the 2nd World Conference. New MI, Simpson JL (eds). Springer 2011.