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Clin Biochem Rev. 2009 May; 30(2): 75–86.
PMCID: PMC2702216

The Clinical and Biochemical Spectrum of Congenital Adrenal Hyperplasia Secondary to 21-Hydroxylase Deficiency

Abstract

21-Hydroxylase Deficiency (21-OH Deficiency) represents the most common form of Congenital Adrenal Hyperplasia (CAH), a complex and heterogenous group of conditions, characterised by defects in one of the five enzymes involved in adrenal steroidogenesis. Defects in this steroidogenic enzyme, the product of the CYP21A2 gene, cause disruption in the pathway involved in cortisol and aldosterone production and consequently, the accumulation of their steroid precursors as well as a resulting adrenocorticotrophic hormone (ACTH)-driven overproduction of adrenal androgens. Treatment with glucocorticoid, with or without mineralocorticoid and salt replacement, is directed at preventing adrenal crises and ensuring normal childhood growth by alleviating hyperandrogenism. Conventionally, two clinical forms of 21-OH Deficiency are described - the classical form, separated into salt-wasting and simple-virilising phenotypes, and the non-classical form. They are differentiated by their hormonal profile, predominant clinical features and age of presentation. A greater understanding of the genotype-phenotype correlation supports the view that 21-OH Deficiency is a continuum of phenotypes as opposed to a number of distinct phenotypical entities. Significant advancements in technologies such as Tandem Mass Spectrometry (TMS) and improvements in gene analysis, such as complete PCR-based sequencing of the involved gene, have resulted in remarkable developments in the areas of diagnosis, treatment and treatment monitoring, neonatal screening, prenatal diagnosis and prenatal therapy.

Introduction

Congenital Adrenal Hyperplasia (CAH, OMIM 201910) describes a group of autosomal recessive disorders characterised by enzyme defects in the steroidogenic pathways involved in the biosynthesis of cortisol, aldosterone and androgens. 21-Hydroxylase (21-OH) is the most common of these enzyme deficiencies being found in up to 95% of cases. Despite its origins in the defect of a single enzyme, CAH, secondary to 21-OH Deficiency, represents a complex disease entity with a high degree of heterogeneity in terms of clinical manifestations. The scientific developments that have contributed to a greater understanding of the condition have been achieved over a number of centuries, and the expansion of our knowledge continues in light of the recent technological advances in the areas of analytical and genomic medicine. Notwithstanding this, glucocorticoid and mineralocorticoid replacement, with the principal aims of preventing acute adrenal crises and maximising childhood growth, continue to be the foundation of management. In order to achieve this, continued clinical research into methods of monitoring the efficacy of treatment and its side-effects remains vital. Arguably the greatest recent inroads have been made in our understanding of the genotype-phenotype correlation in 21-OH Deficiency, and in the application of genetic testing to allow early prenatal diagnosis, and the initiation of prenatal treatment with dexamethasone. The death of male babies with salt-wasting CAH1 remains a major clinical problem which potentially may be addressed by recent advances in neonatal screening, notably the addition of second tier testing of blood spots by TMS. This review discusses the most recent advances in the biochemical and genetic aspects of 21-OH Deficiency and how these have influenced clinical practice. For a more comprehensive discussion of CAH, a number of excellent reviews are recommended.24

Historical Aspects

Since the initial identification of the adrenal gland by the Italian anatomist Bartolomeo Eustaccio in 1563, there has been a gradual accumulation of knowledge that forms the current basis for the diagnosis and management of CAH.5 Addison’s classic description of adrenal insufficiency was published in the mid-nineteenth century while the adrenal gland enlargement associated with CAH was first described in the literature in 1865 by the Neapolitan anatomist De Crecchio.6 The contributions of Reichstein7 and Kendall8 in isolating adrenal steroids earned them the Nobel Prize in the 1930s. Other milestones include the first use of cortisone to treat CAH in 1950,9,10 the elucidation of the role of cytochrome P450 in 21-hydroxylation in 1965,11 and the cloning of the majority of the steroidogenic enzyme genes in the 1980s.12 More recently, enhancements in the techniques for measuring the various steroid precursors by TMS, and in our understanding of the genetics of the condition, have resulted in advancements in the areas of neonatal screening as well as prenatal diagnosis and treatment.

Pathophysiology of 21-OH Deficiency

The adrenal cortex, formed in the fourth week of gestation from coelomic epithelial mesoderm, is functionally secreting steroids by the sixth to seventh week of gestation.13 Biosynthesis (from cholesterol) in the adrenal cortex of the physiologically critical steroids, cortisol, aldosterone, and androgens,14 occurs under the stimulus of the pituitary hormone ACTH with the involvement of five crucial enzymes.15 These steroidogenic enzymes are members of the cytochrome P450 family of oxidases composed of approximately 500 amino acids and a single heme group (so-named [cytochrome P450] because of their ability to absorb light at 450nm in their reduced states).16 This biosynthetic process is stimulated by ACTH which, in turn, is inhibited by cortisol, giving rise to a negative feedback loop. Cortisol insufficiency, regardless of the underlying pathology, results in increased ACTH production. CAH is a term used to describe a group of conditions caused by autosomal recessive defects in one of the five enzymes in the adrenal steroidogenesis pathway (Figure 1), resulting in hyperplasia of the adrenal cortex due to continued stimulation by ACTH. As well as activating steroidogenesis, ACTH is a trophic factor for the adrenal cortex which atrophies when it is absent and undergoes hyperplasia when it is present in excess. Within the framework of the recently proposed classification for Disorders of Sexual Development (DSD), the diverse forms of CAH are represented in both 46XY DSD (undervirilised males) and 46XX DSD (virilised females).17 21-OH Deficiency causes 46XX DSD whereas rarer defects early in the steroidogenic pathway lead to impaired androgen synthesis as well as cortisol deficiency and cause 46XY DSD (Figure 1). The other associated consequences are increased production of the steroid precursors above the block and increased excretion of their urinary metabolites. This occurs in all forms of CAH, except Lipoid CAH which results from abnormalities in the Steroidogenic Acute Regulatory protein (StAR).18 Clinical manifestations of CAH reflect not only cortisol insufficiency but varying degrees of mineralocorticoid deficiency, and overproduction (direct shunting into the androgen biosynthetic pathway) or underproduction (impaired biosynthesis) of androgens. Distinct hormonal profiles and clinical phenotypes accompany each enzymatic defect (Table). A recently described CAH variant, characterised by mutations in the electron donor enzyme P450 oxidoreductase, responsible for transferring electrons to all microsomal cytochrome P450 enzymes, is unique in that female virilisation occurs in the absence of elevated androgen levels, suggesting the possibility of undefined alternative androgen biosynthetic pathways.19,20

Figure 1
Steroidogenesis in the three zones of the adrenal cortex. Shaded boxes represent the five enzyme defects described in the various types of Congenital Adrenal Hyperplasia. Boxes with dashed lines indicate enzymes involved in androgen biosynthesis which ...

The most common form of CAH, accounting for 95% of cases, is caused by deficiency of the 21-OH enzyme which is a cytochrome P450 enzyme responsible for the conversion of 17-hydroxyprogesterone (17-OHP) to 11-deoxycortisol. On a functional level, there is a variable degree of aldosterone and cortisol deficiency, increased production of steroid precursors proximal to the 21-hydroxylation step (17-OHP and progesterone), and increased adrenal androgen production, which can result in almost complete masculinisation of the external genitalia in a 46XX infant with a severe enzyme deficiency.

Genetics of 21-OH Deficiency

The 21-OH gene, which exists in two highly homologous forms, is located on chromosome 6p21.3 within the HLA histocompatibility complex.21 The two 21-OH genes - CYP21A2, encoding the active 21-OH, and the inactive pseudogene CYP21A1P - are approximately 30kb apart. They are arranged adjacent to and alternating with the genes encoding the fourth component of serum complement (C4B and C4A).22 CYP21 gene expression occurs principally in the zona fasciculata and glomerulosa of the adrenal cortex but extra-adrenal 21-hydroxylation by other CYP21 isoenzymes exists to a variable extent.23 Recombination events between the active and the inactive gene occur at a particularly high rate and are responsible for a significant majority (estimated to be up to 95%) of mutations in the active CYP21A2 gene.24 These recombination events occur when a loop forms in the linear DNA of the chromosome. The active CYP21A2 and inactive CYP21P genes then lie against each other. This allows transfer of genetic material between them, or loss on one chromosome and duplication on the other of the genetic material in the loop. This is a normal process in the HLA locus and leads to advantageous, increased immunological diversity over time. It is thus unfortunate that the CYP21A2 gene sits adjacent to the HLA locus and becomes a passive participant in this process. The resulting mutations involving the CYP21A2 gene include deletions (10%), microconversions (75%), and macroconversions (10%). They are associated with variable degrees of impairment in 21-OH activity, ranging from complete inactivation to partially functioning enzymes, translating into a diverse range of disease severity and phenotypes.4,25 Gene conversion refers to the phenomenon whereby deleterious sequences, usually present on the pseudogene, are transferred to the active gene, consequently affecting its ability to encode a fully functional enzyme.26 Gene conversions can be quite large and more than one pseudogene mutation can be transferred, thus it cannot be assumed that, when two mutations are found, they are on different alleles. Genotyping of patients in an Australian cohort showed a greater frequency of deletions and large conversions compared with a previously described worldwide population (Figure 2).27 Approximately 5% of mutations found in patients with 21-OH Deficiency are private family mutations in CYP21A2 itself which are not found in the adjacent pseudogene. Private family mutations refer to mutations that are found solely in a particular family. Copy number variation is also not uncommon at this locus and about 5% of chromosomes contain either no or two copies of CYP21A2, or no or two copies of CYP21A1P. Where more than one copy of CYP21A2 occurs, it is not known whether only one or both copies are functionally attached to the distal promoter and are expressed, complicating genetic analysis. Such situations along with intronic mutations probably account for the 1% of alleles from patients with phenotypic 21-OH Deficiency in whom a genetic mutation is not found with current technology (gene sequencing and Multiplex Ligation-dependent Probe Amplification - MLPA). Compound heterozygotes, who possess two or more different mutations on the two alleles, constitute the main group of patients, and their disease severity is determined by the mutation having the greater residual activity. In individuals with 21-OH Deficiency, it is estimated that 1–2% of affected alleles are spontaneous mutations not present in the parents, necessitating genotyping in the parents for accurate prenatal diagnosis and counselling.28

Figure 2
Schematic representation of the CYP21A2 gene with microconversions causing 21-OH Deficiency, and the frequency (%) of mutations found in a cohort of Australian patients compared with those described in worldwide studies (reference 27). *Mutation associated ...

Clinical Manifestations of 21-OH Deficiency

21-OH Deficiency is a heterogenous condition and debate continues about whether it represents a continuum or a number of distinct phenotypes.4,29 Disease severity is dictated by the least severe CYP21A2 mutation, in terms of its functional effect on 21-OH activity. The most widely accepted classification differentiates the disorder into classical (salt-wasting and simple-virilising) and non-classical (or late-onset) forms. The simple-virilising classical and the non-classical forms can be confidently distinguished only if a detailed examination of the genitalia is performed in the neonatal period. There is a significantly higher incidence of the non-classical form (approximately 1 in 500) compared to the classical form (1 in 7000–15000),3032 with the frequency in certain populations such as Ashkenazi Jews being as high as 1 in 27.30 Due to limitations in the ability of neonatal screening to detect the non-classical form, estimations of its frequency are highly variable. The Yupic Eskimos of Alaska have the highest rate of the classical form with a frequency of 1 in 280.33,34

When considered as a continuum, 21-OH Deficiency can manifest in various clinical scenarios from the newborn period through to childhood and adulthood (Table). Individuals with the classic form of 21-OH Deficiency (C21OHD) typically present in the newborn or early childhood period and possess a hormonal profile characterised by excessive production of cortisol precursors and androgens. In the severe salt-wasting form of C21OHD, there is combined aldosterone and cortisol deficiency (salt-wasting phenotype), while the mildest form of C21OHD is characterised by relative cortisol sufficiency with overproduction of androgens. C21OHD represents the most common cause of ambiguous genitalia in 46XX infants with the underlying mechanism being in utero exposure to excessive levels of androgens during critical periods of genital development. The severity of virilisation of the external genitalia is traditionally classified according to the Prader Staging.35,36 Importantly, the female internal organs are unaffected, Wolffian duct structures are absent, and gonads are not palpable in the labio-scrotal folds. Females with C21OHD are usually diagnosed and treatment commenced soon after birth due to the genital ambiguity, but occasionally, a late diagnosis in a salt-wasting C21OHD female results in presentation with shock, secondary to adrenal crisis. Late diagnosis of salt-wasting C21OHD is more of a concern in male infants however, as they may not have evidence of saltwasting in the first few days of life and the external genitalia are essentially normal. Early subtle evidence of C21OHD in males which include hyperpigmentation and penile enlargement may be missed.37 Salt-wasting C21OHD males typically present in the second week of life in variable degrees of shock with hyponatraemia, hyperkalaemia, weight loss, lethargy and dehydration. Males with non-salt-wasting forms of C21OHD typically present during childhood with early virilisation although a salt-wasting crisis can be precipitated by intercurrent illness at any age.1 The clinical spectrum of non-classical 21-OH Deficiency (NC21OHD), characterised by cortisol sufficiency and hyperandrogenism, ranges from patients with isolated biochemical abnormalities to patients with precocious pubarche, hirsutism, acne, menstrual disturbances, short stature and infertility.38,39

Diagnosis of 21-OH Deficiency

The diagnosis of 21-OH Deficiency is reliant on the measurement of inappropriately elevated levels of 17-OHP, a cortisol precursor, which is normally converted to 11-deoxycortisol by the steroidogenic actions of the 21-OH enzyme. Age-, gestation- and sex-specific reference intervals for both basal and ACTH-stimulated 17-OHP levels are available.4042 The timing of 17-OHP measurements and the use of basal versus ACTH-stimulated values is dependent on the age of presentation and the clinical phenotype. In the clinical scenario of a 46XX full term infant presenting with ambiguous genitalia, a random 17-OHP level of greater than 240 nmol/L on day three of life is diagnostic for C21OHD.43 There is no significant published data comparing 17-OHP levels in salt-wasting and simple-virilising forms of C21OHD, but it is thought that levels are generally lower in simplevirilising forms of C21OHD.37 Additional investigations that may be useful in confirming the diagnosis in difficult cases include a 60-minute 17-OHP level following stimulation with intravenous synthetic ACTH, measurement of a urinary steroid profile4446 and genetic analysis.4749 Serial monitoring of urine sodium concentration can give an early indication of salt-wasting while the results of more complex testing are awaited. 17-OHP levels in premature infants are considerably higher than those seen in term infants and it is important to accurately determine gestation and interpret results using gestation-appropriate reference intervals. Random 17-OHP measurements are not a reliable method for the diagnosis of NC21OHD as a significant proportion of affected individuals will have normal levels. The random level may be more discriminatory when taken before 8am.43 A more specific and sensitive test for the diagnosis and prediction of disease severity of NC21OHD is the measurement of ACTHstimulated (250 μg cosyntropin intravenously) 17-OHP and androstenedione at 60 minutes which can then be plotted on a published nomogram.28 Differentiation of unaffected individuals, heterozygotes, and those with NC21OHD and C21OHD, can be achieved in many cases by comparing basal and ACTH-stimulated 17-OHP levels with those of a well-defined cohort (Figure 3).43,50 A degree of overlap does, however, exist between the various groups. The ratio of 17- OHP increase to cortisol increase from 0 to 30 minutes (Δ17-OHP/Δcortisol) following stimulation with intramuscular synthetic ACTH, has also been suggested as a method for differentiating these groups.51

Figure 3
Simplified nomogram illustrating the relationship between basal 17-OHP levels and 17-OHP levels at 60 minutes following stimulation with synthetic ACTH (250 μg intravenous). Differentiation of normal individuals from carriers is complicated by ...

Genotype-Phenotype Correlation in 21-OH Deficiency

CAH secondary to 21-OH Deficiency can be grouped into three general categories: salt-wasting C21OHD, simplevirilising C21OHD and NC21OHD. NC21OHD and the non-salt-wasting form of C21OHD have a similar degree of elevation in precursor steroids,39 supporting the concept that CAH, secondary to 21-OH Deficiency, represents a continuum of phenotypes as opposed to a number of distinct phenotypes. The correlation between genotype and phenotype has been studied extensively.28,5257 Most patients are compound heterozygotes and therefore the phenotype should be predicted by the least severely affected allele. However, it seems that particular mutations can be associated with more than one phenotype giving rise to genotype-phenotype discordance.28 PCR-sequencing of the entire CYP21A2 gene in a cohort of patients, including exons, splice sites and the proximal promoter, revealed that the I172N mutation, described in the literature as being associated with the simple-virilising C21OHD form, can, in fact, lead to either a salt-wasting or a simple-virilising phenotype.56

A significant difference exists between the degree of genotypephenotype correlation demonstrated in relation to the level of aldosterone and that of genital phenotype.2 We can predict the degree of salt-wasting from genotype more confidently than we can the degree of virilisation. The relationship between genotype and genital phenotype is less predictable due to the numerous genetic factors that regulate androgen biosynthesis and sensitivity. In vitro studies, that have investigated measured residual 21-OH activity associated with specific mutations, have shown a correlation between salt-wasting 21-OH Deficiency and large gene deletions or point mutations associated with no measurable enzyme activity,5862 simple-virilising forms and mutations resulting in residual enzyme activity of 1–2%,24,63,64 and NC21OHD and mutations giving rise to residual enzyme activity as high as 60%.63,6567 There is evidence for strong genotypephenotype concordance in individuals who have the most severe and mildest forms of the condition. Genetic analysis is particularly useful in salt-wasting forms of C21OHD as the clinical diagnosis of salt-wasting is not always clear, and in some cases, salt or mineralocorticoid replacement has been commenced prior to the relevant biochemical investigations being performed. Although the aldosterone/plasma renin activity (PRA) ratio is more sensitive for the identification of salt-wasting than aldosterone or PRA alone, its accuracy is not adequate to reliably distinguish disease phenotypes.68 The discovery of associated adrenal medullary dysfunction in 21-OH Deficiency69 has led to the utilisation of free plasma metanephrine levels as a biomarker for phenotype prediction. It has been shown to be comparable to genotyping for the prediction of salt-wasting (free plasma metanephrine levels ≤18.5 pg/mL) and NC21OHD phenotypes.70

Neonatal Screening in 21-OH Deficiency

The infant mortality rate for CAH in the absence of neonatal screening has been estimated to be as high as 20–40%33 although robust disagreement exists with regard to the true rate of death from undiagnosed 21-OH Deficiency.71 Male infants with salt-wasting forms of 21-OH Deficiency provide insufficient clinical evidence for the timely diagnosis of the condition and can present in the first two weeks of life with severe shock secondary to an adrenal crisis before the results of neonatal screening are available. Simple-virilising CAH in male babies can present with severe salt-wasting crises during intercurrent illness after the first two weeks of life, and this morbidity and mortality should be preventable by neonatal screening.1 The rationale for neonatal screening for CAH is based upon the prevention of this clinical scenario. Additional objectives, argued with less conviction, include the possibility of improved outcome in terms of virilisation and growth with an earlier diagnosis of less severe forms of 21-OH Deficiency, and a reduction in the rate of incorrect sex assignment of virilised 46XX infants with simple-virilising forms of 21-OH Deficiency.72

Neonatal screening for 21-OH Deficiency was first developed in the late 1970s73 and is now established in many countries.74 The conventional method for neonatal screening in CAH utilises 17-OHP immunoassays on filter paper samples of dried blood.33,75,76 Recent advances in TMS have seen a shift in focus toward the development of TMS for cost-effective, high through-put screening.77 One study, comparing the two techniques, found a Positive Predictive Value (PPV), the proportion of individuals with a positive test who actually have 21-OH Deficiency, of 1.0% and 4.5% respectively with immunoassay and TMS.78 These PPVs illustrate the limitations of neonatal screening for 21-OH Deficiency and are due to a number of factors including falsely elevated levels of adrenal hormones caused by stress or a physiological postnatal surge in adrenal steroid production, cross-reactivity with other hormones in immunoassay-based methods, and a lack of appropriate gestation- and weight-based reference intervals. A prematurity index, using a gestational age and birth weight scoring system to calculate a corrected 17-OHP level, was shown to be of limited value.79 The additional analysis of steroid profiles by TMS, in particular, the determination of various steroid ratios, has significantly improved the sensitivity and specificity of the tests. The additional run time required for these additional analyses (in the range of 5–10 minutes) has limited the use of this approach for mass screening77,80,81 but it is eminently applicable for second tier testing following an initial screen by immunoassay.82 Improvements in these technologies are anticipated to appreciably reduce analysis time for steroid profiling and may make the approach more appropriate for use as first tier testing in neonatal screening programs. These neonatal screening methods may fail to identify affected infants with the simple-virilising form of 21-OH Deficiency (these children are not at risk for saltwasting) 83 and those exposed antenatally to exogenous glucocorticoids given to the mother.84 More significantly, the effectiveness of a neonatal screening program in reducing infant mortality in 21-OH Deficiency has been questioned by some researchers.71 However in a number of retrospective studies, females with salt-wasting CAH outnumber males with salt-wasting CAH,85,86 thus providing indirect evidence of unreported deaths in males with salt-wasting CAH. The debate regarding the actual death rate as a result of undiagnosed 21-OH Deficiency inevitably influences any decision on the cost-effectiveness of neonatal screening.71,87,88 At present, no significant economic modelling on the cost-effectiveness of a neonatal screening program for 21-OH Deficiency attempts to accurately quantify and incorporate outcomes other than infant mortality into the analysis, and further research into this area with pilot programs utilising the newer technologies is clearly needed.

Monitoring of Treatment in 21-OH Deficiency

The monitoring of treatment in 21-OH Deficiency should always involve the combined assessment of clinical and biochemical parameters. The principal aims of treatment in 21-OH Deficiency are to allow normal growth and pubertal progression by suppressing excessive adrenal androgen production and to provide appropriate glucocorticoid replacement during periods of stress. Suppression of androgen production may require supraphysiological doses of glucocorticoids – typically in the range of 12–18 mg/m2/day of hydrocortisone89 compared to the physiological cortisol secretion rate of 6 mg/m2/day.9092 Newly-diagnosed infants may initially require substantially higher doses in order to overcome the significant ACTH-driven production of androgens in the early neonatal period, however care must be taken to avoid over-treatment. Hydrocortisone is preferred to cortisone acetate as the latter requires conversion to cortisol for biological activity and the variability of the rate of conversion adversely affects drug efficacy. It is usually given in three divided doses but kinetic studies measuring salivary cortisol and 17-OHP during hydrocortisone therapy provide evidence for splitting the morning dose to achieve suppression of 17-OHP to appropriate levels.93 Longer-acting glucocorticoids, such as prednisone and dexamethasone, are often employed in the treatment of adult patients with 21-OH Deficiency where compliance may be an issue, but are avoided in children due to their perceived heightened growth-suppressing effects. Recently, using lower doses of prednisone and dexamethasone, studies have shown promising results in regard to achieving normal growth.94,95 These studies assumed that the potency of prednisone and dexamethasone, relative to hydrocortisone, was significantly greater than the conventional estimates of four to one and 25–30 to one respectively. Treatment protocols involving the use of a reduced physiological dose of glucocorticoid with oestrogen and androgen blockade via the use of aromatase-inhibitors and anti-androgens respectively, hold promise for further improvements in clinical outcomes, though this strategy does involve polypharmacy. The results of these studies await long-term efficacy and safety outcomes.37

Fludrocortisone is used for mineralocorticoid replacement at a dose independent of body surface area or weight and ranging from 100–200 μg daily. The dose required in early infancy is high relative to body size due to the attenuated action of mineralocorticoids on the immature kidney. Sodium chloride supplementation (1–2 g daily) may also be necessary during this period. Clinical assessment of fluid balance and measurement of PRA (aiming for the mid-reference interval) are utilised to guide adjustments. Measurement of PRA is most useful in preventing over-treatment with mineralocorticoid. There is also evidence that the use of Fludrocortisone in non-saltwasting forms of 21-OH Deficiency can reduce the amount of glucocorticoid required to achieve adequate suppression of adrenal androgen production.89

The recommended methods for assessing efficacy of treatment (the adequacy of adrenal hormone suppression) include early morning (pre-8am) or pre-dose measurement of 17-OHP and androstenedione, measurement of 17-OHP on serial filter paper blood samples collected throughout the day,100 measurement of salivary cortisol and 17-OHP,93,9698 and measurement of urinary metabolites. 17-OHP levels should not be suppressed to the reference interval as achieving this may cause Cushing’s Syndrome. The principal therapeutic objective is to use the lowest glucocorticoid dose required to prevent symptoms related to hyperandrogenism.25,99 A number of studies have shown excellent correlation between 17-OHP levels on filter paper and serum,100,101 allowing for therapeutic adjustments based on diurnal ACTH secretion and the convenience of sample collection at home. Foetal adrenal steroids, however, may cross-react with 17-OHP if they are not specifically removed by a solvent extraction process and therefore filter paper 17-OHP measurements are not reliable for infants less than three months of age. Salivary cortisol levels measured by radioimmunoassays correlate strongly with measured serum levels. The appropriateness of using this method for glucocorticoid dose adjustments is still to be determined as elevated serum 17-OHP levels often correspond with normal salivary cortisol levels prior to the next scheduled glucocorticoid dose.97 Measurement of cortisol in serum and saliva does not appear to correlate with serum 17-OHP levels and is therefore not clinically useful. The presence of blood or serous exudate in the mouth can lead to artificially elevated levels, particularly of cortisol. Comparison of filter paper 17-OHP levels measured by nonchromatographic radioimmunoassay and 24h urinary steroid profiles obtained using gas chromatography showed a close correlation between the individual daily means of serial filter paper 17-OHP measurements and the urine pregnanetriol/tetrahydrocortisone ratio which, according to the authors, supports the use of urinary steroid profiling as a method for monitoring treatment.102 Dehydroepiandrosterone (DHEA) and Dehydroepiandrosterone Sulfate (DHEAS), although quantitatively important adrenal androgens, are easily suppressed in response to glucocorticoid treatment and are of no value in the monitoring of therapy.103

Prenatal Diagnosis of 21-OH Deficiency

In 46XX infants with C21OHD, exposure to excessive adrenal androgens during the critical period of sexual differentiation at seven to twelve weeks, leads to varying degrees of genital masculinisation and ambiguity including clitoral enlargement, labial fold fusion, and rostral migration of the urethral/vaginal perineal orifice104 but normal mullerian structures. Prenatal administration of dexamethasone to affected 46XX foetuses during this critical period (typically before eight weeks gestation) suppresses the active foetal hypothalamicpituitary-adrenal axis and consequently prevents androgen overproduction and inappropriate masculinisation.105 In addition to the prevention of physical masculinisation, there is evidence to suggest that prenatal dexamethasone in 21-OH Deficiency may also attenuate behavioural and psychological masculinisation.106 The evolution of prenatal diagnosis has seen the measurement of hormones in amniotic fluid107 obtained by amniocentesis (15–18 weeks gestation) replaced by gene analysis using foetal cells obtained by chorionic villous sampling (CVS) at 10–12 weeks gestation. The limitations of gene analysis are that both mutations cannot be identified using current technology (gene sequencing and assessment of copy number using MLPA) in 1% of families. Prior genetic analysis of the proband and both parents is required preferably prior to conception. The possibility of contamination with maternal DNA material has to be borne in mind and tested for in such samples. PCR-based analysis of the entire CYP21A2 gene and close examination of microsatellites may improve the accuracy of gene analysis.108 Prenatal treatment was first instituted in 1978. In order to prevent genital virilisation in 46XX foetuses, prenatal treatment with glucocorticoids needs to be commenced before genetic analysis of the foetus is possible. Dexamethasone is preferred as it is not inactivated by the placental 11-beta-hydroxysteroid dehydrogenase type 2 enzyme and binds minimally to cortisol-binding globulin109 thus allowing it to cross the placenta. It is given before eight weeks gestation, on a daily basis at a dose of 20 μg/kg (in three divided doses), normally based on a pre-pregnancy weight.110 At CVS or amniocentesis, dexamethasone is discontinued if the foetus is a male or an unaffected female and is only continued in the case of an affected female. Therefore seven out of eight infants are unnecessarily exposed to dexamethasone until the results of the CVS are available. Treatment is reasonably well tolerated by most mothers but glucocorticoid-related side effects are relatively common. Treatment failure, although rare, does occur and is most commonly related to non-compliance or sub-optimal dosing/duration of therapy – occasionally a cause is not found.99 Prenatal dexamethasone, however, has been implicated in obstetric complications, including intrauterine growth restriction, pre-term labour, pre-eclampsia and chorio-aminionitis.111 There does not appear to be any consistent evidence of significant adverse effects on physical parameters of the developing foetus at birth or on postnatal growth.110 Increased internalising behaviour (shyness)112 and impaired verbal working memory113 have been reported. There is a body of literature, mainly arising from animal studies, that suggests that prenatal glucocorticoids, through foetal programming, may increase the risk of a number of long-term cardiovascular, metabolic, and neuropsychiatric disorders.114 In these animal studies, however, the treatment was administered late in pregnancy and the doses were generally higher than those used in human treatment protocols. Longitudinal follow-up studies are currently being conducted in an attempt to ascertain the nature and extent of long-term consequences of prenatal exposure to glucocorticoids.

Conclusions

21-OH Deficiency, the most common form of CAH, is a complex disorder with its origins in a single adrenal steroidogenic enzyme defect. Patients with 21-OH Deficiency exhibit a continuum of different clinical presentations spanning the entire period from birth to adulthood. Advances in genetic analysis have not only improved our ability to diagnose the less severe forms of the condition but have also allowed a greater insight into the genotype-phenotype correlation in this condition. Neonatal screening programs are being developed throughout the world, and the greater use of TMS as a second tier test following initial immunoassay has resulted in improved reliability and a greatly reduced false positive rate. Prenatal treatment of mothers with glucocorticoids in an effort to minimise genital virilisation of 46XX infants is also a topic of debate, largely because seven out of eight infants are unnecessarily exposed to dexamethasone.

Table.
The clinical spectrum of 21-OH Deficiency (modified from Hughes I, 2007 – reference 29).

Footnotes

Competing Interests: None declared.

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