Neonatal Screening for 21-Hydroxylase Deficient Congenital Adrenal Hyperplasia—The Role of CYP21 Analysis

Allen W. Root

University of South Florida, Tampa and All Children’s Hospital St. Petersburg, Florida 33701

Address correspondence and requests for reprints to Allen W. Root, M.D., Division of Pediatric Endocrinology, Diabetes and Metabolism, All Children’s Hospital, 801 6th Street South, Box 6900, St. Petersburg, Florida 33701.


    Introduction
 Top
 Introduction
 References
 
Neonatal screening for 21-hydroxylase deficient congenital adrenal hyperplasia (21OH-CAH), an autosomal recessive disorder with an incidence of approximately 1 in 15,000 live births in North America, became feasible in 1977 with the development of a microfilter paper radioimmunoassay for measurement of capillary blood concentrations of 17-hydroxyprogesterone (17OHP) by Pang et al. (1). Subsequently, the method has been modified by the use of polyclonal and monoclonal antibodies with differing specificities for 17OHP in single or dual antibody sandwich assays and the use of either radioactive, enzymatic, or fluorometric markers of antibody-antigen interaction. The immunological measurement of 17OHP in neonatal whole blood is relatively nonspecific because the fetal adrenal cortex secretes a large number of compounds that are immunologically similar to and cross-react in immunoassays for 17OHP (2, 3). In addition to the problem of assay specificity, circulating levels of 17OHP decline with gestational maturity and fall rapidly after birth (4). Thus, normal whole blood screening values of 17OHP must be determined for neonates of varying birth weight and gestational duration as well as for postnatal age (5).

Despite these methodological difficulties, neonatal screening programs for 21OH-CAH have been established in approximately 20 states (USA) as well as in many other countries (Switzerland, Italy, France, Sweden, Israel, Japan, to name a few). The neonatal screening programs for 21OH-CAH are designed to 1) identify newborns with the salt-wasting, life-threatening form of this disorder; and 2) prevent or rectify incorrect sex assignment of virilized female neonates. The screening programs have undoubtedly salvaged many infants that might have died or been assigned to the wrong sex. They have also identified, on occasion, neonates with the simple virilizing and nonclassical forms of this disease, although certainly not all such subjects.

The gene (CYP21) encoding 21-hydroxylase or P450c21 and a highly homologous, but inactive, pseudogene (CYP21P) are located on chromosome 6p in the midst of the histocompatibility leucocyte antigen complex (linked to two complement components, C4A and C4B). Deletions of large portions of CYP21 and macroconversions to sequences present in CYP21P account for 30% of the genetic errors leading to the salt-wasting form of 21OH-CAH; a point mutation (A/C->G) within intron 2 is found in 55% of patients with salt wasting and in 25% of subjects with the simple virilizing form of 21OH-CAH. Point mutations in CYP21 result in a base sequence identical to that present at the same site in CYP21P and lead to varying decreases in activity of the protein. Most subjects with 21OH-CAH are compound heterozygotes for mutations of CYP21. Null mutations in CYP21 (i.e. those that completely inactivate the gene product) are associated with the salt-wasting form of 21OH-CAH. While there is general concordance between phenotype and genotype, nonetheless, there may be differing clinical manifestations of 21OH-CAH in patients with the same mutation(s) and even within a sibship (6).

In this issue of JCEM, Nordenstrom et al. (7) (see page 1505) determined the genotype of 91 children with 21OH-CAH detected in a neonatal screening program for this disorder in Sweden, where this study is routinely performed. They collected blood specimens at approximately 8–12 days of age and determined the most common CYP21 mutations in DNA extracted from peripheral leukocytes by a method that permits the analysis to be completed within hours (8). The authors noted that null (deletion, E3 del 8bp, Leu307insT, Gln318stop, Arg356Trp) and intron-2 splice mutations were associated with higher screening 17OHP levels and more severe disease than were the Ile172Asn and Val181Leu mutations. In many instances, however, similar screening 17OHP concentrations were found in infants with the simple virilizing and nonclassical forms of 21OH-CAH as well as in some normal infants (false-positive). Therefore, the investigators recommend genotyping as a useful adjunctive assessment of the neonate with 21OH-CAH, to help define the severity of 21-hydroxylase deficiency. Krone et al. (9) have recently described another method for the rapid analysis of the structure of CYP21 that differentiates between functional and nonfunctional genes and identifies more than 99% of the mutations.

Genotyping of patients with 21OH-CAH is of interest and is important for genetic counseling and evaluation of future pregnancies in the same couple. It may be useful in the evaluation of a patient with a variant of this disorder not otherwise classified. The more mutations identified, the greater will be our understanding of the physiological and functional significance of different sites within the P450c21 protein. However, this writer doubts that immediate genotyping of all neonates with 21OH-CAH is necessarily more helpful than the clinical and laboratory assessment of such infants in determining the severity of the disorder and its appropriate management for the following reasons:

  1. The purpose of neonatal screening for 21OH-CAH (and other congenital disorders) is to identify the newborn at risk for this disease; it is not to diagnose the disorder or to establish its degree of severity. It is the urgent responsibility of the clinician to confirm (or refute) the diagnosis of 21OH-CAH and to determine its management. Variable screening 17OHP concentrations in patients with the same genotype and phenotype may reflect sampling either at a time of peak secretion, during a secretory trough, or in response to the stress of the heel stick. In addition, different assays have individual specificities for and "read" different values of 17OHP. Thus, all infants with screening blood 17OHP values that exceed the threshold level mandate full evaluation to confirm the diagnosis and to identify its seriousness.
  2. As the investigators point out, only the highest screening 17OHP values were useful in detecting the most severely affected newborns with 21OH-CAH; in neonates with somewhat lower or borderline screening 17OHP levels there were overlapping phenotypes and genotypes and variable phenotypes even in infants with the same genotype. It is more rapid and less expensive to measure serum sodium and potassium concentrations and plasma renin activity (PRA) as indices of the salt-wasting form of 21OH-CAH than to await the genotype of each infant. One must also guard against the possibility of a false sense of security if a neonate is found to have other than a null or intron 2 mutation. Because 75% of subjects with 21OH-CAH are at-risk for mineralocorticoid insufficiency, measurement of PRA in all neonates with 21OH-CAH is reasonable to identify those with subclinical salt wasting.
  3. The delay between initial screening and later genotyping (approximately 8–12 days), even with a rapid analytic method, is often beyond that interval necessary for the identification and treatment of the neonate with salt-wasting 21OH-CAH. Rising concentrations of serum potassium are often the initial biochemical sign of mineralocorticoid insufficiency and should be measured serially.
  4. For patients with borderline screening 17OHP levels, serial clinical assessment, determination of basal levels, and if necessary, the 17OHP secretory response to ACTH may help to distinguish between variant forms of 21OH-CAH (10). As the authors point out, genotyping may be especially helpful in identifying the neonate with the nonclassical form of 21OH-CAH, as such children require careful consideration concerning the advisability of glucocorticoid treatment.

Because 21OH-CAH is a continuum of functional disorders, its clinical classification into classical (salt-wasting and simple virilizing) and nonclassical forms remains valid today (11). While genotyping of all patients with mutations in CYP21 may ultimately become a standard of care, particularly if a central analytic facility is established and funded, it is currently an adjunctive study of interest and importance in the appropriate setting and should not supersede good clinical management.

Received March 15, 1999.

Accepted March 17, 1999.


    References
 Top
 Introduction
 References
 

  1. Pang S, Hotchkiss J, Drash AL, Levine LS, New MI. 1977 Microfilter paper method for 17-hydroxyprogesterone radioimmunoassay: its application for rapid screening for congenital adrenal hyperplasia. J Clin Endocrinol Metab. 45:1003–1008.[Medline]
  2. Al Saedi S, Dean H, Dent W, Stockl E, Cronin C. 1996 Screening for congenital adrenal hyperplasia: the Delfia Screening Test overestimates serum 17-hydroxyprogesterone in preterm infants. Pediatrics. 97:100–102.[Abstract]
  3. Lange-Kubini K, Zachmann M, Kempken B, Torresani T. 1996 15-Beta hydroxylated steroids may be diagnostically misleading in confirming congenital adrenal hyperplasia suspected by a newborn screening programme. Eur J Pediatr. 155:923–931.
  4. Al Saedi S, Dean H, Dent W, Cronin C. 1995 Reference ranges for serum cortisol and 17-hydroxyprogesterone levels in preterm infants. J Pediatr. 126:985–987.[Medline]
  5. Allen DB, Hoffman GL, Fitzpatrick P, Laessig R, Maby S, Slyper A. 1997 Improved precision of newborn screening for congenital adrenal hyperplasia using weight-adjusted criteria for 17-hydroxyprogesterone levels. J Pediatr. 130:128–133.[Medline]
  6. Pang S. 1997 Congenital adrenal hyperplasia. Endocrinol Metab Clin NA. 26:853–891.[Medline]
  7. Nordenstrom A, Thilen A, Hagenfeldt L, Larsson A, Wedell A. 1999 Genotyping is a valuable diagnostic complement to neonatal screening for congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. J Clin Endocrinol Metab. 84:1505–1509.[Abstract/Free Full Text]
  8. Wedell A, Luthman H. 1993 Steroid 21-hydroxylase deficiency: two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations. Hum Mol Genet. 2:499–504.[Abstract]
  9. Krone N, Roscher AA, Schwarz HP, Braun A. 1998 Comprehensive analytical strategy for mutation screening in 21-hydroxylase deficiency. Clin Chem. 44:2075–2082.[Abstract/Free Full Text]
  10. New MI, Lorenzen F, Lerner AJ, et al. 1983 Genotyping steroid 21-hydroxylase deficiency: Hormonal reference data. J Clin Endocrinol Metab. 57:320–326.[Abstract]
  11. Hughes IA. 1998 Congenital adrenal hyperplasia—a continuum of disorders. Lancet 352:752–754.