Neonatal Screening for 21-Hydroxylase Deficient Congenital Adrenal HyperplasiaThe Role of CYP21 Analysis
Allen W. Root
University of South Florida, Tampa and
All Childrens 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 Childrens Hospital, 801 6th Street South, Box 6900, St. Petersburg, Florida 33701.
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Introduction
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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 812 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:
- 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.
- 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.
- The delay between initial screening and later genotyping
(approximately 812 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.
- 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.
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