Key words
neonatal screening - congenital adrenal hyperplasia - 21-hydroxylase deficiency -
salt-wasting form - simple virilizing form
Introduction
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder of adrenal
steroidogenesis. More than 90% of CAH cases are caused by deficiency of the
enzyme 21-hydroxylase (21-OHD) [1]
[2]
[3]
[4]
[5]
[6]. It
is characterized by a reduced ability to synthesize cortisol and aldosterone,
coupled with the overproduction of adrenal androgens. Hormone synthesis can be
affected to different degrees that result in a wide spectrum of severity and
clinical onset during the neonatal period, childhood or adulthood [1]
[2]
[3]
[4]
[5]
[6]
[7]. Classics forms include salt-wasting (SW)
forms, for which there is a high risk of life-threatening adrenal insufficiency
during the first month of life, and simple-virilizing (SV) forms. In both cases,
female neonates present with markedly virilized external genitalia. Salt wasting CAH
is a rapidly evolving and life-threatening disorder. Babies with untreated, severe
CAH typically present in the first month of life with vomiting, weight loss,
dehydration, and shock and may die if the diagnosis and treatment are delayed.
Nonclassic forms can manifest with hyperandrogenism later in life and do not warrant
early recognition through neonatal screening.
Despite the fact that CAH is one of the most common inborn endocrine disorders, some
patients are not identified, or may even die, in an acute salt-losing crisis.
Neonatal screening program for 21-OHD began in 1977, when Pang et al. developed a
specific radioimmunoassay to analyze 17-hydroxyprogesterone (17-OHP) in dried blood
spot specimens collected on filter paper cards [8]. The objectives of 21-OHD screening are to detect newborns with the
classic salt-wasting form before potential life-threatening shock develops, to
prevent or rectify incorrect sex assignment of virilized female neonates, and to
anticipate the diagnosis of boys with simple virilizing forms [1]
[2]
[9]
[10].
However, screening remains controversial, with some main arguments against its
routine use. One of them is the proportion of cases for which screening contributes
to diagnosis is unclear, as most cases in females are easy to detect clinically and
salt wasting can occur before the screening results are obtained. Screening programs
do not confirm the diagnosis of 21-OHD and therefore cases must be evaluated
individually. Some newborns present elevated 17-OHP levels in consecutive blood
determinations that decrease spontaneously within the first months of life and are
not diagnosed with CAH. This issue represents a disadvantage in terms of the
psychological consequences for the patient’s family and the cost to the
health system. It is known that in the group of preterm neonates, 17-OHP levels are
inversely proportional to gestational age and birth weight [11]. To minimize this limitation, new cutoff
levels adjusted for both gestational age and birth weight have been stablished and
new methods of analysis are used to decrease cross-reactivity. These anti-screening
theses are no longer accepted. Neonatal CAH screening should not be viewed as a
cost. Because delays in diagnosis may result in death, especially in 46, XY infants,
and may lead to sexual identity preference problems in 46, XX cases.
Neonatal screening for 21-OHD was included in the neonatal screening program of the
Madrid region of Spain in 1990, with a 21-OHD incidence of 1/19.211 [12].
The main objective of this study was to analyze the clinical suspicion and where the
patients were when they received the result of the neonatal screening for 21-OHD.
We
retrospectively collected real-life screening data and clinical data for affected
neonates to determine whether screening had facilitated case detection before
clinical diagnosis.
Patients and Methods
Study population
The present data were derived from a retrospective analysis of a group of
patients with classical 21-OHD patients discovered by newborn screening in
Madrid, Spain.
In a retrospective study covering the period from 1990 to 2015, we analyzed the
clinical suspicion and where the patients were when they received the result of
the neonatal screening for 21-OHD and examined the time elapsing before
diagnosis of CAH patients with follow-up in our hospital. CAH patients with
classical forms were diagnosed according to clinical (SW and SV form) and
biochemical data (increased 17-OHP and adrenal androgens, cortisol and
aldosterone deficiency) and confirmed by molecular analysis. The patients were
cared for in a single institution, over a period of 25 years, ensuring a fairly
uniform approach and the continuity of care, thus making the comparisons more
reliable.
Variables
The clinical data evaluated were where patients were when they received the
results of newborn screening program, birth weight and length, diseases during
the neonatal period and family history. The study variables were initial
screening levels of 17-OHP, age at diagnosis by newborn screening, values for
pH, glucose, electrolytes and serum 17-OHP determined at first evaluation, age
of onset of treatment and CYP21A2 analysis.
Screening program
17-OHP was analyzed using dried blood spot specimens (Whatman #903) collected at
48 hours of life. Levels of 17-OHP were determined by fluoroinmmunoassay
(AutoDELFIA; PerkinElmer Life and Analytical Sciences, Wallac Oy, Turku,
Finland).
Values higher than the 99th percentile for gender, born age and
weight born are considered pathological. The 17-OHP cutoff limit for a positive
test in neonates’ weight-term-adequate for gestational age is
15 nmol/l (4.95 ng/ml). Children with
17-OHP>30 nmol/l (9.9 ng/ml) are
recalled immediately to the Pediatric Endocrinology Center for a clinical
evaluation. If 17-OHP levels are between 15 and 30 nmol/l, a
second analysis is performed. Preterm neonates and those who are small for
gestational age have different threshold values, which are adjusted for
gestational age and weight, as established by our laboratory.
Parents of newborns with a positive result are contacted by telephone and
referred to the Pediatric Endocrinology Department for clinical evaluation. A
pediatric endocrinologist then provides confirmatory examination and testing of
all at-risk infants to provide the final diagnosis of CAH.
Statistical analyses
Statistical analyses were performed using SPSS 15.5. Qualitative variables are
expressed as the frequency and comparisons were performed using the x2-test.
Quantitative variables are expressed as the median and mean plus standard
deviation (SD). Comparisons were performed using the Mann–Whitney
U-test. Statistical significance was set at p <0.05 for all
analyses.
Results
Patients
During the study period, a total of 1 594 481 neonates were screened. We analyzed
46 newborns (32 boys and 14 girls) affected by classical forms for 21-OHD for
whom the initial evaluation and further monitoring were performed at our
hospital. Of these neonates, 36 were affected by SW forms and 10 by SV
forms.
Family and perinatal history
Median gestational age was 39.7 weeks (range 38–40.2). Five patients
presented preterm delivery. Three of them were born at 36 weeks, one at 34
weeks, and one girl at 31 weeks (950 g), who had received prenatal
treatment with dexamethasone for a previous affected sibling. No differences
were found in gestational age according to sex and clinical form. The Z-score of
weight (3210 g; 3000–3550) and length (50 cm;
48.5–51) at birth in our patients did not differ from that found in the
general population.
Six patients (12.2%) were found to have a history of consanguinity in the
parents. Two families (4.3%) had a child previously die of unknown cause
in the first 3 months of life. In both families, the child included in the study
had a form of salt loss.
Four (8.16%) patients had a previous affected sibling who had been the
index case in the family. Of the 4 families that after having a first affected
child had a second child also affected by the disease, 3 families requested
treatment and prenatal diagnosis and one of them refused treatment and prenatal
diagnosis. Three pregnant women received prenatal treatment with dexamethasone.
In one of them, treatment was suspended after the male fetal sex was known and
in the remaining two families dexamethasone was maintained until the end of
gestation due to the affected female fetal sex.
Clinical suspicion before the results of neonatal screening
In 38 (82.6%) patients (30 SW and 8 SV), the disease had not been
suspected before the result of newborn screening but only in 8 (17.4%)
patients, the disease had been suspected before the result of newborn screening
([Table 1]). Of these 30 patients
with SW form, 24 were boys and 6 were girls. Six of the 8 patients affected by
SV form without clinical suspicion of the disease were boys and 2 were girls. In
almost all (30 of 32) of the boys, the disease had not been clinically
suspected. In 6 of 14 girls, sex assignments were incorrect until the screening
result was known.
Table 1 Patients with/without clinical suspicion
for 21-hydroxylase deficiency.
|
Patients
|
SW
|
SV
|
|
Clinical suspicion
|
8 (17.4%)
|
6
|
2
|
|
♂ 1
|
♀ 5
|
♂ 1
|
♀ 1
|
|
Without clinical suspicion
|
38 (82.6%)
|
30
|
8
|
|
♂ 24
|
♀ 6
|
♂ 6
|
♀ 2
|
SW: Salt-wasting form; SV: Simple-virilizing form.
The 8 patients with clinical suspicion of the disease, 6 of them were affected by
SW form. The disease had been suspected by previous relative affected in 4
patients (2 girls with SW form, 1 boy with SW form and 1 boy with SV form). In 4
females the suspicion was made because of ambiguous genitalia at birth (3 were
affected with SW form and 1 with SV form) ([Fig. 1]). The disease was clinical suspected in 6 of 14 girls and 2
of 32 boys, that is, the disease is suspected in females but not in males (p
<0.001).
Fig. 1 Patients with/without clinical suspicion before the
neonatal screening result for 21-hydroxylase deficiency.
Clinical situation on recall
The clinical situation when the result of neonatal screening was obtained in 38
patients without clinical suspicion was 30 patients (78.9%) were at home
without suspicion of illness, 3 (7.9%) were at home but with hospital
follow-up due to clinical suspicion of illness and 5 (13.2%) newborns
were admitted to the hospital for different reasons before the screening results
were available ([Fig. 2]).
Fig. 2 Where were patients when they received the neonatal
screening result for 21-hydroxylase deficiency?
-
Patients at home without clinical suspicion of disease (n=30): 23
patients affected by SW form (22 boys and 1 girl in with incorrect sex
assignment at birth) and 7 by SV form (6 boys and 1 girl with Prader
II).
-
Patients at home in follow-up for suspected illness (n=3): 2
females with SW form with incorrect sex assignment at birth like males
with cryptorchidism at birth and 1 female with SV form under study by
ambiguous genitalia classified as Prader III.
-
There were 25 patients with SW (22 boys and 3 girls) in their homes with
morbidity and mortality risk like healthy neonates.
-
Hospitalized patients (n=5): all of them affected by SW form, 3
of them were admitted with suspicion of sepsis (2 girls and 1 boy), 1
boy with jaundice and 1 girl with hypoglycemia.
Age at diagnosis by newborn screening for 21-OHD
The median age at diagnosis by Neonatal screening Program was 8.5 days (range,
6–10.5 days). The median age of the boys and girls at detection was 9
days (range, 8–12 days) and 7 days (range, 3–9 days),
respectively, and was not significantly different. The median age at detection
in patients with SW form was earlier (8 days; range, 6–9 days) than in
those with SV form (18 days; range, 14.5–31.5), being this statistically
significant difference (p <0.001) as detailed in [Fig. 3].
Fig. 3 Age at diagnosis by neonatal screening for 21-hydroxylase
deficiency according to sex and clinical form.
As shown in [Table 2], 39%
patients affected by SW were diagnosed in the first week of life and 58%
in the second week of life. Thirty percent patients affected by SV form were
diagnosed in the first 15 days of life (20% in the first week and
10% in the second). Forty percent patient affected by SV form were
diagnosed between 16 and 30 days of life and 30% beyond the month of
life. The diagnosis is later in SV forms because it requires several
determinations of 17-OHP to arrive at the confirmation of the diagnosis.
Neonatal Screening allowed the diagnosis of the SV form in boys in the first
month of life (18 days; range, 14.5–31.5 days).
Table 2 Number of patients diagnosed with 21-hydroxylase
deficiency according to age.
|
≤ 7 days of life
|
8–15 days of live
|
16–30 days of live
|
≥31 days of life
|
n
|
|
SW form
|
14 (38.9%)
|
21 (58.3%)
|
1 (2.7%)
|
0
|
36 (100%)
|
|
SV form
|
2 (20%)
|
1 (10%)
|
4 (40%)
|
3 (30%)
|
10 (100%)
|
|
Total
|
16 (34.8%)
|
22 (47.8%)
|
5 (10.8%)
|
3 (6.5%)
|
46 (100%)
|
SW: Salt-wasting form; SV: Simple-virilizing form.
Values of 17-OHP in dried blood spot specimens from
neonatal screening
17-OHP initial concentrations detected using dried blood spot specimens was
422.5 nmol/l (range, 294.0–622.0 nmol/l)
in SW forms and 58 nmol/l (range,
38.5–93 nmol/l) in SV forms ([Fig. 4]). Neonates with SW forms had
higher 17-OHP concentrations than SV forms (p <0.001). The highest
values of 17-OHP in absorbent paper were obtained at 2 days
(2–6) in SW forms at 18 days of life in SV forms, this difference being
statistically significant (p <0.001). All patients affected by SW forms
were identified when performing the first screening test, however, most of the
children affected by the SV forms required several repetitions of the screening
test (2 to 4 repetitions) because slightly elevated values in some patients.
Fig. 4 17-Hydroxyprogesterone initial concentrations in dried
blood spot specimens.
Physical examination
All of patients included were diagnosed by neonatal screening program of classic
forms of 21-OHD. The most common symptom and sign of neonates with 21-OHD was
hyperpigmentation (71.7%). The most frequently affected
hyperpigmentation areas were the genitals in 26 (78.8%), nipples in 2
(6.1%) and both areas (genitals and nipples) in 5 (15.1%)
patients. Poor feeding was noted in 15 patients affected by SW form.
All girls with 21-OHD, regardless of type, had atypical external genitalia,
clitoromegaly and variable degrees of posterior labial fusion at the time of
diagnosis except 2 of the 14 girls born with normal female genitals because they
had been treated prenatally with dexamethasone. An incorrect assignment of sex
at birth had been performed in 6 of the 14 girls (42.8%).
Biochemical findings
CAH patients tended to present hyponatremia and hyperkalemia, although no
relevant differences were observed in pH values. [Table 3] shows biochemical and neonatal
anthropometry data according to clinical form. The median baseline 17-OHP level
was 311 ng/ml (range, 69.3–479.5 ng/ml)
in SW form and 234 ng/ml (range,
160–276 ng/ml) in SV form, which was not statistically
significantly different.
Table 3 Biochemical data and neonatal anthropometry for
21-hydroxylase deficiency patients according to clinical
form.
|
SW
|
SV
|
p-Value
|
|
Age at diagnosis, days
|
8.00; 6.00–9.00
|
18.00; 14.50–3150
|
<0.05
|
|
Screening 17-OHP, nmol/l (Median, p25-p75)
|
422.5 (294.0–622.0)
|
58.0 (38.5–93.0)
|
<0.05
|
|
Sodium, mmol/l
|
128.3±7.5
|
132.5±1.0
|
<0.05
|
|
Potassium, mmol/l
|
6.7±1.3
|
5.3±0.4
|
<0.05
|
|
pH
|
7.35±0.08
|
7.35±0.06
|
|
|
Glycemia, mg/dl
|
90.7±28.4
|
81.8±5.6
|
|
|
Serum 17-OHP, ng/ml (Median, p25–p75)
|
311.0 (69.3–479.5)
|
234.0 (160–276)
|
|
|
Birth weight, SD
|
0.14±1.20
|
0.10±0.88
|
|
|
Birth length, SD
|
0.12±1.41
|
0.05±1.53
|
|
SW: Salt-wasting form; SV: Simple-virilizing form; 17-OHP:
17-Hydroxyprogesterone.
Molecular analysis of the 21-OH gene
Molecular analysis of the 21-OH gene was performed in all patients included. The
most frequent type of genetic alteration detected was
deletions/conversions found 26/92 of alleles (28.26%).
The most frequent point mutation detected was
c.293–13 C>G in intron 2, found 21/92 of alleles
(22.82%).
Four patients with SW presented a homozygous genotype, in one of them there was
no history of consanguinity. Homozygosis could be explained because both parents
had a mutation considered frequent in the general population
(c.293–13 C>G).
In 6 of 46 patients there were consanguinity history, 3 presented a homozygous
genotype and another 3 were compound heterozygotes.
In our cohort there is a good genotype phenotype correlation (94%) except
in 3 patients ([Table 4]), in which the
expected phenotype based on the genotype (severe mutation in one allele and
slight mutation in the other) would correspond to a non-classical form but the
clinical behavior is in a way SV. This phenotype is based on visible signs of
virilization from the neonatal stage (macrogenitalism), hyperpigmentation of
nipples and genitals, elevation of 17-OHP greater than
100 nmol/l in neonatal screening detection and confirmed in
venous blood and even elevation of plasma renin activity in the pubertal stage
in one of them that needed to add to the treatment fludrocortisone.
Table 4 Allele mutation in congenital adrenal hyperplasia
patients without good genotype phenotype correlation.
|
Clinical form
|
Mother’s allele mutation
|
Father’s allele mutation
|
|
SV
|
c.293–13 C>G
|
c.844 G>T (p.Val282Leu)
|
|
SV
|
c.1069 C>T (p.Arg357Trp)
|
c.844 G>T (p.Val282Leu)
|
|
SV
|
c.1360 C>T (p.Pro454Ser)
|
c.92 C>T (p.Pro31Leu)
|
SW: Salt-wasting form. SV: Simple-virilizing form.
Two new mutations not previously described in the literature have been found in
two patients. The intronic variant c.292+5 G>A which in
cis with the slight mutation p.Val282Leu constitutes a serious allele with PS1
and the mutation c.518 T>A (p.Ile173Asn) associated with SV
form.
Treatment
The age of onset of hydrocortisone treatment coincides with the age at diagnosis
in the SW forms at 8 days (range, 6–9 days), since the treatment begins
on the day the diagnosis is made. In the SV forms, the treatment is delayed up
to 26 days (range, 22–64 days), since the diagnosis in this case may not
be as obvious and the differential diagnosis between simple virilizing form and
transient elevations of 17-OHP has to be made.
Of the 10 patients with SV form, 3 required treatments with mineralocorticoid
from the beginning due to a sustained elevated PRA. In these patients the
treatment with fludrocortisone began at 16.5 days (range, 9–20
days).
Discussion
21-OHD Neonatal Screening Programs have allowed the early diagnosis of the classic
form with saline loss (before its clinical expression) favoring that the age of
initiation of treatment is increasingly early, so that the adrenal crisis can be
prevented and therefore the mortality and morbidity derived from the pathology
itself as well as shortening the time of incorrect assignment of sex at birth. Early
diagnosis and treatment are crucial to prevent these life-threatening crises and
their irreversible consequences such as intellectual disability due to brain damage
secondary to hyponatremia. The European Society of Pediatric Endocrinology and the
American Society of Pediatric Endocrinology (Lawson–Wilkins) establish that
21-OHD neonatal screening is recommended with a grade of recommendation
++ and level of evidence I [13]
[14].
The probability of death due to an adrenal crisis in the neonatal period in the
absence of screening is controversial but most studies establish that between
0–4% of children with saline loss due to 21-OHD would die despite
being born in highly qualified health centers [15].
In this study, 36 of 46 patients (78%) had SW 21-OHD, which is similar to
other studies reported in Europe and the United States [16]
[17].
A key aspect of our study is the possibility of having the results of the screening
test in time to prevent the occurrence of the salt loss crisis, which usually occurs
between the 2nd and 3rd week of life. The age at diagnosis of patients diagnosed by
screening our sample (8.5 days; 6.0–10.5) allows early diagnosis and thus
the establishment of treatment in time to avoid the salt loss crisis, adjusting to
the fundamental objective of a neonatal screening program on its ability to reduce
morbidity/ mortality.
In the studies published in the literature there is a great variability in the age
of
children at the time of definitive diagnosis of the classic form of the disease
(note that some authors present the data in means and others in medians). The age
of
diagnosis ranges from 6 days to about a month. The study by Chu et al. (Taiwan)
[18] establishes an average age at the
time of diagnosis by screening of 11.6 days (4–20) and average age at the
time of definitive diagnosis of 14.8 days (5–31). Steigert et al.
(Switzerland) [19], describe a mean age at
onset of treatment of 6.7 days (1–22) in children diagnosed by screening.
Gruñeiro-Papendieck et al. (Argentina) [20] places the average age at diagnosis at 6 days, Van der Kamp et al.
(Netherlands) [21] reports an average age of
treatment initiation in areas with 21-OHD screening at 7 days of life (1–31)
and in areas without 21-OHD screening at 14 days of life (0–115). Brosnan et
al. (Arkansas, Oklahoma and Texas, USA) [16]
describe a mean age at diagnosis by screening in men with SW of 12 days in screening
areas and 26 days in areas without screening. The age at diagnosis by screening in
the study by Cartigny-Maciejewski et al. (Lille, France) [22] was 18 days on average (5–90) and 9
days on average. The median age at the time of diagnosis by screening for children
with SW form reported by Therrell et al. in Texas [9] was 11 days (0–40) and for children with SV form 34 days
(0–111). In Sweden, Thilén et al. [23] establish a median age at diagnosis of 9 days in areas with screening
and 21 days in areas without screening and in Emilia-Romagna (Italy), Balsamo et al.
[24], report a median age at diagnosis by
screening of 20 days (12–150) in the period between 1980 and 1983 and 11
days (5–35) in the period between 1991 and 1995. In Israel, Sack et al.
[25] reported an age of initiation of
treatment before the month of life in areas with screening and in Wisconsin, Allen
et al. [26], reveal that the results of the
screening were obtained between the 5th and 9th day of life. In Spain, the average
age of diagnosis reported by the 5 Detection Centers that perform 21-OHD screening
is 10.9 days (5 percentile: 7.3 percentile 99: 15.9) [27], higher than that found in our sample (8.5
days; 6.0–10.5).
There was no clinical suspicion of 21-OHD in 82.6% (n=38) patients
diagnosed by neonatal screening, this figure exceeds 47% described by
Thilén [23] and 73% of the
Therrell study [9]. Of the patients without
clinical suspicion of any disease, 30 patients (79%) were SW forms with
potential risk of adrenal crisis. Among the 30 SW forms without suspicion of
disease, 24 were boys and 6 were girls. The remaining 8 (21%) patients
without clinical suspicion of 21-OHD affected by SV form (6 boys and 2 girls) the
benefit obtained from the screening has been early diagnosis, shortening the study
time of the girl with virilization of the genitals (diagnosis at 18 days of life)
and prevention of early pseudopuberty.
The disease was suspected in 17.4% (n=8) of the patients. The reason
for suspicion was the ambiguous genitalia in women (3 with SW 1 with SV form)
followed by family history in 4 cases (in 2 of them with prenatal diagnosis).
Therefore, as Therrell et al. describes, the cases detected before knowing the
results of the screening were due to the involvement of a sibling or to certain
signs of virilization in girls [9].
Taking these results into account, we can say that of the 46 patients affected by
21-OHD, 30 (65%) affected by saline loss with risk of mortality from adrenal
crisis, would have obtained an absolute benefit of the screening. In the
retrospective study carried out by Steigert et al. [19], he found that of the 31 patients with confirmed 21-OHD, 15 of them
(48.4%) would be those really benefited from the screening, since in them,
the diagnosis had not been suspected by the clinic. They would also have obtained
relative benefits, the 6 women with incorrect sex assignment at birth, the 8
patients affected by SV form without clinical suspicion of disease because thanks
to
the screening, the time was shortened until the definitive diagnosis was
reached.
Newborn girls with 21-OHD are recognized earlier due to their genital ambiguity. The
clinical examination makes it possible to suspect, prior to the result of the
screening, the classic forms of the disease in some women with ambiguous genitals,
consequently the joint use of the clinical examination and the screening test is a
very useful strategy to improve the early diagnosis of the illness. Men with SW
forms would be the most benefited by this screening because they do not have
clinical signs that warn of the disease before salt-wasting. In the absence of
screening, there is a percentage of cases with SW form that would die without ever
being diagnosed [28]
[29].
In contrast to those diagnosed in the prescreening era, most patients detected by
neonatal screening were asymptomatic or had mild symptoms upon diagnosis, especially
among males. The data here confirm those previous studies [23]
[30]
[31].
There are no studies in the literature that analyze where patients were when they
received the result of neonatal screening. In this study, 79% (n=30)
were at home without suspicion of any disease and 7.91% (n=3) were
at home pending study for clinical suspicion of any disease (ambiguous genitalia,
cryptorchidism) and 13.2% (n=5) were admitted to the hospital for
reasons unrelated to CAH (sepsis, jaundice, hypoglycemia).
It is important to highlight that 69.4% of the patients
(n=25/36) with SW form identified by neonatal screening were in
their homes with potential risk of death and that 6 women had been incorrectly
labeled as men at birth (3 were in their homes as healthy men without any clinical
suspicion, 2 were in their homes pending study for hypospadias, and 1 of them was
hospitalized for ambiguous genitals).
In conclusion, this study allows us to detect that in the majority of patients with
21-OHD, the diagnosis by screening was anticipated to the clinical suspicion of the
disease even in female patients with ambiguous genitalia. The Neonatal Screening
Program allows the identification of affected by CAH newborns and avoids the
establishment of severe dehydration and shock conditions; reduce the time of
incorrect sex assignment at birth in virilized girls and detect boys with simple
virilizing form early.
