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Original Studies |
Department of Pediatrics (A.N., A.L.) and PKU Screening Laboratory (A.N., L.H.A.L.), Huddinge University Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden; the Department of Pediatrics, Ryhov (A.T.), S-551 85 Jökoping, Sweden; and the Department of Molecular Medicine, CMM:02, Karolinska Hospital (A.W.), S-171 76 Stockholm, Sweden
Address all correspondence and requests for reprints to: Anna Nordenström, M.D., Department of Pediatrics, Huddinge University Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The syndrome comprises a very wide spectrum of severity (1). The most severe form, with complete absence of 21-hydroxylase function, leads to acute circulatory collapse, usually during the first weeks of life. This salt-wasting (SW) crisis is due to a severe lack of mineralocorticoid, which causes loss of sodium in the kidney. Children with the SW form of the disease are exposed to excessive androgen levels during embryogenesis. The girls present with virilization of the external genitalia, i.e. clitoromegaly, fusion of the labia majora, and a common urethral and vaginal opening. A slightly less severe form of 21-OHD, with prenatal virilization but without life-threatening salt loss, is usually referred to as the simple virilizing (SV) form of CAH. Finally, milder forms of 21-OHD are associated with slightly elevated androgen levels, which are not sufficient to cause prenatal genital malformations. These forms of the disease may present as precocious pseudopuberty or growth acceleration in childhood or may cause menstrual disturbances, hirsutism, and infertility in adult women. They are often referred to as nonclassical (NC) CAH.
CAH due to 21-OHD is inherited as an autosomal recessive trait, and the molecular genetics of the disorder has been studied extensively. The 21-hydroxylase gene (CYP21) is located on the short arm of chromosome 6, between the human leukocyte antigen class I and class II gene clusters, in tandem with a highly homologous pseudogene (CYP21P) (3, 4). This genomic structure predisposes to misalignment during meiosis, which may result in recombination events between the CYP21 and CYP21P genes. This can lead to deletion of CYP21 as well as to an exchange of sequences between the two genes (5, 6). Deletion of CYP21 together with nine smaller, pseudogene-derived sequence aberrations are responsible for around 95% of all affected Scandinavian CYP21 alleles (7) and are also responsible for the majority of CAH patients in other ethnic groups (8, 9, 10, 11). The remaining 5% of cases are due to rare, mostly population-specific mutations. In Scandinavia, we have identified 12 such rare mutations to date. Five are nonsense, splice, or frameshift mutations and have therefore not been assayed functionally in vitro (12, 13, 14). Seven are missense mutations that have displayed variable degrees of impaired enzyme function after expression in cultured cells (15, 16, 17).
We have found clear relationships between clinical disease severity and the CYP21 mutations (7). In short, one group of mutations completely inactivates the gene and is referred to as Null mutations. Homozygosity or compound heterozygosity for any of these mutations is associated with the most severe form of CAH, with salt loss and prenatal virilization of external genitalia in females. The I2 splice mutation can be regarded as slightly less severe. It is associated with severe prenatal virilization in females, but a small number of children with this mutation have escaped salt loss. The Ile172Asn mutation is associated with the simple virilizing phenotype, although around 10% of cases with Ile172Asn develop salt-wasting symptoms, and the degree of virilization can vary. Finally, the Val281Leu mutation has in our experience not been found in any patient with virilized genitalia. Instead, it is associated with mild, late-onset symptoms. In conclusion, we find genotyping of CYP21 mutations to be very useful for the prediction of clinical outcome in CAH patients. Similar experiences were reported previously (11), whereas others have concluded that the phenotype is not always concordant with the genotype (10).
Neonatal screening for CAH started on a national basis in Sweden in 1986 (18). Elevated levels of 17-hydroxyprogesterone (17-OHP) are used as an indicator of the disease (19, 20). By the end of 1997, more than 1.3 million Swedish children had been screened. The prevalence of CAH among these subjects was 1 in 9800, and 85% of the patients had the SW form of the disease (21). No children with other causes of CAH, such as 3ß-hydroxysteroid or 11ß-hydroxylase deficiency, were diagnosed during these years.
There are several obvious advantages of neonatal screening for CAH, such as earlier diagnosis, avoidance of salt crises, and an earlier correct gender assignment (22, 21). At the same time, however, it has raised a few novel questions, such as how to interpret a neonatal 17-OHP screening value in terms of disease severity. In particular, we have seen a tendency for overtreatment of mildly affected cases that have been diagnosed neonatally through screening. This is a risk when treatment is initiated before symptoms have had time to develop and, thus, the disease severity is not clinically assessed. On the other hand, it is one of the benefits of screening that salt loss and its potentially harmful effects on central nervous system development can be avoided. To evaluate genotyping as a diagnostic complement to the neonatal screening for CAH, we have compared CYP21 genotypes and neonatal screening values of 17-OHP for 91 Swedish patients diagnosed with CAH between the years 1986 and 1997. Comparisons were also made to screening values of 17-OHP for 141 false positive cases.
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Subjects and Methods |
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17-OHP was analyzed in filter paper blood spots collected on day 3, 4, or 5 after birth using RIA (1986–1990) or fluoroimmunoassay (1991–1997) (Delphia, Wallac, Finland) (19, 18). The cut-off limit for a positive test has gradually been lowered from 200 nmol/L in 1986 to 150 nmol/L in 1988, and, since 1991, it has been defined as 75 nmol/L plasma (assuming a hematocrit of 50%) for full-term infants. For infants born before the 37th week of gestation, a cut-off limit of 200 nmol/L after ether extraction was used. Recall was on the eighth day of life (median) (21). A second filter paper sample was requested for all cases with a positive screening value. The second sample was usually analyzed about 1 week after the first screening sample. Children in whom 17-OHP levels had decreased convincingly in the second sample (>20%) and/or in whom subsequent clinical examination excluded CAH were defined as false positive cases.
CYP21 genotyping
CYP21 mutation analysis was carried out using allele-specific PCR from genomic DNA prepared from venous blood samples (13). This detects the 95% of alleles that carry any of the common pseudogene-derived mutations. Additional rare alleles were characterized by direct DNA sequencing (12). Genotypes were divided into four groups depending on the mildest mutation: Null, I2 splice, Ile172Asn, and Val281Leu.
Statistical analysis
The Kruskal-Wallis test, paired t test, Mann-Whitney U ranking sum test, and the SPSS computer program (SPSS, Inc., Chicago IL) were used in the statistical analysis.
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Results |
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The genotypes of the remaining 91 full-term infants who were diagnosed
with CAH are summarized in Table 1
. The
Null genotype group consisted of 22 infants. The I2 splice group
comprised 26, and the Ile172Asn group consisted of 30
infants. Thirteen infants belonged to the mildest genotype group,
represented by the Val281Leu mutation. A second sample was
obtained before steroid treatment was initiated for 19 infants in the
Ile172Asn group and 6 in the Val281Leu group.
The second sample was obtained at about 8–12 days of age.
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The 17-OHP screening values for the CAH children ranged from 41–1371
nmol/L plasma. Screening values of 17-OHP were significantly different
among the three genotype groups, Null, I2 splice, and
Ile172Asn+Val281Leu (P < 0.05;
Fig. 1). The two mildest groups did not
differ from each other, but were different from the false positive
group. Therefore, 17-OHP values representing the second sample, from
the recall filter papers, were compared among the
Ile172Asn, Val281Leu, and false positive groups
(Fig. 2). The groups representing the
Ile172Asn and Val281Leu mutations were then
significantly different from each other (P < 0.05) as
well as from the false positive group.
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As a rule, the full-term infants with false positive screening tests
had only moderately elevated 17-OHP levels, in the same range as those
in patients with genotypes associated with the SV or NC forms of the
disease (Fig. 1
). Among the children with false positive tests, there
were 2 infants for whom the second sample showed an increased 17-OHP
level compared to the first (97 to 154 and 104 to 175 nmol/L) and 4 who
had unchanged levels in the second sample. In 3 of these 6 cases,
including the 2 with increased values, CYP21 mutations were
excluded by genotyping. In an additional 7 infants, the 17-OHP value
did not decrease convincingly. Thus, in 10.3% of the false positive
cases (13 of 126), the second sample was not conclusive. A relatively
large group of these infants (45%) was born in gestational week 37.
This is in keeping with the fact that immature infants have higher
levels of 17-OHP in the screening. The symptoms of these children are
not known to the screening laboratory in any great detail. However, a
large proportion of these children seem to have been under perinatal
stress, e.g. they had respiratory distress, intracranial
hemorrhage, sepsis, or cardiac malformation with cardiac failure.
The genotypes of six children diagnosed with CAH between 1988 and 1997
despite negative screening values were also determined and compared to
their neonatal 17-OHP screening values (Table 2). Four of these children with false
negative screening tests had genotypes associated with the mildest form
of CAH (mutation Val281Leu). None of these had any symptoms
from birth. One boy had the Pro30Leu mutation. He was
diagnosed neonatally because his elder sister was known to be affected
with CAH. The sister, who was born before the neonatal screening
program was initiated, was detected due to growth acceleration,
premature pubarche, and modest clitoral enlargement at 3.8 yr of age.
The Pro30Leu mutation is known to be associated with
nonclassical CAH, but it usually produces somewhat more symptoms,
including slight clitoromegaly, compared to the Val281Leu
mutation (24). One girl had the Ile172Asn mutation, which
is associated with the simple virilizing form of CAH. When she was
diagnosed at 2.5 yr of age, signs of prenatal virilization were found
(clitoromegaly and moderate labial fusion). These malformations, which
had been missed neonatally, were subsequently surgically corrected.
Thus, the severity of symptoms of the false negative children at
diagnosis was in conformity with their genotypes.
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Discussion |
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, a severely elevated screening value of 17-OHP was clearly associated
with a genotype in which Null or I2 splice was the mildest mutation. In
fact, apart from one child with an Ile172Asn/Null genotype
who had a screening value of 852 nmol/L, all children in the
Ile172Asn and Val281Leu groups had values below
400 nmol/L. The girl with a level of 852 nmol/L was initially taken for
a boy and started to show reduced serum sodium levels before treatment
was initiated; she thus belonged to the most severely affected children
seen in the Ile172Asn genotype group. For the above
reasons, we believe that a screening value above 500 nmol/L can be
regarded as evidence of the most severe forms of CAH. However, with
regard to lower screening levels of 17-OHP, no safe conclusions about
disease severity can be drawn, even though the genotypes were
significantly different from each other when grouped. For example, a
value around 150–200 nmol/L was seen in relation to all degrees of
disease severity, from salt wasting to mild CAH (genotype groups I2
splice, Ile172Asn, and Val281Leu), and could
even constitute a false positive test. For infants with moderately
elevated 17-OHP levels, it is important that the diagnosis is also
indicated by other laboratory tests or clinical signs before treatment
is started to avoid excess use of steroids. One consequence of introducing neonatal screening for CAH that must be considered is the fact that patients who would otherwise be diagnosed due to late-onset symptoms will now be identified from birth. It cannot be taken for granted that these patients will only benefit from being treated from this early age, before the onset of symptoms. In fact, we have seen a tendency for overtreatment of these mildly affected cases from birth because the degree of disease severity has not been clear. Verifying a mild genotype (e.g. the Val281Leu mutation) means that the risk of salt loss is extremely low. In addition, there have been observations indicating that early growth may not be increased in these patients until after 18 months of age (25). Thus, with careful clinical monitoring, including measurements of bone age, height, growth velocity, and steroid levels, glucocorticoid replacement therapy can be kept to a minimum in children in whom the mildest mutations are identified. In addition to these somatic examinations, however, it may also be important to consider possible influences of excess steroid hormones on behavior (26).
The three genotype groups, Null, I2 splice, and Ile172Asn+Val281Leu, were already significantly different from each other in the first screening sample (days 3–5). The Ile172Asn and Val281Leu groups could clearly be separated by the second measurement, which was obtained around days 8–12. Thus, a more obvious increase in 17-OHP evolves over time in the moderate Ile172Asn group compared to the milder Val281Leu group. Increasing 17-OHP values with time was also seen in the screening program in Texas, where two screening samples were taken routinely. A relatively large proportion of patients affected by milder forms of CAH was identified with the second sample, which was obtained at 2 weeks of age (27).
We also compared 17-OHP levels after subdividing the patients according to the different genotypes within the four major groups, i.e. we compared individuals who were homozygous and hemizygous for the mutations. Hemizygosity (I2 splice/Null: median, 746 nmol/L; Ile172Asn/Null: median, 222 nmol/L) was associated with higher screening values than homozygosity (I2 splice/I2 splice: median, 710 nmol/L; Ile172Asn/Ile172Asn, median, 152 nmol/L), indicating a gene dosage effect. These differences were not statistically significant, however.
The relatively large interindividual spread in screening values is not surprising, as it is well known that individual 17-OHP values are affected by factors such as concurrent illness and stress. One patient in the I2 splice/Null subgroup was delivered by elective cesarean section because he was in the breech position. This patient had the lowest 17-OHP screening value within his subgroup (186 nmol/L). This is not surprising because it has been reported that early corticosteriod levels are influenced by birth conditions (28).
The patients who had the Pro30Leu mutation, which is
known to be associated with nonclassical CAH (24), were not included in
our calculations because only two cases were found with genotypes
including this mutation. These two patients were both males, both had a
Pro30Leu/Null genotype, and both were born in 1991. Their
screening values were considerably different from each other; one had a
value of 41 nmol/L and was thus a false negative case (Table 2),
whereas the other one had a value of 419 nmol/L. We have previously
found that some patients with Pro30Leu have gene
conversions in their promoter, changing CYP21 sequences into
pseudogene-derived sequences, whereas others retain the
CYP21 promoter (unpublished). As the CYP21P
promoter is known to be partially active compared to that of
CYP21 (29), patients with Pro30Leu in
combination with the promoter mutation can be expected to be more
severely affected than those with Pro30Leu alone. However,
we sequenced the CYP21 genes of the two boys who had
Pro30Leu up to 800 bases upstream of translation
initiation. Both patients had the CYP21P promoter in the
CYP21 allele that carried Pro30Leu,
i.e. the complete 5'-parts of CYP21 were
converted into CYP21P in both cases. Thus, we could not find
any explanation for the large difference in their screening values.
For 13 infants with positive screening samples who were later shown to be unaffected, i.e. in 10.3% of the false positive cases, the second screening sample was inconclusive. As CYP21 genotyping identifies 95% of all alleles causing CAH, and the majority of the remaining 5% are Null mutations, mutation analysis is a reliable as well as rapid way of discarding or verifying the diagnosis of CAH in these cases. It is important to shorten the time of uncertainty as much as possible to minimize the negative psychological effects on the family of a false positive screening result (30).
In Sweden, genotyping is performed for most, if not all, children
in whom positive screening samples of 17-OHP are found. We find this to
be an ideal diagnostic complement, both in providing prognostic
information on the degree of disease severity as well as in
discriminating against false positive cases. However, as shown in Table 2, some patients with mild and even moderate forms of the disease will
invariably escape diagnosis in the screening unless it is designed to
produce unacceptable numbers of false positive results. Obviously, the
well-being of these children will continue to be dependent on the
perceptiveness of clinicians who are skilled at identifying symptoms
and signs of androgen excess.
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Acknowledgments |
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Footnotes |
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Received September 1, 1998.
Revised January 13, 1999.
Accepted January 25, 1999.
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