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Original Studies |
Department of Pediatrics, New York University Medical Center (D.C., N.U., R.D., S.O.), New York, New York 10016; Department of Pediatrics, North Shore University Medical Center (P.W.S., N.D.), New York, New York 11030; and the Department of Medicine, Cornell University Medical Center (J.I.-M.), New York, New York 10021
Address all correspondence and requests for reprints to: Sharon E. Oberfield, M.D., Babies and Children’s Hospital, Box 50, Department of Pediatrics, 3959 Broadway, New York, New York 10032.
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Abstract |
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Steroidogenic profiles were obtained for the three affected siblings. The available clinical history of the two affected aunts was retrieved. Genotyping was performed on several members of the kindred. Detailed sequencing of the entire CYP21 gene of two clinically dissimilar subjects in this family was undertaken to explore the possibility of other mutations or polymorphisms.
PCR with ligase detection reaction analysis of CYP21 revealed that the affected family members III-2, III-3, III-4, II-3, and II-4, all were compound heterozygotes carrying the intron 2 point mutation known to interfere with splicing (nucleotide 656 A to G) and the exon 4 point mutation causing a nonconservative substitution of asparagine for isoleucine at codon 172 (I172N). Detailed sequencing of the gene was performed for the two most phenotypically dissimilar subjects. A single silent polymorphism was found in the third nucleotide for codon 248 in patient II-4, but not in patient III-4, and no additional mutations were found.
Classic congenital adrenal hyperplasia remains a difficult diagnosis to make in the absence of newborn screening because of the variability of phenotypic expression. Likewise, the variable degree of genital ambiguity in affected females in this family serves to question universal advocacy of prenatal steroid treatment in pregnancies at risk for congenital adrenal hyperplasia. Extensive molecular exploration did not provide an explanation of the phenotypic heterogeneity and supports the possibility of influences other than the CYP21 gene for the observed divergence.
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Introduction |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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Case Report |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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The proband, III-2, is the second of four children born to parents
of nonconsanguineous Irish-English-Italian ancestry (Fig. 1
). The family history is significant for
two paternal aunts (II-3 and II-4) with classic CAH due to
21-hydroxylase deficiency, one of whom (II-4) has the severe
salt-wasting form. They were cared for by the same pediatrician as the
proband. III-2 first came to medical attention at age 4 1/2 yr for
evaluation of acne. He was subsequently referred by the dermatologist
for a comprehensive endocrinological evaluation upon finding, in
addition to the acne, tall stature, a large phallus for his age, pubic
hair, as well as a younger sibling (III-3) with a history of premature
pubarche. Their mother (II-1) was 7 months pregnant at the time of
their (III-2 and III-3) diagnosis of CAH. At birth, III-4 was
discharged from the nursery as a normal healthy newborn girl despite a
high index of suspicion for the disorder. On day of life 5, she was
evaluated by the authors and found to have subtle signs of
virilization.
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This male child was born by cesarian section (breech); his weight and length were 3.88 kg and 53.3 cm, respectively. He was healthy during infancy and was never hospitalized for dehydration. Linear growth acceleration was noted from age 2 months at the 90th percentile to more than the 97th percentile by age 3 yr. Acne had been noted by his mother since the age of 2 1/2 yr. Concern about the large size of the genitalia and the presence of pubic down prompted the initial endocrine evaluation.
Physical examination revealed a height age of 7 3/4 yr (125.7 cm) and a weight age of 8 3/4 yr (27.2 kg) at chronological age 4 1/2 yr. Blood pressure was normal. Cystic acne was present on his forehead and upper back. A few long, dark, curly hairs (Tanner II) were seen at the base of his phallus. The stretched penile length was 7 x 2.5 cm. Testes were Prader 2 mL bilaterally.
Laboratory data are shown in Table 1. The bone age was close to 12 yr
according to the standards of Greulich and Pyle. The predicted height
of 157.5 cm (62 in.) was in marked contrast to the adjusted midparental
height of 181.6 cm (71.5 in.).
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This female child was born full term by vaginal delivery; her weight and length were 3.23 kg and 55.8 cm, respectively. The pregnancy was significant for maternal use of 200-mg progesterone suppositories each day during weeks 11–15. This was prescribed because of bleeding at week 11. The child’s growth was always along the 90th percentile, and she had no history of salt craving. Pubic down was noted at the age of 2 1/2 yr along with a slight growth acceleration.
Initial evaluation at age 2 yr 11 months revealed a height age of 3 3/4 yr (100.3 cm) and a weight age of approximately 4 yr (17 kg). Blood pressure was normal. She had moderate acne of the forehead. There was a 1.5-cm erectile clitoris with two separate perineal orifices (urethral and vaginal). There was partial fusion of the labia minora in the posterior region of the introitus. She had early Tanner II pubic hair and Tanner I breast tissue.
Laboratory data are shown in Table 1
. The bone age according to the
standards of Greulich and Pyle was 6 yr, with a predicted height of
143.5 cm (56.5 in.), which is 25.4 cm (10 in.) less than her
adjusted midparental height.
Sibling III-4
The mother (II-1) was 7 months pregnant at the time the diagnosis of CAH was made in her children, III-2 and III-3. No prenatal testing was performed. The parents also did not wish to know the sex of the child by ultrasound. The pregnancy was without complications; she did, however, receive 25 mg progesterone, im, every day during weeks 7–14 because of three spontaneous abortions between the births of III-3 and III-4.
At the time of delivery, the neonatal staff were alerted to the possibility of the child being affected. Two independent experienced pediatricians and an attending neonatologist assessed the genitalia as consistent with that of a normal newborn female. Birth weight was 3.37 kg (>50th percentile), and length was 50.8 cm (75th percentile). The infant was discharged home on the second day of life.
On day 5 of life, when 17OHP was reported to be elevated (Table 1), she
was admitted for further endocrinological evaluation and initiation of
treatment. Electrolytes were normal. She had been thriving at home and
breast-feeding without difficulty. Weight was 3.4 kg. She appeared well
hydrated, alert, and vigorous. The clitoris was enlarged, measuring 1.5
cm erect with a redundant clitoral hood (6). Mild posterior labial
fusion was suggested by a foreshortened introitus with increased
anogenital distance (7). A urethral opening was identified separate
from the vagina. Pelvic ultrasonography identified the left adrenal
gland as measuring 1.6 x 1.7 cm and the right as measuring
1.6 x 1.4 cm and confirmed the presence of a uterus, although the
ovaries were not identified.
The clinical findings of clitoromegaly and mild posterior labial fusion and the biochemical abnormalities of a moderately elevated 17OHP in the presence of a blunted cortisol response to ACTH stimulation were consistent with the diagnosis of CAH due to steroid 21-hydroxylase deficiency (8).
All three children were classified as simple virilizers and responded to glucocorticoid therapy alone with a decline in 17OHP, adrenal androgens, and PRA to within normal limits for age.
The affected paternal aunts
In the preceding generation there are two affected females, II-3
and II-4 (Fig. 1). II-3 has received cortisone acetate since her
diagnosis in early infancy. By report, she had minimal clitoral
enlargement, but had surgical correction as a young child for posterior
labial fusion. She never had evidence of salt wasting. She is married,
conceived while receiving glucocorticoid therapy, and delivered four
healthy children, two vaginally and a set of twins by cesarian section.
Her adult height is 155 cm (5 ft, 1 in.).
II-4 had a salt-wasting crisis early in infancy with a serum sodium of
132 mEq/L and a potassium of 8.0 mEq/L, requiring treatment with
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-fludrocortisone (Florinef). Her history is significant for
multiple hospitalizations for dehydration and adrenal crises in
childhood despite treatment with both cortisone acetate and Florinef.
Similar to her sister, she also had a minimally enlarged clitoris, but
only underwent surgical correction of labial fusion. She has not
married and has no children. She is 149 cm (4 ft, 11 in.) tall. Her
medical history is significant for a debilitating seizure disorder.
Other than the two paternal aunts that were known to have the disorder, there are no additional family members with suggestive histories. There is no history of infertility, except that II-5 has required clomiphene induction and, as stated above, II-1 has had three miscarriages between the births of her third and fourth children. There are no reports of hirsutism, acne (9), or premature adrenarche (10).
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Materials and Methods |
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17OHP of the cord blood for III-4 was measured by minor modification of previously published standard RIA methods after column extraction with Celite by the Pediatric Endocrine Laboratory of New York University/Bellevue Hospital Center (New York, NY). All other steroid hormones were measured using RIAs by Endocrine Sciences (Calabassas Hills, CA).
Oligonucleotides, reagents, and standard methods for detecting mutations in CYP21
Oligonucleotide primers were synthesized on a model 394 automated DNA synthesizer (Applied Biosystems Division, Perkin-Elmer Corp., Foster City, CA). Fluorescent label was attached to the 5'-end of oligonucleotides using 6-carboxyfluorescein or tetrandrine amidites. Gene-specific PCR amplifications and ligase detection reactions (LDR) were performed as described by Day et al. (11). Taq DNA polymerase, Stoffel fragment, and other PCR reagents were obtained from the Applied Biosystems Division of Perkin-Elmer. The methods used for detection of mutations in CYP21 are standard in our laboratory and were described previously (11).
Microsatellite PCR amplifications
The sequences of the microsatellite primers used for
amplification of loci D6S273 and tumor necrosis factor- (TNF)
have been described previously (12, 13). All of the forward primers
were 5'-labeled with a fluorescent dye to allow automated analysis on a
fluorescent DNA sequencer. The TNF primer was labeled with fluorescent
dye, 6-carboxyfluorescein, and the D6S273 primer was labeled with the
fluorescent tetrandrine dye. PCR and gel electrophoresis were performed
as described by Day et al. (14). Data were collected using
an Applied Biosystems sequencer (Applied Biosystems, Foster City, CA)
running Genescan software. The sizes of amplification products were
determined relative to the ROX 100 or TAMRA 350 markers.
DNA sequencing of PCR products
DNA sequencing was performed in the most (II-4) and the least
(III-4) affected family member using an Applied Biosystems 377
four-color automated sequencer. PCR products that were to be sequenced
were purified using the Wizard PCR Preps DNA Purification System
(Promega, Madison, WI) according to the manufacturer’s instructions.
Purified PCR product (100 ng) and appropriate PCR primer used as
sequencing primer (5 pmol) were added to a total volume of 12 µL, and
the product was sequenced using the Applied Biosystems dye terminator
chemistry following the manufacturer’s instructions. The sequences of
the primers used for sequencing are provided in Table 2. Both strands were sequenced, and the
exons, introns, and a 227-bp region of the promoter immediately
upstream of the transcriptional start site and known to be important in
transcriptional regulation (15) were sequenced for patients II-4 and
III-4.
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Results |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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). Thus, in this pedigree, the genotypes
are clear, and the alleles segregate as expected between generations.
Because mutations were clearly identified on both maternal and paternal
alleles, there was no concern about deletion of CYP21, and duplication
of CYP21 is rare and unlikely to contribute to phenotype (16). Interestingly, the paternal aunts, II-3 and II-4, were also compound heterozygotes for the same two CYP21 mutations as the three proband children, III-2, III-3, and III-4. In light of these findings and the dissimilar phenotypes of II-4 and III-4, the entire CYP21 in both these family members was sequenced, including a portion of the 5'-promoter region. A single silent polymorphism was found in the third nucleotide for codon 248 in patient II-4’s maternal haplotype, but not in III-4 (CTC is changed to CTG, both of which encode the wild-type leucine residue and therefore would not affect enzyme function). There were no additional mutations found.
Family member II-5 was at first typed as nucleotide 656 G homozygous
despite sequencing in both directions, a finding incompatible with her
normal phenotype. Microsatellite typing with primers for chromosome 6
markers D6S273 and TNF indicated that she was, in fact,
heterozygous, sharing the affected haplotype of her mother, I-2, but
also carried the same unaffected haplotype as her father, I-3. These
data combined indicated that one CAH allele had not been amplified
during PCR (14).
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Discussion |
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TopAbstractIntroductionCase ReportMaterials and MethodsResultsDiscussionReferences |
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Although the genotypes of the affected members, II-3 and II-4, as well as those of III-2, III-3, and III-4 were identical, the mother of our proband, III-2, was not related to her husband. They are all of mainly Irish ancestry and shared the common nucleotide 656 G mutation with the paternal grandmother, I-2, and the paternal aunts, II-3 and II-4; haplotype sharing is frequent in homogeneous ethnic populations. Only II-4 had suffered severe salt-wasting episodes in infancy requiring treatment with a mineralocorticoid in addition to glucocorticoid. The degree of genital virilization was also divergent among these four female family members, with the youngest two having only minimally affected genitalia that will not require surgical intervention, in contrast to the severely affected salt-wasting aunt, II-4, and the multiparous aunt, II-3, both of whom have had genital surgery.
In subjects III-3 and III-4, the impact of prenatal exposure to progesterone on genital development cannot be resolved. Progesterone has been associated with increased clitoral size (28), although others have reported no evidence of fetal virilization with its use (29). The fact that our patients were only minimally virilized, probably indicates that this drug exposure did not pose a confounding problem in genital development.
This family is also informative with respect to recent controversy regarding prenatal treatment with dexamethasone in pregnancies at risk for CAH. Some centers prescribe prenatal glucocorticoid treatment in an attempt to reduce the degree of genital ambiguity in potentially affected females with CAH. The short and long term safety of such treatment to the mother and to the affected and nonaffected fetuses is the subject of continued debate (30). Based on the experience gained with this family, we advocate caution in advising prenatal treatment for all female fetuses at risk. The last child (III-4) had only mildly affected genitalia and may have been regarded as a successful treatment outcome had the mother received intrapartum dexamethasone therapy. However, as no treatment was rendered due to the late diagnosis of the proband, the "outcome" was equally "successful." Thus, this family represents an albeit small but ideal study of the natural history of genital virilization in CYP21-identical girls with CAH.
The reliance on clinical diagnosis as opposed to mass newborn screening may result in significant mortality (in the case of an unidentified salt-wasting male infant) and morbidity (i.e. incorrect sex assignment) (31). Although more regions are introducing 17OHP screening of newborns, the debate of the cost vs. benefit continues. The cost of a screening 17OHP can be as low as 30 cents. The northern Italian Emilia-Romagna region report confirms that during a period without newborn screening, many affected infants were not identified based on clinical survey alone (32). False positive reports are most often associated with prematurity or hyperbilirubinemia (33). Whereas prevention of death of undiagnosed males is the primary purpose of newborn screening programs for CAH, we speculate that the predicted final height of III-2 and III-3 might have been improved had the children been identified by newborn screening, thus prompting earlier treatment (34, 35, 36, 37).
In summary, the clinical presentations of this family underscore several difficult issues of management and diagnosis of children with classic CAH. This family was fortunate in that the children, III-2, III-3, and III-4, did not have the salt-wasting form of the disease, which is present in 75% of CAH patients and is a significant cause of mortality. The mild genito-urinary abnormalities despite clear biochemical evidence of hyperandrogenism of the female probands, III-3 and III-4, evokes further discussion of the benefit of prenatal therapy. A molecular explanation of the divergence of phenotypes across two successive generations was not found and therefore supports possible influences other than the CYP21 gene (27).
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Acknowledgments |
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Footnotes |
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Received January 6, 1998.
Revised March 2, 1998.
Accepted March 9, 1998.
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