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Original Studies |
Department of Pediatrics, Weill Medical College of Cornell University (B.I.C., R.S.N., S.N., M.I.N., R.C.W.), New York, New York 10021; Fondation Jean Dausset, Centre d’Étude du Polymorphisme Humain (L.P.), 75010 Paris, France; Baker Medical Research Institute (K.M.C.), 8008 Melbourne, Australia; and Childrens Hospital Los Angeles (T.F.R.), Los Angeles, California 90027
Address all correspondence and requests for reprints to: Maria I. New, M.D., Department of Pediatrics, Division of Pediatric Endocrinology, Weill Medical College of Cornell University, 525 East 68th Street, Room M-622, New York, New York 10021. E-mail: minew{at}mail.med
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Abstract |
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1) in the CYP11B1 gene in this family. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The CYP11B1 gene, which encodes the 11ß-hydroxylase enzyme, is comprised of nine exons. It is located on chromosome 8q24.3 (5), about 40 kb from the highly homologous gene CYP11B2 (6, 7), which encodes aldosterone synthase (8). Mutations in the CYP11B1 gene have been identified throughout the coding region, but there is clustering around exons 2, 6, 7, and 8, suggestive of mutational hot spots (9, 10). These mutations have been identified from diverse ethnic backgrounds, with the highest incidence among a highly inbred group of Moroccan (Sephardic) Jews. This parallels the finding that nonclassical 21-OHD is found most commonly among the Eastern European (Ashkenazi) Jews (1 in 27 in a heterogeneous population of New York City) (11). However, unlike 21-OHD CAH, where the majority of mutations are deletions or gene conversions from the neighboring CYP21P pseudogene, the majority of the mutations found in CYP11B1 are deleterious random point mutations of the CYP11B1 gene (12). Although there is clustering of mutations in some exons, mutation analysis is relatively more difficult compared to that for CYP21, in which approximately 10 known mutations account for 84–96% of the mutations causing 21-OHD CAH (13). Gene conversions do occur between CYP11B1 and CYP11B2 (10, 14), however, the resulting converted gene would also be expected to be functional, as both encoded enzymes have 11ß-hydroxylase activity.
In 11ß-OHD CAH, the experience in prenatal treatment is comparatively limited: to date, there have been five families reported to have undergone prenatal diagnosis and/or treatment. In 1989, Bouchard reported the first prenatal treatment with dexamethasone of an affected female (15). Other attempts followed thereafter. Two families were reported by Curnow et al. (9), one was reported by Geley et al. (16), and one was recently reported by Cerame et al. (17). In Bouchard’s first case, treatment was initiated late, was interrupted in midpregnancy, and eventually was discontinued based on normal steroid concentrations in the amniotic fluid. The result was a failure, as the baby was born severely virilized. In the latter four cases, the fetus was either a heterozygote or unaffected. We report here the first successful prenatal treatment of an affected female with 11ß-OHD CAH (family 1). The newborn had normal female external genitalia.
In addition, we report clinical and molecular analysis of a family who has two 11ß-OHD-affected sons (family 2) and wishes to have a third child. Preconception counseling is part of our program of prenatal diagnosis and treatment. In preparation for early prenatal treatment, molecular analysis was initiated, and a novel mutation in the CYP11B1 gene was found.
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Subjects and Methods |
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Subjects
Family 1 is a consanguineous Yemenite Muslim family (see
pedigree, Fig. 1
). The index case (IIa)
was highly virilized prenatally and was born with ambiguous genitalia
(Prader IV). She was diagnosed late in infancy. At the time of her
diagnosis, the mother was pregnant with a second child. At 34 weeks
gestation, hormonal diagnosis was made on this second pregnancy based
on measurement of an elevated urinary tetrahydro-11-deoxycortisol (THS)
level of 1.43 mg/24 h (normal, <0.050 mg/24 h). Due to the late stage
of gestation, prenatal treatment was not initiated. The second female
child (IIb) was also born with ambiguous genitalia (Prader IV). The
third pregnancy (IIc) in this family was prenatally treated with
dexamethasone starting at the fifth week of gestation, blind to the
gender and genetic status of the fetus, according to the established
21-OHD CAH algorithm (3). The mother was recently pregnant again for
the fourth time, and dexamethasone treatment was initiated at the fifth
week of gestation just as in her third pregnancy. Karyotyping of the
fourth pregnancy revealed that the child (IId) was male; therefore,
dexamethasone treatment was discontinued at week 11 of gestation.
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). Clinical diagnosis of 11ß-OHD was
made at age 18 months in the index case (IIa). He presented with
virilized and enlarged genitalia, penile stretched length of 9.5 cm
with a diameter of 2.5 cm, and testicular volume of 2 cc bilaterally.
He had Tanner stage 3 pubic hair. He was muscular and had a deep voice.
His body growth was rapid, and his height and weight were both above
the 95th percentile. His blood pressure was elevated above the 95th
percentile for age (130/90 mm Hg; normal, 106/59). His bone age was 7
yr at a chronological age of 19 months. The younger brother (IIb), who
was born 4 yr later, had enlarged and pigmented genitalia at birth. At
1 month of age, his penile stretched length was 4.5 cm with a diameter
of 1.3 cm. Testicular volume was 1.5 cc bilaterally. Blood pressure was
normal, and the remainder of his exam was unremarkable.
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For family 1, DNA analyses was performed by PCR amplification of exons 3 and 5, followed by direct sequencing, as previously described, using peripheral blood or chorionic villous tissue (9).
For family 2, DNA was prepared from peripheral blood following standard
methods. Genomic DNA was amplified using PCR and primer specific for
the CYP11B1 gene, as described by White et al.
(12). PCR was performed in two steps. First-step PCR primers were as
described by White et al. (12) (Table 1). The second step PCR was performed
using a forward primer containing an M13 sequence on the 5'-end and a
reverse primer modified with biotin on the 5'-end (Table 1). In the
first PCR, 100–500 ng genomic DNA were denatured for 10 min at 98 C.
The following reagents were added to the denatured DNA: 50 mmol/L KCl,
10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 0.01%
gelatin, 0.75 U Taq polymerase (Life Technologies, Inc., Grand Island, NY), 200 µmol/L deoxy-NTP, and 0.3
µmol/L of each primer in a final volume of 50 µL. The samples were
denatured at 94 C for 2 min. Four cycles, consisting of 95 C for
45 s, 66 C for 1.5 min, and 72 C for 3 min, were performed,
followed by 30 cycles consisting of 95 C for 1 min, 66 C for 1 min, and
72 C for 2.5 min. A final cycle consisted of 94 C for 1 min, 66 C for
1.5 min, and 72 C for 10 min.
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). The remaining
reagents were identical to those of the first PCR. The samples were
denatured for 1 min at 95 C, followed by four cycles of 1 min at 95 C,
30 s at 45 C, and 10 min at 72 C; 30 cycles of 30 s at 95 C,
30 s at 45 C, and 1 min at 72 C; 1 cycle of 1 min at 95 C, 1.5 min
at 45 C, and 10 min at 72 C. For exons 7–9, the PCR conditions were
the same, except the annealing temperature was 58 C. The PCR products
were analyzed by agarose gel electrophoresis and ethidium bromide
staining. CYP11B1 exon fragments were sequenced using solid phase, single strand sequencing with a Taq FS Dye Primer Kit (PE Applied Biosystems, Foster City, CA) containing M13 primers. Single stranded DNA from the PCR fragments were purified with streptavidin-bound magnetic beads as described by the manufacturer (Dynal, A.S., Oslo, Norway). After denaturation to remove the nonbiotinylated DNA strand, the bead-bound DNA strand was sequenced following the procedure described in the sequencing manual supplied with the sequencing kit (PE Applied Biosystems). The sequencing products were analyzed on a PE Applied Biosystems 373A automated sequencer.
Hormonal assays
For family 1, PRA and hormone analyses were performed as previously described (18, 19, 20, 21). The hormonal analysis for family 2 was performed by Endocrine Sciences, Inc. (Calabasas Hills, CA).
Prenatal treatment
The same algorithm for the prenatal diagnosis and treatment of 21-OHD CAH was used for prenatal treatment of 11ß-OHD (3). Labial fusion occurs before the eighth week of gestation; therefore, once pregnancy is confirmed in women found to be at risk for having a fetus with classical CAH, they are treated immediately with dexamethasone (20 µg/kg·day in three divided doses), blind to the status of the fetus. Chorionic villus sampling (CVS) at approximately 10 weeks or amniocentesis at approximately 14 weeks provides tissue for DNA analysis and karyotyping to determine the sex of the fetus. The dexamethasone therapy is discontinued if the fetus is a male or if DNA analysis indicates a female is either heterozygous or homozygous normal. Thus, only affected female fetuses are treated until term (one of eight), and the others (seven of eight) are treated only until the sex or unaffected diagnosis has been established. At birth, diagnosis is confirmed clinically, hormonally [by measurement of serum deoxycorticosterone (DOC) and 11-deoxycortisol (compound S) levels], and genetically.
In family 1, prenatal treatment with dexamethasone was initiated for the third pregnancy during the fifth week of gestation. A home pregnancy test kit and a prescription for dexamethasone had been made available to the mother soon after the second child was discharged from the hospital. For the fourth pregnancy, dexamethasone treatment was likewise initiated in the fifth week of gestation. Prenatal karyotyping was performed by CVS.
Family 2 has undergone genetic testing and counseling to be prepared for early prenatal treatment in the event of another pregnancy.
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Results |
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All of the three affected girls studied in family 1 (IIa–IIc) have the typical elevations in levels of DOC for patients with 11ß-OHD, but their PRA levels were not suppressed, which may be secondary to the transient end-organ resistance to the effects of aldosterone that is common in premature babies. Their blood pressures were normal. ACTH stimulation revealed normal levels of 17-hydroxyprogesterone, ruling out 21-OHD. The DOC response was characteristic of 11ß-OHD. The heterozygous son (IId) had near-normal values for DOC, and his PRA was higher, as expected in newborns. His blood pressure was also normal.
DNA analysis on the two older sisters revealed a homozygous missense
mutation in exon 5 (T318M), where a threonine (ACG) is converted to a
methionine (ATG) (9). Prenatal karyotyping showed that the third fetus
was a female. Diagnosis using DNA was attempted, but the results were
uncertain. Nevertheless, dexamethasone treatment was initiated at 5
weeks gestation and continued to term. Postnatally, DNA sequencing
revealed that this offspring had the T318M mutation, and therefore,
prenatal treatment had indeed been indicated (see Fig. 3). The mother tolerated the treatment
very well, except for the development of some violaceous striae at 7
months gestation, which improved after reduction of the dexamethasone
dose to 16 µg/kg·day. The pregnancy went to term, and the baby was
born with completely normal female external genitalia. Her DOC levels
rose significantly in response to ACTH, confirming that she was
affected (Table 2). This child is now 4 yr of age and has been growing
close to her expected target percentile. She maintains good hormonal
control and remains normotensive. She is well adjusted psychologically
and has attained excellent developmental milestones.
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Family 2
As part of our program of prenatal diagnosis and treatment, we also offer preconceptual counseling. Recently, we have also performed DNA analysis on a new family with two male offspring affected with 11ß-OHD. Because another pregnancy is desired in the near future, genotyping was initiated so that prenatal diagnosis could be performed swiftly in the event of pregnancy.
We detected a novel single base pair deletion in codon 394 of the
CYP11B1 gene. Both affected sons were found to be homozygous
for the deletion, whereas both parents were heterozygous (Fig. 4). This mutation changes the normal
sequence from AAC to AC, R3941. A previously reported 2-bp insertion
in the same codon resulted in premature termination at codon 469 and
was shown to have no in vitro enzymatic activity due to
disruption of the heme-binding domain (22). The novel single base pair
deletion found in the patients results in premature termination at
codon 429, which completely eliminates the heme-binding domain. Hence,
this mutation should also result in no enzymatic activity of
11ß-hydroxylase.
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Discussion |
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Although most agree that prenatal treatment offers great promise, opinions have differed concerning the present status of treatment (for review, see Refs. 2, 23, 24), that is, whether it should be considered the standard of care or experimental. The largest human studies have shown prenatal dexamethasone treatment to be effective and safe for both mother and child even for treatment throughout the course of the pregnancy, provided patients and physicians adhere to the recommended protocol (3, 4, 25, 26). Now over 400 pregnancies have been studied, with successful outcome in 23 affected females treated properly.
Several studies report the successful application of this approach by administering dexamethasone to the mother as soon as pregnancy is recognized (3, 4, 25, 26). Dexamethasone is the chosen treatment because it is not bound by corticosteroid-binding globulin and also is not metabolized by placental 11ß-hydroxysteroid dehydrogenase. If treatment is started in time and is not interrupted, and if the dose of dexamethasone is correct, virilization is prevented in females in most cases, and genitoplasty is not required in the newborn (1, 2, 3, 4, 25, 26). Prenatal treatment has been shown to be safe in both mother and child (1, 2, 3, 4, 25, 26). In a recent report, no significant or enduring side-effects were noted (including low birth weight, fetal wastage, or reported school performance) in those who were prenatally treated (4). In several reports, infants who were prenatally treated with dexamethasone were not different in auxological parameters from those not treated (3, 4, 25, 26). Cognitive and behavioral studies have begun, but they need to be continued and expanded (27, 28). Our data demonstrate the efficacy of prenatal treatment in reducing virilization of the genitalia in females affected with CAH due to 21-OHD. Our preliminary follow-up studies indicate no detrimental effects on cognition or behavior, but the data are few. Forest et al., in a multicenter study in France (25, 26), demonstrated normal growth, development, and intelligence in children followed past 10 yr of age. No statistically significant differences have been found for the presence of striae, edema, hypertension, or gestational diabetes by report in mothers treated with dexamethasone compared to mothers who were not treated; however, treated mothers did exhibit statistically significant greater weight gain (4).
As prenatal treatment of CAH began only 10 yr ago, it is agreed that long term follow-up (3) is required for both fetuses treated briefly and to term, and studies should be carried out in closely monitored settings to assess safety. Nevertheless, the possibility of eliminating the need for surgical reconstruction in these female infants and the accompanying reduction in emotional trauma to patient, parents, and family have generated great enthusiasm for prenatal treatment.
The birth of a child with ambiguous genitalia is a very traumatic event in a family. Females born with ambiguous genitalia who are not treated prenatally suffer consequences of the genital ambiguity even if the diagnosis is made at birth. They must have difficult genital surgery and frequent examinations of their genitalia during childhood. Because this new case demonstrates that prenatal dexamethasone treatment is effective, we will continue to study the long term results in the prenatal treatment of 11ß-OHD. Parents at risk for having children with 11ß-OHD and who are planning on having more children should prepare early with genetic testing, as did family 2.
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Acknowledgments |
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Footnotes |
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Received December 23, 1998.
Revised May 11, 1999.
Accepted May 17, 1999.
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References |
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His) associated
with steroid 11ß-hydroxylase deficiency in Jews of Moroccan origin. J Clin Invest. 87:1664–1667.
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