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Prenatal diagnosis and treatment of 21-hydroxylase deficiency
J. Steroid Bioehem. Molec. Biol. Vol. 45, No. 1-3, pp, 75-82, 1993 Printed in Great Britain. All rights reserved
PRENATAL
0960-0760/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
DIAGNOSIS
AND
21-HYDROXYLASE
TREATMENT
OF
DEFICIENCY
MAGUELONE G. FOREST,l* MICHEL DAVID2 and YVES MOREL~ qNSERM-U.329 and 2Pediatric Clinic, H6pital Debrousse, 69322 Lyon cedex 05, France
Summary--Prenatal diagnosis of 21-hydroxylase deficiency, the most common cause of congenital adrenal hyperplasia (CAH), has benefited from the advances in endocrinologic and molecular genetic studies. In 1976, prenatal diagnosis of the disease was first attempted by measuring 17-hydroxyprogesterone in the amniotic fluid in the second trimester of pregnancy. Discovery of a close linkage between HLA and the disease gave a second approach for prenatal diagnosis, the latter being made by linkage study of the haplotypes of the index case in a given ~family. Diagnosis was later made directly by molecular biology. Currently, the studies of the C4-CYP21B gene locus by Southern blotting and the CYP21B gene mutations by PCR methods simplify the diagnositic procedure of an early and accurate prenatal diagnosis in the first trimester. In these conditions all families are now informative. Moreover, using a direct genetic analysis associated with the possibility of detecting the heterozygotes in a non-related CAH population, a prenatal diagnosis can be done in a family without a previously CAH affected child. From our results in a series of 274 pregnancies at risk for CAH in whom prenatal diagnosis has been made by these different approaches, it can be concluded that steroid analysis in the amniotic fluid is an accurate method but provides only a late (second trimester) diagnosis, while an early and accurate diagnosis now relies on adequate molecular genetic studies on chorion villus biopsies. In the aim to prevent the virilization of the external genitalia in CAH female fetuses, prenatal treatment was instituted in our group in 1979 by giving dexamethasone to the mother. This prenatal treatment appears safe for the fetus and the child and is effective in preventing virilization of CAH affected females. Although the degree of prevention is not always complete in all cases, the advantages of prenatal treatment are prevailing over the complications observed in a few mothers.
INTRODUCTION
made in an affected child (proband or index case).
Steroid 21-hydroxylase (21-OH) deficiency is the most common and well characterized inborn error of steroid metabolism. The world incidence of the classic form occurs in about 1 of 14,200 live births [1], aside from some populations with much higher incidence. This is an autosomic recessive disease leading to congenital adrenal hyperplasia (CAH). In C A H affected subjects prenatal hypersecretion of adrenal androgens virilizes the affected female fetuses, but does not interfere with a normal sexual differentiation in males. Prenatal diagnosis of the affection is warranted because sexual ambiguity may potentially result in erroneous sex assignment in genetic females, and neonatal mortality from adrenocortical insufficiency in both sexes. Prenatal diagnosis is only made in families known to be at risk, i.e. in whom the diagnosis of a given enzyme defect has previously been
Sexual ambiguity in C A H affected females is a severe complication of the disease, leading to psychological disturbances of the child and parents, necessitating difficult reconstructive surgery. This is why prenatal treatment has been proposed [2] in the aim to prevent the in utero virilization of C A H female fetuses.
PRENATAL DIAGNOSIS OF 21-OH DEFICIENCY
Attempted in 1965 [3], on the findings of elevated steroid levels in the amniotic fluid (AF) in the case of an affected fetus, the prenatal diagnosis of the disease has used three successive and/or combined approaches: hormonal measurements, H L A typing and molecular genetic techniques. Prenatal hormonal diagnosis
Proceedings of the Symposium: Adrenal Steroids: Current Views and New Frontiers, Heidelberg, Germany, 4-6 June 1992. *To whom correspondence should be addressed.
Excessive production of 170t-hydroxyprogesterone (OHP) is the classical biological marker of the enzyme defect, although it has 75
76
MAGUELONEG, FORESTet al.
been k n o w n for years that 21-deoxycortisol is also selectively elevated in this disease [4]. Prenatal h o r m o n a l diagnosis was initiated in 1975 when Frasier et al. [5] reported the finding o f elevated levels o f O H P in the A F obtained by amniocentesis in the second trimester (16-19 weeks). A F measurements o f A4-androstene dione (A 4) [6, 7], 21-deoxycortisol [8], or testosterone (T) in females, seem to be useful confirmatory tests. The simplicity and reliability o f a prenatal diagnosis o f the classical form o f 21-OH deficiency by steroid analysis is well established [7, 9, 10]. The prenatal diagnosis is fully informative even when the index case is deceased. However, A F levels o f O H P m a y not be significantly elevated in cases of simple virilizing or non-classical forms o f 21-OH deficiency [9, 10], and A F steroid levels are normal in heterozygote carriers [7].
Serological H L A genotyping of fetal cells The CYP21 genes (pseudogene C Y P 2 1 A and functional gene CYP21B) are located on the short arm o f c h r o m o s o m e 6 in tandem with the
C4A and C4B genes o f the fourth c o m p o n e n t o f the complement, in close linkage with the genes o f the major h u m a n histocompatibility complex ( H L A ) [see review in il]. This is why H L A typing has been used as a marker for transmission of the C A H trait, and soon afterwards this technique has provided a second approach for indirect prenatal diagnosis o f 21-OH deficiency [12]. The method has the advantage o f making it possible to predict a heterozygous fetus, but necessitates the previous H L A - t y p i n g o f the family (index case and parents). It is accurate except when an i n t r a - H L A recombination occurs [13] (1% in our families [11]) or when the index case is misdiagnosed as having 21-OH deficiency[9]. However, H L A - t y p i n g is not informative in families where there is antigen sharing in the parents [13] and caution must be exercised in interpreting results if multiple births are expected. Serological H L A - t y p ing o f the fetus is difficult because of poor antigen expression in fetal cells. H L A - D R typing of fetal cells has been made possible only recently by preactivating cultured fetal cells by g a m m a interferon or other H L A class II antigen
C4A 21A C4B 21B 1~
CHROMOSOMES (n = 355)
NORMAL GENES
F-go
2 3 --I
~
MUTATIONS in the CYP21B Gene
4 --~q~-'~
~'-
5
-II - - r-7
53%-
with C4A and CYP21A DELETIONS q
J
3,4%
with C4 and CYP21A DUPLICATIONS
18%
with C4B and C YP21 A DELETIONS
t1]--
with other abnormalities
7 6,1%
1,7%
i i
~
~
'
~
]
-
ABNORMALITIES ~ of the / GENE CONVERSION
1]"'-
CeteYP2cltaBblG?bN~y/30kbDELETION
10,1%1 11,8%123,9%
Fig. 1. Frequency of different lesions of the C4-CYP21 gene units determined by Southern blotting studies in 355 CAH chromosomes. The top diagram shows the normal arrangement of these genes: from left to right, gene for C4A (hatched), CYP21A (the pseudogene 21-OHA) (solid black), C4B (dotted), and CYP21B (the functional 21-OHB gene) (open). Lines 2 to 9 represent the various pathological patterns observed. Individual point mutations are indicated by a vertical line in the CYP21B gene. Lines 3-6 show rearrangements of the CYP21A pseudogene and C4 genes associated with point mutations of the CYP21B gene. Lines 3 and 4 show deletion of the CYP21A pseudogene associated with a deletion of either the C4A gene (line 3) or the C4B gene (line 4), but both do not occur. Line 5 shows a duplication of the CYP21A pseudogene associated with a point mutation in CYP2 ! B. Other rearrangements of the CYP21A and C4 genes may also occur with point mutations of the CYP21B gene. Lines 7-9 show lesions in the CYP21B gene that can be detected by Southern blotting. CYP21B conversions, where the 5' end of the 21B gene acquires CYP21A sequences, can occur with or without conversion of the C4B gene. Line 8 describes a deletion of about 30-kb of DNA extending from the middle of the CYP21A pseudogene to the homologous point in the CYP21B gene. A rare, newly described deletion spares the 3' end of CYP21A, but eliminates C4B and CYP21B (line 9). Adapted from [11].
77
Prenatal diagnosis and treatment of CAH
2a6"eAPlI
T InsertionI SW form I
1237 Val~Glu I
8 bp deletion 1239 Met-'Lys I
SW form
1
2
13
I SW form 61I 4 5
30Pro Leul 1172,,e Asn NC form SV form
I I 71 I
I SW form
I
I st°p
I
Ic°d°n 3181 9 I18 10
I
281 Val ~ Leu NC form
356 Arg -~ Trp I SV form
I
All these mutations are present in the pseudogene CYP21A A phenomenon termed MICRO CONVERSION Fig. 2. Point mutations in the CYP21Bgene. The 10 exons (black bars) and 9 introns (lines)of the human 21-OHB gene are drawn to scale, the 5' and 3' untranslated regions, 1 and 10, respectively,are indicated by narrower bars. The location and the nature of each mutation known to be associated with CAH due to 21-hydroxylasedeficiencyare indicated, as well as the observedassociation with a givenclinical form of the disease: SW = classical salt-wasting; SV = classical simple virilizing;NC = non-classical. Adapted from [11]. inducing agents[14]. First trimester prenatal diagnosis can be made by HLA-typing of samples obtained by chorion villus biopsy [15]. Finally, improvement of diagnostic accuracy has been obtained by applying molecular genetic techniques to HLA-typing [16].
Molecular genetic diagnosis The last and theoretically the most logical approach to prenatal diagnosis is the use of 21-OH probes for direct molecular genetic diagnosis of the disease. However, it is now clear that genetic lesions resulting in 21-OH deficiency are heterogeneous, being most frequently (about 70%) point mutations not evidenced by restriction fragment length polymorphism (RLFP) studies[ll]. Experience of Southern blotting studies applied to prenatal diagnosis of 21-OH deficiency have been scarce [17, 18] but promising in a number of families. The combined use of appropriate D N A probes for R L F P studies of class I (HLA-A, -B or -C) [16], class II (HLA-DR, -DP and -DQ)[18], complement C4 (C4A/C4B)[19], and 21-OH [18, 19] genes has considerably increased the accuracy of recognition of the allelic genes transmitted from the parents and present in the fetal genotype. Southern blotting studies of both the CYP21B and the duplicated C4-CYP21 regions have shown some complex arrangements and were fully informative for prenatal diagnosis in 60% of our CAH familes (Fig. 1)[11].
A major advance was the recent finding that all 9 known mutations of the CYP21B gene causing CAH (Fig. 2) are also present in the CYP21A pseudogene and could be detected after specific PCR amplification of specific fragments of the functional CYP21B gene [11, 20]. Thus the studies of the C4-CYP21 gene loci by the Southern blotting and/or that of the CYP21B point mutations by the PCR method on fetal DNA extracted from chorion villus biopsy simplify the procedure. This is currently the best way of making an early and accurate prenatal diagnosis, providing prior identification of the genetic lesions of the CYP21 gene in the parents.
Our experience Since 1979, our managrnent of prenatal diagnosis of 21-OH deficiency has followed the technical advances described above. We have performed a prenatal diagnosis in 210 untreated pregnancies. In 198 of them, diagnosis was made by measuring AF steroid hormones. AF levels of OHP and A4 found in 41 affected fetuses were well above that of the 174 controls, and both clearly had a diagnostic value whatever the sex (Table 1). In addition T levels were significantly elevated in CAH female fetuses. Thus when a female sex was found, the observation of high T levels had an additive diagnositic value. Mean AF levels of 17-hydroxypregnenolone were higher in CAH fetuses than
78
MAGUELONEG. FOREST et al.
Table I. Mean (_+SD) AF steroid levels(nmol/l)in 41 pregnanciesassociatedwith a CAH affectedfetus Sex (n) Testosterone Andostenedione OHP A5-OHP CAH F(18) 0.78+0.42* 15.63+_8.72 27.42+11.63 7.96+5.50 (13 20 WA) M (19) 1.52 -t- 1.64 21.41 ± 13.09 37.57 + 21.85 5.86 ± 3.48 ? (4) 1.12 + 0.39 25.93 ± 12.88 60.05 ± 42.80 5.59 ± 1.28 Controls F (75) 0.16 + 0.07* 1.66 + 0.84* 3.52 ± 1.29 3.77 + 1.86 (16-20 WA) M (75) 0.81 ± 0.37 2.69 ± 1.38 3.14 + 0.95 3.30 + 1.40 Controls F (7) 0.22 ± 0.07* 0.73 ± 0.42* 4.27 ± 1.21 3.70 _+1.27 (13-15 WA) M (8) 0.67_+0.08 1.46_+0.57 3.03_+0.59 3.35_+ 1.27 Values observedin 174 controlsat 2 differentgestationalages are given for comparison. *Significantlylower in females that in males of the same age group. OHP = 17 hydroxyprogesterone;ALOHP= 17 hydroxypregnenolone. in controls. H o w e v e r , there was a large o v e r l a p between the 2 groups, a n d this p a r a m e t e r was therefore not helpful in the p r e n a t a l diagnosis. The 157 o t h e r cases h a d n o r m a l A F levels (results n o t shown) a n d thus were d i a g n o s e d as being unaffected. As observed in a previous series[7], A F steroid h o r m o n e s were n o t different from controls a n d n o t different between heterozygotes a n d unaffected hom o z y g o t e s (pre- or p o s t n a t a l identification by HLA-typing). M o r e recently, A F levels o f 21-deoxycortisol were m e a s u r e d by a specific r a d i o i m m u n o a s s a y using an a n t i b o d y kindly p r o v i d e d by D r Vecsei[21]. As shown in T a b l e 2, o u r results confirm that this p a r a m e t e r is o f value in the p r e n a t a l diagnosis o f 21-OH deficiency [8]. P r e n a t a l diagnosis o f C A H affected fetuses was confirmed after birth, by clinical, h o r m o n a l a n d / o r H L A - t y p i n g investigations in all but one case. The latter was a male infant whose p a r e n t s refused p o s t n a t a l investigations, a n d was lost to follow-up. A m o n g the unaffected fetuses there was a p p a r e n t l y one false negative, a male infant with n o r m a l A F levels who d e v e l o p e d a severe salt losing C A H after birth. The A F was sent to us by mail, a n d the possibility o f a h u m a n error (wrong sample) could not be eliminated. Results o f p r e n a t a l diagnosis b y H L A - t y p i n g o f a m n i o t i c cells were not as accurate. In the 198 u n t r e a t e d pregnancies, H L A - t y p i n g was not d o n e (n = 105) o r not i n f o r m a t i v e (n = 10) in 58% o f the cases. W h e n p e r f o r m e d , H L A typing confirmed the p r e d i c t i o n o f steroid analysis in 74 pregnancies, b u t was inconclusive (n = 4) or gave a w r o n g diagnosis (n = 5) in 1% o f the cases. In the latter, the d i s c o r d a n t predic-
tion m a d e by A F steroid analysis was revealed to be correct after birth. M o r e recently, p r e n a t a l diagnosis was m a d e by m o l e c u l a r b i o l o g y on c h o r i o n villus b i o p s y in 12 a d d i t i o n a l pregnancies. All were predicted unaffected. Prediction was confirmed 8 times after birth while p r e g n a n c y is still in progress in the last 4.
P R E N A T A L T R E A T M E N T OF 21-OH DEFICIENCY
Patients and methods
This r e p o r t u p d a t e s the results o f the F r e n c h multicentric study o f p r e n a t a l t r e a t m e n t o f 2 1 - O H deficiency a n d concerns a total o f 64 treated pregnancies in 55 m o t h e r s at risk, o f which 44 in 38 familes have been r e p o r t e d previously [2, 22]. Families were n u m b e r e d chronologically, while two successive p r e g n a n cies are identified by family n u m b e r a n d a or b: this was the case for 8 m o t h e r s at risk (family nos 3, 4, 6, 13, 17, 9, 25, a n d 36). The very first trial o f p r e n a t a l t r e a t m e n t was m a d e by giving h y d r o c o r t i s o n e (40 then 50 rag/ day) to the mother, p r o t o c o l s and results o f which have been r e p o r t e d in detail previously [2]. The following 54 m o t h e r s received dexamethasone. Rationale and protocols of t r e a t m e n t were as r e p o r t e d previously [2, 22]. The daily dose o f d e x a m e t h a s o n e o f 0.5 mg every 12h used in the first 28 pregnancies (Nos 1-23 a n d 35) was increased to 0.5 mg every 8 h in the following 15 m o t h e r s (Nos 19b, 24-34, 36a, 36b a n d 37)[22]. Since then, that is to say in 20 a d d i t i o n a l pregnancies (Nos 17b, 25b a n d 38-55), the m o t h e r s were given d e x a m e t h a s o n e , either 0 . 5 m g every 8 h or 20-25 p g/kg o f m a t e r n a l b o d y weight in 3 Table 2. Mean (±SD) AF levels(pmol/l)of 21-deoxycortisolin 17 divided doses. D e x a m e t h a s o n e is available, pregnancies at risk at least in F r a n c e as 2 different p r e p a r a t i o n s , Pregnancies Fetus (n) Mean + SD (Range) (dexamethasone acetate) or Untreated Unaffected 9 115 ___31 (82-173) dectancyl ~ CAH 5 950 ___482 (558-1746) d e c a d r o n ~ ( d e x a m e t h a s o n e ) which might not be Prenatal Rx Unaffected 3 98 -+41 (70-145) Untreated Controls 24 108 + 54 (36-263) equivalent in term o f p h a r m a c o k i n e t i c s . In this study, d e x a m e t h a s o n e acetate has been used Values observedin controls are given for comparison.
Prenatal diagnosis and treatment of CAH
in all but 2 cases (associated with an unaffected fetus). The degree of fetal suppression was assessed by measuring A F steroid levels at the time of prenatal diagnosis, and in the case of affected females by measuring maternal serum estriol. Measurement of AF levels of steroid hormones, determination of H L A antigens, and/or molecular biology studies have been performed as reported previously [11, 20, 22].
Prenatal diagnosis and maternal tolerance of treatment Prenatal treatment must be started early, prior to the stage when the genital anlage is sensitive to the action of androgens, i.e. before any type of prenatal diagnosis is feasible. Treatment is thus systematic and not necessary in 7 out of 8 cases. Decision to stop treatment is based on the results of prenatal diagnosis, treatment being stopped in males and unaffected females. In our protocol, it was decided not to interrupt treatment at the time of prenatal diagnosis, and the latter has relied on HLA-typing of fetal cells until the recent development of adequate molecular biology techniques. At times, diagnoses based on HLA-typing have been difficult (Table 3). The decision to stop treatment was not a problem in the case of a male fetus, but was made erroneously once in the case of a female fetus. This was possibly a reason for the failure of the treatment, although we believe that it was rather due to late onset of the treatment (case 9 in [22]).
79
The other erroneous prenatal diagnoses had no regrettable consequences (Table 3), in particular in the case in which the prediction of an affected female was not confirmed by postnatal hormonal studies (normal ACTH test); this mother was treated until term; she complained of excessive weight gain, but her pregnancy was uneventful and her child is doing very well. Previous studies have shown that prenatal treatment was well tolerated in all mothers, although some did complain of weight gain, usually kept under control after salt and caloric restriction [22]. In the next 20 pregnancies of the present series, maternal complications were recorded in three occasions. Two mothers presented an increase in weight and appetite, mood fluctuations, pedal and leg edema, with striae and an increase in blood pressure in one or general discomfort in the other. The dose of dexamethasone was decreased before knowing the results of prenatal diagnosis, which predicted an unaffected fetus in both and was thus stopped. In a third mother (case 52)[23], prenatal diagnosis predicted a CAH female, and for the first time we observed that fetal suppression was not achieved at the time of prenatal diagnosis, since AF levels of OHP, A4 and T were very high. The dose of dexamethasone was increased from 1.5 to 2 mg/day. Fetal suppression was subsequently apparently achieved since maternal estriol levels decreased. However, treatment was not well tolerated (as above), progressively decreased and stopped 2/3 weeks before term. There has been a few reports of maternal complications, a report of
Table 3. Prenatal diagnosis in treated pregnancies, based on fetal HLA-typing and karyotyping CAH Prediction of a male fetus = treatment stopped H L A prediction ---confirmed - - n o t confirmed H L A not done (postnatal diagnosis) Final diagnosis in males (n = 28)
Unaffected
2 I 1b 4
18 (1~)r 6~ 24
Prediction of a female fetus: treatment continued til term in fetuses c H L A prediction --confirmed 4 - - n o t confirmed I
Discordance with
--inconclusive --steroids - m o l e c u l a r biology
H L A not done Final diagnosis in females (n ~ 30)
2h Ih (1) I c,h 9
,---,
16 (l')S id
(1) V T + I~ 21
Figures in parentheses represent a prediction not confirmed by postnatal studies "Not H L A identical to proband, mother was later diagnosed as non-classical CAH. bRefusal of prenatal diagnosis. CProband deceased, or mother being the index case. aRecombination. eDiagnosis made on molecular genetic studies. rCase reported in details in Ref. [27]. SCase 4b, see text for more details. hTreatment continued until term.
80
MAGUELONE G . FOREST e t al. Table 4. Results of prenatal treatment (our experience and literature review): (l) successful treatments, i.e. female fetuses with normal external genitalia (n = 8) Authors
Dose dex (mg)
[Refs] case number
David and Forest Nivelon et al. David et al. This report
Onset Rx (WA) Interrupted
Index case a
Treated fetus a
[2] = case 4b of [22] [23] = case 19b of [22] [28] Case 50
0.5 0.5 0.5 0.5
x x x x
12 h 8h 8h 8h
5.7 5.6 6th 7th
No No No No
Stage 4 Stage 4 Male Stage 3~
Normal Normal Normal Normal
[29] [29] [31] [32]
0.5 0.5 0.5 0.5
x x x x
12 h 12h 12 h 12 h
7th 5th 9th 9th
No No No No
Stage 3 Male Ambiguous Clitoromegaly ~
Normal Normal Normal Normal
Romer et al. Odink et al. Laforgia et a l ? Speiser et al. b
'Genitalia, Prader stage. bLikely the same case? CSimple virilizing, all other cases (index or treated fetus) were salt losers.
a single case [24], 1 out of 3 [25] and 3 out of 36 [26].
Mean birth weights, lengths and head circumference were normal.
Outcome of pregnancies
Results of treatment
As previously reported [22], a fetal death 18 weeks after stopping treatment was recorded, and likely occurred for obstetrical reasons independent of the prenatal treatment. An unexplained stillbirth at 33 weeks of gestation was observed in the only large series of prenatal treatment other than ours reported in the literature [26], and was not attributed to dexamethasone treatment (discontinued at 19th week). In our series, two early miscarriages were recorded (Nos 43a and 46) among the 20 additional treated pregnancies, i.e. a total of 6 early miscarriages out of a total of 64 treated pregnancies (an incidence of 9%). These likely represent spontaneous abortions (14% in previously untreated pregnancies in the other mothers), rather than a complication of prenatal treatment. Moreover, 4 of the 6 mothers who had a miscarriage became pregnant again and had an uneventful treated pregnancy (until term in 3 cases). No birth defects occurred in the infants born after prenatal treatments of various duration.
In our series 9 girls were CAH affected. One of them was diagnosed after birth as having a 'mild' form of the disease (case 4b). She was the sister of the case born with normal genitalia in our first report [2]. Prenatal diagnosis predicted an unaffected heterozygote. However, she had an accelerated growth and by 2 years of age presented typical hormonal abnormalities. In this, as in another family reported previously [27], one parent (the father in the present case), presented an undiagnosed non-classical form of the disease. These two cases further illustrate the difficulty of making a prenatal diagnosis by HLA-typing when one of the parents is also CAH affected. Molecular genetic studies now obviate this difficulty. Treatment was fully or partially successful, yet judged very satisfactory by the physicians and the parents, in 6 CAH females. Conditions of prenatal treatment in these cases and those reported in the literature are detailed in Tables 4 and 5, respectively. Even when the success was
Table 5. Results of prenatal treatments (our experience and literature review): (2) satisfactory treatments of partial successes, i.e. slight fetal virilization (n = 8) Authors David and Forest Loeuille et al.
[Refs] case number
Dose dex (mg)
Onset a Rx (WA)
Interrupted
[2] = case 4b of [22] [33] = case 13b of [22]
40, then 50 mg per day of F 0.5 × 12 h
9th 3.2
No No
Stage 4 Stage 4
Mild clitoromegaly Mild post-fusion
9th 5th 7.5th 1" at 16w 8th 8th T at 16w 8th ~. at 17w
5 Days 5 Days No
Stage 4 Not said Stage 4
Mild posterior fusion Stage 2 Posterior fusion
No No?
Male Stage 4
Minimal virilization ~ Mild virilization ~
No
Stage 4
Mild clitoromegaly
Petersen et al. D6rr et al. Shulman et al.
[34] [25] [35]
Shapiro et al. Pang et al.
[36] [24]
Speiser et al.
[32]
0.5 x 0.5 x 0.5 × 0.5 × 0.5 × 1.0 x 1.0x 0.25 x 0.25 x
12 h 8h 12 h 8h 6h 24 h 12h 6h 8h
' R x = treatment; W A or w = weeks of amenorrhea. bGenitalia, Prader stage. CDescribed as 'mild or minimal' posterior labial fusion and clitoromegaly.
Index case b
Treated fetus b
Prenatal diagnosis and treatment of CAH
81
Table 6. Results of prenatal treatments(our experiencesand literature review):(3) unsuccessfultreatments,i.e. virilizedfemale fetuses (n = 6) Authors
Forest et al. This report
[Refs] case number
Case 9 in [21] Case 52
Dose dex (mg)
Onset Rx (WA)
Interrupted
0.5 x 12 h 0.5 x 8 hb 1' then 0.5 × 24h 1.0 x 24h 1.0 x 24 h 0.5 x 8 h
8-12th 6th
At 26 w 12-24h
Index case' Treated fetus"
Stage 4 Male
Stage 4 Stage 3/4
Knorr et al. [37] 12th From 15 to 21 w Stage 4 Stage 4 D6rr et aL [38] 10th 5 Days Stage 4 Stage 4 D6rr et al. [38] 10th 5 Days Stage 4 Stage 4 D6rr et al. [25] 5th At 26 w Stage 4 Not said aGenitalia,Prader stage. bDaily dose increased to 2 mg at 20 WA because of high OHP levelsin AF, and decreased to 1.5mg at 32 weeks, to 1mg at 36 weeks (compliance?)because treatment was not well tolerated. n o t said to be ' c o m p l e t e ' all girls h a d 2 s e p a r a t e o p e n i n g s for the u r e t h r a a n d the v a g i n a . T h i s f a v o u r a b l e a n a t o m i c a l s i t u a t i o n was f o u n d very satisfactory b y p e d i a t r i c u r o l o g i s t s u r g e o n s , as it necessitates at the m o s t a simple surgical p r o c e d u r e at p u b e r t y . I n 2 cases in o u r series, t r e a t m e n t was u n s u c cessful ( T a b l e 6). I n the first i n s t a n c e (case 9) the failure o f t r e a t m e n t , as discussed a b o v e , was a t t r i b u t e d to b o t h a late start a n d the d i s c o n t i n u a t i o n o f t r e a t m e n t at 26 weeks. I n the s e c o n d case (No. 52), also discussed a b o v e , fetal a d r e n a l s u p p r e s s i o n was n o t achieved at the time o f p r e n a t a l d i a g n o s i s (14 weeks). This case is very similar to two o t h e r cases, the single case r e p o r t e d b y P a n g e t al. [24] a n d o n e o f the cases r e p o r t e d b y D 6 r r e t al. [25]. T h e three cases have in c o m m o n the a p p a r e n t failure o f a d r e n a l s u p p r e s s i o n with a dose o f d e x a m e t h a s o n e w h i c h was efficient in o t h e r m o t h e r s , t o g e t h e r with a p o o r m a t e r n a l t o l e r a n c e o f the t r e a t m e n t , the cause o f r e d u c i n g progressively the dose o f d e x a m e t h a s o n e a n d even d i s c o n t i n u i n g treatm e n t b e f o r e term. T h e r e a s o n s for these s y m p t o m s a n d / o r failure o f a d r e n a l s u p p r e s s i o n are u n c l e a r . It is possible t h a t they result f r o m i n d i v i d u a l v a r i a t i o n s in d e x a m e t h a s o n e m e t a b olism l e a d i n g to lowered p l a c e n t a l t r a n s f e r a n d i n c r e a s e d m a t e r n a l levels o f d e x a m e t h a s o n e , a n d / o r i n c r e a s e d m a t e r n a l sensitivity to glucocorticoids. T h e few o t h e r u n s u c c e s s f u l treatm e n t s are early trials a n d likely e x p l a i n e d b y insufficient a n d / o r daily g l u c o c o r t i c o i d dose ( T a b l e 6). IN CONCLUSION P r e n a t a l t r e a t m e n t o f C A H by d e x a m e t h a s o n e offers the a d v a n t a g e o f p r e v e n t i n g genital a m b i g u i t y , a v o i d i n g plastic surgery in m o s t cases, a n d p r e v e n t i n g the risk o f sex m i s a s s i g n m e n t . T h e l o n g t e r m studies in this series h a v e s h o w n t h a t this f o r m o f p r e n a t a l t r e a t m e n t is
safe for the fetus a n d the child. T h e o c c u r r e n c e o f m a t e r n a l c o m p l i c a t i o n s in s o m e i n s t a n c e s suggests t h a t the m i n i m a l effective dose s h o u l d be used, a n d u n n e c e s s a r y t r e a t m e n t s s h o u l d be s t o p p e d as early as possible. W i t h the a d v e n t o f reliable a n d a c c u r a t e m o l e c u l a r genetic techniques, a n early a n d safe p r e n a t a l d i a g n o s i s is n o w available, best p e r f o r m e d w h e n e v e r possible in the first trimester.
REFERENCES
1. Pang S., Wallace M. A., Hofman L., Thuline H. C., Dorche C., Lyon I. C. T., Dobbins R. H., Kling S., Fujieda K. and Suwa S.: Worldwide experience in newborn screening for classical congenital adrenal hyperplasia due to 2 l-hydroxylase deficiency. Pediatrics 81 (1988) 866-874. 2. David M. and Forest M. G.: Prenatal treatment of congenital adrenal hyperplasia resulting from 21hydroxylase deficiency. J. Pediat. 105 (1984) 799-803. 3. Jeffcoate T. N. A., Fleigner J. R. H., Russel S. H., Davis J. C. and Wade A. P.: Diagnosis of the adrenogenital syndrome before birth Lancet ii (1965) 553 555. 4. Franks R. C.: Plasma 17-hydroxyprogesterone, 21deoxycortisol and cortisol in congenital adrenal hyperplasia. J. Clin. Endocr. Metab. 39 (1974) 1099-1102. 5. Frasier S. D., Thorneycroft I. H., Weiss B. A. and Horton R.: Elevated anmiotic fluid concentration of 17ct-hydroxyprogesterone in congenital adrenal hyperplasia. J. Pediat. 86 (1975) 310-312. 6. Pang S., Levine L. S., Cederquvist L. L., Fuentes M., Riccardi V. M., Holcombe J. H., Nitowsky H. M., Sachs G., Anderson C. E., Duchon M. A., Owens R., Merkatz 1. and New M. I.: Amniotic fluid concentration of As and A4steroids in fetuses with congenital adrenal hyperplasia due to 21-hydroxylase deficiency and in anencephalic fetuses. J. Clin. Endocr. Metab. 51 (1980) 223 229. 7. Forest M. G.: Pitfalls in prenatal diagnosis of 21-hydroxylase deficiency by amniotic fluid steroid analysis? Ann. N.Y. Acad. Sci. 458 (1985) 130-147. 8. Gueux B., Fiet J., Couillin P., Raux-Demay M. C., Mornet E., Galons H., Vilette J. M., Bou6 J. and Dreux C.: Prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia by simultaneous radioimmunoassay of 21-deoxycortisol and 17-hydroxyprogesterone in amniotic fluid. J. Clin. Endocr. Metab. 66 (1988) 534-537. 9. Pang S., Pollack M. S., Loo M., Green O., Nussbaum R., Clayton G., Dupont G. and New M. I.: Pittfalls of prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia. J. Clin. Endocr. Metab. 61 (1985) 89-97.
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10. Hughes I. A., Dyas J., Riad-Fahmy D. and Laurence K. M.: Prenatal diagnosis of congenital adrenal hyperplasia: reliability of amniotic fluid analysis. J. Med. Genet. 24 (1987) 344-347. 11. Morel Y. and Miller W. I.: Clinical and molecular genetics of congenital adrenal hyperplasia due to 21hydroxylase deficiency. In Advances in Human Genetics 20 (Edited by H. Harris and K. Hirschhorn). Plenum Press, New York, Vol. 20 (1991) pp. 1 68. 12. Pollack M. S., Levine L. S., Pang S., Owwens R. P,, Nitowsky H. M., Maurer D., New M. I., Duchon M,, Merkatz 1. R., Sahs G. and Dupont B.: Prenatal diagnosis of congenital adrenal hyperplasia (21hydroxylase deficiency) by HLA typing. Lancet i (!979) 1107-1108.
13. Forest M. G., B6tuel H., Couillin P. and Bou6 A.: Prenatal diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency by steroid analysis in the amniotic fluid of mid-pregnancy; comparison with HLA-typing in 17 pregnancies at risk for CAH. Prenatal Diagnoses 1 (1981) 197-207. 14. Mauer D. H., Hanke J. H., Mickelson E., Rich R. R. and Pollack M. S.: Differential presentation of HLADR, DQ, and DP restriction elements by interferongamma-treated dermal fibroblats. J. Immun. 139 (1987) 715 723. 15. Callaway C., Falcon C., Grant G., Maurer D. H., Auerbach A. D., Rosenwaks Z. and Pollack M. S.: HLA typing used with cultured amniocytes and chorionic villus cells for early prenatal diagnosis or parentage testing without one parent's availability. Hum. lmmun. 16 (1986) 200 204. 16. Mornet E., Bou6 J., Raux-Demay M., Couillin P., Oury J. F., Dumez Y., Dausset J., Cohen D. and Bou6 A.: First trimester prenatal diagnosis of 21-hydroxylase deficiency by linkage analysis to HLA-DNA probes and by 17-hydroxyprogesterone determination. Hum. Genet. 73 (1986) 358-364. 17. Strachan T., Sinnott P. J., Smeaton I. S., Dyer P. A. and Harris R. Prenatal diagnosis of congenital adrenal hyperplasia Lancet ii (1987) 1272-1273. 18. Raux-Demay M., Mornet E., Bou6 J., Couillin P., Oury J. F., Ravise N., Deluchat C. and Bou6 A.: Early prenatal diagnosis of 21-hydroxylase deficiency using amniotic fluid 17-hydroxyprogesterone determination and DNA probes. Prenatal Diagnoses 9 (1989) 457-466. 19, Whitehead A. S., Woods D. E. and Fleishnick E.: DNA polymorphism of the C4 genes. A new marker for analysis of the major histocompatibility complex. New Engl. J. Med. 310 (1984) 888-891. 20. Morel Y., Murena M., Forest M. G., Nicolino M. and David M.: Frequency of the 8 known deleterious point mutations of the CYP21B gene in more than 100 families with 21-hydroxylase deficiency (CAH): implications for prenatal diagnosis. Endocrine Soc., 73rd A. Meet., Atlanta, GA (1991) Abstr. 1379. 21. Milewicz A., Vecsei P., Korth-Schiiltz S., Haack D., R6sler A., Lichtwald K., Kewicka S. and Mittelstaedt G. V.: Development of plasma 21-deoxycortisol radioimmunoassay and application to the diagnosis of patients with 21-hydroxylase deficiency. J. Steroid Biochem. 21 (1984) 185-191. 22. Forest M. G., B&uel H. and David M.: Prenatal treatment in congenital adrenal hyperplasia due to 21-hydroxylase deficiency: up-date 88 of the French multicentric study, Endocrine Res. 15 (1989) 277-301. 23. Nivelon J. L., Forest M. G., Morel Y., Huet F., Nivelon-Chevallier A., Franqois C. and Chouchane M.: Traitement pr6natal de l'hyperplasie surr6nale cong~nitale par d~ficit en 21-hydroxylase. A p r o p o s
de 9 grossesses trait+es. Ann Pkdiat. (Paris) (1993). In press. 24. Pang S., Pollack M. S., Marshall R. N. and lmmken L. D.: Prenatal treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. New Engl. J. Med. 322 (1990) 111 115. 25. D6rr H. G., Sippel W. G., Bidlingmaier F. and Knorr D.: Experience with intrauterine therapy of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Endocrine Soc. 70th A. Meet., New Orleans, LA (1988) Abstr. 3. 26. Karaviti L. P., Mercado A. B., Mercado M. B., Speiser P. W., Buegeleisen M., Crawford C., Antonina L., White P. C. and New M. I.: Prenatal diagnosis/ treatment in families at risk for infants with steroid 21-hydroxylase deficiency (congenital adrenal hyperplasia). J. Steroid Biochem. Molec. Biol. 41 (1992) 445 -45 I. 27. Morel Y., David M., Forest M. G., Bdtuel H., Hauptman G., Andr~ J., Bertrand J. and Miller W. L.: Gene conversion and rearrangements cause discordance between inheritance of forms of 2 l-hydroxylase deficiency and HLA types. J. Clin. Endocr. Metab. 68 (1989) 592 599. 28. Forest M. G., David M., B&uel H., Morel Y. and Contamin B.: Prenatal treatment of congenital adrenal hyperplasia (CAH): successful prevention of sexual ambiguity. Horm. Res. 33 (1990) 43. 29. Romer T. E. and Ginalska-Malinowska M.: Sucessful prenatal treatment of congenital adrenal hyperplasia resulting from 21-hydroxylase deficiency, is prenatal diagnosis in a mother at risk essential? Endokrynologia Polska 38 (1987) 125 130. 30. Odink R. J. H., Bou6 A. and Jansen M.: The value of chorion villus sampling in early detection of 21-hydroxylase deficiency. Pediat. Res. 23 (1988) 131. 31. Laforgia N., Yang S. Y., Khan R., Kato K., White P. C., Elias S., Schriock E., Speiser P. W., Simpson J. L. and New M. I.: Prenatal diagnosis with molecular probes and successful treatment of classical steroid 21-hydroxylase deficiency (CAH). Pediat. Res. 25 (1989) 87A. 32. Speiser P. W., Laforgia N., Kata K., Pareira J., Khan R., Yang S. Y., Whorwood C., White P. C., Elias S., Schriock E., Schriock E., Simpson J. L., Taslimi M., Najjar J., May S., Mills G., Crawford C. and New M. I.: First trimester prenatal treatment and molecular genetic diagnosis of congenital adrenal hyperplasia (21-hydroxylase deficiency). J. Clin. Endocr. Metab. 70 (1990) 838-848. 33. Loeuille G. A., David M. and Forest M. G.: Prenatal treatment of congenital adrenal hyperplasia. Report of a new case. Eur. J. Pediat. 149 (1990) 237-240. 34. Petersen K. E., Damkjaer Nielsen M., Buus O. and Couillin P.: Congenital adrenal hyperplasia (CAH): prenatal treatment. Pediat. Res. 20 (1986) 1201. 35. Schulman D. I., Mueller O. T., Gallardo L. A., Stiff D. and Oster H.: Treatment of congenital adrenal hyperplasia (CAH): prenatal treatment. Pediat. Res. 20 (1989) 1201. 36. Shapiro E., Santiago J. V. and Crane J. P.: Prenatal fetal suppression following in utero diagnosis of congenital adrenal hyperplasia. J. Urol. 142 (1989) 663 666. 37. Knorr D., Bidlingmaier F., D6rr H. G. and Kuhnle U.: Prenatal treatment of a girl with 21-hydroxylase deficiency. Pediat. Res. 19 (1985) 623. 38. Ddrr H. G., Sippel W. G., Haack D., Bidlingmaier F. and Knorr D.: Pitfalls of prenatal treatment of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Pediat. Res. 20 (1986) 1182.
PRENATAL
0960-0760/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
DIAGNOSIS
AND
21-HYDROXYLASE
TREATMENT
OF
DEFICIENCY
MAGUELONE G. FOREST,l* MICHEL DAVID2 and YVES MOREL~ qNSERM-U.329 and 2Pediatric Clinic, H6pital Debrousse, 69322 Lyon cedex 05, France
Summary--Prenatal diagnosis of 21-hydroxylase deficiency, the most common cause of congenital adrenal hyperplasia (CAH), has benefited from the advances in endocrinologic and molecular genetic studies. In 1976, prenatal diagnosis of the disease was first attempted by measuring 17-hydroxyprogesterone in the amniotic fluid in the second trimester of pregnancy. Discovery of a close linkage between HLA and the disease gave a second approach for prenatal diagnosis, the latter being made by linkage study of the haplotypes of the index case in a given ~family. Diagnosis was later made directly by molecular biology. Currently, the studies of the C4-CYP21B gene locus by Southern blotting and the CYP21B gene mutations by PCR methods simplify the diagnositic procedure of an early and accurate prenatal diagnosis in the first trimester. In these conditions all families are now informative. Moreover, using a direct genetic analysis associated with the possibility of detecting the heterozygotes in a non-related CAH population, a prenatal diagnosis can be done in a family without a previously CAH affected child. From our results in a series of 274 pregnancies at risk for CAH in whom prenatal diagnosis has been made by these different approaches, it can be concluded that steroid analysis in the amniotic fluid is an accurate method but provides only a late (second trimester) diagnosis, while an early and accurate diagnosis now relies on adequate molecular genetic studies on chorion villus biopsies. In the aim to prevent the virilization of the external genitalia in CAH female fetuses, prenatal treatment was instituted in our group in 1979 by giving dexamethasone to the mother. This prenatal treatment appears safe for the fetus and the child and is effective in preventing virilization of CAH affected females. Although the degree of prevention is not always complete in all cases, the advantages of prenatal treatment are prevailing over the complications observed in a few mothers.
INTRODUCTION
made in an affected child (proband or index case).
Steroid 21-hydroxylase (21-OH) deficiency is the most common and well characterized inborn error of steroid metabolism. The world incidence of the classic form occurs in about 1 of 14,200 live births [1], aside from some populations with much higher incidence. This is an autosomic recessive disease leading to congenital adrenal hyperplasia (CAH). In C A H affected subjects prenatal hypersecretion of adrenal androgens virilizes the affected female fetuses, but does not interfere with a normal sexual differentiation in males. Prenatal diagnosis of the affection is warranted because sexual ambiguity may potentially result in erroneous sex assignment in genetic females, and neonatal mortality from adrenocortical insufficiency in both sexes. Prenatal diagnosis is only made in families known to be at risk, i.e. in whom the diagnosis of a given enzyme defect has previously been
Sexual ambiguity in C A H affected females is a severe complication of the disease, leading to psychological disturbances of the child and parents, necessitating difficult reconstructive surgery. This is why prenatal treatment has been proposed [2] in the aim to prevent the in utero virilization of C A H female fetuses.
PRENATAL DIAGNOSIS OF 21-OH DEFICIENCY
Attempted in 1965 [3], on the findings of elevated steroid levels in the amniotic fluid (AF) in the case of an affected fetus, the prenatal diagnosis of the disease has used three successive and/or combined approaches: hormonal measurements, H L A typing and molecular genetic techniques. Prenatal hormonal diagnosis
Proceedings of the Symposium: Adrenal Steroids: Current Views and New Frontiers, Heidelberg, Germany, 4-6 June 1992. *To whom correspondence should be addressed.
Excessive production of 170t-hydroxyprogesterone (OHP) is the classical biological marker of the enzyme defect, although it has 75
76
MAGUELONEG, FORESTet al.
been k n o w n for years that 21-deoxycortisol is also selectively elevated in this disease [4]. Prenatal h o r m o n a l diagnosis was initiated in 1975 when Frasier et al. [5] reported the finding o f elevated levels o f O H P in the A F obtained by amniocentesis in the second trimester (16-19 weeks). A F measurements o f A4-androstene dione (A 4) [6, 7], 21-deoxycortisol [8], or testosterone (T) in females, seem to be useful confirmatory tests. The simplicity and reliability o f a prenatal diagnosis o f the classical form o f 21-OH deficiency by steroid analysis is well established [7, 9, 10]. The prenatal diagnosis is fully informative even when the index case is deceased. However, A F levels o f O H P m a y not be significantly elevated in cases of simple virilizing or non-classical forms o f 21-OH deficiency [9, 10], and A F steroid levels are normal in heterozygote carriers [7].
Serological H L A genotyping of fetal cells The CYP21 genes (pseudogene C Y P 2 1 A and functional gene CYP21B) are located on the short arm o f c h r o m o s o m e 6 in tandem with the
C4A and C4B genes o f the fourth c o m p o n e n t o f the complement, in close linkage with the genes o f the major h u m a n histocompatibility complex ( H L A ) [see review in il]. This is why H L A typing has been used as a marker for transmission of the C A H trait, and soon afterwards this technique has provided a second approach for indirect prenatal diagnosis o f 21-OH deficiency [12]. The method has the advantage o f making it possible to predict a heterozygous fetus, but necessitates the previous H L A - t y p i n g o f the family (index case and parents). It is accurate except when an i n t r a - H L A recombination occurs [13] (1% in our families [11]) or when the index case is misdiagnosed as having 21-OH deficiency[9]. However, H L A - t y p i n g is not informative in families where there is antigen sharing in the parents [13] and caution must be exercised in interpreting results if multiple births are expected. Serological H L A - t y p ing o f the fetus is difficult because of poor antigen expression in fetal cells. H L A - D R typing of fetal cells has been made possible only recently by preactivating cultured fetal cells by g a m m a interferon or other H L A class II antigen
C4A 21A C4B 21B 1~
CHROMOSOMES (n = 355)
NORMAL GENES
F-go
2 3 --I
~
MUTATIONS in the CYP21B Gene
4 --~q~-'~
~'-
5
-II - - r-7
53%-
with C4A and CYP21A DELETIONS q
J
3,4%
with C4 and CYP21A DUPLICATIONS
18%
with C4B and C YP21 A DELETIONS
t1]--
with other abnormalities
7 6,1%
1,7%
i i
~
~
'
~
]
-
ABNORMALITIES ~ of the / GENE CONVERSION
1]"'-
CeteYP2cltaBblG?bN~y/30kbDELETION
10,1%1 11,8%123,9%
Fig. 1. Frequency of different lesions of the C4-CYP21 gene units determined by Southern blotting studies in 355 CAH chromosomes. The top diagram shows the normal arrangement of these genes: from left to right, gene for C4A (hatched), CYP21A (the pseudogene 21-OHA) (solid black), C4B (dotted), and CYP21B (the functional 21-OHB gene) (open). Lines 2 to 9 represent the various pathological patterns observed. Individual point mutations are indicated by a vertical line in the CYP21B gene. Lines 3-6 show rearrangements of the CYP21A pseudogene and C4 genes associated with point mutations of the CYP21B gene. Lines 3 and 4 show deletion of the CYP21A pseudogene associated with a deletion of either the C4A gene (line 3) or the C4B gene (line 4), but both do not occur. Line 5 shows a duplication of the CYP21A pseudogene associated with a point mutation in CYP2 ! B. Other rearrangements of the CYP21A and C4 genes may also occur with point mutations of the CYP21B gene. Lines 7-9 show lesions in the CYP21B gene that can be detected by Southern blotting. CYP21B conversions, where the 5' end of the 21B gene acquires CYP21A sequences, can occur with or without conversion of the C4B gene. Line 8 describes a deletion of about 30-kb of DNA extending from the middle of the CYP21A pseudogene to the homologous point in the CYP21B gene. A rare, newly described deletion spares the 3' end of CYP21A, but eliminates C4B and CYP21B (line 9). Adapted from [11].
77
Prenatal diagnosis and treatment of CAH
2a6"eAPlI
T InsertionI SW form I
1237 Val~Glu I
8 bp deletion 1239 Met-'Lys I
SW form
1
2
13
I SW form 61I 4 5
30Pro Leul 1172,,e Asn NC form SV form
I I 71 I
I SW form
I
I st°p
I
Ic°d°n 3181 9 I18 10
I
281 Val ~ Leu NC form
356 Arg -~ Trp I SV form
I
All these mutations are present in the pseudogene CYP21A A phenomenon termed MICRO CONVERSION Fig. 2. Point mutations in the CYP21Bgene. The 10 exons (black bars) and 9 introns (lines)of the human 21-OHB gene are drawn to scale, the 5' and 3' untranslated regions, 1 and 10, respectively,are indicated by narrower bars. The location and the nature of each mutation known to be associated with CAH due to 21-hydroxylasedeficiencyare indicated, as well as the observedassociation with a givenclinical form of the disease: SW = classical salt-wasting; SV = classical simple virilizing;NC = non-classical. Adapted from [11]. inducing agents[14]. First trimester prenatal diagnosis can be made by HLA-typing of samples obtained by chorion villus biopsy [15]. Finally, improvement of diagnostic accuracy has been obtained by applying molecular genetic techniques to HLA-typing [16].
Molecular genetic diagnosis The last and theoretically the most logical approach to prenatal diagnosis is the use of 21-OH probes for direct molecular genetic diagnosis of the disease. However, it is now clear that genetic lesions resulting in 21-OH deficiency are heterogeneous, being most frequently (about 70%) point mutations not evidenced by restriction fragment length polymorphism (RLFP) studies[ll]. Experience of Southern blotting studies applied to prenatal diagnosis of 21-OH deficiency have been scarce [17, 18] but promising in a number of families. The combined use of appropriate D N A probes for R L F P studies of class I (HLA-A, -B or -C) [16], class II (HLA-DR, -DP and -DQ)[18], complement C4 (C4A/C4B)[19], and 21-OH [18, 19] genes has considerably increased the accuracy of recognition of the allelic genes transmitted from the parents and present in the fetal genotype. Southern blotting studies of both the CYP21B and the duplicated C4-CYP21 regions have shown some complex arrangements and were fully informative for prenatal diagnosis in 60% of our CAH familes (Fig. 1)[11].
A major advance was the recent finding that all 9 known mutations of the CYP21B gene causing CAH (Fig. 2) are also present in the CYP21A pseudogene and could be detected after specific PCR amplification of specific fragments of the functional CYP21B gene [11, 20]. Thus the studies of the C4-CYP21 gene loci by the Southern blotting and/or that of the CYP21B point mutations by the PCR method on fetal DNA extracted from chorion villus biopsy simplify the procedure. This is currently the best way of making an early and accurate prenatal diagnosis, providing prior identification of the genetic lesions of the CYP21 gene in the parents.
Our experience Since 1979, our managrnent of prenatal diagnosis of 21-OH deficiency has followed the technical advances described above. We have performed a prenatal diagnosis in 210 untreated pregnancies. In 198 of them, diagnosis was made by measuring AF steroid hormones. AF levels of OHP and A4 found in 41 affected fetuses were well above that of the 174 controls, and both clearly had a diagnostic value whatever the sex (Table 1). In addition T levels were significantly elevated in CAH female fetuses. Thus when a female sex was found, the observation of high T levels had an additive diagnositic value. Mean AF levels of 17-hydroxypregnenolone were higher in CAH fetuses than
78
MAGUELONEG. FOREST et al.
Table I. Mean (_+SD) AF steroid levels(nmol/l)in 41 pregnanciesassociatedwith a CAH affectedfetus Sex (n) Testosterone Andostenedione OHP A5-OHP CAH F(18) 0.78+0.42* 15.63+_8.72 27.42+11.63 7.96+5.50 (13 20 WA) M (19) 1.52 -t- 1.64 21.41 ± 13.09 37.57 + 21.85 5.86 ± 3.48 ? (4) 1.12 + 0.39 25.93 ± 12.88 60.05 ± 42.80 5.59 ± 1.28 Controls F (75) 0.16 + 0.07* 1.66 + 0.84* 3.52 ± 1.29 3.77 + 1.86 (16-20 WA) M (75) 0.81 ± 0.37 2.69 ± 1.38 3.14 + 0.95 3.30 + 1.40 Controls F (7) 0.22 ± 0.07* 0.73 ± 0.42* 4.27 ± 1.21 3.70 _+1.27 (13-15 WA) M (8) 0.67_+0.08 1.46_+0.57 3.03_+0.59 3.35_+ 1.27 Values observedin 174 controlsat 2 differentgestationalages are given for comparison. *Significantlylower in females that in males of the same age group. OHP = 17 hydroxyprogesterone;ALOHP= 17 hydroxypregnenolone. in controls. H o w e v e r , there was a large o v e r l a p between the 2 groups, a n d this p a r a m e t e r was therefore not helpful in the p r e n a t a l diagnosis. The 157 o t h e r cases h a d n o r m a l A F levels (results n o t shown) a n d thus were d i a g n o s e d as being unaffected. As observed in a previous series[7], A F steroid h o r m o n e s were n o t different from controls a n d n o t different between heterozygotes a n d unaffected hom o z y g o t e s (pre- or p o s t n a t a l identification by HLA-typing). M o r e recently, A F levels o f 21-deoxycortisol were m e a s u r e d by a specific r a d i o i m m u n o a s s a y using an a n t i b o d y kindly p r o v i d e d by D r Vecsei[21]. As shown in T a b l e 2, o u r results confirm that this p a r a m e t e r is o f value in the p r e n a t a l diagnosis o f 21-OH deficiency [8]. P r e n a t a l diagnosis o f C A H affected fetuses was confirmed after birth, by clinical, h o r m o n a l a n d / o r H L A - t y p i n g investigations in all but one case. The latter was a male infant whose p a r e n t s refused p o s t n a t a l investigations, a n d was lost to follow-up. A m o n g the unaffected fetuses there was a p p a r e n t l y one false negative, a male infant with n o r m a l A F levels who d e v e l o p e d a severe salt losing C A H after birth. The A F was sent to us by mail, a n d the possibility o f a h u m a n error (wrong sample) could not be eliminated. Results o f p r e n a t a l diagnosis b y H L A - t y p i n g o f a m n i o t i c cells were not as accurate. In the 198 u n t r e a t e d pregnancies, H L A - t y p i n g was not d o n e (n = 105) o r not i n f o r m a t i v e (n = 10) in 58% o f the cases. W h e n p e r f o r m e d , H L A typing confirmed the p r e d i c t i o n o f steroid analysis in 74 pregnancies, b u t was inconclusive (n = 4) or gave a w r o n g diagnosis (n = 5) in 1% o f the cases. In the latter, the d i s c o r d a n t predic-
tion m a d e by A F steroid analysis was revealed to be correct after birth. M o r e recently, p r e n a t a l diagnosis was m a d e by m o l e c u l a r b i o l o g y on c h o r i o n villus b i o p s y in 12 a d d i t i o n a l pregnancies. All were predicted unaffected. Prediction was confirmed 8 times after birth while p r e g n a n c y is still in progress in the last 4.
P R E N A T A L T R E A T M E N T OF 21-OH DEFICIENCY
Patients and methods
This r e p o r t u p d a t e s the results o f the F r e n c h multicentric study o f p r e n a t a l t r e a t m e n t o f 2 1 - O H deficiency a n d concerns a total o f 64 treated pregnancies in 55 m o t h e r s at risk, o f which 44 in 38 familes have been r e p o r t e d previously [2, 22]. Families were n u m b e r e d chronologically, while two successive p r e g n a n cies are identified by family n u m b e r a n d a or b: this was the case for 8 m o t h e r s at risk (family nos 3, 4, 6, 13, 17, 9, 25, a n d 36). The very first trial o f p r e n a t a l t r e a t m e n t was m a d e by giving h y d r o c o r t i s o n e (40 then 50 rag/ day) to the mother, p r o t o c o l s and results o f which have been r e p o r t e d in detail previously [2]. The following 54 m o t h e r s received dexamethasone. Rationale and protocols of t r e a t m e n t were as r e p o r t e d previously [2, 22]. The daily dose o f d e x a m e t h a s o n e o f 0.5 mg every 12h used in the first 28 pregnancies (Nos 1-23 a n d 35) was increased to 0.5 mg every 8 h in the following 15 m o t h e r s (Nos 19b, 24-34, 36a, 36b a n d 37)[22]. Since then, that is to say in 20 a d d i t i o n a l pregnancies (Nos 17b, 25b a n d 38-55), the m o t h e r s were given d e x a m e t h a s o n e , either 0 . 5 m g every 8 h or 20-25 p g/kg o f m a t e r n a l b o d y weight in 3 Table 2. Mean (±SD) AF levels(pmol/l)of 21-deoxycortisolin 17 divided doses. D e x a m e t h a s o n e is available, pregnancies at risk at least in F r a n c e as 2 different p r e p a r a t i o n s , Pregnancies Fetus (n) Mean + SD (Range) (dexamethasone acetate) or Untreated Unaffected 9 115 ___31 (82-173) dectancyl ~ CAH 5 950 ___482 (558-1746) d e c a d r o n ~ ( d e x a m e t h a s o n e ) which might not be Prenatal Rx Unaffected 3 98 -+41 (70-145) Untreated Controls 24 108 + 54 (36-263) equivalent in term o f p h a r m a c o k i n e t i c s . In this study, d e x a m e t h a s o n e acetate has been used Values observedin controls are given for comparison.
Prenatal diagnosis and treatment of CAH
in all but 2 cases (associated with an unaffected fetus). The degree of fetal suppression was assessed by measuring A F steroid levels at the time of prenatal diagnosis, and in the case of affected females by measuring maternal serum estriol. Measurement of AF levels of steroid hormones, determination of H L A antigens, and/or molecular biology studies have been performed as reported previously [11, 20, 22].
Prenatal diagnosis and maternal tolerance of treatment Prenatal treatment must be started early, prior to the stage when the genital anlage is sensitive to the action of androgens, i.e. before any type of prenatal diagnosis is feasible. Treatment is thus systematic and not necessary in 7 out of 8 cases. Decision to stop treatment is based on the results of prenatal diagnosis, treatment being stopped in males and unaffected females. In our protocol, it was decided not to interrupt treatment at the time of prenatal diagnosis, and the latter has relied on HLA-typing of fetal cells until the recent development of adequate molecular biology techniques. At times, diagnoses based on HLA-typing have been difficult (Table 3). The decision to stop treatment was not a problem in the case of a male fetus, but was made erroneously once in the case of a female fetus. This was possibly a reason for the failure of the treatment, although we believe that it was rather due to late onset of the treatment (case 9 in [22]).
79
The other erroneous prenatal diagnoses had no regrettable consequences (Table 3), in particular in the case in which the prediction of an affected female was not confirmed by postnatal hormonal studies (normal ACTH test); this mother was treated until term; she complained of excessive weight gain, but her pregnancy was uneventful and her child is doing very well. Previous studies have shown that prenatal treatment was well tolerated in all mothers, although some did complain of weight gain, usually kept under control after salt and caloric restriction [22]. In the next 20 pregnancies of the present series, maternal complications were recorded in three occasions. Two mothers presented an increase in weight and appetite, mood fluctuations, pedal and leg edema, with striae and an increase in blood pressure in one or general discomfort in the other. The dose of dexamethasone was decreased before knowing the results of prenatal diagnosis, which predicted an unaffected fetus in both and was thus stopped. In a third mother (case 52)[23], prenatal diagnosis predicted a CAH female, and for the first time we observed that fetal suppression was not achieved at the time of prenatal diagnosis, since AF levels of OHP, A4 and T were very high. The dose of dexamethasone was increased from 1.5 to 2 mg/day. Fetal suppression was subsequently apparently achieved since maternal estriol levels decreased. However, treatment was not well tolerated (as above), progressively decreased and stopped 2/3 weeks before term. There has been a few reports of maternal complications, a report of
Table 3. Prenatal diagnosis in treated pregnancies, based on fetal HLA-typing and karyotyping CAH Prediction of a male fetus = treatment stopped H L A prediction ---confirmed - - n o t confirmed H L A not done (postnatal diagnosis) Final diagnosis in males (n = 28)
Unaffected
2 I 1b 4
18 (1~)r 6~ 24
Prediction of a female fetus: treatment continued til term in fetuses c H L A prediction --confirmed 4 - - n o t confirmed I
Discordance with
--inconclusive --steroids - m o l e c u l a r biology
H L A not done Final diagnosis in females (n ~ 30)
2h Ih (1) I c,h 9
,---,
16 (l')S id
(1) V T + I~ 21
Figures in parentheses represent a prediction not confirmed by postnatal studies "Not H L A identical to proband, mother was later diagnosed as non-classical CAH. bRefusal of prenatal diagnosis. CProband deceased, or mother being the index case. aRecombination. eDiagnosis made on molecular genetic studies. rCase reported in details in Ref. [27]. SCase 4b, see text for more details. hTreatment continued until term.
80
MAGUELONE G . FOREST e t al. Table 4. Results of prenatal treatment (our experience and literature review): (l) successful treatments, i.e. female fetuses with normal external genitalia (n = 8) Authors
Dose dex (mg)
[Refs] case number
David and Forest Nivelon et al. David et al. This report
Onset Rx (WA) Interrupted
Index case a
Treated fetus a
[2] = case 4b of [22] [23] = case 19b of [22] [28] Case 50
0.5 0.5 0.5 0.5
x x x x
12 h 8h 8h 8h
5.7 5.6 6th 7th
No No No No
Stage 4 Stage 4 Male Stage 3~
Normal Normal Normal Normal
[29] [29] [31] [32]
0.5 0.5 0.5 0.5
x x x x
12 h 12h 12 h 12 h
7th 5th 9th 9th
No No No No
Stage 3 Male Ambiguous Clitoromegaly ~
Normal Normal Normal Normal
Romer et al. Odink et al. Laforgia et a l ? Speiser et al. b
'Genitalia, Prader stage. bLikely the same case? CSimple virilizing, all other cases (index or treated fetus) were salt losers.
a single case [24], 1 out of 3 [25] and 3 out of 36 [26].
Mean birth weights, lengths and head circumference were normal.
Outcome of pregnancies
Results of treatment
As previously reported [22], a fetal death 18 weeks after stopping treatment was recorded, and likely occurred for obstetrical reasons independent of the prenatal treatment. An unexplained stillbirth at 33 weeks of gestation was observed in the only large series of prenatal treatment other than ours reported in the literature [26], and was not attributed to dexamethasone treatment (discontinued at 19th week). In our series, two early miscarriages were recorded (Nos 43a and 46) among the 20 additional treated pregnancies, i.e. a total of 6 early miscarriages out of a total of 64 treated pregnancies (an incidence of 9%). These likely represent spontaneous abortions (14% in previously untreated pregnancies in the other mothers), rather than a complication of prenatal treatment. Moreover, 4 of the 6 mothers who had a miscarriage became pregnant again and had an uneventful treated pregnancy (until term in 3 cases). No birth defects occurred in the infants born after prenatal treatments of various duration.
In our series 9 girls were CAH affected. One of them was diagnosed after birth as having a 'mild' form of the disease (case 4b). She was the sister of the case born with normal genitalia in our first report [2]. Prenatal diagnosis predicted an unaffected heterozygote. However, she had an accelerated growth and by 2 years of age presented typical hormonal abnormalities. In this, as in another family reported previously [27], one parent (the father in the present case), presented an undiagnosed non-classical form of the disease. These two cases further illustrate the difficulty of making a prenatal diagnosis by HLA-typing when one of the parents is also CAH affected. Molecular genetic studies now obviate this difficulty. Treatment was fully or partially successful, yet judged very satisfactory by the physicians and the parents, in 6 CAH females. Conditions of prenatal treatment in these cases and those reported in the literature are detailed in Tables 4 and 5, respectively. Even when the success was
Table 5. Results of prenatal treatments (our experience and literature review): (2) satisfactory treatments of partial successes, i.e. slight fetal virilization (n = 8) Authors David and Forest Loeuille et al.
[Refs] case number
Dose dex (mg)
Onset a Rx (WA)
Interrupted
[2] = case 4b of [22] [33] = case 13b of [22]
40, then 50 mg per day of F 0.5 × 12 h
9th 3.2
No No
Stage 4 Stage 4
Mild clitoromegaly Mild post-fusion
9th 5th 7.5th 1" at 16w 8th 8th T at 16w 8th ~. at 17w
5 Days 5 Days No
Stage 4 Not said Stage 4
Mild posterior fusion Stage 2 Posterior fusion
No No?
Male Stage 4
Minimal virilization ~ Mild virilization ~
No
Stage 4
Mild clitoromegaly
Petersen et al. D6rr et al. Shulman et al.
[34] [25] [35]
Shapiro et al. Pang et al.
[36] [24]
Speiser et al.
[32]
0.5 x 0.5 x 0.5 × 0.5 × 0.5 × 1.0 x 1.0x 0.25 x 0.25 x
12 h 8h 12 h 8h 6h 24 h 12h 6h 8h
' R x = treatment; W A or w = weeks of amenorrhea. bGenitalia, Prader stage. CDescribed as 'mild or minimal' posterior labial fusion and clitoromegaly.
Index case b
Treated fetus b
Prenatal diagnosis and treatment of CAH
81
Table 6. Results of prenatal treatments(our experiencesand literature review):(3) unsuccessfultreatments,i.e. virilizedfemale fetuses (n = 6) Authors
Forest et al. This report
[Refs] case number
Case 9 in [21] Case 52
Dose dex (mg)
Onset Rx (WA)
Interrupted
0.5 x 12 h 0.5 x 8 hb 1' then 0.5 × 24h 1.0 x 24h 1.0 x 24 h 0.5 x 8 h
8-12th 6th
At 26 w 12-24h
Index case' Treated fetus"
Stage 4 Male
Stage 4 Stage 3/4
Knorr et al. [37] 12th From 15 to 21 w Stage 4 Stage 4 D6rr et aL [38] 10th 5 Days Stage 4 Stage 4 D6rr et al. [38] 10th 5 Days Stage 4 Stage 4 D6rr et al. [25] 5th At 26 w Stage 4 Not said aGenitalia,Prader stage. bDaily dose increased to 2 mg at 20 WA because of high OHP levelsin AF, and decreased to 1.5mg at 32 weeks, to 1mg at 36 weeks (compliance?)because treatment was not well tolerated. n o t said to be ' c o m p l e t e ' all girls h a d 2 s e p a r a t e o p e n i n g s for the u r e t h r a a n d the v a g i n a . T h i s f a v o u r a b l e a n a t o m i c a l s i t u a t i o n was f o u n d very satisfactory b y p e d i a t r i c u r o l o g i s t s u r g e o n s , as it necessitates at the m o s t a simple surgical p r o c e d u r e at p u b e r t y . I n 2 cases in o u r series, t r e a t m e n t was u n s u c cessful ( T a b l e 6). I n the first i n s t a n c e (case 9) the failure o f t r e a t m e n t , as discussed a b o v e , was a t t r i b u t e d to b o t h a late start a n d the d i s c o n t i n u a t i o n o f t r e a t m e n t at 26 weeks. I n the s e c o n d case (No. 52), also discussed a b o v e , fetal a d r e n a l s u p p r e s s i o n was n o t achieved at the time o f p r e n a t a l d i a g n o s i s (14 weeks). This case is very similar to two o t h e r cases, the single case r e p o r t e d b y P a n g e t al. [24] a n d o n e o f the cases r e p o r t e d b y D 6 r r e t al. [25]. T h e three cases have in c o m m o n the a p p a r e n t failure o f a d r e n a l s u p p r e s s i o n with a dose o f d e x a m e t h a s o n e w h i c h was efficient in o t h e r m o t h e r s , t o g e t h e r with a p o o r m a t e r n a l t o l e r a n c e o f the t r e a t m e n t , the cause o f r e d u c i n g progressively the dose o f d e x a m e t h a s o n e a n d even d i s c o n t i n u i n g treatm e n t b e f o r e term. T h e r e a s o n s for these s y m p t o m s a n d / o r failure o f a d r e n a l s u p p r e s s i o n are u n c l e a r . It is possible t h a t they result f r o m i n d i v i d u a l v a r i a t i o n s in d e x a m e t h a s o n e m e t a b olism l e a d i n g to lowered p l a c e n t a l t r a n s f e r a n d i n c r e a s e d m a t e r n a l levels o f d e x a m e t h a s o n e , a n d / o r i n c r e a s e d m a t e r n a l sensitivity to glucocorticoids. T h e few o t h e r u n s u c c e s s f u l treatm e n t s are early trials a n d likely e x p l a i n e d b y insufficient a n d / o r daily g l u c o c o r t i c o i d dose ( T a b l e 6). IN CONCLUSION P r e n a t a l t r e a t m e n t o f C A H by d e x a m e t h a s o n e offers the a d v a n t a g e o f p r e v e n t i n g genital a m b i g u i t y , a v o i d i n g plastic surgery in m o s t cases, a n d p r e v e n t i n g the risk o f sex m i s a s s i g n m e n t . T h e l o n g t e r m studies in this series h a v e s h o w n t h a t this f o r m o f p r e n a t a l t r e a t m e n t is
safe for the fetus a n d the child. T h e o c c u r r e n c e o f m a t e r n a l c o m p l i c a t i o n s in s o m e i n s t a n c e s suggests t h a t the m i n i m a l effective dose s h o u l d be used, a n d u n n e c e s s a r y t r e a t m e n t s s h o u l d be s t o p p e d as early as possible. W i t h the a d v e n t o f reliable a n d a c c u r a t e m o l e c u l a r genetic techniques, a n early a n d safe p r e n a t a l d i a g n o s i s is n o w available, best p e r f o r m e d w h e n e v e r possible in the first trimester.
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