Prenatal Diagnosis of Congenital AdrenalHyperplasia (21-Hydroxylase Deficiency) in Croatia

M. Dumic,1 L. Brkljacic,1 V. Plavsic,1 R. Zunec,1 J. Ille,1 R.C. Wilson,2 I. Kuvacic,1 A. Kastelan,1 andM.I. New2*1Department of Pediatrics, University Hospital, Zagreb, Croatia2Department of Pediatrics, Division of Endocrinology, The New York Hospital-Cornell Medical Center,New York, New York

We report on the prenatal diagnosis of con-genital adrenal hyperplasia due to 21-hydroxylase in 20 at-risk pregnancies (16salt-wasting and 4 simple virilizing fami-lies). We have diagnosed 3 affected fetuses (2males and 1 female), 3 healthy homozygotes(2 males and 1 female), and 14 healthy het-erozygotes (7 females and 7 males). Thesedata were collected over 4 years.

In 16 fetuses, the diagnosis was made withmeasurements of 17-hydroxyprogesterone(17-OHP) and D-4-androstenedione (D) inamniotic fluid (AF), human leukocyte anti-gen (HLA) typing of amniotic cells, as well askaryotypes between the 16th and 18thweeks of gestation. In 4 fetuses, DNA analy-sis of amniotic cells was also performed. In 3pregnancies in which affected fetuses weresuspected (on the basis of HLA typing andmeasurements of 17-OHP and D concentra-tions in AF), the fetuses were electivelyaborted between the 17th to 19th weeks ofgestation by parental decision. In allaborted fetuses, diagnosis was confirmedwith HLA typing, autopsy findings of hyper-plastic adrenal glands, and ambiguous geni-talia in female fetuses.

Postnatal diagnosis was confirmed inhealthy fetuses with HLA typing and serummeasurements of 17-OHP concentrations,and in 4 of them with DNA analysis. In 3 ofthe 4 families, DNA analyses revealed thefollowing mutations: in Family 1, the indexcase mutation was Intron 2, Exon 3/Exon 6,and the fetus was Normal/Exon 6; in Family2, the index case mutation was Ex1 Int2 Ex3/Int2, and the fetus was Ex1 Int2 Ex3/Normal;and in Family 3, the index case mutation

was Ex8356/Ex8356, and the fetus was Ex8356/Normal.

We also report one case of prenatal diag-nosis and treatment. Dexamethasone 0.5 mgBID (20 mg/kg/d) was given starting at 6thweek of gestation. Prenatal diagnosis sug-gested, but did not prove, that the femalefetus was a heterozygote as the fetus lackedthe paternal mutation Ex8318. No mutationwas found in the mother. The fetus, themother, and the affected sib shared a haplo-type, further suggesting heterozygosity. Theunaffected status was confirmed postna-tally. Am J. Med. Genet. 72:302–306, 1997.© 1997 Wiley-Liss, Inc.

KEY WORDS: congenital adrenal hyperpla-sia; 21-hydroxylase mutation;prenatal diagnosis; prenataltreatment

INTRODUCTION

Prenatal diagnosis of congenital adrenal hyperplasia(CAH) due to 21-hydroxylase deficiency (21-OHD) wasinitially attempted by hormonal assay of amniotic fluid(AF) obtained by amniocentesis during the second tri-mester [Jeffcoate et al., 1965; Merkatz et al., 1969; Fra-sier et al., 1975; Gueux et al., 1988]. The hormone thatgave the most accurate result was 17-hydroxyproges-terone (17-OHP). This method was valuable only in thesalt-wasting form, as AF concentrations of 17-OHP arenormal in the simple virilizer and nonclassical forms[Pang et al., 1985]. However, assay of D-4-androstene-dione (D) demonstrates elevated levels in the simple-virilizing form of CAH.

When human leukocyte antigen (HLA) was found tobe linked with CAH due to 21-OHD, diagnoses weremade by using HLA genetic linkage marker analysis[Levine et al., 1978; Mornet et al., 1987; Strachan et al.,1987; Speiser et al., 1990]. This method resulted inmany diagnostic errors due to recombination or haplo-type sharing between parents. In order to make thediagnoses more precise, the analysis of DNA was added

*Correspondence to: Dr. Maria I. New, Department of Pediat-rics, Division of Endocrinology, The New York Hospital-CornellMedical Center, 525 East 68th Street, Room M-420, New York,NY 10021.

Received 18 December 1996; Accepted 28 April 1997

American Journal of Medical Genetics 72:302–306 (1997)

© 1997 Wiley-Liss, Inc.

to the prenatal diagnostic techniques [Wilson et al.,1995].

The algorithm for prenatal diagnosis [Mercado et al.,1995] is shown in Figure 1. Recommended treatment isdexamethasone (20 mg/kg/d in 3 divided doses) beforethe 10th week of gestation if the pregnancy is at risk forCAH, blind to the status of the fetus. The fetal sex(karyotype) is then determined from tissue obtained bychorionic villus sampling (CVS) or amniotic cells. If thefetus is a male, treatment is discontinued. If the fetusis a female, treatment continues until DNA analysisreveals the fetus to be unaffected. If affected, treatmentis continued until birth. Results have shown the treat-ment to be safe and effective [Mercado et al., 1995].

Since 1987, the families at risk for CAH due to 21-OHD have been offered prenatal diagnosis in the De-partment of Pediatrics, University Hospital Rebro,Zagreb. The present study reports our experience with20 prenatal diagnoses (4 of which were also based onDNA analysis) and one prenatal treatment with dexa-methasone from the 6th to 16th week of gestation.These data were collected over 4 years.

MATERIALS AND METHODS

AF for radioimmunoassay of 17-OHP and D; amnio-cytes for karyotype; HLA-A,B, and C typing [Pollack etal., 1979; Terasaki et al., 1978]; DNA analysis of theHLA-DR locus [Olerup and Zetterquist, 1992]; and di-rect DNA analysis of the 21-OH gene [Wilson et al.,1995] were obtained by amniocentesis performed be-tween the 16th and the 18th week of gestation. Steroiddetermination, karyotyping, and HLA typing were per-formed in Zagreb; samples for DNA analysis were di-vided and analyzed in Zagreb and New York.

Subjects

An index case (sib) was present in each of the fami-lies studied. Thus, the fetus was at increased geneticrisk for CAH due to 21-OHD. A total of 20 at-risk preg-nancies (16 salt-wasting and 4 simple-virilizing fami-lies) were monitored. Prepregnancy counseling in theDepartment of Pediatrics, University Hospital Rebro,Zagreb, was provided for each of the families. In the

Fig. 1. Algorithm for prenatal diagnosis and treatment of steroid 21-OHD. (Reproduced from Mercado et al., 1995, with permission of thepublisher.)

Prenatal Diagnosis of Congenital Adrenal Hyperplasia 303

first 17 families, in which only prenatal diagnosis wasoffered, the parents elected to abort should the fetusprove to be affected (regardless of the sex). Of the last3 families in which prenatal therapy was offered, one ofthem decided to accept dexamethasone treatment.

RESULTS

Using the concentrations of 17-OHP and D in AF andHLA genotyping of amniotic cells, prenatal diagnosis of

CAH due to 21-OHD was accomplished in 20 Croatianfamilies. We have diagnosed 3 affected fetuses (2 fe-males and 1 male), 3 healthy homozygotes (2 males and1 female), and 14 healthy heterozygotes (7 females and7 males). Prenatal prediction was confirmed postna-tally in all of the 17 unaffected fetuses with HLA typ-ing, normal plasma levels of 17-OHP and D, and in 4 ofthem with DNA analysis (Table I). The prenatal diag-noses in 3 fetuses affected with CAH due to 21-OHDare summarized in Table II. All 3 pregnancies were ter-

TABLE I. Prenatal Diagnosis in 17 Families at Risk for CAH (21-OH Deficiency) in Which an Unaffected Fetus Was Predicted*

CaseIndex case

HLA haptotype

Prenatal testsa

Postnatal HLA typingPostnataldiagnosisHLA

17-OHP(nmol/l)

D(nmol/l) Karyotype

1. A9 B7 DR2 A9 B7 10.59 1.50 46, xy A9 B7 DR2 HealthyA1 B8 DR3 A2 B27 A2 B27 DR2 heterozygote

2. A2 B35 DR5 A2 B13 9.08 0.59 46, xy A2 B13 DR7 HealthyA2 B21 DR7 A2 B21 A2 B21 DR7 heterozygote

3. A3 Bx DR7 A2 B17 4.51 0.87 46, xy A2 B17 DR6 HealthyA25 B18 DR7 A25 B18 A25 B18 DR7 heterozygote

4. A1 B35 DR1 A2 B15 8.95 2.37 46, xy A2 B15 DR6 HealthyA2 B22 DR5 A32 B16 A32 B16 DR2 homozygote

5. A1 B35 DR6 A1 B18 10.44 4.47 46, xx A1 B18 DR2 HealthyA3 B47 DR7 A9 B47 A9 B47 DR4 homozygote

6. A32 B21 DR7 A3 B35 4.00 1.96 46, xx A3 B35 DR14 HealthyA3 B47 DR7 A3 B47 A3 B47 DR7 heterozygote

7. A2 B5 DR6 A2 B5 6.17 1.71 46, xy A2 B5 DR6 HealthyA3 B35 DR5 A3 B5 A3 B5 DR8 heterozygote

8. A2 B22 DR2 A2 B22 11.50 3.63 46, xy A2 B22 DR2 HealthyA32 B5 DR2 A3 B5 A3 B5 DR1 heterozygote

9. A32 B21 DR7 A3 B35 8.17 1.39 46, xx A3 B35 DR6 HealthyA3 B47 DR7 A3 B47 A3 B47 DR7 heterozygote

10. A32 B38 DR6 A32 B38 9.32 2.41 46, xx A32 B38 DR6 HealthyA1 B22 DR5 A2 B15 A2 B15 DR6 heterozygote

11. A2 B5 DR6 A2 B5 10.98 1.57 46, xx A2 B5 DR6 HealthyA31 B40 DR4 A28 B35 A28 B35 DR8 heterozygote

12. A2 B27 DR2 A26 B16 6.42 2.57 46, xy A26 B16 DR5 HealthyA9 B35 DR1 A9 B35 A9 B35 DR1 heterozygote

13. A2 B12 DR11 A28 B35 3.22 0.42 46, xy A28 B35 DR8 HealthyA2 B12 DR11 A2 Bx A2 Bx DR3 homozygote

14. A2 B21 DR7 DQ2 A3 B12 DR4 9.32 2.02 46, xy A3 B12 DR4 DQ3 HealthyA33 B17 DR13.2 DQ3 A33 B17 DR13.2 A33 B17 DR13.2 DQ3 heterozygote

15. A9 B21 DR11 DQ3 A9 B21 DR11 4.58 2.68 46, xx A9 B21 DR11 DQ3 HealthyA9 B35 DR15 DQ1 A30 B35 DR11 A30 B35 DR11 DQ3 heterozygote

16. A11 B5 DR14 DQ1 A11 B5 DR14 8.22 4.19 46, xx A11 B5 DR14 DQ1 HealthyA11 B5 DR14 DQ1 A11 B5 DR15 A11 B5 DR15 DQ1 heterozygote

17. A31 B12 DR13 DQ1 A26 B38 DR16 2.80 0.40 46, xx A26 B38 DR16 DQ1 HealthyA2 B5 DR7 DQ2 A2 B5 DR7 A2 B5 DR7 DQ2 heterozygote

*Subjects 4, 7, 8, and 12 have th simple-virilizing form and subjects 1, 2, 3, 5, 6, 9, 10, 11, 13, 14, 15, 16, and 17 have the salt-wasting form of CAH.aPrenatal tests: HLA, HLA typing of cultured AF cells; 17-OHP, normal range in AF: 0.93–12.83 nmol/l; D, normal range in AF: 0.98–7.73 nmol/l.

TABLE II. Prenatal Diagnosis in 3 Families at Risk for CAH (21-OH Deficiency) in Which an Affected Fetus Was Predicted*

CaseIndex case

HLA haplotype

Prenatal testsa

PostnatalHLA typing

PostnataldiagnosisHLA

17-OHP(nmol/l)

D(nmol/l) Karyotype

1 A32 B21 DR7 A32 B21 84.96 43.47 46, xx A32 B21 DR7 AffectedA3 B47 DR7 A3 B47 A3 B47 DR7

2 A11 B5 DR13 A11 B5 DR13 23.3 16.5 46, xy A11 B5 DR13 AffectedA2 B22 DR11 A2 B33 DR11 A2 B22 DR11

3 A2 B12 DR11 A2 B12 13.71 3.18 46, xx A2 B12 DR11 AffectedA3 B47 DR7 A3 B47 A3 B47 DR7

*All affected subjects have the salt-wasting form of CAH.aPrenatal tests: HLA, HLA typing of cultured AF cells; 17-OHP, normal range in AF: 0.93–12.83 nmol/l; D, normal range in AF: 0.98–7.73 nmol/l.

304 Dumic et al.

TABLE III. HLA Typing and 21-OH Mutations in the Last Four Prenatal Tests*

Case

Father Mother HLA genotype 21-OHase mutation

HLA 21-OH mutation HLA 21-OH mutation Index case Fetus Index Fetus

1 A2 B21 DR7 (a) Int2 Ex3 Ex8318 A33 B17 DR13.03 (c) Ex6 a b Int2 Ex3 Ex8318 NormalA3 B12 DR4 (b) A1 B40 DR14 (d) c c Ex6 Ex6

2 A9 B21 DR11 (a) Ex1 Int2 Ex3 A9 B35 DR15 (c) Int2 a a Ex1 Int2 Ex3 Ex1 Int2 Ex3A2 B15 DR13 (b) A30 B35 DR1 (d) c d Int2 Normal

3 A11 B5 DR14 (a) Ex8356 A11 B5 DR14 (c) Ex8356 a a Ex8356 Ex8356A2 B38 DR10 (b) A11 B5 DR15 (d) c d Ex8

356Normal

4 A31 B12 DR13 (a) Ex8318 A2 B5 DR7 (c) Not detected a b Ex8318 NormalA26 B38 DR16 (b) A1 B13 DR4 (d) c c Not detected Not detected

*All affected subjects have the salt-wasting form of CAH.

Fig. 2. HLA typing (loci HLA-A/HLA-B) and oligotyping revealed thatthe fetus has inherited the parental b/c haplotypes and is predicted to beheterozygous for 21-OHD. This is confirmed by DNA analysis of the CYP21gene.

Fig. 3. Oligotyping was used for prenatal diagnosis because fetal cellsdid not grow. The fetus inherited a paternal (a) haplotype and a maternal(d) haplotype and is predicted to be heterozygous for 21-OHD. This isconfirmed by DNA analysis of the CYP21 gene. Prenatal diagnosis wasconfirmed by postnatal HLA typing and DNA analysis of the newborn, whowas born with normal female genitalia.

Fig. 4. The female fetus at birth had normal genitalia. HLA typing andoligotyping revealed that the fetus has inherited parental a/d haplotypesand is heterozygous for 21-OHD. Prenatal diagnosis was confirmed bypostnatal HLA typing of the newborn. The proband has the same HLAhaplotype on both alleles because of haplotype sharing in the parents ow-ing to consanguinity in the grandparents. Also, only the maternal DR15antigen could be used for diagnosis as the class A antigens are the same onboth alleles.

Fig. 5 The female fetus was born with normal female genitalia and wasHLA identical to her unaffected female sib. The maternal mutation in theCYP21 gene was not detected, but the fetus did not inherit the paternalmutation, thus confirming that the fetus was not affected. Dexamethasonetreatment in the mother was stopped after prenatal diagnosis becauseoligotyping was performed simultaneously with amniocentesis.

Figs. 2–5. Pedigrees of families at risk of steroid 21-OHD in which prenatal diagnosis was carried out by HLA genotyping and DNA analysis of theCYP21 gene.

minated between the 17th and the 19th weeks of ges-tation by parental decision. In each aborted fetus, di-agnosis was confirmed with HLA typing, autopsy find-ings of hyperplastic adrenal glands, and ambiguousgenitalia in female fetuses.

In 4 of the 20 families (no. 14, 15, 16, 17), directmolecular analysis of the 21-OH locus has been used inaddition to hormone determination. Results are pre-sented in Table III and Figures 2–5. The mother (Fig.5) was treated from the 6th week of gestation withdexamethasone 0.5 mg twice per day (20 mg/kg/d), al-though the recommended treatment was 3 times daily.Treatment was discontinued at the 16th week of ges-tation when prenatal diagnosis suggested that the fe-tus was heterozygous and unaffected, since the pater-nal mutation was lacking, and the fetus, the mother,and the affected sib shared a haplotype. During the 10weeks of treatment, the mother did not have any sideeffects or complications. A female newborn was born atterm with normal female external genitalia, confirmingthat the prenatal prediction was correct. She is now an8-month-old healthy female infant.

DISCUSSION

In this study, prenatal diagnosis, performed by mea-suring 17-OHP and D concentrations in AF and by HLAtyping of amniotic cells, was shown to be safe and ac-curate in CAH due to 21-OHD. Prenatal prediction wasconfirmed postnatally in all 20 cases (100%).

A newly developed rapid allelic specific polymerasechain reaction was used for DNA analysis in 5 fetuses,and prenatal treatment with dexamethasone was em-ployed in one of them. In this family (Fig. 5), prenataldiagnosis predicted that the fetus was unaffected, sincethe paternal mutation Ex8318 was not detected. There-fore, treatment was discontinued. The absence of a mu-tation in the mother, despite the finding of an affectedoffspring (index case), can be explained by either 1) ade novo mutation in the fetus or 2) an undetected mu-tation in the mother, the index case, and fetus. Thelatter possibility occurred in 6% of data in New Yorkand in 12% of data in Lyon, France [Mornet et al.,1991]. The mutations found in Croatian patients werenot different from those found in other European eth-nic groups [Mornet et al., 1991].

As CVS will be developed in Croatia, we are prepar-ing to analyze DNA from CV samples taken in the10th–12th weeks of gestation to make the diagnosis ofall 3 forms of CAH (salt-wasting, simple-virilizing, andnonclassical). A recent publication by Simpson et al.[1995] indicates that limb reduction defects are not as-sociated with CVS when performed at 10–12 weeks.DNA analysis of CV tissue will be more reliable andwill reduce the period of unnecessary therapy with

dexamethasone in male and healthy female fetuses.We anticipate that even greater numbers of familieswill accept prenatal diagnosis, as well as prenataltherapy, for CAH due to 21-OHD in the future.

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