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How a Patient Homozygous for a 30-kb Deletion of the C4-CYP 21 Genomic Region Can Have a Nonclassic Form of 21-Hydroxylase Deficiency

Department of Pediatrics (D.L., A.G., D.S., H.K.), Virchow-Klinikum, Charité, Humboldt University, 13353 Berlin, Germany; and Laboratoire de Biochimie Endocrinienne (V.T., Y.M.), INSERM Unité 329, Université de Lyon et Hôpital Debrousse, 69322 Lyon Cedex 05, France

Address all correspondence and requests for reprints to: Prof. Dr. Annette Grüters-Kieslich, Charité Kinderklinik, Campus Virchow Humboldt Universität, Augustenburger Platz 1, D-13353 Berlin, Germany. E-mail: annette.grueters{at}charite.de

	Abstract

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A case of nonclassic (NC) 21-hydroxylase deficiency, with a moderately elevated 17-hydroxyprogesterone level (145 nmol/L in filter paper blood spot), was detected in newborn screening. The newborn’s phenotype was female, with no sign of virilization. Confirmatory diagnosis revealed elevated serum levels of 17-hydroxyprogesterone and of 21-desoxycortisol, whereas cortisol, PRA, and electrolytes were normal. Hydrocortisone substitution was considered at the age of 6 months, when virilization became obvious. For clinical reasons, this case had to be classified as late-onset congenital adrenal hyperplasia (CAH) with unusually early manifestation. However, the diagnosis of classic 21-hydroxylase deficiency was obtained by Southern blotting studies, showing that she was homozygous for the 30-kb deletion, including the 3' end of CYP21P pseudogene, the C4B gene, and the 5' end of the functional CYP21 gene. Further studies, using PCR and sequencing, were conducted to explain the discrepancy between this genotype, usually associated with a classic salt-wasting form, and the girl’s phenotype. Typically, patients homozygous for the 30-kb deletion encoding classic CAH possess a unique CYP21P/21 hybrid gene with the junction site located after the third exon, yielding a nonfunctional pseudogene. The girl in question, however, was heterozygous for the 8-bp deletion, suggesting that the chimeric pseudogene on one allele had a junction site before the third exon. She was compound heterozygous for a 30-kb deletion encoding classic CAH on the paternal allele, and a 30-kb deletion encoding NC CAH on the maternal allele. This novel maternal CYP21P/21 hybrid gene is characterized by a junction site before intron 2 and differs from the normal CYP21 gene only by the P30L mutation in exon 1 and the promoter region of the CYP21P pseudogene. Because the P30L mutation has been described to result in an enzyme with 30–60% activity of the normal P450c21 enzyme, and the CYP21P promoter reduced the transcription to 20% of normal, this puzzling phenotype of a NC CAH with early onset may be fully explained by the genotype of the patient and considered as an intermediate form between the simple virilizing and NC form.

	Introduction

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CONGENITAL ADRENAL HYPERPLASIA (CAH) belongs to a group of autosomal recessive inborn errors of adrenocortical steroidogenesis (1). Steroid 21-hydroxylase deficiency (21-OHD) accounts for some 95% of cases. A variety of phenotypes has been observed, ranging from female pseudohermaphrodism with salt-wasting (SW), evident in the first weeks of life, to asymptomatic forms ascertained only by genetic studies of affected families. Clinically, almost all forms can be allocated to three forms; the SW, the simple virilizing, and the nonclassic (NC) forms (2). The SW and the simple virilizing form are characterized by prenatal virilization, resulting in genital ambiguity at birth in newborn females. The NC forms, in contrast, are characterized by virilization of postnatal onset or the lack of almost any symptom until adulthood.

The 21-hydroxylase gene (CYP21) is located on the short arm of chromosome 6 and is duplicated with its pseudogene (CYP21P) in tandem with C4 genes. High homology of these duplicated regions predisposes to misalignment during meiosis, resulting in rearrangement (2, 3, 4). Genetic lesions causing 21-OHD are either large gene deletions, large gene conversions, small gene conversions, or (rarely) point mutations (5, 6, 7, 8). Because there is a relatively good correlation between genotype and phenotype, molecular genetic studies may be clinically valuable, either in confirming or establishing the diagnosis and as basis for genetic counseling (7, 9). However, some discrepancies between genotype and phenotype have been reported (10, 11); for instance, the mild mutation 281L has been found to be associated with an SW form (11). In our and other’s experience, this discrepancy may be explained by the simultaneous occurrence of one or more additional CYP21 mutations on the allele carrying the mild mutation. Nevertheless, some other discrepancies have not yet been resolved: the absence of demonstrable CYP21 mutations on one or two alleles in 21-OHD after extensive sequencing (12 and unpublished own results, Morel and Tardy), or the improvement of aldosterone biosynthesis in an SW form homozygous for a 30-kb deletion of the CYP21 gene (10).

Newborn screening can detect severe forms before an adrenal crisis occurs, especially in affected boys who are phenotypically normal at birth (13). Nevertheless, some cases of the NC form have been detected by neonatal screening and confirmed by molecular studies, but the number of detected cases depends on the cut-off limit of 17-hydroxyprogesterone (17-OHP) (9, 14, 15, 16, 17).

This report describes a girl with an NC phenotype, who is apparently homozygous for a 30-kb deletion of the C4B/CYP21 genomic region, which usually encodes classic CAH, as defined by Southern blot analysis (2, 3, 18). She was detected because of moderately elevated 17-OHP levels in a newborn screening program and showed no virilization or other symptoms at birth. In the postnatal period, however, some clinical symptoms emerged. Additional molecular studies revealed a novel deletion of the C4-CYP21 region on one allele, explaining this intermediate form of 21-OHD.

	Materials and Methods

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Neonatal screening and steroid assay

The newborn screening for CAH, by determination of 17-OHP in filter paper blood spots on the third to fifth day of life, was established in Berlin in 1992 (19). The fluoroimmunoassay measures 17-OHP by specific antibody without prior extraction (DELFIA Neonatal 17OHP-kit, EG&G Wallac, Inc., Turku, Finland). The recall limit of the screening assay in full-term newborns is 60 nmol/L. Newborns with classic CAH were found to have elevated 17-OHP levels, between 150–900 nmol/L (19, 20). Baseline and stimulated (0.25 mg ACTH_1–24 iv_, Synacthen, Ciba-Geigy GmbH, Wehr, Germany) serum steroid hormones were measured by specific RIA without prior extraction: Cortisol (DPC, Bad Nauheim, Germany), 17-OHP (IBL/RSL, Carson, LA), 4-androstenedione (DSL, Sinsheim, Germany) and dehydroepiandrosterone-sulfate (IBL/RSL), PRA by RIA after dialysis (21). 17-OHP, aldosterone, and 21-desoxycortisol levels were verified by tritiated RIA after chromatography (22, 23).

Molecular analysis of the C4 and CYP21 genes

The informed consent to perform extensive molecular studies and to publish the data were obtained from the parents.

Human leukocyte DNA was digested with TaqI restriction enzyme, and Southern blotting studies were conducted as previously described (18). Blots were probed with a mixture of two fragments: a 500-bp BamHI-KpnI fragment of the C4 complementary DNA, and a 3.1-kb genomic EcoRI- BamHI fragment of the 5.5-kb BglII-BamHI fragment encompassing the entire CYP21B gene cloned in the BamHI site of bluescript SK⁺ plasmid (2).

PCR amplification of CYP21 gene fragments

Each primer has been numbered according to the Higashi’ functional CYP21 sequence (24): the first number denotes the nucleotide at the 5' end and the second number this at the 3' end.

Nonselective amplification of exon 3 of CYP21 and CYP21P genes was performed using these two oligonucleotides: primer A: 5'-GTCTAAGAACTACCCGGACCTGTC-3' (+678/+701); and primer B: 5'-CTTCTTGTGGGCTTTCCAGAGCAG-3' (+741/+718).

Two other primers with a common sequence for both CYP21 genes located at the 5' and 3' ends, respectively, have been used: primer C: 5'-TCCCAAGGCCAATGAGACTGGTGT-3' (-176/-153); and primer D: 5'-CTGAGGTACCCGGCTGGCATCGGT-3' (+2740/+2717).

To amplify the functional CYP21 gene, but not the CYP21P pseudogene, the 3' end of these following primers exhibited some mismatches (underlined nucleotides) with the CYP21P pseudogene sequence, in particular around the 8-bp deletion located in exon 3, and could not amplify the corresponding fragment of the pseudogene: primer E: 5'-GGCTTTCCAGAGCAGGGAGTAGTC-3' (+732/+709); primer F: 5'-GGACCTGTCCTTGGGAGACTA-3' (+693/+713); and primer G: 5'-TCGGTGGGAGGGTACCTGAA-3' (-123/-104).

Primer H (5'-CCGGACCTGTCGTTGGTCTCT-3') (+691/+730) could amplify specifically the CYP21P pseudogene.

One hundred-microliter reactions contained 750 ng genomic DNA, 50 µM of each primer, 200 µmol/L of each deoxynucleotide triphosphate, 2.5 U Taq polymerase, and 1x Taq reaction buffer (Eurobio, les Ulis, Paris). The concentration of MgCl₂ was optimized for each amplified fragment; 5% dimethylsulfoxide was added. The PCR program, on a GeneAmp 9600 thermocycler (Perkin-Elmer Corp., Norwalk, CT), was 95 C for 5 min, followed by 30 cycles of 30 sec at 95 C, 30 sec at 56 C (60 C for exon 3 to 3' end), and 30 sec at 72 C, with a 72 C final extension for 10 min in the last cycle. Direct sequencing of PCR products was performed with a 373A model automatic sequencer (PE Applied Biosystems, Foster City, CA), as previously described (25).

	Case Report

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The patient is the first child of nonconsanguineous parents of German origin. There is no family history of CAH, of virilization in female family members, or of impaired fertility. Pregnancy and delivery were uneventful, and birth weight and length were normal (3600 g, 52 cm).

In the filter paper specimen for screening on the fifth day of life of the girl, an elevated 17-OHP level of 145 nmol/L was found. The control examination on day 17 of life, in filter paper blood spot, reconfirmed the elevation of 17-OHP (197 nmol/L) and led to the girl’s referral to the Department of Pediatric Endocrinology for further examinations. At the age of 6 weeks, she was lacking any sign of virilization, but subsequent serum analysis of adrenal steroids again revealed increased levels of 17-OHP (Table 1) and of adrenal androgens, in the presence of normal values for cortisol, sodium (145 mmol/L), potassium (4.6 mmol/L), and PRA (2.1 pmol/sec/L). At the age of 3 months, an ACTH-stimulation test was performed, although there were still no signs of virilization (Table 1). Baseline cortisol levels were still normal, obviously due to maximum stimulation by endogenous ACTH, and did not further respond to stimulation with exogenous ACTH, whereas baseline levels of the precursors 17-OHP and 21-deoxycortisol were clearly elevated and rose markedly after ACTH stimulation.

	Results

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Southern blot analysis suggested that the patient was homozygous for the classic 30-kb deletion. In Fig. 1, the CAH girl‘s pattern, chromosomes a and d, shows an absence of 5.4- or 6.0-kb bands corresponding to C4B gene and an absence of 3.7-kb and 2.4-kb bands. This result suggests that the deletion includes the 5' end of the CYP21 gene, the C4B gene, and the 3' end of the CYP21P pseudogene (18). Because this hybrid gene is nonfunctional because of the location of the crossing-over event between the CYP21P and CYP21 genes downstream from the 8-bp deletion in exon 3 (e.g. haplotype III, Fig. 2), homozygosity of this deletion would have resulted in a severely affected phenotype with prenatal virilization and SW. However, this girl’s attenuated clinical form suggested a different localization of the crossing-over event in at least one allele. Interestingly, the mother’s chromosomal pattern was characterized as a combination of a 30-kb deletion (chromosome d) and the presence of three C4/CYP21 gene units, with a duplication of the long C4B gene, two CYP21P pseudogenes, and a normal CYP21 gene (chromosome c); clinically, CAH was not suspected.

View larger version (44K): [in this window] [in a new window]   Figure 1. Southern blots of TaqI digested genomic DNA, after hybridization with a mixture of two fragments: a 500-bp BamHI-KpnI fragment of the C4 complementary DNA, and a 3.1-kb genomic EcoRI-BamHI fragment encompassing the entire CYP21 gene (18 ). The CAH girl seems homozygous for a 30-kb deletion (chromosomes a and d): absence of the C4B gene (no 5.4- or 6.0-kb bands), of 5' end of the CYP21 gene (no 3.7-kb band), and of 3' end of the CYP21P pseudogene (lack of 2.4-kb band). The father’s pattern (Fa), showing decreased intensities of 6.0-kb, 3.7-kb, and 2.4-kb bands, is heterozygous for this 30-kb deletion with a normal chromosome b. The mother’s pattern (Mo) is attributable to a combination of a 30-kb deletion (chromosome d) and the presence of three C4/CYP21 gene units with a duplication of the long C4B gene, two CYP21P pseudogenes, and a normal CYP21 gene (chromosome c). 1 and 2, DNA pattern of normal subjects.

View larger version (24K): [in this window] [in a new window]   Figure 2. Structure of the normal CYP21 (I) and two chimeric CYP21P/21 genes (II, maternal gene; III, paternal gene), determined by PCR and sequencing. Oligonucleotide primers A, B, C, and D (white triangles) have common sequences for both CYP21 genes; primers E, F, and G (black triangles) have specific sequences for CYP21 gene; primer H (square) has specific sequences for CYP21P gene. Fragments with solid lines, but not those with dotted lines, have been amplified. The precise junction site between the CYP21P pseudogene and the CYP21 gene was determined by sequencing the amplified fragments. Boxes represent the exons; black and dashed boxes or lanes represent sequences of the CYP21 gene and the CYP21P pseudogene, respectively; gray boxes and lanes located between the CYP21P and CYP21 gene correspond to sequences common to both genes.

View larger version (89K): [in this window] [in a new window]   Figure 3. Pattern of the amplification of exon 3, with primers A and B having a common sequence for both CYP21 genes. The pattern of the CAH girl is compared with other patterns: 1) normal subject; 2) patient homozygous for a classic CAH deletion; and 3) subject heterozygous for a deletion of the CYP21P. The 64-bp and 56-bp fragments correspond to the CYP21 and CYP21P genes, respectively (see text), and both are present in our patient.

View larger version (54K): [in this window] [in a new window]   Figure 4. The sequence of the junction site of the CYP21P/21 hybrid gene, shown from the nucleotide +85 (middle of exon 1) to +470 (beginning of intron 2). The nucleotides and the amino acids have been numbered according the Higashi’ functional CYP21 sequence (24 ). The lines are termed as follows; P450c21 corresponds to the amino acid sequence of the functional protein, CYP21 to the functional CYP21 sequence, CYP21P to the pseudogene CYP21P sequence, CYP21P/21 to the hybrid gene (II, Fig. 2), and mutant P450c21 to the protein deduced from the hybrid gene. Only the nucleotide alterations in the CYP21P and CYP21P/21 hybrid genes from the CYP21 counterpart are indicated under the corresponding nucleotides in the CYP21 gene. A gap, represented by bars, in intron 2 of the functional CYP21 is introduced to minimize the consequence of the TGTT insertion in the CYP21P. Amino acids are identical for proteins deduced from the CYP21 and hybrid CYP21P/21 sequences, except one amino acid caused by the P30L mutation.

	Discussion

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The initial mismatch between genotype and phenotype was resolved by further molecular genetic studies. Of the two 30-kb deletions, only one resulted in the nonfunctional CYP21P/21 hybrid gene, the junction site being located downstream from exon 3, which homozygously would have resulted in a severe phenotype. The location of this unequal crossing-over event differs from those previously described (3, 26, 27) and is located downstream from codon 114 (exon 3) and upstream from codon 172 (exon 4). To date, the recombination site has been characterized only in the HLA-Bw47-linked hybrid CYP21P/21 and was located downstream from position 3474 of intron 7 and upstream from codon 318 (exon 8) (27, 28). Similarly, in our experience, including 200 large deletions and gene conversions, most crossing-over events have occurred between exon 6 and exon 8; but, less frequently, recombination sites may also be located between exons 3 and 4, as described for the present haplotype III (Fig. 3).

The maternal allele carried a novel hybrid gene CYP21P/21, with the junction site in intron 2, containing two deleterious mutations, P30L and the pseudogene promoter. The mutant enzyme encoded by the maternal allele will be expressed only at a low level, because the CYP21P promoter has only 20% of the transcriptional activity of the CYP21 promoter (29, 30). Because the other mutation has also been shown to result in a P450c21 protein, retaining only partial activity of about 40% (31), the defect on the second allele left exons 3–10 intact and yielded a 21-hydroxylase activity estimated at below 20%. This is lower than the activities described to result from the point mutations typical for NC-CAH (5, 32) and fully explains the unusually early onset of the NC adrenal hyperplasia.

The extensive studies were necessary to convince the parents of the persistence of the initially mild disease caused by a genetic defect and requiring a permanent therapy and additionally allowed for appropriate genetic counseling for further offspring in this family. The management of a pregnancy with a fetus with an NC defect of 21-hydroxylase considerably differs from a case with a fetus with a classic defect. In pregnancies with severe forms of classic CAH, prenatal therapy may be offered to reduce virilization of the newborn female (33, 34); but, because this treatment is still controversial because of the lack of data on long-term safety (35), it must be conducted within the framework of prospective, centralized, long-term studies. In late-onset forms, however, prenatal therapy and diagnostics as amniocentesis or chorionic villus sampling are definitely not indicated. Nevertheless, although this intermediate form has not virilized the female fetus in utero, this family has to be provided genetic counseling as previously described (7), because the father carries a null mutation. Thus, although V281L has not been associated with the duplication of the C4-CYP21 region on the maternal allele (chromosome c, Fig. 1), an ACTH test should be proposed to the mother. If the peak level of 17-OHP is consistent with an NC-CAH, molecular studies should be performed to identify the nature of the mutation on chromosome c. Only if a severe mutation is detected should prenatal diagnostics be proposed.

In conclusion, when genotype does not match phenotype in CAH, extensive molecular studies may explain the discrepancy, safeguard appropriate genetic counseling of the family, and prevent unnecessary prenatal diagnostics.

	Acknowledgments

 
We thank Prof. W. G. Sippell for the chromatographic determination of steroid hormones.

	Footnotes

 
¹ Contributed equally to this work and should be considered as an equal first author.

Received April 21, 2000.

Revised July 13, 2000.

Revised August 21, 2000.

Accepted August 27, 2000.

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