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A Rare Duplicated 21-Hydroxylase Haplotype and a de Novo Mutation: A Family Analysis

Department of Internal Medicine III, Division of Clinical Endocrinology and Metabolism, University of Vienna, A-1090 Vienna, Austria

Address all correspondence and requests for reprints to: Sabina M. Baumgartner-Parzer, Ph.D., Department of Internal Medicine III, Division of Endocrinology and Metabolism, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: sabina.baumgartner-parzer{at}akh-wien.ac.at.

	Abstract

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21-Hydroxylase (21-OH) genotyping was performed in clinically unaffected family members of a congenital adrenal hyperplasia (CAH) index patient (Prader stage 3), who is a compound heterozygous carrier of the I172N (exon 4) and the intron2 splicing mutations. Whereas the latter mutation could be traced to the father, the exon 4 aberration represents a de novo mutation (accounting for 1% of CAH alleles) harbored on an unaffected allele, which was inherited from the mother. Although clinically and biochemically unaffected, the patient’s brother was found to be compound heterozygous for intron2splice (paternal allele) and Q318X in exon 8 (maternal allele). As shown by PCR-based sequence and Southern blot analysis, the maternal haplotype, inherited by the brother, has a duplicated CYP21B (functional) gene, one of which carries a Q318X mutation. This duplicated Q318X-affected haplotype is the first of its kind among 800 alleles screened for 21-OH deficiency in our laboratory and has to date been reported only in three Swedish CAH patients, all of them bearing an intron2splice and a Q318X mutation. This family analysis highlights the complexity of the CYP21/CYP21P(pseudogene) loci and the difficulties of 21-OH genotyping.

	Introduction

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CONGENITAL ADRENAL HYPERPLASIA (CAH) is a recessively inherited disorder (1, 2), in more than 90% of patients caused by lesions in the steroid 21-hydroxylase gene (21-OH; CYP21), resulting in androgen overproduction and inefficient cortisol and aldosterone synthesis (3, 4).

Forming tandem repeats with the upstream located complement C4A and C4B genes (5), the functionally active (CYP21B) and the 98% homologous, but inactive (CYP21P, CYP21A), pseudogene lie within the human leukocyte antigen (HLA) I/II clusters on the short arm of chromosome 6. The majority of mutations causing 21-OH deficiency result from either unequal crossing over during meiosis, resulting in a complete CYP21B deletion, or gene conversion events transferring deleterious mutations of the CYP21A pseudogene to the functional CYP21B gene (1, 5, 6). The high variability in CYP21A/B copy numbers as well as the existence of more than one mutation per allele (1, 5, 6, 7, 8, 9) further complicate genotyping.

In the course of 21-OH genotyping, routinely performed in our laboratory (8, 9, 10, 11), a female presenting with severe CAH was found to be heterozygous for the I172N (exon 4) and intron2splice mutations. Segregation analysis performed in the parents and the brother revealed the latter and the mother to carry a duplicated CYP21B gene, bearing a Q318X point mutation. Such a 21-OH defect constellation had to date only been reported in three Swedish CAH patients (12).

	Subjects and Methods

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Subjects

The female index patient was referred to our department at the age of 18 yr. Diagnosed with CAH 2 months after birth, she had surgical correction of the sinus urogenitalis at the age of 1 yr. The diagnosis had been based on the clinical manifestation (Prader stage 3, without salt loss) and was confirmed by detection of the relevant steroid metabolites in plasma [17-hydroxyprogesterone (17-OHP), 20,513 ng/dl (622 nmol/liter); normal range, 35–375 ng/dl (1.1–11.4 nmol/liter); testosterone, 229 ng/dl (7.95 nmol/liter); normal range, <50 ng/dl (1.74 nmol/liter)] and urine [pregnantriol, 63.44 mg/24 h (1880 µmol/liter); normal range, 0.36–1.38 mg/24 h (10–41 µmol/liter)], determined as described previously (13, 14). HLA typing was performed according to standard methods.

The parents and the brother were clinically asymptomatic, and their basal 17-OHP and testosterone plasma concentrations were within the normal range. In the case of the brother, an ACTH (0.25 mg, iv) stimulation test was performed, inducing a rise in 17-OHP from basal [109 ng/dl (3.3 nmol/liter)] up to 495 ng/dl (15 nmol/liter). The patient’s mother achieved menarche at 18 yr of age. She is not hirsute, is 173 cm of height, has regular menses, and required 4 and 2 yr of unprotected intercourse to achieve her first and second pregnancies, respectively. Written informed consent for mutation analysis was obtained from all individuals tested.

21-OH genotyping

Genomic DNA was extracted from peripheral blood leukocytes (8, 9, 10, 11). Genotyping for large gene deletions and conversions was performed by Southern blot analysis as described previously (8, 9, 10). In brief, TaqI-digested DNAs, immobilized on nylon membranes, were hybridized with a [³²P]deoxy-CTP-labeled CYP21 probe (American Type Culture Collection, Manassas, VA).

The common mutations P30L, I2Splice, I172N, Cluster E6, V281L, F307insT, G291S, Q318X, R356W, G424S, and P453S were detected by direct sequencing (using [³³P]dideoxy-NTP terminators and a cycle sequencing kit) of three fragments (I–III), specifically amplified by selective PCR primers as previously described (8, 9, 15), differentiating CYP21B from CYP21A by the 8-bp deletion located in exon 3 of CYP21A. The GenBank accession number is NM 000500.

	Results

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21-OH genotyping

The index patient presenting with classical CAH (Prader stage 3, without salt loss) was found to be a compound heterozygous carrier of the I172N (exon 4) and intron2splicing mutation. The latter mutation was found in both the father and the brother, whereas the exon 4 mutation was not detectable in any of the studied family members. The mother carries a heterozygous Q318X (exon 8) mutation (associated with HLA A2, B50, Cw6), whereas the brother is compound heterozygous for the intron2splice and Q318X mutations.

Based on the results of pedigree analysis, the allele carrying the intron2splicing mutation and linked to the A24, B41, Cw7 HLA extended haplotype was of paternal origin (Fig. 1A). The HLA haplotype analysis indicated that the proband and her brother inherited different maternal alleles.

View larger version (35K): [in this window] [in a new window]   Figure 1. A, Pedigree of the CAH index patient (a/c, •) and sequence analysis of the intron2splice location. Functionally normal CYP21B genes are indicated by open symbols; those affected by genetic aberrations are indicated by black symbols. The presence of three CYP21B genes [in the mother (c/d) and the brother (a/d)] is mirrored by the symbols divided by 3. Arrowheads indicate the heterozygous nucleotide mutation (G) from the normal (A, C) haplotypes to C/G in the father and A/G in the index patient, whereas three bases (C/A/G) are present in the brother. Double intensity of the A vs. C haplotype indicates the constellation C/A/A in the mother. #, Association of the respective HLA haplotype with the intron2splice and the Q318X mutation, respectively. B, TaqI restriction fragments (Southern blot analysis) representing the functional CYP21B (3.7-kb band) and the pseudogene CYP21A (3.2-kb band). The intensity of the band is assumed to correspond to the respective gene’s copy number: lane 1, control DNA: CYP21B/CYP21A ratio of approximately 1:1, suggesting the presence of two CYP21B and two CYP21A genes; lane 2, control DNA: heterogeneous CYP21B deletion; lane 3, father: heterogeneous CYP21A deletion; lane 4, mother: three CYP21B and two CYP21A genes; lane 5, brother: three CYP21B genes and one CYP21A gene; and lane 6, index patient: heterogeneous CYP21A deletion.

Such assumptions are in line with the results of Southern blot analysis, showing increased intensity of the 3.7-kb TaqI band (in the mother and brother) representing the functional CYP21B gene (Fig. 1B) compared with the respective bands detected in the index patient, the father, and two control DNAs. The CYP21B/CYP21A ratio (index patient and father, 2:1; brother, 3:1; mother, 3:2) indicates 1) a heterozygous deletion of the CYP21A pseudogene in the index patient and the father, 2) the presence of three CYP21B and 1 pseudogenes in the brother, and 3) the presence of three CYP21B and two CYP21A pseudogenes in the mother (Fig. 1B).

	Discussion

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Segregation analysis in the reported CAH family revealed the index patient’s father and mother to be heterozygous (carriers) for the intron2splice and the Q318X mutation, respectively. As can be deduced from HLA and intron 2 haplotypes, the index patient inherited the intron2-bearing paternal allele and the unaffected maternal allele, on which she harbored a de novo mutation in exon 4 (Ile172N).

The patient’s brother presented an equally rare constellation, exhibiting compound heterozygosity for the intron2splice and the Q318X mutation, the latter located on a duplicated 21-OH B gene inherited from the mother.

As this family is the first presenting with a duplicated CYP21B gene of more than 800 alleles genotyped in our laboratory, we assume that this haplotype, previously only reported for three Swedish CAH patients (12, 16), is also extremely rare in the Austrian CAH population. Of note, in the Swedish patients (12) as well as in our patients the duplicated CYP21B gene carried a Q318X mutation. As the family is of Croation origin, any Swedish ancestors are extremely unlikely.

21-OH genotyping in this CAH family revealed the index patient to carry a de novo mutation, accounting for 1% of CAH alleles (1), and the mother and the brother to carry a rare duplicated CYP21B haplotype bearing a Q318X mutation. It appears that the brother’s unaffected CYP21B gene exhibits enough 21-OH activity to account for his normal biochemical and clinical phenotype (1, 3, 9). It remains unknown whether there is any relationship of the mother’s rare CYP21 haplotype and her late menarche or her unfulfilled wish for pregnancy for 4 and 2 yr (17, 18). In conclusion, this family analysis emphasizes the complexities of 21-OH genotyping.

	Acknowledgments

 
We thank Susanne Pauschenwein, Angelika Freudenthaler, Karin Pölzler, and Rita Lang for expert technical assistance.

	Footnotes

 
Abbreviations: CAH, Congenital adrenal hyperplasia; HLA, human leukocyte antigen; 21-OH, 21-hydroxylase; 17-OHP, 17-hydroxyprogesterone.

Received October 11, 2002.

Accepted March 10, 2003.

	References

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Baumgartner-Parzer, S. M. Articles by Vierhapper, H. Search for Related Content PubMed PubMed Citation Articles by Baumgartner-Parzer, S. M. Articles by Vierhapper, H. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Substance via MeSH Genetics Home Reference