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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-0779
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 11 4510-4513
Copyright © 2006 by The Endocrine Society

BRIEF REPORT

Divergent Phenotype of Two Siblings Human Leukocyte Antigen Identical, Affected by Nonclassical and Classical Congenital Adrenal Hyperplasia Caused by 21-Hydroxylase Deficiency

O. Porzio, V. Cunsolo, M. Malaponti, E. De Nisco, A. Acquafredda, L. Cavallo, M. Andreani, E. Giardina, M. Testi, M. Cappa and G. Federici

Departments of Internal Medicine (O.P., V.C., M.M., E.D.N., G.F.) and Biopathology and Diagnostic Imaging (E.G.), University of Rome "Tor Vergata," 00133 Rome, Italy; Immunogenetics Laboratory (M.A., M.T.), Mediterranean Institute of Hematology (MIH) Foundation, 00133 Rome, Italy; Endocrinology and Diabetology Unit and Research Laboratory (M.C., G.F.), Bambino Gesù Children’s Hospital, 00165 Rome, Italy; and Division of Paediatrics B. Trambusti (A.A., L.C.), University of Bari, 70126 Bari, Italy

Address all correspondence and requests for reprints to: Ottavia Porzio, M.D., Department of Internal Medicine, University of Rome "Tor Vergata," Via di Montpellier 1, 00133 Rome, Italy. E-mail: porzio{at}uniroma2.it.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders most often caused by enzyme 21-hydroxylase deficiency. Most mutations causing enzymatic deficiency are generated by recombinations between the active gene CYP21 and the pseudogene CYP21P. Only 1–2% of affected alleles result from spontaneous mutations. The phenotype of CAH varies greatly, usually classified as classical or nonclassical, depending on variable degree in 21-hydroxylase activity. Here we report a divergent phenotype of two human leukocyte antigen identical siblings, affected by nonclassical and classical CAH caused by 21-hydroxylase deficiency due to different genotype.

Patients and Methods: Using direct sequencing method and Southern blot, we studied two children (one male and one female), affected, respectively, by nonclassical and classical CAH and their parents.

Results: The mother was heterozygous for the Q318X mutation, and the father was heterozygous for the V281L mutation. The brother was a compound heterozygote for the mutations V281L and Q318X, whereas the proband was compound heterozygote for the Q318X mutation and a large conversion. The two children are human leukocyte antigen identical (A*02;B*14;DRB1*01/A*33;B*14;DRB1*03).

Conclusions: Different phenotype of the proband is the result of compound heterozygosity for the maternal mutation Q318X and a de novo large conversion.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
CONGENITAL ADRENAL HYPERPLASIA (CAH) due to deficiency of adrenal enzyme 21-hydroxylase (21-OH) is an autosomal recessive inherited disorder of steroid metabolism. 21-OH is a cytochrome P-450 enzyme that catalyzes the conversion of 17-hydroxyprogesterone (17-OHP) to 11-deoxycortisol, a precursor of cortisol, and the conversion of progesterone to deoxycorticosterone, a precursor of aldosterone. The phenotype of CAH varies greatly, depending on the degree in the impairment of 21-OH activity. A severe form with a defect in cortisol biosynthesis, with or without aldosterone deficiency, is termed classical CAH (salt wasting or simple virilizing type, respectively); a mild or asymptomatic form is called nonclassical (NC) CAH, generally associated with signs of postnatal androgen excess.

The 21-OH gene (CYP21) is located on chromosome 6p21.3 within the human leukocyte antigen (HLA) histocompatibility complex, together with a highly homologous inactive pseudogene (CYP21P). The two genes are located in tandem repeats, with the genes encoding the fourth component of serum complement (C4A and C4B). CYP21 and CYP21P genes consist of 10 exons and show a high homology with a nucleotide identity of 98% in their exon and 96% in their intron sequences (1, 2). Most mutations causing 21-OH deficiency arise from recombinations between CYP21 and CYP21P: when deleterious sequences normally present in the pseudogene are transferred to the active gene, the latter becomes incapable of encoding a normal enzyme. This process, called gene conversion, represents approximately 75% of the deleterious mutations. About 20% are meiotic recombinations that delete a 30-kb gene segment that encompasses the 3' end of the CYP21P, all of the adjacent C4B gene, and the 5' end of the CYP21, producing a nonfunctional chimeric pseudogene (2). The remaining 5% are de novo mutations that do not originate from the pseudogene, and they are unique within single families.

CYP21 mutations can be grouped into three categories according to the level of enzymatic activity: the first group consists of deletions or nonsense mutations such as Q318X (3) that totally ablate enzyme activity generally related to salt-wasting disease (SW). The second group of mutations yields enzymes with 1–2% of normal activity, with adequate aldosterone synthesis, characteristically found in patients with simple virilizing disease. The final group includes mutations such as V281L (3) and P30L (3) that produce enzymes retaining 20–60% of normal activity, associated with the NC disorder. Most patients are compound heterozygotes, having different mutations of the CYP21 gene on each allele: the clinical expression of CAH is reported to be correlated with the less severely mutated allele (3, 4). Several studies have suggested high concordance rates between genotype and phenotype in patients with the most severe and mildest forms of the disease but less genotype-phenotype relation in moderately affected patients (4, 5). Herein we describe two children with discordant phenotype, one with NC and one with SW disease, despite being HLA identical: molecular genetic analysis of the family has shown that different phenotype is the result of a de novo large conversion in the SW patient. Large conversion is located on the paternal allele carrying the V281L mutation and determines the substitution of a mild with a severe mutation, modifying the phenotype from mild to severe form.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

A Caucasian girl was delivered after an uneventful pregnancy from nonconsanguineous parents: she presented with ambiguous genitalia (Prader stage 3) and dehydration, with hyponatremia and hyperkalemia. The proband had high basal 17-OHP (>5000 ng/dl), testosterone (>1000 ng/dl), and {Delta}4-androstenedione (>1000 ng/dl), and she was diagnosed as SW CAH. The karyotype was 46,XX. Her brother was born 2 yr before, without signs of SW or clinical abnormalities: his auxological parameters were normal and his bone age was correspondent to chronological age. Endocrine evaluation performed at the sister’s birth had showed high basal 17-OHP (2410 ng/dl) that rose to 5000 after ACTH stimulation. Testosterone, {Delta}4-androstenedione, cortisol levels, and plasma renin activity were normal.

PCR, sequencing, and Southern blot

Informed consent for molecular analyses was obtained. Genomic DNA was prepared from peripheral blood leukocytes by standard procedures (6). CYP21 was amplified with gene-specific PCR primers (Table 1Go); the fragments were amplified in a volume of 100 µl containing 200–500 ng genomic DNA, 10 mM Tris (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates; and 10 pmol/µl of the respective primers (Table 1Go), 0.75 U Taq polymerase and dimethylsulfoxide (5%). Amplifications were performed by 35 cycles of denaturation at 94 C for 1 min, annealing at 62 C for 40 sec (with primers D and H) and 56 C for 30 sec (with primers A and E), and extension at 72 C for 1 min. PCR products were directly sequenced using a CEQ 2000 XL DNA analysis system (Beckman Coulter, Fullerton, CA).


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  		TABLE 1. CYP21 gene primers used in PCR and sequencing

 
For Southern blot analysis, 10 µg of genomic DNA was digested by TaqI and BglII restriction enzymes. BglII- and TaqI-digested fragments were electrophoresed on 0.8% agarose gels, transferred to nylon membranes, and hybridized with a 32P-labeled complementary DNA pC21/3C (American Type Culture Collection, Manassas, VA).

HLA molecular typing

HLA class I (HLA-A and HLA-B) typing was performed using a reverse dot blot technique with the automated InnoLipa system (Innogenetics, N.V., Zwijndrecht, Belgium). HLA class II (DRB1) typing was performed using sequence-specific primers (PCR-SSP) Dynal Allset (Invitrogen, Carlsbad, CA).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
In the present study, we carried out mutation analysis of the functional CYP21 gene in two siblings of the same family presenting a different phenotype, i.e. NC- and SW-CAH. Both children were HLA genotypically identical (A*02;B*14;DRB1*01/A*33;B*14;DRB1*03); their mother was HLA-A*33;B*14;DRB1*03/A*01;B*08;DRB1*03 and their father was HLA-A*02;B*14;DRB1*01/A*01;B*57;DRB1*13. Molecular genetic analysis of the CYP21 gene showed that the mother carried a heterozygous Q318X mutation, whereas the father was heterozygous for the V281L mutation. The elder child was compound heterozygous for the two different mutations on each chromosome, the paternal mutation V281L and the maternal mutation Q318X. When we analyzed the sister’s DNA, we found it to be homozygous for mutation Q318X, whereas the exon 7 (V281L) mutation was not detectable. Southern blot analysis showed a decreased intensity of the 3.7-kb band and an increased intensity of the 3.2-kb band in TaqI digestion of the proband DNA, with normal BglII intensity, representing the pattern of the heterozygous gene conversion involving the TaqI site in CYP21 gene. Southern blot analysis of the father and son was normal (Fig. 1Go), whereas the mother had a decreased intensity of the 3.2-kb TaqI and 12-kb BglII bands, diagnostic for the heterozygous deletion of the CYP21P.


Figure 1
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  		FIG. 1. Southern blot hybridization of TaqI- and BglII-digested DNA with CYP21 probe. Lanes 1 and 2, Normal; lane 3, decreased intensity of the 3.2-kb TaqI and 12-kb BglII bands, representing the pattern of the heterozygous for CYP21P deletion; lane 4, decreased intensity of the 3.7-kb band and an increased intensity of 3.2-kb band in TaqI digestion of the proband DNA, with normal BglII intensity, representing the pattern of the heterozygous gene conversion involving the TaqI site in CYP21 gene; lanes 5 and 6, normal Southern blot analysis of the brother’s and father’s DNA, respectively; lane 7, Southern blot analysis of the mother’s DNA, heterozygous for CYP21P deletion.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
CAH is one of the most frequent inborn errors of steroid metabolism, and 21-OH deficiency accounts for 90–95% of all cases. The mutations that cause 21-OH deficiency can be large gene rearrangements or point mutations. The presence of several mutations results in most of the patients being compound heterozygotes; in general, the clinical form correlates with the mutated allele with higher enzymatic activity (4, 7). Although genotype usually predicts phenotype, some discrepancies in genotype-phenotype correlation have been reported (5, 8): this disparity is explained in patients carrying intron 2 (A or C to G) by alternative splicing, resulting in the production of variable levels of enzymatic activity (9). Some reports have showed the presence of additional mutations on the same allele, not detected in the screening studies (10) or substitutions in the transcriptional regulatory regions (11). Finally, germline mutations may explain some reports of discordant genotype-phenotype (12).

In this report, we describe an Italian family composed of two heterozygous parents and two affected children with NC and SW CAH, respectively. Before molecular biological techniques were available, their different phenotype would have been called variable genetic expression: in fact, this substantial difference is clinically unexplainable because two HLA-identical siblings inherited the same paternal and maternal chromosome 6. Close genetic linkage between the HLA complex located on the short arm of chromosome 6 and 21-OH deficiency was first described in 1977 (13), and subsequently genetic linkage disequilibrium between 21-OH deficiency and HLA alleles has been repeatedly demonstrated (13, 14) so that HLA typing was the main way to perform prenatal diagnosis before cloning of CYP21 (15). For classical 21-OH deficiency, the most significant association is with HLA-A3;B47;DR7. The NC form is often associated with HLA-B14;DR1 and particularly with the V281L mutation in CYP21 (16). Finally, HLA-A1;B8;DR3 is negatively associated with 21-OH deficiency because this haplotype has a C4A null allele and is associated with deletion of the CYP21P genes (17).

Based on the results of pedigree analysis, both children have paternal HLA-A*02;B*14;DRB1*01 haplotype, but only the father and son are heterozygotes for V281L mutation; in fact, sequencing analysis does not detect the exon 7 mutation in the sequence of sister’s DNA. The mother and her children have the HLA haplotype A*33;B*14;DRB1*03 and all carry the nonsense mutation in codon 318 (Q318X). When Southern blot analysis was performed, the presence of a large conversion was revealed only in the CYP21 gene of the proband, whereas the father and brother were normal. The mother was heterozygous for the deletion of the CYP21P, as described in literature in association with her HLA-A*01;B*08;DRB1*03 haplotype (18). As can be deduced from HLA haplotypes, the SW patient inherited the Q318X-bearing maternal allele and the V281L-bearing paternal allele, on which she harbored a de novo rearrangement. Large conversion determines the loss of most of the CYP21 gene, including the exon 7 sequence-bearing V281L mutation, rendering the functional gene totally incapable of encoding an otherwise partially active enzyme.

Both de novo deletions and de novo gene conversions have been documented (4, 18, 19); the latter usually involve the intron2 nt 656 g mutation and comprise approximately 1% of 21-OH deficiency alleles. De novo recombinations involving CYP21 have also been documented by PCR in sperm and leukocytes (20). Unequal crossing over is detected only in sperm (1 in 105 to 106 genomes), confirming that this process takes place only during meiosis. Gene conversion, however, takes place at equal frequencies in somatic cells and gametes, suggesting that gene conversions occur mainly in mitosis. The frequency of gene conversion observed in these studies (~1 in 104) is consistent with the reported rate of de novo gene conversions in patients with 21-OH deficiency. In our report, we describe a de novo large conversion on one allele carrying a mild mutation, which determines the substitution with a severe mutation, modifying the phenotype from NC to SW form.

In conclusion, this family analysis emphasizes the complexities of 21-OH genotyping and the importance of molecular genetic analysis for both clinical and prenatal diagnosis of 21-OH deficiency.


   Acknowledgments
 
We thank Prof. Giuseppe Novelli for helpful discussions.


   Footnotes
 
Disclosure statement: The authors have nothing to disclose.

First Published Online August 15, 2006

Abbreviations: CAH, Congenital adrenal hyperplasia; HLA, human leukocyte antigen; NC, nonclassical; 21-OH, 21-hydroxylase; 17-OHP, 17-hydroxyprogesterone; SW, salt-wasting disease.

Received April 10, 2006.

Accepted August 4, 2006.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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  8. Ordonez-Sanchez ML, Ramirez-Jimenez S, Lopez-Gutierrez AU, Riba L, Gamboa-Cardiel S, Cerrillo-Hinojosa M, Altamirano-Bustamante N, Calzada-Leon R, Robles-Valdes C, Mendoza-Morfin F, Tusie-Luna MT 1998 Molecular genetic analysis of patients carrying steroid 21-hydroxylase deficiency in the Mexican population: identification of possible new mutations and high prevalence of apparent germ-line mutations. Hum Genet 102:170–177[CrossRef][Medline]
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