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A Novel Missense Mutation, GLY424SER, in Brazilian Patients with 21-Hydroxylase Deficiency¹

Unidade de Endocrinologia do Desenvolvimento e Laboratório de Hormônios e Genética Molecular da Disciplina de Endocrinologia-LIM/42, Faculdade de Medicina da Universidade de São Paulo, Hospital das Clínicas, Sao Paulo, Brazil

Address all correspondence and requests for reprints to: Berenice B. Mendonca, M.D., Faculdade de Medicina da Universidade de São Paulo, Divisão de Endocrinologia, Hospital das Clínicas, Caixa Postal 3671, Sao Paulo CEP 01060–970, Brazil. E-mail: beremen{at}usp.br

	Abstract

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A previous screening of 17 mutations in 130 Brazilian patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency did not identify mutations in 20% of the alleles. To diagnose these alleles we sequenced the entire CYP21 gene of one Mulatto patient with the simple virilizing form, who had only the R356W mutation in a heterozygous state. We identified a heterozygous GA transition in codon 424. This mutation leads to a substitution of glycine by serine in a conserved region where glycine is conserved in at least 4 species. This novel mutation eliminates 1 of the restriction sites of the BanI enzyme, which made its screening possible for the whole series. The G424S mutation was found in a compound heterozygous state in 5 families; 4 presented the simple virilizing form, and 1 presented the nonclassical form. Interestingly, 3 of 5 families have a Mulatto origin. This mutation was not identified in 118 CYP21 alleles of normal individuals, ruling out the possibility of a polymorphism, or in 80 pseudogenes, indicating a casual mutagenic event and not a microconversion event. All patients with the G424S mutation presented CYP21P and C4A gene deletions and human leukocyte antigen DR17 on the same haplotype, suggesting a linkage disequilibrium and a probable founder effect. Search for the G424S mutation in other populations will reveal whether it is restricted to the Brazilian patients or if it has a wider ethnic distribution.

	Introduction

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CONGENITAL adrenal hyperplasia (CAH) is one of the most frequent inborn errors of metabolism. It has an autosomal recessive pattern of inheritance, and 95% of CAH cases are due to 21-hydroxylase (CAH-21OH) deficiency (1). This defect is caused by large mutations (deletions or conversions) or point mutations in the CYP21 gene that encodes the enzyme. These mutations cause different degrees of impairment of enzyme activity, which are responsible for the wide spectrum of clinical manifestations in CAH-21OH (2, 3, 4, 5).

Several population studies using techniques for the identification of large mutations and 7–15 point mutations identified 73–95% of the affected alleles (2, 3, 4, 5, 6); thus, the remaining alleles must carry new mutations or regulatory abnormalities. The study of 17 mutations (15 point mutations and 2 large mutations) in 130 Brazilian patients with CAH-21OH identified 80% of the mutant alleles (7, 8). In this report we describe a new mutation found after sequencing the CYP21 gene of a Mulatto patient with the simple virilizing (SV) form. This mutation was also identified in 4 other Brazilian families with CAH-21OH.

	Subjects and Methods

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Informed consent for the study was obtained from all patients’ parents or tutors.

Patients 1–5 are Brazilian girls (four Mulattos and one Caucasian) who presented ambiguous genitalia at birth (Prader III) without salt-wasting crisis and were diagnosed with the SV form. Patients 4 and 5 are siblings. Patient 6 is the affected brother of patient 1 and presented with precocious pubarche (Tanner II), penile enlargement, and advanced bone age (11 yr) at a chronological age of 3 yr and was also diagnosed with the SV form. Patient 7 is a Caucasian girl with precocious pubarche (Tanner III), normal external genitalia, and advanced bone age (9 yr) at age 4 yr and was diagnosed with the late-onset form. The patients’ hormonal data at diagnosis are shown in Table 1.

Human leukocyte antigen (HLA)-DRB typing was performed by low resolution PCR amplification with sequence specific primers according to the method of Olerup and Zetterquist (12). The amplification of DR3 comprehends DRB1_*0301 and includes several other rare alleles, such as DRB1_*0304 and DRB1_*0306, seldom found in the Brazilian population. We have named this group of alleles collectively DR17.

	Results

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Sequencing the 2.3-kb PCR-amplified fragment of patient 1 identified a GA transition in codon 424 that results in substitution of the amino acid glycine by the amino acid serine and eliminates a BanI restriction site (Fig. 1). This mutation, found in a heterozygous state, was inherited from the father and was also present in the affected brother (patient 6). Digestion of the 0.61-kb fragment from patient 1, her brother, and her father originated 5 fragments of 27, 154, 198, 239, and 437 bp (Fig. 2). The latter resulted from the loss of 1 BanI restriction site between fragments of 198 and 239 bp. This mutation was also found in a heterozygous form in 5 other patients after the screening of 128 CAH-21OH patients through BanI digestion and was inherited from 1 of the parents in all cases. All alleles with G424S mutation also carry the CYP21P and C4A deletions (7) and were positive for HLA-DR17. The genotypes of the 7 patients are shown in Table 1. Screening of 59 normal individuals (118 CYP21 genes) as well as 80 pseudogenes (20 normal individuals and 20 patients) did not identify this mutation.

View larger version (20K): [in this window] [in a new window]   Figure 1. DNA sequence of CYP21 gene of patient 1 showing the G to A transition at nucleotide 2494 in the heterozygous form, which results in the glycine to serine mutation at codon 424.

View larger version (119K): [in this window] [in a new window]   Figure 2. BanI restriction of the 0.61-kb PCR fragment, containing codon 424. Patient 1 (P) and her father (F) are heterozygous for the G424S mutation, which eliminates one restriction site, producing a fragment of 437 bp in addition to the normal ones. The mother (M), who does not have the mutation, presents a normal restriction pattern of the fragment. L, One-kilobase ladder; A, nondigested 0.61-kb PCR fragment.

	Discussion

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We had previously studied the presence of large rearrangements (7) and 15 point mutations (8) in 130 CAH-21OH patients, and we did not identify mutations in 20% of the alleles (13% in the classical form and 31% in the nonclassical form). In an attempt to diagnose these alleles, we sequenced the CYP21 gene of a female patient with the SV form of CAH-21OH. She presented the mutation R356W in heterozygous form, which was inherited from her mother and was also present in the patient’s affected brother (case 6). Sequencing of exon 10 identified a GA transition in codon 424, in heterozygous form, resulting in the substitution of glycine by serine, also identified in the patient’s affected brother and normal father. This mutation eliminates a BanI restriction site, and this approach was used as an easier and faster screening compared to sequencing. The presence of this mutation was screened through enzyme restriction in another 128 CAH-21OH patients and was found in heterozygous form in 5 of them. The absence of this mutation in 59 normal individuals ruled out the possibility of polymorphism. Additional evidence that G424S is a mutation and not a polymorphism is that this gene region is very conserved. This glycine is conserved in at least 4 different species, human, bovine, murine, and swine (13, 14, 15, 16), and is near the Cys⁴²⁸ residue, which is a ligand for the heme prosthetic group (17). G424S was found in patients with the SV form, and therefore, it should confer severe impairment of enzymatic activity. In vitro expression studies will determine the enzymatic activity of this mutation and its association to the SV form. The late-onset form exhibited by patient 7 is a consequence of the presence of V281L in the other allele, which confers moderate impairment of enzymatic activity (2).

The G424S was not found in 80 pseudogenes, indicating a casual mutagenic event and not a microconversion event. Interestingly, 3 of 5 families with this mutation have a Mulatto origin.

All seven patients had the G424S mutation on the same haplotype, along with deletion of the CYP21 pseudogene and the C4A gene, and HLA-DR17, suggesting a founder effect. These events are in linkage disequilibrium in the same way as the association of V281L and CYP21P and C4B duplication in the nonclassical form (17).

Alteration in the number of copies of the CYP21/C4 unit may cause unequal cross-over and, therefore, microconversion events. These events might explain the de novo I2 splice mutation in patients 2, 4, and 5.

A search for the G424S mutation in other populations will reveal whether it is restricted to the Brazilian patients or has a wider ethnic distribution.

	Acknowledgments

 
The authors thank Ms. Maria A. Medeiros for technical assistance, and Ms. Sonia Strong for the English review.

	Footnotes

 
¹ This work was supported in part by grants from FAPESP—Fundação de Amparo à Pesquisa No Espado de São Paulo [95/8325–6 (to A.E.C.B.) and 98/00243–9 (to T.A.S.S.B.)].

² A.E.C.B. and T.A.S.S.B. contributed equally to this work.

Received January 28, 1999.

Revised May 17, 1999.

Accepted April 19, 1999.

	References

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