0021-972X/99/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 1999 by The Endocrine Society
Vol. 84, No. 8 Printed in U.S.A.
A Novel Missense Mutation, GLY424SER, in Brazilian Patients with 21-Hydroxylase Deficiency* ˆ NIA A. S. S. BACHEGA†, ELIANA T. FRAZZATTO, ANA ELISA C. BILLERBECK†, TA MIRIAN YUMIE NISHI, ANNA CARLA GOLDBERG, MARIA LUCIA C. MARIN, GUIOMAR MADUREIRA, OSMAR MONTE, IVO J. P. ARNHOLD, AND BERENICE B. MENDONCA Unidade de Endocrinologia do Desenvolvimento e Laborato´rio de Hormoˆnios e Gene´tica Molecular da Disciplina de Endocrinologia-LIM/42, Faculdade de Medicina da Universidade de Sa˜o Paulo, Hospital das Clı´nicas, Sao Paulo, Brazil ABSTRACT 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 G3 A 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
C
ONGENITAL 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 (2–5). Several population studies using techniques for the identification of large mutations and 7–15 point mutations identified 73–95% of the affected alleles (2– 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.
Received January 28, 1999. Revision received May 17, 1999. Accepted April 19, 1999. Address all correspondence and requests for reprints to: Berenice B. Mendonca, M.D., Faculdade de Medicina da Universidade de Sa˜o Paulo, Divisa˜o de Endocrinologia, Hospital das Clı´nicas, Caixa Postal 3671, Sao Paulo CEP 01060 –970, Brazil. E-mail:
[email protected]. * This work was supported in part by grants from FAPESP—Fundac¸a˜o de Amparo a` Pesquisa No Espado de Sa˜o Paulo [95/8325– 6 (to A.E.C.B.) and 98/00243–9 (to T.A.S.S.B.)]. † A.E.C.B. and T.A.S.S.B. contributed equally to this work.
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. (J Clin Endocrinol Metab 84: 2870 –2872, 1999)
Subjects and Methods 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 saltwasting 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. Genomic DNA from patients and their parents (except the parents of patient 3) was obtained from peripheral blood leukocytes by standard procedures (9) and submitted to PCR for the amplification of a 2.3-kb fragment specific for the CYP21 gene. This specificity was achieved by using a primer located in exon 3 that does not contain the eight-nucleotide deletion specific for the pseudogene. The primers used were P4 and P55 (10). For amplification of the pseudogene, primer P55 was substituted for another (P56) containing the 8-bp deletion in exon 3 (10). The amplified fragment was submitted to automated sequencing in the Perkin Elmer Corp. ABI PRISM 310 sequencer (Foster City, CA) using the dideoxynucleotide terminator methodology (11). The 2.3-kb amplified fragment was submitted to a second round of PCR using internal primers P11 and P45 (10), and the resulting amplified fragment (0.61 kb) was restricted by BanI endonuclease (New England Biolabs, Beverly, MA) according to the manufacturer’s specifications. The restriction of this fragment from normal individuals results in four fragments: 27, 154, 198, and 239 bp. 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.
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TABLE 1. Clinical data and genotype of seven patients with 21-hydroxylase deficiency and the G424S mutation Patient no.
Clinical form
Sex
1 2 3 4 5 6 7
SV SV SV SV SV SV LO
F F F F F M F
Prader stage
17OHP (ng/mL)
D4 (ng/mL)
T (ng/dL)
III III III III III
150 157 90 124 140 188 26
1.8 3.9 11.0 29.0 31.0 12.1 1.7
45 20 217 508 464 116 26
I
PRA (ng/mL z h)
12.3 11.0 2.6 6.8
Genotype
CYP21P CYP21P CYP21P CYP21P CYP21P CYP21P CYP21P
Del, Del, Del, Del, Del, Del, Del,
G424S/R356W G424S, I2 sp/I2 sp G424S, I2 sp/I172N G424S, I2 sp/I2 sp G424S, I2 sp/I2 sp G424S/R356W G424S/CYP21P Dup, V281L
SV, Simple virilizing form; LO, late onset form; F, female; M, male; 17OHP, basal 17-hydroxyprogesterone; D4, androstenedione; T, testosterone; CYP21P Del, pseudogene deletion; sp, splice; Dup, duplication. Patients 1 and 6 and patients 4 and 5 are siblings.
FIG. 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.
Results
Sequencing the 2.3-kb PCR-amplified fragment of patient 1 identified a G3 A 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. Patients 2, 4, and 5 (Table 1) also presented the mutation I2 splice in a homozygous state (8), although only one of the parents in each family showed this mutation. The G424S mutation was carried by the parent who did not have the I2 splice mutation. The results of the I2 splice mutation in these families were confirmed with different methodologies (allele-specific PCR and sequencing) using different templates. Discussion
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 G3 A 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 (13–16), and is near the Cys428 residue, which is a ligand for the heme prosthetic
FIG. 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.
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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.
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