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Molecular Genotyping in Brazilian Patients with the Classical and Nonclassical Forms of 21-Hydroxylase Deficiency¹

Unidade de Endocrinologia do Desenvolvimento e Laboratório de Hormônios e Genética Molecular da Disciplina de Endocrinologia (T.A.S.S.B., A.E.C.B., G.M., J.A.M.M., I.J.P.A., B.B.M.), Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo 01060–970, Brazil; Serviço de Endocrinologia Pediátrica (C.A.L.), Departamento de Pediatria da Santa Casa, São Paulo, Brazil; Departamento de Clínica Médica I (M.V.L.), Universidade Federal do Rio Grande do Norte, Natal, Brazil 59010-180

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

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The aim of our study was to determine, by allele-specific PCR, the frequency of point mutations in 130 Brazilian patients with the classical and nonclassical forms of 21-hydroxylase deficiency and to correlate genotype with phenotype. The most frequent mutations were I2 splice (41.8% in salt wasting), I172N (32.6% in simple virilizing), and V281L (40.2% in late onset form). The frequency of the 9 most common point mutations was similar to that reported for other countries, except for Del 8 nt and Cluster, which were less frequent in the classical form. Rarer mutations such as P453S, G291S, I7 splice, W405X, R483P, and R483frameshift were rarely found or were absent. The 93 fully genotyped patients were classified into 3 mutation groups, based on the degree of enzymatic activity (group A, <2%; group B, 2%, and group C, >18%). In group A, 62% of the cases presented the salt wasting form; in group B, 96% the simple virilizing form; and in group C, 88% the late onset form. We diagnosed 80% of the affected alleles after screening for large rearrangements and 15 point mutations. The absence of previously described mutations in 20% of the affected alleles suggests the presence of new mutations in our population.

	Introduction

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CONGENITAL adrenal hyperplasia (CAH) caused by 21-hydroxylase (21OH) deficiency is the most frequent inborn metabolism error, and it accounts for 90–95% of CAH cases (1, 2). The spectrum of clinical manifestations includes a severe form (classical; with prenatal virilization of external genitalia in the female fetus and postnatal virilization in both sexes, with or without salt losing) and a milder form (nonclassical). In this form, patients remain asymptomatic or develop symptoms during childhood or at puberty (1).

The structural gene (CYP21) for the 21OH enzyme is located in the HLA class III region on the short arm of chromosome 6 (6p21.3), as well as the pseudogene (CYP21P), which is 98% identical. Both genes alternate in tandem with the C4B and C4A genes (3, 4, 5). Because of the high homology and tandem-repeat organization of CYP21 and C4 genes, this region of the genome is subject to unequal cross-over events and gene conversions (6, 7). The mutations that account for 21OH deficiency can be CYP21 deletions, large gene conversions, or point mutations. The frequency of CYP21 deletions and large gene conversions ranges from 20–33% in several studies of Caucasian populations (8, 9, 10, 11). Point mutations represent approximately two-thirds of affected alleles, and approximately 23 point mutations have been described (11, 12). Because of the presence of several mutations, most of the patients are compound heterozygotes, and the clinical form correlates to the mutated allele with higher enzymatic activity (9, 11, 12, 13, 14, 15).

We report the frequency of 15 point mutations, in a large Brazilian cohort with classical and nonclassical forms of CAH-21OH, and their correlation with phenotype.

	Subjects and Methods

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Patients

All families gave their informed consent for the genetic study. We studied 130 (97 Caucasian, 24 Brazilian Creoles, 7 Blacks, and 2 Asians) CAH-21OH patients (110 genetic females) from 116 families, 4 of them related. There was one affected subject in 106 families, 2 affected subjects in 8 families (2 of these families included one affected parent), and 4 affected subjects in 2 families (1 of these families with 1 affected parent). Five families were consanguineous. We also studied 132 parents and 22 normal siblings. The patients were classified in 1 of the 3 clinical forms of CAH-21OH, defined according to standard criteria (16). Among the 130 patients, 29 presented the salt wasting (SW) form; 51, the simple virilizing (SV) form; and 50, the late onset (LO) form. The SW form was characterized by extremely elevated concentrations of 17OHP and plasma renin activity (PRA), hyperkalemia, hyponatremia, and dehydration in the first months of life. All females had ambiguous genitalia, graded according to Prader staging. The SV form was characterized by ambiguous genitalia in females, sexual precocity in males without salt wasting, and elevated 17OHP and PRA levels. Height and bone age were advanced. The LO form was characterized in girls by normal external genitalia or mild clitoral enlargement and, in both sexes, by precocious pubarche and stimulated 17OHP more than 12 ng/mL (mean + 2 SD of ACTH-stimulated 17OHP levels in obligate heterozygotes studied in our laboratory).

Hormone assays

Serum hormones were measured by RIA (17). PRA was measured with commercial kits from CIS-Bio International, Gif-Sur-Yvette, France.

Genotyping of mutations in the CYP21 gene

DNA samples were obtained from peripheral blood leukocytes by standard procedures. Allele-specific PCR, as described by Wilson et al. (18), was used for the determination of 8 point mutations (P30L, I2 splice, Del 8nt, I172N, Cluster, V281L, Q318X, R356W). Positive and negative control DNA were used in all reactions. Allele-specific-PCR methodology, described by Wedell et al. (19), was used for identification of the T insertion on exon 7, P453S, G291S, I7 splice, W405X, R483P, and R483frameshift.

Genotype categories

The patients were divided into three different genotype groups, according to the impairment of enzymatic activity, as described by Speiser et al. (9). Group A included patients who were homozygous for mutations that predict 0% overall activity (subgroup A1) and patients who were homozygous for I2 splice or compound heterozygous with mutations from group A1 [low, but measurable, enzymatic activity (subgroup A2)]. Group B included patients who were homozygous for I172N (2% of the enzymatic activity) or compound heterozygous with mutations from group A. Group C included patients who were homozygous for P30L, V281L, and P453S (20–50% of enzymatic activity) or compound heterozygous with mutations from groups A or B.

Statistical analysis

The -square test was used to analyze the association of each mutation with each clinical form. Differences in basal levels of 17OHP, testosterone, and androstenedione among the three genotype groups were evaluated using the Kruskal-Wallis test.

	Results

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In the 228 nonrelated alleles, the most frequent point mutations were I2 splice (20.6%), I172N (14%), and V281L (18%). In the SW form, the most frequent mutation was I2 splice (41.8%); in the SV form, the most frequent one was I172N (32.6%), followed by I2 splice (16.3%). In the LO form, the most frequent mutation was V281L (40.2%), followed by I2 splice (5.7%) and P30L (4.6%). There was a significant association of I2 splice, I172N, and V281L with SW, SV, and LO forms, respectively (P < 0.0001). Point mutations were present in 158 alleles. Ten (6.3%) of these alleles presented 2 or 3 point mutations: I2 splice+V281L (2 alleles), I2 splice+InsT, P30L+R356W, Ins T+Q318X+R356W, Q318X+R356W, I172N+Cluster, I2 splice+R356W, I172N+V281L, and I2 splice+Del 8nt. The distribution of alleles with 1 point mutation, according to the clinical form, is shown in Table 1 and is compared with other populations in Table 2.

When searching for the mutations of index cases in their parents, we diagnosed three asymptomatic affected mothers, two homozygous for V281L and the other with the V281L/R356W genotype. The latter mother had three affected children, one with SV form and two with LO form.

Considering our previous study for large mutations (20) and the present one, we diagnosed 80% of all alleles, being 93% in SW, 83% in SV, and 69% in LO form. Ninety-three patients had mutations identified in both alleles (Table 3). From these patients, 34 presented group A genotype (21 with SW and 13 with SV form); 26 patients presented group B genotype (25 with SV and 1 with LO form); 33 patients presented group C genotype (29 with LO and 4 with SW form). Virilization of the external genitalia ranged in group A from Prader I to V; and in group B, from Prader I to IV (Table 3).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We report the frequencies of CYP21 point mutations in a large Brazilian cohort with 21OH deficiency with the classical and nonclassical forms. The Brazilian population is ethnically heterogenous, consisting mainly of Portuguese ancestry and interracial marriages with Blacks and Native Indians. The most frequent mutations in our series were those also present in the pseudogene, similar to previous reports (8, 9, 11, 13, 14, 21, 22), except for Del 8nt mutation, which is normally associated with the classical form and was detected only in heterozigosity in the LO form. According to the clinical form, the most frequent mutation in SW form was I2 splice; in SV form, I172N; and in LO form, V281L. Reports, which studied approximately 10 mutations, comprising large and point mutations, diagnosed 77.6–95% of the alleles (8, 9, 11, 14, 21). Even though we screened for 17 mutations, we had a significant number of nonidentified alleles (20%). Higher numbers of undiagnosed alleles were also observed in other Latin-American studies (12, 22). Because rare mutations were also scarcely found, or even absent, in our alleles, we suppose that new mutations might be present in our population, as observed in a Mexican study (12). The highest frequency of undiagnosed alleles in our series was found in the LO form, as well as in other large series (8, 21). However, we did not rule out the presence of other rare mutations, such as P105L, R339H, or promoter mutations that have been found in the LO form (23). On the other hand, the lowest value of ACTH-stimulated 17OHP levels in LO patients, in whom mutations in both alleles were determined, was 17 ng/mL. These data suggest that the cutoff value of 12 ng/mL for stimulated 17OHP levels might have overestimated the diagnosis of nonclassical form of CAH-21OH. Genotyping patients with LO form will support the definition of a new cutoff value for ACTH-stimulated 17OHP levels.

We observed a good correlation between genotype and phenotype, also found in other populations (9, 11, 13, 14, 15). The worst correlation occurred in group A, where the mutations I2 splice and R356W that are generally associated with the SW form were also present in 10 patients with SV form. The association between I2 splice and SV form has been previously described in other reports (13, 14), and it can be explained by different degrees of normally spliced messenger RNA. Discordance between genotype and phenotype was found in 4 patients with SW form and with a mild mutation in 1 of the alleles. We cannot rule out either the presence of an additional new mutation in these alleles or of regulatory abnormalities. Because of some discordance between phenotype and genotype, a phenotype prediction must be made with caution in prenatal diagnosis based on molecular genetic analysis of fetal DNA. We also observed a good correlation of genotype with 17OHP, testosterone, and androstenedione levels, which reflects the degree of enzymatic activity impairment. The intensity of hyperandrogenism was correlated with the severity of neonatal external genitalia virilization found in group A.

When studying available parents, we incidentally found three asymptomatic affected mothers. Parents that are compound heterozygotes for mutations with different enzymatic activities can generate offspring with different genotypes; and, consequently, this can explain different phenotypes among siblings.

Our data suggest the presence of new mutations in Brazilian patients with different clinical forms of CAH-21OH.

	Acknowledgments

 
The authors thank Dr. Robert C. Wilson and Dr. Yves Morel for kindly providing the positive mutant controls for the allele-specific PCR, Ms. Sonia Strong for the review of English, Ms. Miriam Y. Nishi and Maria Aparecida Medeiros for technical support, and Mr. José Arnaldo Soares Vieira for paternity tests.

	Footnotes

 
¹ This work was supported by a grant from FAPESP 95/8325-6 (A.E.C.B.), and T.A.S.S.B. was supported by a scholarship from CNP 140.745/93-9. T.A.S.S.B. and A.E.C.B. contributed equally to this work.

Received April 20, 1998.

Revised August 13, 1998.

Accepted September 9, 1998.

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