The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 9 3357-3360
Copyright © 1998 by The Endocrine Society
Salt-Wasting Congenital Adrenal Hyperplasia: Detection of Mutations in CYP21B Gene in a Chilean Population1
Carlos E. Fardella,
Helena Poggi,
Pedro Pineda,
Julia Soto,
Isabel Torrealba,
AndreÍna Cattani,
Eveline Oestreicher and
Arnaldo Foradori
Department of Endocrinology (C.E.F., A.C., E.O.) and The Research
and Development Unit of the Associated Unit of Clinical Laboratories
(H.P., J.S., A.F.), Faculty of Medicine, Catholic University of Chile;
and Department of Endocrinology of the Clinical Hospital at University
of Chile (P.P.), Endocrinology Service (I.T.), Sotero del Rio and Luis
Calvo Mackenna Hospitals, Public Health Services, Santiago, Chile
Address all correspondence and requests for reprints to: Carlos E. Fardella, Department of Endocrinology, Pontificia Universidad Catolica de Chile, Lira 44, Santiago, Chile.
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Abstract
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The steroid 21-hydroxylase deficiency (21OHD) is the most frequent
cause of congenital adrenal hyperplasia. We have characterized the
disease-causing mutations in the 21-hydroxylase genes of 63 patients
with salt-wasting congenital adrenal hyperplasia from a Chilean
population of Hispanic origin, a group that has been scarcely
evaluated. Using allele-specific PCR, lesions were identified in 97
chromosomes out of 126 tested (77%). The most frequent findings were
the gene deletion or large gene conversion (LGC) = 22.9%, I2
splice = 19%, R357W = 12.7%, and Q319X = 10.5%. We
did not find alleles with the mutation F308insT and we found three
alleles with the cluster E6. The frequency of the point mutation R357W
was at least two times more frequent than the one found in Caucasians
populations, but similar to that communicated in Asian populations;
this finding may be explained by the Asian ancestry of our
South-Amerindian population. The frequency of Q319X was also high,
similar only to those patients studied in Italy and in a neighboring
Argentinian population. In summary, this is a genetic characterization
of 21OHD made in an almost pure Hispanic population in Latin America.
The high frequency of deletion of CYP21B gene, I2 splice, R357W, and
Q319X mutations probably reflects the European-Caucasian-Spanish
influence of the conquerors, mixed with Amerindians of Asian ancestry
and modulated by other European immigrations.
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Introduction
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THE steroid 21-hydroxylase deficiency
(21OHD) compromises about 95% of all cases of congenital adrenal
hyperplasia (CAH) and has an overall incidence of about 1 in 13,000
live births (1, 2, 3). About two thirds of patients have salt loss, making
it the most common congenital salt-wasting (SW) disease. Adrenal
21-hydroxylase activity is catalyzed by the cytochrome P450c21, encoded
by a gene termed CYP21B, to distinguish it from the duplicated but
nonfunctional P450c21A gene (4, 5). The genetics of P450c21 are unusual
and complicated. Random deletions and de novo mutations
almost never occur, instead, gene conversion accounts for about 85% of
all mutant P450c21 alleles. In these gene conversions, all or part of
the CYP21B gene is replaced by, or converted to, the sequence of the
corresponding sequence of the CYP21A gene (3, 6, 7).
Genetic disorders in the P450c21 that reduce more than 99% of the
enzyme activity results in deficient synthesis of cortisol and, in the
majority of cases, also causes SW and virilization. Clinically, it has
been shown that deletion of the P450c21 gene and the aberrant splicing
in intron 2 are the most frequent cause of the SW form. However,
several other mutations also result in a complete inactivation of
P450c21 (3, 6, 8). Because it has been demonstrated that ethnic
differences may determine changes in the pattern of mutations, we
decided to evaluate the frequency of the principal mutations described
as causing the SW form in a Chilean population of Hispanic origin, a
group that has been scarcely evaluated. The knowledge of the relative
frequencies of point mutations might be useful to delineate appropriate
strategies for molecular diagnosis and treatment to prevent a birth
defect (9, 10, 11, 12).
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Patients and Methods
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Patients
Sixty three patients with SW CAH (25 males and 38 females) and
their parents, when available, were studied. These patients were
unrelated and had no known consanguinity. All patients were diagnosed
as having SW by onset of hyperkalemia (6–9 mmol/L), hyponatremia
(118–125 mmol/L), and dehydration in the first month of life that
required glucocorticoid and mineralocorticoid treatment. All patients
had elevated levels of 17-hydroxyprogesterone (30–1029 ng/mL),
diagnostic for steroid 21OHD. All females were virilized in
utero and were born with ambiguous genitalia. Informed consent was
obtained from all participants according to the International
Guidelines for Biomedical Research Involving Human Subjects, CIOMS,
WHO, Geneva, Switzerland, 1982. The protocol was approved by the
Research Commission of the School of Medicine at Catholic University of
Chile.
Methods
Genomic DNA was isolated from the citrated blood of 63 unrelated
SW CAH patients and their parents as previously described (13).
Genotyping was performed by allele-specific PCR as was described by
Wedell and Luthman (14).
A first round of amplification using specific primers to amplify the
CYP21B gene was carried out. The specific primers were synthesized
based on the 8-bp deletion in exon 3 present only in the pseudogene
(CYP21A). The PCR reactions rendered two fragments, one encompassing
exons 1–3 and the other exons 4–10 of the CYP21B gene. These
fragments were used in a second round of amplification to detect the
different mutations. For each mutated position, primers specific for
the normal and mutant alleles were synthesized. Using this method, we
studied the most frequent gene microconversions reported in Caucasian
populations with SW CAH (Fig. 1
). The
presence of deletion or apparent large gene conversion (LGC) was
suspected when the above reactions failed to generate the expected
fragment and confirmed performing another PCR with specific primers
(14). All the samples were studied for each mutation. In all
amplifications we used a positive control of each mutation generously
provided by Dr. Wedell. The sequence of all primers used and the PCR
conditions were extensively described by Wedell and Luthman (14).
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Figure 1. Diagram of CYP21 B gene showing 10 exons
(bars) and localization of different mutations studied.
Abbreviations: I2 splice, an A >G change in second intron that
create an aberrant splice acceptor sequence; I173N, an isoleucine to
asparagine change at codon 173; R357W, an arginine to triptophan change
at codon 357; Q319X, a glutamine to stop codon change at codon 319;
F308insT, a T insertion at codon 308; and cluster E6, an
isoleucine-valine-methionine to asparagine-glutamine-lysine change at
codons 237–238-240.
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The parents’ genotype were also analyzed to establish the
segregation of the mutated allele. When discrepancies appeared between
the children’s and parent’s genotype, a paternity testing was carried
out (15). Heterozygous CYP21 deletion or LGC was inferred when the
affected child appeared homozygous for a given mutation but only one
parent carried the mutation. When the children were homozygous for a
given mutation and the parents were not available, we considered one
allele an uncertain allele. The frequency of the different mutations
was calculated taking into account the number of uncertain alleles
involving the mutation (16).
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Results
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We studied 126 chromosomes corresponding to 63 patients with SW
CAH and their parents (both parents were available for analysis in 60%
of cases). The mutated alleles were identified in 97 chromosomes
(77%); 8 of them were uncertain alleles (I2 splice or
deletion = 5, Q319X or deletion = 2, cluster E6 or
deletion = 1). The most frequent findings were: deletion or
LGC = 22.9%, I2 splice = 19.0%, and R357W = 12.7%. We
did not find alleles with the mutation F308insT, and we found three
alleles with the cluster E6. The frequency of the mutations analyzed in
this study, and the frequencies of the same mutations found in other
populations are shown in Table 1.
The complete genotype was determined in 41/63 patients (65.1%) and one
allele in 15/63 patients (23.8%). More than one mutation by allele was
found in only 2 patients. The most frequent genotypes corresponded to
homozygous deletion (9/63 = 14.3%) and homozygous to I2 splice
(7.9%); the other genotypes founded are listed in Table 2. In 7/63 (11.1%) patients, the two
alleles remained not characterized, but hemizygosity cannot be excluded
with the method used.
The parents’ genotypes permitted us to establish the
segregation of the mutated allele in every case. However, in one case
the patient was a compound heterozygote for Q319X and R357W, the mother
being a carrier of Q319X but the father a carrier of normal alleles.
Paternity testing by DNA analysis was carried out, demonstrating that
the assumed father was not the biological progenitor.
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Discussion
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The present study showed that the most common lesions found in our
Chilean population with SW 21OHD corresponded to deletion or LGC of
CYP21B gene and to the point mutations I2 splice, R357W, and Q319X.
These lesions in the CYP21B gene explain more than 70% of all cases of
SW 21OHD studied.
The frequency of deletion and the aberrant I2 splice found in our study
was similar to that previously described in Caucasians (16, 17, 18, 19, 20, 21, 22, 23, 24) or
Asian populations (25, 26, 27), in which these lesions constituted between
40–70% of the genetic defects found in the SW form of 21OHD. However,
our results differ from those found in a Mexican population, in which
the deletion represented less than 1% of the disease alleles (28). The
frequency of the point mutation R357W (12.7%) was twice as high as
that described in Caucasians populations, but similar to that
communicated in Asian populations from Japan and Taiwan (Table 1
). The
frequency of Q319X was also high (10.5%), similar only to those
patients studied in Italy and in a neighboring Argentinian population
(20, 21, 29). The low frequency of I173N is probably explained by the
fact that we did not include patients with the simple virilizant form
of classic 21OHD, in which this mutation is more prevalent (3, 6, 8).
The lesions F308insT and cluster E6 were extremely uncommon and appear
to explain only a small percentage of SW 21OHD in our population. A
similar low frequency of these two mutations had been communicated in
all the other populations studied (16, 17, 18, 19, 20, 21, 22, 23, 25, 27). In 23% of the
chromosomes, none of the five point mutations or a deletion of the
21-hydroxylase gene were found.
The allele frequency of the different mutations studied probably
reflects the biracial mixture of Chilean population, with Caucasian
genes coming from the Spanish conquerors and a gene pool derived from
the native Amerindians (Mapuches) (30). Moreover, analysis of the
mitochondrial DNA support the idea that Amerindians had an Asian origin
and derived from a small number of maternal lineages (31). Thus, we
hypothesized that the high frequency of the point mutation R357W found
in this study, as well in the Asian population, may be explained by
the Asian ancestry of our South-Amerindian population. Similarities in
the genotype distributions between Amerindian and Asian populations
have also been described for other studies (32, 33). The high frequency
of Q319X found in our population, as well as in Argentina and in Italy,
probably is the result of the Italian immigration that occurred in
these countries. The high frequency of deletion and I2 splice is
expected if we consider previous studies done worldwide. The low
frequency of deletion reported in the Mexican study was attributed by
the authors to the missed detection of salt wasters (28).
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Acknowledgments
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We thank pediatric endocrinologists Drs. F. Ugarte, A.
Cortínez, and M. E. Willshaw. We also thank Dr. Anna Wedell,
who provided us with positive controls for genotyping CAH patients.
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Footnotes
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1 This work was supported by Research and Development funds of the
Associated Unit of Clinical Laboratories, Catholic University and by
Chilean grant Fondecyt 1951094. 
Received December 18, 1997.
Revised May 19, 1998.
Accepted May 26, 1998.
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Abstract
Introduction
