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Journal of Clinical Endocrinology and Metabolism
Copyright © 1998 by The Endocrine Society
Vol. 83, No. 9
Printed in U.S.A.
COMMENTS
Salt-Wasting Congenital Adrenal Hyperplasia: Detection
of Mutations in CYP21B Gene in a Chilean Population*
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
ABSTRACT
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) 5 22.9%, I2
splice 5 19%, R357W 5 12.7%, and Q319X 5 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
T
HE 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 (1–3). 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
Received December 18, 1997. Revision received May 19, 1998. Accepted May 26, 1998.
Address all correspondence and requests for reprints to: Carlos E.
Fardella, Department of Endocrinology, Pontificia Universidad Catolica
de Chile, Lira 44, Santiago, Chile.
* This work was supported by Research and Development funds of
the Associated Unit of Clinical Laboratories, Catholic University and by
Chilean grant Fondecyt 1951094.
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 SouthAmerindian 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 EuropeanCaucasian-Spanish influence of the conquerors, mixed with Amerindians of Asian ancestry and modulated by other European immigrations. (J Clin Endocrinol Metab 83: 3357–3360, 1998)
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 –12).
Patients and Methods
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.
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Vol 83 • No 9
COMMENTS
Methods
Results
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).
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).
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 5, Q319X or deletion 5 2, cluster E6 or deletion 5 1). The most frequent
findings were: deletion or LGC 5 22.9%, I2 splice 5 19.0%,
and R357W 5 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 5 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.
Discussion
FIG. 1. Diagram of CYP21 B gene showing 10 exons (bars) and localization of different mutations studied. Abbreviations: I2 splice, an
A3.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.
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 –24) or Asian populations (25–27), 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,
TABLE 1. Mutation frequencies on affected alleles and comparison with other populations
Study
Total (no.) of
chromosomes
Del/LGC
(%)
I2 splice
(%)
R357W
(%)
Q319X
(%)
I173N
(%)
Cluster E6
(%)
F308insT
(%)
Present study
Argentine (29)a
USA (18)a
USA (17)a
Italy (20, 21)a
Spain (22)a
France (23)a
Sweden (16)a
Taiwan (25)a
Japan (27)a
126
48
92
254
90
40
84
186
13
70
22.9
25.0
39.1
30.3
22.4
27.5
36.9
35.3
42.9
ND
19.0
25.0
28.3
46.1
38.9
32.5
22.6
41.3
35.7
32.9
12.7
2.1
7.6
4.3
1.1
5.0
ND
3.8
14.3
18.6
10.5
18.8
6.5
5.1
12.2
5.0
4.8
3.8
0.0
2.9
7.1
0.0
7.6
2.8
3.3
0.0
8.3
17.4
0.0
2.9
2.4
0.0
5.4
ND
0.0
0.0
4.8
1.1
0.0
2.9
0
ND
3.3
0.8
1.1
2.5
1.2
0.5
0.0
0.0
ND, Not determined. a Percentages were recalculated taking into account only patients with SW CAH. Number of reference is in parentheses.
COMMENTS
3359
letion reported in the Mexican study was attributed by the
authors to the missed detection of salt wasters (28).
TABLE 2. Genotypes grouped according to complete or
incomplete identification
Genotype
No. of index
patients
%
Del or LGC/Del or LGC
I2 splice/I2 splice or Del or LGCa
Del or LGC/R357W
Del or LGC/I2 splice
I2 splice/Q319X
I2 splice/I2 splice
R357W/Q319X
Del or LGC/Q319X
Q319X/Q319X or Del or LGCa
R357W/I172N
I172N/R357W 1 Q319X
I2 splice/R357W
Q319X/I172N
I2 splice/Cluster E6
I172N/Cluster E6
I2 splice/I172N
Cluster E6/Cluster E6 or Del or LGCa
I2 splice/ND
R357W/ND
I172N/ND
R357W 1 Q319X/ND
Q319X/ND
9
5
4
3
3
2
2
2
2
2
1
1
1
1
1
1
1
5
5
3
1
1
14.3
7.9
6.4
4.8
4.8
3.2
3.2
3.2
3.2
3.2
1.6
1.6
1.6
1.6
1.6
1.6
1.6
7.9
7.9
4.8
1.6
1.6
Del or LGC, CYP21 deletion or apparent large conversion.
Homozygosity could not be distinguished from hemizygosity in
cases in which parents were unavailable (uncertain alleles).
a
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 –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 de-
Acknowledgments
We thank pediatric endocrinologists Drs. F. Ugarte, A. Cortı́nez, and
M. E. Willshaw. We also thank Dr. Anna Wedell, who provided us with
positive controls for genotyping CAH patients.
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