The Journal of Clinical Endocrinology & Metabolism Vol

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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 1 368-374
Copyright © 2004 by The Endocrine Society
Molecular Genetic Analysis of Tunisian Patients with
a Classic Form of 21-Hydroxylase Deficiency:
Identification of Four Novel Mutations and High
Prevalence of Q318X Mutation
Maher Kharrat, Véronique Tardy, Ridha M’Rad, Faouzi Maazoul, Lamia Ben
Jemaa, Mohamed Refaï, Yves Morel and Habiba Chaabouni
Laboratoire de Génétique Humaine (M.K., R.M., F.M., L.B.J., M.R., H.C.), Faculté de
Médecine de Tunis, 1006, Tunisie; and Laboratoire de Biochimie Endocrinienne
(V.T., Y.M.), Institut National de la Santé et de la Recherche Médicale unité 329,
Université de Lyon et Hôpital Debrousse, 69322 Lyon Cedex 05, France
Address all correspondence and requests for reprints to: Pr. Habiba Chaabouni, Laboratoire de
Génétique Humaine, Faculté de Médecine de Tunis, 1006, Tunisie. E-mail: habiba.chaabouni@rns.tn .
Abstract
Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders
mainly due to defects in the steroid 21-hydroxylase (CYP21) gene. To determine the
mutational spectrum in the Tunisian CAH population, the CYP21 active gene was
analyzed in 51 unrelated patients using our cascade strategy (digestion by restriction
enzyme, sequencing). All patients had a classical form of 21-hydroxylase deficiency.
Mutations were detected in over 94% of the chromosomes examined. The most
frequent mutation in the Tunisian CAH population was found to be Q318X, with large
prevalence (35.3%), in contrast to 0.5–13.8% described in other series. Incidence of
other mutations does not differ, as previously described: large deletions (19.6%),
mutation in intron 2 (17.6%), and I172N (10.8%). Four novel mutations were found in
four patients with the salt-wasting form. These four novel mutations include three
point mutations that have not been reported to occur in the CYP21P pseudogene:
R483W, W19X, 2669insC, and one small conversion of DNA sequence from exon 5
to exon 8. Our results have shown a good genotype/phenotype correlation in the case
of most mutations. This is the first report of screening for mutations of 21hydroxylase gene in the Tunisian population and even in the Arab population.
Introduction
CONGENITAL ADRENAL HYPERPLASIA (CAH) is an autosomal recessive
disease. The most common form (95%) is due to 21-hydroxylase deficiency (21OHD) resulting from molecular defect in the steroid 21-hydroxylase (CYP21) gene (1,
2). There are three major disease phenotypes depending on the specific mutation in
the gene coding for 21-hydroxylase, CYP21. In the classic salt-wasting (SW) form,
the most severe form, patients suffer from renal salt loss due to the lack of aldosterone
as well as virilization due to accumulated adrenal androgen. In the classic simple
virilizing (SV) form, patients also undergo virilization. In the nonclassic form, patients
lack the neonatal symptoms and present with late-onset androgen excess, such as
pseudoprecocious puberty and hirsutism. The incidence of the classic forms of 21OHD is 1/14,000 in the Caucasian population (3), thereby being one of the most
frequent autosomal recessive disorders, and three fourths of classical cases are SW
(4).
The gene encoding 21-hydroxylase, CYP21 is located in a locus with a complicated
structure. It is found on the short arm of chromosome 6 (band 6p21.3) together with a
highly homologous inactive pseudogene, CYP21P. The two genes are located in
tandem repeats with the genes encoding the fourth component of serum complement
(C4A and C4B) (5, 6). The CYP21 and CYP21P genes consist of 10 exons and show a
high homology with a nucleotide identity of 98% in their exon and 96% in their intron
sequences (7, 8). The proximity and the high degree of homology between the two
genes are believed to be the main reason for unequal crossover and gene conversionlike events, which give rise to mutations in CYP21 (9, 10). Approximately 95% of all
disease-causing mutations in CYP21 are either deletion/conversion (large deletions) or
any of nine point mutations that have been transferred from CYP21P into the active
CYP21 (11, 12). The remaining 5% of the disease-causing mutations are rare and
unique for single families or are considered as population specific. These uncommon
alleles do not originate from the pseudogene. Large deletions are characterized by a
single nonfunctional chimeric gene (CYP21P/CYP21) having CYP21P sequences at
the 5' end and CYP21 sequences at the 3' end (13).
The incidence of the CYP21 mutations in 21-OHD has been extensively studied in the
last years. No significant difference has been observed in the Caucasian population.
Large deletions account for 25% of 21-OHD alleles, depending on ethnic group, and
about 75% of chromosomes encoding CAH seem to carry point mutations. The most
frequently reported mutation found in patients with classic forms is an A or C G
change in the second intron affecting pre-mRNA splicing (IVS2–13A/C G), this
mutation has been detected in patients affected with either the SW or SV form of the
disease (14).
In this report, we study the genetic aspect of the classical form of CAH in a Tunisian
population. Our aims are to identify mutations in the CYP21 gene, determine their
frequency, and correlate genotype with phenotype.
Patients and Methods
Patients
Informed consent for mutation analysis was obtained from all patients and family
members. Molecular analysis concerned 51 unrelated Tunisian CAH patients that
were referred to the Department of Congenital and Hereditary Diseases of the Charles
Nicolle Hospital in Tunis. All patients (10 males and 41 females) had a classical form
of 21-OHD (44 patients with the SW form and 7 with the SV form) (Table 1 ).
View this table: TABLE 1. Genotype and phenotype of 51 Tunisian affected
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CAH with SW was characterized by onset of hyperkaliemia, hyponatremia, and
dehydration in the first month of life. All females had ambiguous genitalia, grade III–
IV on the scale of Prader (15). All patients with the SV form were females with
ambiguous genitalia (from posterior fusion of the labioscrotal folds to stage IV of
Prader) and with no evidence of sodium depletion.
Consanguinity was present in 31 families (60.8%), absent in 15 families, and
unknown in five families. Consanguineous families were unrelated and originated
from different regions of the country: 41.9% from Southern, 22.6% from Central, and
35.5% from Northern Tunisia. Two patients died at very early life, during the first
month. Blood samples were obtained from all patients and from their parents if
available.
Molecular genetic studies
In this study, CYP21 was amplified with gene-specific PCR primers. Restriction
enzyme digestion of PCR-amplified DNA was used to detect the presence of six more
common point mutations. Rare mutations were detected by direct sequencing of all the
exons of the CYP21 gene. Large deletions and the 8-bp-deletion located in exon 3
were detected by PCR method.
PCR amplification
Genomic DNA was prepared from peripheral blood leukocytes by standard
procedures (16). Amplifications were performed in a vol of 100 µl containing
approximately 0.7 µg of genomic DNA, 150 ng of each nucleotide primer, 0.2 mM of
each deoxynucleotide triphosphate, 1.5 mM of MgCl2, 1x PCR buffer (Eurobio, Paris,
France), and 2.5 U of Taq DNA polymerase (Eurobio). Thirty cycles of amplification
were used, each consisting of a denaturation step for 60 sec at 95 C, annealing step for
60 sec at 58 C, and extension step for 60 sec at 72 C. The amplification products were
analyzed by 1% agarose gel electrophoresis with presence of ethidium bromide stain.
Detection of six point mutations
Because they were the more common within different reported populations, we
studied the following mutations: the P30L in exon 1, the splice site mutation in intron
2 (IVS2–13A/C G), the I172N in exon 4, the cluster of three mutations (I236N,
V237E, M239K) in exon 6 designed as cluster E6, the V281L in exon 7, and the
Q318X stop codon in exon 8.
For analysis of these mutations, four different specific amplifications using CYP21
gene-specific primers were carried out on genomic DNAs followed by digestion with
appropriate restriction enzymes according to the manufacturer’s protocol (Table 2 ).
To obtain selective CYP21 amplifications, we used specific primers that are unable to
amplify pseudogene. A list of PCR primers is reported in Table 3 . The primers were
used to amplify the four different regions shown on Fig. 1 .
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TABLE 2. PCR fragments and appropriate restriction enzymes
used for the detection of six point mutations
View this table: TABLE 3. PCR primers used for amplification of CYP21
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FIG. 1. Schematic diagram of primer positions in
the CYP21 gene (see Table 3 ) and localization
of four new mutations. The numbered boxes
represent the exons. A, Amplification strategy
for PCR-RFLP analysis. B, Amplification
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strategy for PCR-direct sequence.
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Detection of large deletions and 8-bp-deletion
The presence of an 8-bp-deletion in exon 3 or large deletions in homozygous state
were suspected when the above reactions failed to generate the expected fragment.
These genetic defects were confirmed by nonselective amplification of exon 3 of
CYP21 and CYP21P genes with primers P7 and P8 (Table 3 ). The result of PCR
gives a single band of 56-bp with absence of the band of 64-bp normally present, as
described (17). Then, we realized a second PCR (from intron 2 to exon 6) to
differentiate between large deletions and the 8-bp-deletion using selective
amplification of CYP21 gene with specific primers P3 and P9. The absence of a 789bp band confirms a large lesion.
Detection of rare point mutations by direct sequencing
If no mutation was detected on at least 1 allele, direct sequencing of two independent
PCR amplifications of the CYP21 gene, using selective primers (Fig. 1 and Table 3
), was performed with a 373A model automatic sequencer (PE Applied Biosystems,
Forest City, CA) using the dideoxynucleotide terminator methodology, as previously
described (18, 19).
Once a deletion or point mutation was identified for a patient, segregation of the
corresponding mutation was studied in both parents, every time available.
Results
We screened for six point mutations, large deletions, and noncommon mutations using
restriction fragment length polymorphism (RFLP) methods, PCR, and sequencing of
CYP21 gene, respectively. Mutations were found in 94.1% (96/102) of the disease
chromosomes studied corresponding to 51 unrelated Tunisian patients clinically
diagnosed as having a classical form of 21-OHD. The frequency of the mutations
analyzed in this study and the frequencies of the same mutations found in other
populations are shown in Table 4 . Chromosomes were affected by one of these
mutations in 36 patients (70.6%), nine patients (17.6%) were compound heterozygous
with different mutations on each chromosome, one patient (2%) was homozygous for
two different mutations, four patients (7.8%) had only one copy of one mutation, and
one patient (2%) harbored none of the tested mutations.
View this TABLE 4. Distribution of mutations obtained in 51 Tunisian
patients with classic form and comparative frequencies with
table:
[in this previous reports (Refs. 22 ,24 ,26 ,27 ,28 , and 29 )
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The molecular defects detected were distributed as follows: Q318X in exon 8
(35.3%), large lesion of the CYP21 gene (19.6%), IVS2–13A/C G in intron 2
(17.6%), I172N in exon 4 (10.8%), and R356W in exon 8 (2.9%). Del 8-bp and cluster
E6 were not detected in this study.
Direct sequencing revealed three novel point mutations never described: the first is
W19X in exon 1 (1 allele) that is a non-sense mutation, the second is a frame shift
mutation due to insertion of C in 2669 position in exon 10 (1 allele), and the third one
is R483W in exon 10 (2 alleles) that is a missense mutation. In addition we revealed
one allele with a novel small conversion DNA sequence from exon 5 to exon 8,
generated by transfer of deleterious mutations from the CYP21P to the functional
CYP21 gene, confirmed by direct sequencing. The patient carrying the small
conversion is a compound heterozygous having the Q318X mutation on her other
allele (data not shown).
Two families in this series had one allele with more than one mutation. Patient 36 had
the Q318X and IVS2–13A/C G mutations both on his maternal and paternal allele,
and patient 16 carried the IVS2–13A/C G and P30L on her paternal allele. These
complex alleles probably resulted from large conversions or multiple mutations
events.
The distribution of mutation frequencies in the Tunisian population is significantly
different from those previously reported in all parts of the word (20). The Q318X is
very frequent (35.3%), whereas R356W (2.9%) is lower than in Western countries and
other populations.
Thirteen of the 36 alleles (83.3%) with single Q318X mutation were linked to a
polymorphism (601 C G) in intron 2, revealed by AciI enzyme that we use for
screening P30L mutation. This polymorphism was detected only on chromosomes
carrying Q318X mutation.
SW forms were predominant, representing more than 86% of reported classical forms.
Q318X was present in 21 of 44 (47.7%) SW cases. Homozygoty for this mutation
(13/44 = 29.5%) was the most frequent genotype. In the SV form, all the patients
carry at least one I172N mutation. Five of seven affected children were homozygous
for this mutation; I172N was not observed in SW cases. All remaining mutations were
associated with the SW phenotype.
Discussion
Molecular analysis of CYP21 gene was performed to determine genetic aspects of 51
unrelated Tunisian patients with a classical form of 21-OHD. This study is the first
report about distribution of mutations causing 21-OHD in the Tunisian population and
even in the Arab population. The rate of consanguinity is 60.8%, no other population
reported in literature has so high a rate. Consanguineous families were unrelated and
originated from different regions of Tunisia. In this study, we screened for six of the
most common mutations, large deletions, and rare mutations using RFLP, PCR, and
direct sequencing methods, respectively. Identifying these mutations, we were able to
characterize 94.1% of chromosomes. Our results indicate that four mutations (Q318X,
IVS2–13A/C G, I172N, and large deletions) represent about 87% of those causing
severe forms of 21-OHD in the Tunisian population.
The most frequent mutation in Tunisian classical forms was Q318X, with 35.3%, in
contrast to 0.5–13.8% described in other series (Table 4 ). This lesion constituted
38.6% of CYP21 gene defects in the SW form. However, our results differ from all
those reported in other populations (Table 5 ).
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TABLE 5. Frequency of Q318X mutation in patients with SW
forms in different populations
The reason for the high frequency of the Q318X mutation is still unknown. However,
a founder effect could be partly responsible for the observed distribution of this
mutation in our population. Indeed, we have detected linkage desequilibrium between
this mutation and a CYP21 gene polymorphism (601 C G in intron 2) in 83.3% of
alleles, a loci probably due to the antiquity of the founder chromosomes. It would be
interesting to search for this mutation in other Arab populations, to clarify the history
of this particular founder chromosome.
The subsequent most frequent mutation was large deletions (19.6%), followed by
IVS-13A/C G (17.6%) and I172N (10.8%). The R356W mutation (2.9%) was less
frequent than other reports (Table 4 ). Some alleles harbor more than one mutation;
we found such alleles (Q318X+IVS2–13A/C G and IVS2–13A/C G+P30L). Cluster
E6 and del 8-bp, which are two mutations associated with the SW form (21, 22), were
absent in our study. Absence of V281L mutation may be explained by the fact that we
did not include patients with the nonclassic form.
Four novel mutations were revealed; they have never been previously reported
elsewhere. In patient 38, we detected a novel C-to-T transition in codon 483 that
results in substitution of the amino acid arginine (CGG) by the amino acid tryptophan
(TGG) (Fig. 2 ). The R483W mutation was found in homozygous state and was
carried by both parents. The amino acid R483 is positioned in a conserved C-terminal
region of the 21-OH protein, and it has been shown that another mutation in this
position, R483P, results in an increased degradation pattern of the enzyme (23). In
patient 37, we detected a novel insertion of C in 2669 position (2669insC) at exon 10,
causing a frame shift mutation (Fig. 3 ). This mutation, found in heterozygous state,
was inherited from the father. In patient 43, we detected a novel G-to-A transition,
changing codon 19 from tryptophan (TGG) to the stop codon (TAG), which results in
mutation of W19X (Fig. 4 ). This mutation was found in heterozygous state and was
inherited from the mother. Neither of these three point mutations has been observed in
CYP21P pseudogene; and thus, the presence of each in the patients mutant CYP21
gene does not appear to be the result of gene conversion. However, in patient 39, we
detected a novel small conversion from exon 5 to exon 8. This patient, who carried the
Q318X mutation inherited from his mother, had a transfer of deleterious mutations
from CYP21P to CYP21 gene allele by gene conversion on her paternal chromosome.
All novel mutations were found in patients with the SW form; and therefore, they
should confer no enzymatic activity.
FIG. 2. DNA sequence of CYP21 gene of patient
16 showing the C-to-T transition at nucleotide
2668 in homozygous form, which results in the
arginine-to-tryptophan mutation at codon 483.
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FIG. 3. DNA sequence of CYP21 gene of patient
17 showing the insertion of C in 2669 position
at exon 10, causing a frame shift mutation in
heterozygous form.
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FIG. 4. DNA sequence of CYP21 gene of patient
28 showing the G-to-A transition at nucleotide
56 in the heterozygous form, which results in
the tryptophan-to-stop codon at position 19.
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Generally the severity of the phenotype depends on mutations present on both alleles.
Large deletions, Q318X and R356W mutations found in our series, were always
associated with the SW form, whereas the IVS2–13A/C G mutation was found in
either SW or SV, and I172N mutation was associated with SV (24, 25).
In conclusion, the methods described in this study are suitable for genetic screening,
including the prenatal diagnosis of disease. It is the first time that so large a
prevalence of Q318X mutation has been reported. Therefore, a screening for this
mutation in the Arab population should be evaluated.
Footnotes
M.K. and V.T. have contributed equally to this work and could be considered as first
authors.
Abbreviations: CAH, Congenital adrenal hyperplasia; CYP21, steroid 21-hydroxylase
gene; 21-OHD, 21-hydroxylase deficiency; RFLP, restriction fragment length
polymorphism; SV, simple virilizing; SW, salt wasting.
Received June 19, 2003.
Accepted September 16, 2003.
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