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The Journal of Clinical Endocrinology & Metabolism 89(1):368 –374
Copyright © 2004 by The Endocrine Society
doi: 10.1210/jc.2003-031056
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
Congenital adrenal hyperplasia (CAH) is a group of autosomal
recessive disorders mainly due to defects in the steroid 21hydroxylase (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 dele-
tions (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 21-hydroxylase gene in the Tunisian population
and even in the Arab population. (J Clin Endocrinol Metab 89:
368 –374, 2004)
C
ONGENITAL ADRENAL HYPERPLASIA (CAH) is an
autosomal recessive disease. The most common form
(95%) is due to 21-hydroxylase deficiency (21-OHD) 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 21-OHD 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 conversion-like 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 C3 G change in the
second intron affecting pre-mRNA splicing (IVS2–13A/C3 G),
this mutation has been detected in patients affected with either
the SW or SV form of the disease (14).
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.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the endocrine community.
368
Kharrat et al. • Genetic Analysis of Tunisian 21-OHD Patients
J Clin Endocrinol Metab, January 2004, 89(1):368 –374 369
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).
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
TABLE 1. Genotype and phenotype of 51 Tunisian affected patients
Patient no.
Genotype mutations
(Fc/Met)
Phenotype
Sex
Prader Stage
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/Q318X
Q318X/In2
R356W/Q318X
P30L⫹In2/Q318X
LD/Q318X
LD/LD
LD/LD
LD/LD
LD/LD
LD/LD
LD/LD
LD/LD
LD/LD
LD/LD
LD/In2
In2/In2
In2/In2
In2/In2
In2/In2
In2/In2
In2/In2
In2/In2
R356W/R356W
Q318X⫹In2/Q318X⫹In2
2669insC/In2
R483W/R483W
Small conv/Q318X
Q318X/ND
Q318X/ND
ND/Q318X
ND/W19X
ND/ND
I172N/I172N
I172N/I172N
I172N/I172N
I172N/I172N
I172N/I172N
Q318X/In2
I172N/Q318X
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SW
SV
SV
SV
SV
SV
SV
SV
F
M
F
F
F
F
M
F
F
M
F
F
F
F
F
F
F
M
M
F
F
M
F
F
F
F
F
F
M
F
M
F
F
F
F
M
F
F
F
F
F
F
F
M
F
F
F
F
F
F
F
IV
IV
IV
IV
IV
IV
IV
IV
III
IV
III–IV
?
III
IV
IV
?
17OHP
(ng/ml)
Nf
Nf
12.7a
32a
952
⬎125
Inc
440
Nf
Inc
Nf
⬎275
Nf
Nf
Nf
Inc
Inc
⬎125
156
Nf
Nf
a
IV
IV
?
?
III
III
III
IV
IV
?
IV
IV
?
IV
IV
IV
IV
III
II
II
II
II
III
III–IV
?
440
Nf
Nf
Inc
Inc
121
Inc
⬎25
Inc
202
Nf
Inc
Inc
⬎275
36a
88
411
Nf
12.7a
105
Nf
230
⬎25
⬎25
75
⬎125
Nf
Inc
104
Consanguinity
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
?
No
No
No
No
Yes
Yes
Yes
Yes
Yes
No
No
No
?
No
Yes
Yes
Yes
Yes
Yes
?
?
Yes
Yes
No
Yes
Yes
No
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
No
?
In2, IVS2-13A/C3 G mutation; Inc, increased; LD, large deletions or large gene conversions; ND, no mutation detected; Nf, 17OHP not
furnish; Fc, father’s chromosome; Mc, mother’s chromosome; F, female; M, male.
a
17OHP under treatment with hydrocortisone.
370
J Clin Endocrinol Metab, January 2004, 89(1):368 –374
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 ␮l containing approximately 0.7 ␮g of genomic DNA, 150 ng of
each nucleotide primer, 0.2 mm of each deoxynucleotide triphosphate, 1.5 mm of MgCl2, 1⫻ 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/C3 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.
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 789-bp band confirms a large
lesion.
Kharrat et al. • Genetic Analysis of Tunisian 21-OHD Patients
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.
The molecular defects detected were distributed as follows: Q318X in exon 8 (35.3%), large lesion of the CYP21 gene
(19.6%), IVS2–13A/C3 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.
TABLE 3. PCR primers used for amplification of CYP21 gene
Primers
Sequence (5⬘–3⬘)
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
GGACCTGTCCTTGGGAGACTAC
GCCGTGTGGTGCGGTGGGGCAAGGCTA
AGGTCAGGCCCTCAGCTGCCTTCA
GGCTTTCCAGAGCAGGGAGTAGTC
TCTCCGAAGGTGAGGTACCAG
TCGGTGGGAGGGTACCTGAA
GTCTAGGAACTACCCGGACCTGTC
CTTCTTGTGGGCTTTCCAGAGCAG
AGCTGCATCTCCACGATGTGA
CTGAGGTACCCGGCTGGCATCGGT
Underlined primers are reverse primers. Underlined bases confer
CYP21 specificity to the primer. The sequences selected for specificity
were: the normal counterparts of the 8-bp deletion in exon 3, codons
236 –239 in exon 6 that differentiate CYP21 from CYP21A by four
deoxynucleotides, or nucleotides 620 – 630 in intron 2 that differentiate CYP21 from CYP21A by four deoxynucleotides.
TABLE 2. PCR fragments and appropriate restriction enzymes used for the detection of six point mutations
PCR
Forward primers
Reverse primers
Mutations
Enzymes
Fragment I (1460 bp)
P1 at exon 3
P2 at exon 8
V281L
Cluster E6
Q318X
BsiHKA I
Nde II
Pst I
Fragment II (128 bp)
Fragment III (330 bp)
Fragment IV (854 bp)
P3 at intron 2
P1 at exon 3
P6 at promoter
P4 at exon 3
P5 at exon 4
P4 at exon 3
IVS2-13 A/C3 G
I172N
P30L
Alu I
Bsr I
Aci I
Kharrat et al. • Genetic Analysis of Tunisian 21-OHD Patients
J Clin Endocrinol Metab, January 2004, 89(1):368 –374 371
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 strategy for PCR-direct sequence.
TABLE 4. Distribution of mutations obtained in 51 Tunisian
patients with classic form and comparative frequencies with
previous reports (Refs. 22, 24, 26, 27, 28, and 29)
Frequency
found (%)
Reported
frequency (%)
36
20
18
11
3
2
1
1
0
0
0
0
4
6
35.3
19.6
17.6
10.8
2.9
2.0
1.0
1.0
0
0
0
0
3.9
5.9
3–13.8
12–33
20 –31
2–12
4 –17
96
102
94.1
Mutations
SW
SV
Total
Q318X
Large deletions
IVS-13A/C3 G
I172N
R356W
R483W
W19X
2669insC
del 8-bp
E6 cluster
P30L
V281L
Complex alleles
Not detected
34
20
17
0
3
2
1
1
0
0
0
0
4
6
2
0
1
11
0
0
0
0
0
0
0
0
0
0
Total detected
Total
82
88
14
14
0–6
0 –5
0–6
0 –2
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/
C3 G mutations both on his maternal and paternal allele,
and patient 16 carried the IVS2–13A/C3 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 C3 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 21OHD 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/
C3 G, I172N, and large deletions) represent about 87% of
372
J Clin Endocrinol Metab, January 2004, 89(1):368 –374
Kharrat et al. • Genetic Analysis of Tunisian 21-OHD Patients
TABLE 5. Frequency of Q318X mutation in patients with SW forms in different populations
a
Country
Chromosomes studied
Affected chromosomes with Q318X
% of alleles
Ref.
Tunisia
Argentina
Japan
Lebanon
Chile
Mexico
Spain
Italy
Hungary
Southern Germany
Brazil
USA
Finland
Sweden
88
48
52
28
126
42
120
90
101
184
58
254
72
186
34
9
6
3
13
4
11
8
8 to 13a
14
3
13
2
1
38.6
18.8
11.5
10.7
10.5
9.5
9.2
8.9
8.0 to 13.0
7.6
5.2
5.1
2.8
0.5
Present study
27
28
30
31
32
33
22
34
35
36
37
38
39
Parents of patients were not studied.
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.
FIG. 2. DNA sequence of CYP21 gene of patient 16 showing the Cto-T transition at nucleotide 2668 in homozygous form, which results
in the arginine-to-tryptophan mutation at codon 483.
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).
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 C3 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/C3 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/C3 G
and IVS2–13A/C3 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
FIG. 4. DNA sequence of CYP21 gene of patient 28 showing the Gto-A transition at nucleotide 56 in the heterozygous form, which
results in the tryptophan-to-stop codon at position 19.
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,
Kharrat et al. • Genetic Analysis of Tunisian 21-OHD Patients
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.
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/C3 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.
Acknowledgments
Received June 19, 2003. Accepted September 16, 2003.
Address all correspondence and requests for reprints to: Pr. Habiba
Chaabouni, Laboratoire de Génétique Humaine, Faculté de Médecine de
Tunis, 1006, Tunisie. E-mail: [email protected].
M.K. and V.T. have contributed equally to this work and could be
considered as first authors.
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