Download Identification of a cystic fibrosis mutation W19X in Tunisia

Current practice
Ann Biol Clin 2013; 71 (2): 223-6
Identification of a cystic fibrosis mutation W19X
in Tunisia
Identification de la mutation mucoviscidosique W19X en Tunisie
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 17/06/2017.
Monia Boudaya
Sondess Hadj Fredj
Hajer Siala
Amina Bibi
Taieb Messaoud
Laboratoire de biochimie et de biologie
moléculaire, Hôpital d’enfants de Tunis,
Tunisie
<[email protected]>
Abstract. Cystic fibrosis (CF) is a common and serious condition with autosomal recessive inheritance. It is caused by mutations in the cystic fibrosis
transmembrane conductance regulator gene (CFTR). The frequencies of mutations vary according to the ethnic origin of populations. We describe in this
study a patient with cystic fibrosis. She was homozygous for a new nonsense
mutation identified for the first time in Tunisia: W19X, which expected to cause
significant morbidity. This mutation appears to be specific to Tunisian population, although, it has identified only in CF Tunisian patients. The information
provided by our study contributes to defining the molecular spectrum of CF in
Tunisia, to improve genetic testing and prenatal diagnosis.
Key words: cystic fibrosis, Tunisian patient, CF mutation, W19X
Résumé. La mucoviscidose encore appelée fibrose kystique du pancréas, est la
plus fréquente des maladies autosomiques récessives graves. Elle est causée par
des mutations au niveau du gène CFTR. Les fréquences des mutations mucoviscidosiques varient selon l’origine ethnique des populations. Nous décrivons
dans cette étude, une patiente atteinte de mucoviscidose. Elle était homozygote pour une mutation non-sens identifiée pour la première fois en Tunisie :
la W19X, qui a entraîné une morbidité importante. Cette mutation semble être
spécifique à la population tunisienne, puisqu’elle n’a été identifiée que chez des
patients d’origine tunisienne. Les informations fournies par notre étude contribuent à définir le spectre de mutations de la mucoviscidose en Tunisie, afin
d’améliorer le dépistage génétique et le diagnostic prénatal.
doi:10.1684/abc.2013.0790
Article received May 8, 2012,
accepted September 5, 2012
Mots clés : gène CFTR, patient tunisien, mutation mucoviscidosique, W19X
Cystic fibrosis (CF) is a common and serious condition with
autosomal recessive inheritance. It is caused by mutations
in the cystic fibrosis transmembrane conductance regulator
gene (CFTR) on chromosome 7q31 and is characterized by
malfunction of chloride ion channels and of transport pathway regulation [1, 2]. CFTR protein is primarily expressed
in the apical membrane of exocrine epithelial cells. Classic
CF is characterized by failure to thrive, recurrent bacterial endobronchitis, progressive decline of lung function,
exocrine pancreatic dysfunction [3]. The phenotype is variable; it ranges from mild with limited manifestations to
rapid deterioration and death within the first year of life.
The severity of clinical manifestations depends on the set
of CFTR mutations, modifying genes, and other variables.
To date, more than 1.800 sequence variants have been
reported to the cystic fibrosis mutation database but the
type and the frequency of the mutation change according to
the geographic and ethnic origin. In the present study, we
report a W19X mutation in a Tunisian CF patient and her
clinical presentations.
Studied case
One patient was examined in this study, which have cystic
fibrosis clinical manifestations. The proband CF patient was
two years old female infant; she is the first child of seconddegree consanguineous parents from Mareth located at the
region of Gabes in South-Est of Tunisia. She was born at
term (38 week of gestation) with a birth weight of 2,850 g.
She was firstly admitted to the regional hospital of Gabes
for anemia and generalized edema with hypoproteinemia.
To cite this article: Boudaya M, Fredj SH, Siala H, Bibi A, Messaoud T. Identification of a cystic fibrosis mutation W19X in Tunisia. Ann Biol Clin 2013; 71(2): 223-6
doi:10.1684/abc.2013.0790
223
Current practice
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Then, she was repeatedly hospitalized for severe dehydration and dyspnea but without chronic diarrhea. The spittle
cytology examination revealed Pseudomonas aeruginosa
and the radio chest showed a thoracic distension. Later,
she was referred to the Children hospital in Tunis for a
sweet test. A high chloride concentration was obtained on
two occasions. Her parents were healthy and have not any
clinical manifestation of CF.
Results
The 27 coding exons of the CFTR gene and their intronexon junctions, of the studied patient, were PCR amplified
and screened by DHPLC and/or DGGE.
Based on the DHPLC elution profiles of the heterozygous
parents and the normal control, the only detectable difference was noted in the 189 pb region encompassing the exon
2 (figure 1). Patterns of wild-type genotype and CF mutant
genotypes could not be distinguished. However, only by
additional heteroduplex-based assay of mixing the proband
sample and the standard wild-type control, similar profile to those observed in heterozygous parents were noted.
These results clearly indicate that proband was effectively
homozygous for this variation.
Nucleotide direct sequencing of the PCR-amplified region
in both forward and reverse strands revealed the presence of
a homozygous substitution of a guanine (G) by an adenine
(A) at nucleotide position 189, which causes the conversion
of the tryptophan to a stop codon, this mutation is W19X
(figure 2).
The family of our patient benefited of a prenatal diagnosis
performed on amniotic fluid collected at 14 SA showed
that the fetus is heterozygous for the W19X mutation
(figure 3).
Methods
10 mL of venous blood samples of the proband mucoviscidosic patient and her parents were obtained. Genomic DNA
was extracted according to salting-out extraction method
[4]. Samples were collected after obtaining the informed
consent from parents.
Fetal DNA was extracted from amniotic fluid by the
addition of a lysis solution subsequently subjected to a
temperature of 95◦ C for 10 min followed by 10 min on
ice.
Amplification of all CFTR exons analyzed, including flanking intronic regions, was performed using CFTR gene
specific primers, which are commonly used in many studies
for CFTR analysis [5, 6].
The PCR products were analyzed by DGGE (denaturing
gradient gel electrophoresis) [7] for the exons (5, 11, 17b,
19, 20 and 21) and by DHPLC (denaturing high performance liquid chromatography) for the remaining exons [6].
Samples with altered variation, were sequenced on an ABI
Prism 310 Genetic Analyzer (Applied BioSystems, USA)
using a Big-Dye-Terminator cycle sequencing Ready Reaction Kit. Both forward and reverse strands were sequenced
[8].
Discussion
The W19X mutation appears to be severe, since the patient
associates infection by Pseudomonas aeruginosa at an early
age, while 80% of CF patients are colonized with Pseudomonas aeruginosa by eight years of age [9], dyspnea
and severe hypotrophy <4 DS.
mV
10
0
0
1
2
3
4
T+/- : Témoin hétérozygote
TN : Témoin normal
5
6
7
min
LA : Liquide amniotique
PP: Propositus
Figure 1. The DHPLC profile of the exon 2.
224
Ann Biol Clin, vol. 71, n◦ 2, mars-avril 2013
W19X cystic fibrosis mutation in Tunisia
60
T
T
T
A
G
C
T
G
A
C
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G
70
C A
G
A C
C A
A
T
T
T
T
A
Figure 2. Profile of the strand direct sequencing of the W19X mutation in a homozygous state.
60
T
T
T
A
G
C
T
G
G
R
A
70
C
C
A
G
A
C
C
A
A
T
T
T
T
A
Figure 3. Profile of the strand direct sequencing of the W19X mutation in a heterozygous state.
This nonsense mutation is rare because it has been identified
only by Bozon in 1998, in one CF case whose parents are
from Tunisia (cystic fibrosis mutation database). Indeed, in
two Tunisian studies involving 368 CF patients, the W19X
mutation was not identified [10, 11].
At the southern region of Tunisia (Gabes, Mednine and
Tataouine), 3 mutations have been identified N1303K,
G542X and F508del. The latter one, is the most common
and accounts for 68.75% [11]. The W19X mutation, and
given its identification in one patient born in Gabes, it has
not been identified.
Few works have been developed on the molecular study of
the CFTR gene in Tunisia.
Ann Biol Clin, vol. 71, n◦ 2, mars-avril 2013
5 new mutations have been identified in our population
whose the 2766 del8 mutation appears to be specific to the
Tunisian patients and accounts for 1.85% [10].
The results obtained in this study serve to broaden the spectrum of mutation in our population.
Conclusion
This is the first genotype–phenotype correlation study on
the W19X mutation. Our results showed that patient carrying the W19X allele in a homozygous status have a
severe CF phenotype. Determination of the common CFTR
225
Current practice
mutations in a specific population allows confirming the
clinical diagnosis and facilitates the development of a
rapid and accurate assay for prenatal diagnosis and carrier
detection.
Conflicts of interests:
none.
Copyright © 2017 John Libbey Eurotext. Téléchargé par un robot venant de 88.99.165.207 le 17/06/2017.
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