Clin Genet 2007: 72: 218–223
Printed in Singapore. All rights reserved
# 2007 The Authors
Journal compilation # 2007 Blackwell Munksgaard
CLINICAL GENETICS
doi: 10.1111/j.1399-0004.2007.00854.x
Short Report
Cystic fibrosis in a southern Brazilian
population: characteristics of 90% of the
alleles
Faucz FR, Gimenez J, Ramos MD, Pereira-Ferrari L, Estivill X, Raskin
S, Casals T, Culpi L. Cystic fibrosis in a southern Brazilian population:
characteristics of 90% of the alleles.
Clin Genet 2007: 72: 218–223. # Blackwell Munksgaard, 2007
Cystic fibrosis (CF) is a genetic disease that frequently leads to death in
infancy among Europeans and their descendants. The goals of the
present study were to analyze the molecular aspects of CFTR gene
characterizing mutations, their frequencies, and the haplotypes formed
by four CFTR gene intragenic markers, IVS8-6(T)n, IVS8CA, IVS17bTA
and IVS17bCA, in a southern Brazilian population of Caucasian origin.
DNA samples from 56 non-related CF patients were analyzed using
scanning techniques (single strand conformation polymorphism and
denaturing gradient gel electrophoresis), restriction fragment length
polymorphism and direct DNA sequencing to identify the mutations.
Our results revealed a total of 25 different CF mutations representing
nearly 90% of CF alleles, two being novel mutations. Microsatellite
haplotypes were defined for CF and normal alleles. The mutational
spectrum and the associated haplotypes described for the first time in this
study should prove relevant for genetic counselling and CF population
screening in Brazil. Moreover, our results suggest the presence of
a major Mediterranean component in the contemporary Brazilian CF
patient pool.
FR Faucza,b,c, J Gimenezc,
MD Ramosc, L Pereira-Ferrarib,
X Estivilld, S Raskina,b,
T Casalsc and L Culpib
a
Laboratory of Molecular Genetic,
Department of Genetics, Pontifı́cia
Universidade Católica do Paraná,
PUCPR, Curitiba, Brazil, bDepartment of
Genetics, Universidade Federal do
Paraná, Curitiba, Brazil, cMedical and
Molecular Genetics Center, IDIBELL.
Hospital Durán i Reynals, Barcelona,
Spain, and dGenes and Diseases
Programme, Center for Genomic
Regulation, Biomedical Research Park,
Barcelona, Spain
Key words: Brazilian CF population –
CFTR gene – CFTR haplotypes – cystic
fibrosis – mutation scanning
Corresponding author: Fábio Rueda
Faucz, PhD, Pontifı́cia Universidade
Católica do Paraná, PUCPR, Programa
de Pós-graduac
xão em Ciências da
saúde, CCBS, Rua Imaculada
Conceic
xão, 1155,
Prado Velho, 80215-901 Curitiba,
Paraná, Brazil.
Tel.: 15541 32712285;
Fax: 15541 32711657;
e-mail: [email protected]
Received 13 March 2007, revised and
accepted for publication 11 May 2007
Cystic fibrosis (CF) is the most common lethal
genetic disorder among Caucasians with an
incidence of about one in 2500 live births (1).
More than 95% of CF mutations have been
characterized in several genetically homogeneous
populations (2–5). However, in heterogeneous
populations, the number of different mutations is
very large (6–8). A worldwide survey showed that
66% of the CF alleles carried the F508del
mutation (9). Interestingly, the frequency of the
F508del mutation differs between populations,
218
ranging from 26% in Algeria and Venezuela, 47%
in Brazil and 87% in Denmark (7, 10, 11). In the
past years, at least 1400 sequence alterations have
been reported (12) demonstrating the high CFTR
molecular heterogeneity.
Because geographic differences in the frequency
of CF mutations affect the false-negative rate in
CF diagnosis, CF carrier testing, and neonatal
screening, such data should be provided for
different regions and ethnic groups in each
country. We previously reported the mutation
CF in a southern Brazilian population
heterogeneity in Brazilian CF patients by direct
analysis of F508del and four other common
mutations (G542X, N1303K, G551D and
R553X). These five mutations represented 56%
of CF alleles in Brazil and their frequencies varied
from state to state (7, 13).
Several highly polymorphic microsatellite and
diallelic markers have been described within the
CFTR gene and have been used in CF genetic
testing (14). Their analysis allows for prenatal and
carrier diagnosis in CF families and generates
CFTR haplotypes that are strongly associated with
specific mutations (15, 16). In particular, haplotypes from three microsatellite markers (IVS8CA,
IVS17bTA and IVS17bCA) have been described in
association with specific mutations (15, 17).
Herein, we provide the updated data on the
spectrum of mutations in a southern Brazilian CF
population after screening the 27 exons of CFTR
gene and their flanking sequences. In addition to
mutation analysis, we performed for the first time
in this population an association study including
polymorphic intragenic markers. On the other
hand, this is a blind study, parallel to that
performed by Raskin et al. (7) in which 70 CFTR
mutations were directly tested in a subgroup of the
present series (with a total of 21 shared patients).
In summary, we have improved the genetic testing
in Brazilian CF families combining direct and
indirect analysis and thus, increased the detection
rate of CF alleles.
Materials and methods
CF families
A total of 56 non-related Brazilian CF patients and
their parents, born in two southern states of Brazil
[22 in the state of Santa Catarina (SC) and 34 in the
state of Paraná (PR)] were selected for this study.
The two states studied are highly representative of
the southern Brazilian population, characterized
by a European origin relatively free of admixture
(18). All patients were of Caucasian origin with
a male proportion of 54%. The mean age was
6.7 years and ranged from 2 months to 32 years.
The age of diagnosis varied from the neonatal
period to 4 years and 3 months. Ten patients with
meconium ileus were diagnosed at birth. All
patients had a minimum of two positive chloride
sweat tests (.60 mEq/l) ranging from 60 to
154 mEq/l. Chronic lung infection was identified
in 53 out of 56 CF patients. Pancreatic sufficiency
was less frequent (14.3%) than insufficiency (PI;
85.7%). Infertility was determined in the two adult
male patients included in this series.
Twenty-one out of 56 patients were previously
analyzed for 70 common mutations (7). This blind
replicated study allowed us to evaluate the two
different strategies.
All patients’ parents were born in Brazil.
Parents’ alleles were also analyzed. Their nonCF alleles were assessed as normal CFTR genes.
Detection of mutations and polymorphisms
Genomic DNA was isolated from peripheral
blood lymphocytes according to standard protocols. Direct analysis of the F508del mutation was
carried out in all samples (19). Single-strand
conformation polymorphism (SSCP) analysis
was performed for 13 exons of the CFTR gene
(1, 2, 4, 6a, 6b, 7, 10, 13, 16, 17a, 19, 22 and 24)
(20), and denaturing gradient gel electrophoresis
(DGGE) analysis for the remaining exons (21).
When abnormal band patterns were detected,
direct DNA sequencing was carried out.
Microsatellites IVS8CA, IVS17bTA, and
IVS17bCA were analyzed in a multiplex polymerase chain reaction (PCR) as previously
described (22), with the exception of the extension
temperature (from 74°C to 65°C) and the number
of cycles (25 instead of 30). Migration of fragments was performed in a 6% acrylamide gel.
Through segregation studies, we were able to
establish the association between CF and normal
alleles with their intragenic microsatellite haplotypes. For CF patients, showing homozygosity in
mutations and microsatellites haplotype, special
attention was given to investigating the possibility
of parent’s consanguinity (microsatellites haplotype, questionnaire and surname records) and
gross deletions (microsatellites haplotype).
The polymorphic (T)n locus, a polythymidine
tract (alleles 5T, 7T and 9T) adjacent to the
acceptor splice site of intron 8 was also analyzed,
using the allele specific PCR assay described by
Friedman et al. (23).
Nomenclature
For mutation nomenclature, we have followed
recommendations from the Cystic Fibrosis
Genetic Analysis Consortium (12) and/or that
recommended by the Human Genome Variation
Society (HGVS) (24).
Results
CFTR mutations
We identified 25 mutations accounting for
88.4% of CF alleles. Nine mutations showed a
frequency higher than 1%, F508del (45.5%),
G542X (6.3%), N1303K (4.5%), G85E, R334W
and R1162X (3.6%), 2183AA.G and W1282X
219
220
Exon 11
Exon 21
Exon 3
Exon 7
Exon 19
Exon 13
Exon 20
Exon 11
Exon 1
Exon 2
Intron 4
Intron 5
Intron 5
Exon 7
Intron 10
Intron 10
Intron 11
Exon 12
Exon 12
Intron 12
Exon 17b
Exon 17b
Intron 19
Exon 20
G542X
N1303K
G85E
R334W
R1162X
2183 AA.G
W1282X
R553X
S4X
232del18
62111G.T
71111G.T
71115G.A
R347P
1717-1G.A
1717-8G.A
1812-1G.A
A561E
E585X
189811G.A
G1069R
Y1092X
3849110 kb C.T
W1282G
Unknown
112
100
6.25
4.46
3.57
3.57
3.57
2.68
2.68
1.78
0.89
0.89
0.89
0.89
0.89
0.89
0.89
0.89
0.89
0.89
0.89
0.89
0.89
0.89
0.89
0.89
11.60
45.54
%
16-7-17 (1)/16-29-14 (1)/16-31-13 (1)/17-30-13
(1)/17-31-13 (20)/17-32-13 (7) 23-31-13 (15)/23-32-14
(1)/23-46-13 (1)/25-30-13 (1)/26-31-13 (1)/unknown (1)
23-32-13 (1)/23-33-13 (5)/23-34-13 (1)
16-30-13 (1)/23-30-13 (1)/23-31-13 (3)
16-24-13 (4)
16-34-13 (1)/(16-48-13) (1)/17-33-13 (1)/17-41-13 (1)
17-31-13 (4)
16-31-13 (2)/16-31-14 (1)
17-7-17 (3)
17-44-11 (1)/17-47-11 (1)
(16-__-13) (1)
21-36-13 (1)
__-34-13 (1)
16-25-13 (1)
__-7-17 (1)
16-32-13 (1)
16-7-17 (1)
16-33-13 (1)
16-31-14 (1)
16-44-13 (1)
Unknown (1)
16-45-13 (1)
17-30-13 (1)
16-30-13
16-7-17 (1)
16-32-14 (1)
16-7-17 (1)/16-29-13 (2)/16-30-13 (1)/16-31-13
(1)/16-32-13 (3)/16-33-13 (1) 16-34-13 (1)/16-38-16
(1)/18-35-13 (2)
Haplotypes IVS8CA, IVS17bTA, IVS17bCA (n)
9T (7)
9T (4)/7T (1)
7T (4)
7T (3)/unknown (1)
7T (4)
7T (2)/unknown (1)
7T (2)/9T (1)
7T (1)/unknown (1)
Unknown (1)
Unknown (1)
Unknown (1)
7T (1)
Unknown (1)
7T (1)
7T (1)
9T (1)
9T (1)
7T (1)
7T (1)
7T (1)
Unknown (1)
7T (1)
7T (1)
7T (1)
Unknown (13)
9T (44)/7T (3) unknown (4)
(T)n locus (n)
Ôn’, the total number of chromosomes bearing each haplotype or (T)n locus; Ôunknown’, used when the haplotype/(T)n locus cannot be characterized; Ô_’, used when a specific
allele of the haplotype cannot be characterized.
a
Numbers for microsatellites IVS8(CA)n, IVS17b(TA)n, and IVS17b(CA)n indicate the numbers of repeats.
Total
Exon 10
DF508
(7)
(5)
(4)
(4)
(4)
(3)
(3)
(2)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(1)
(13)
27/24 (51)
Exon/intron
Mutation
5/2
2/3
2/2
1/3
1/3
1/2
1/2
2/0
1/0
0/1
1/0
1/0
1/0
0/1
1/0
1/0
1/0
1/0
1/0
0/1
1/0
1/0
1/0
1/0
13/0
Chromosomes Parana
State/Santa Catarina
State (total)
Table 1. Frequencies of the CFTR mutations, their microsatellite haplotypes and IVS8-6(T)n alleles in the Brazilian CF patientsa
Faucz et al.
CF in a southern Brazilian population
(2.7%) and R553X (1.8%). These nine mutations
represented 74.1% (83/112) of the CF alleles. The
remaining 16 mutations showed frequencies lower
than 1% and accounted for 14.3% of CF alleles
(Table 1).
Two novel mutations were identified. One was
detected by DGGE analysis and direct sequencing
of exon 20 showing a substitution of T to G at
nucleotide 3976, named W1282G, (p.Trp1282Gly).
The second novel mutation corresponded to an
18-bp in-frame deletion named 232del18 or c.100117del (p.Leu34_Gln39del) (HGVS nomenclature), which was detected by SSCP analysis and
direct sequencing of exon 2. The two patients with
novel mutations carried the F508del mutation in
trans. Both patients are women, 18 and 15 years
old, respectively, with positive sweat test ranging
from 93 to 110 mEq/l. They presented typical CF
symptoms with PI and Pseudomonas aeruginosa
colonization.
Two CF mutations were characterized in 45 out
of 56 patients, leading to 34 different genotypes.
Among these, homozygosity for F508del was the
most frequent (n ¼ 12). Nine patients were partially characterized (four F508del; five with several
different mutations 2183AA.G, 62111G.T,
N1030K, G1069R and R553X). No mutation
was identified in the two remaining patients despite
the fact that both fulfilled CF clinical criteria. One
presented P. aeruginosa infection, PI and two
positive sweat tests (above 88 mEq/l), while the
other was diagnosed earlier on with meconium
ileus with other clinical findings, P. aeruginosa
infection, nasal polyps and two positive sweat tests
(above 75 mEq/l).
(T)n locus
This sequence in intron 8 [IVS8-6(T)n] was
analyzed in 88 CF alleles. The 5T allele was
undetected. The frequencies of the other two
alleles were, 67% (59/88) for the 9T allele and 33%
(29/88) for the 7T allele.
Microsatellite haplotypes
CFTR microsatellite haplotypes (IVS8CA,
IVS17bTA and IVS17bCA) were determined in
112 CF alleles and 110 normal alleles.
Microsatellite analysis of the CF alleles allowed
us to define the haplotype-mutation association.
Forty-four haplotypes were identified associated
with 25 different mutations and nine haplotypes
with yet unknown mutations (Table 1). In addition, 48 haplotypes were found associated to the
normal alleles (Table 2).
As expected, several haplotypes were found to
be associated to the old mutations. Specifically,
Table 2. Frequencies of microsatellites haplotype in normal
chromosomes
Microsatellite haplotype
16-7-17
16-30-13
16-31-13
16-32-13
17-7-17
16-46-13
Eleven different microsatellite
haplotypes
Thirty-one different
microsatellite haplotypes
Total
a
Numbera
Total (%)
14
13
11
8
7
4
2
12.7
11.8
10.0
7.3
6.4
3.6
20.0
1
28.2
110
100.0
Number of times that each microsatellite haplotype appears.
the F508del mutation was linked to 12 different
haplotypes (Table 1).
Mutations G85E, R334W, R553X, 62111G.A,
1717-8G.A, G1069R and W1282G were associated with haplotypes not observed in the normal
CFTR genes.
Discussion
In this study, we performed CFTR gene analysis,
including coding regions and exon–intron boundaries, in two southern Brazilian populations, SC
and PR which present a primarily European
origin. Non-CF and CF alleles of 56 unrelated
CF patients and their parents were analyzed to
determine the mutation and haplotype spectrum
in these regions.
Twenty-five mutations accounting for 88.4% of
CF alleles were found along the CFTR gene
(Table 1). Nine common mutations (.1%) were
characterized, the most prevalent being the
F508del (45.5% of the CF alleles). In addition,
16 other mutations were characterized, including
two novel mutations, W1282G and 232del18.
Nowadays, the Brazilian population constitutes
a highly heterogeneous population with a trihybrid composition (native Indians, Africans and
European descendants) in which individuals of
European origin represent 55% of the total (25).
The population from the state of SC has the lowest
admixture rate in Brazil (5.3%), with 93% of
European origin. Similarly, in the state of PR,
75% of the population has European origin (25).
Consequently, CFTR molecular heterogeneity is
expected. Previously, such heterogeneity was
indeed identified in Brazilian CF patients of
European origin by the screening of five common
mutations (F508del, G542X, N1303K, G551D
and R553X) (13). These mutations represented
only 56% of CF alleles in Brazil and their
frequencies varied from state to state (7, 26, 27).
221
Faucz et al.
Herein, we have identified 25 different mutations in 99 CF alleles, and nine different haplotypes associated with yet unknown mutations,
supporting the high molecular heterogeneity of
the population studied.
The mutation spectrum of CF in Brazil indicates
strong influence of a European component;
therefore, we should evaluate the European
ancestry taking into account our results. Our
mutational spectrum showed a certain similarity
with that reported in the Italian population
(F508del, 48.9%; G542X, 5.9%; N1303K, 5.9%;
and 2183AA.G, 2.6%) (28). This figure could
not be observed with other populations that
contributed to the ethnic composition of the
Brazilian population. For example, common
mutations in the Portuguese population show
quite different frequencies (G542X, 1.3%;
N1303K, 0.7%, and 2183AA.G, 0%) (10). The
fact that the mutation spectrum is closer to that
found in Italian patients than in Portuguese
patients is intriguing, although the Portuguese
influence could be more subtle, considering that
A561E, the second most common mutation in
Portugal (3.2%) (10) was also found in our series
(0.9%). This Italian influence in the genetic pool
of southern Brazilian population has been previously documented (29).
Homozygosity for microsatellite haplotypes was
detected in five patients. In all cases, the genotype
was confirmed by a segregation study in each
family. Parents’ consanguinity was reported in
patients’ homozygous for the 2183AA.G mutation. Three other patients were homozygous for
the common mutations: two with F508del and one
with R1162X. The CF mutations in the fifth
patient were not characterized. The segregation
studies allowed us to reduce suspicion of a gross
rearrangement, although not absolutely discarded
in patients with unknown mutations (30).
Combining direct mutation analysis and microsatellite haplotypes, prenatal diagnosis can now
be offered for almost all Brazilian CF families. It is
important to note that in cases without identified
CF mutations, the clinical findings must strongly
support the linkage analysis for pre-natal diagnosis. In these families, genetic counselling must take
into account that some CF phenotypes might not
be related to CFTR pathology (31).
The duplicate analyses of 21 patients permitted
us to compare our strategy with that applied by
Raskin et al. (7). Firstly, all CF mutations
identified by direct analysis were also detected in
our study. In addition, we identified four mutations previously undetected representing an
increase in detection level in both states (PR
80.9% vs 73.0% and SC 100.0% vs 94.8%). These
222
results indicate a higher sensitivity using our
strategy. Moreover, our study suggests that
a strategy combining direct analysis for nine
common mutations and intragenic markers allow
us to take advantage to the haplotype-mutation
association which could, thus, lead to specific
analysis improving the mutation detection rate
and limiting the CFTR gene scanning to uncharacterized CF patients.
This work has led to the characterization of
almost 90% of CF mutations in a southern
Brazilian population from the states of SC and
PR. We now have important tools to offer genetic
testing for CF families in these regions. Moreover,
it is now possible to plan a Neonatal Screening
Program that would allow for early diagnosis and
better follow-up of CF patients. However, further
comprehensive CFTR analysis is still necessary to
understand the complex heterogeneity of the
Brazilian CF population.
Acknowledgements
This research was supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq and International Cystic
Fibrosis (Mucoviscidosis) Association (both through a grant to
Fábio Rueda Faucz); and grants Fondo de Investigaciones
Sanitarias/Fondo Europeo de Desarrollo Regional (FIS/
FEDER) PI050804 and ISCiii C03/07 from Spain. We thank
the CF patients and their families, as well as Associacxão
Brasileira de Apoio a Mucoviscidose (ABRAM, Brazilian CF
Foundation) for their efforts in encouraging this work.
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