Hum Genet (1994) 94:291-294
Juan Rios 9 Omar Orellana 9 Manuel Aspillaga
Isabel Avendano 9 Isabel Largo 9Nora Riveros
CFTR mutations in Chilean cystic fibrosis patients
Received: 5 June 1993 / Revised: 1 February 1994
An analysis of five of the most common cystic
A b s t r a c t
fibrosis (CF) mutations worldwide (AF-508, R-553X, G-
551D, N-1303K and G-542X) was performed in 36 Chilean
patients. Polymerase chain reaction (PCR) amplification
of the DNA followed by allele specific restriction enzyme
analysis was used for detection. The overall frequencies
of the mutations in the chromosomes analyzed were
29.2% for AF-508 and 4.2% for R-553X (n = 72). The G-
542X, G-551D and N-1303 K mutations were absent in
the Chilean sample. Our data suggest however that AF-
508 is not the most common CF mutation in Chilean pa-
tients. AF-508 and R-553X account for only 33.4% of the
alleles; 66.6% of them do not respond to the probes used
and still remain uncharacterized.
Introduction
Cystic fibrosis (CF) is the most common lethal autosomal,
recessive disorder in Caucasians, affecting approximately
1 in every 2,500 newborns. Striking differences in the in-
cidence of CF occur in Asians and native African popula-
tions (< 1 : 100,000) (Tsui 1992; Beaudet 1992; Mclntosh
and Cutting 1992). There is no information available
about the incidence of CF in most Latin American coun-
tries.
The defective gene in CF encodes a protein named cys-
tic fibrosis transmembrane conductance regulator (CFTR).
Approximately 67% of the CF chromosomes analyzed
worldwide have a 3-bp deletion in the gene resulting in
the loss of a phenylalanine at amino acid position 508
(AF-508). More than 100 additional mutations causing the
disease have been found. The relative frequencies of the
J. Rios. O. Orellana 9N. Riveros (1~)
Departamento de Bioquimica, Facultad de Medicina,
Universidad de Chile, Casilla 70086, Santiago 7,
Chile
M. Aspillaga 9I. Avendano 9I. Largo
Departamentos de Pediatria y Broncopulmonar,
Hospital Luis Calvo Mackenna, Santiago, Chile
9 Springer-Verlag 1994
most common mutations vary among racial groups (Col-
lins 1992).
The Chilean population arose from a mixture of Cau-
casians from Spain and Amerindians derived from Mongo-
lian populations. The latter show a very low CF frequency.
The percentage contribution of Amerindian genes to the
Chilean population is thought to be approximately 30%,
based upon ABO, Rh and other genetic markers (Valen-
zuela 1988; Cifuentes et al. 1988). This leads to a pre-
dicted CF frequency of 1:4,000 newborns. At the Luis
Calvo Mackenna Hospital, 16-20 new patients are diag-
nosed in a year. This value is far below the estimated dis-
ease frequency, of 50 for the 200,000 live briths per year
in Chile (Tagle 1988; Valenzuela 1988).
There is no information about the molecular basis and
the real incidence of CF in Chile. In this report we describe
the analysis of a sample of Chilean patients for some of
the most common CF mutations, specifically AF-508, R-
553X, G-551D, N-1303K and G-542X (Tsui 1992).
This work provides the basis for CF DNA analysis in
Chile, where this disease is most likely underdiagnosed.
Materials and methods
Patients
All of the patients are registered at and attended the Luis Calvo
Mackenna Children Hospital where the National Center for CF di-
agnosis was recently located. The diagnosis of CF is based on both
clinical features and abnormally elevated sweat electrolyte con-
centration (> 60 mEq/1).
The ABO, Rh and MNSs systems were typed on blood samples
of CF individuals and their families in order to establish the degree
of Amerindian mixture of the group under study. Birthplaces and
surnames of patients, their parents and grandparents were traced in
order to establish the ancestries.
Analysis of mutations
The analysis was performed on 25 families with at least one child
diagnosed with CF and also on I 1 additional patients suspected of
having CF. Venous blood was drawn from patients and their rela-
tives and genomic DNA was isolated by DTAB and CTAB deter-
292

gent treatment (Gustincich et al. 1991). Amplification of the sam- Table 1 Genotypes of Chilean cystic fibrosis (CF) patients. The
ples was accomplished by the polymerase chain reaction (PCR) notation "other" means that the chromosome must bear a mutation
and was carried out in a Genetic Thermal Cycler, Precision Scien- distinct from AF-508, G-551D, G-542X, N-1303K and R-553X
tific GTC-I apparatus, using the same conditions for all the muta-
tions under study. 100 ng of DNA was added to a 50-btl amplifica- Genotype No. of CF patients Frequency (%)
tion mixture containing 25 pmol of each of the appropriate primers
and 1.5 U of Taq polymerase. Primers described by Friedman et al. AF-508/AF-508 2 5.6
(1991) were used for the analysis of the different mutations. Am- AF-508/R-553X 1 2.8
plification began with a 5 min incubation at 94 ~ C, and proceeded AF-508/other l5 41.6
with 35 cycles, each consisting of 2 rain at 50~ 1 rain at 72~ R-553X/other 2 5.6
and 1 min at 94~ finally 1 cycle of 2 rain at 50~ and ] of 10
rain at 72~ completed the amplification. Other/other 16 44.4
n = 36
Restriction enzyme digestion and electrophoresis

After PCR amplification 20 btl of the reaction mixture was incubated

overnight with the appropriate restriction enzyme. The digested
samples were electrophoresed at 230 V/cm for 4 h on a 12% poly-
acrylamide gel. The gel was stained with ethidium bromide and
analyzed under UV light.
For AF-508 detection, a 219-bp segment of the CFTR was am-
plified. In one of the PCR primers a cytosine residue, normally
present in the gene, was replaced by a guanine. This change gener-
ates in normal and non-AF-508 chromosomes a specific recogni-
tion site for the Mbol restriction enzyme. Amplified material from
genomic DNA digested with MboI gave two fragments (202-bp
and 17-bp) in both cases. Heterozygotes for AF-508 gave the 219-
bp and 202-bp fragments and a heteroduplex band. The AF-508 al-
lele remained uncut.
Primer sequences:
F-5" > G C A C C A T T A A G A A A A T A T G A T < 3"
R-5" > C A T T C A C A G T A G C T T A C C C A < 3"
The R-553X mutation destroys a HincII site normally present in
the gene. Primers used for its detection yield a 425-bp amplifica-
tion product. This product digested with Hinc|I gave 239-bp and a
186-bp fragment in normal and non-R-553X chromosomes. The
mutated allele was not cleaved.
Primer sequences:
F-5" > C A A C T G T G G T T A A A G C A A T A G T T G < 3"
R-5" > G C A C A G A T T C T G A G T A A C C A T A A T < 3"
to c a r r y the A F - 5 0 8 a n d / o r R - 5 5 3 X m u t a t i o n . E v e r y de-
tected m u t a t i o n was present in at least one parent o f the pa-
tient. S i x t e e n patients m u s t carry u n i d e n t i f i e d m o l e c u l a r
lesions d i f f e r e n t f r o m the G - 5 5 1 D , G - 5 4 2 X or N - 1 3 0 3 K
mutations.
D N A a n a l y s i s o f the A F - 5 0 8 / R - 5 5 3 X patient is s h o w n
in Fig. 1. T h e 4 2 5 - b p P C R p r o d u c t f r o m e x o n 11, d i g e s t e d
w i t h HincII y i e l d e d 2 3 9 - b p and 186-bp f r a g m e n t s in the
n o n - R - 5 5 3 X a l l e l e (A, lane 1). In the m u t a t e d allele it re-
m a i n e d uncut. A s can be d e d u c e d f r o m the D N A e l e c -
t r o p h o r e t i c m i g r a t i o n pattern, the p a t i e n t is h e t e r o z y g o u s
for R - 5 5 3 X (A, l a n e 4). T h e o t h e r c h r o m o s o m e carries the
A F - 5 0 8 m u t a t i o n (B, l a n e 2). T h e 2 1 9 - b p P C R p r o d u c t o f
e x o n 10, o b t a i n e d b y use o f the a p p r o p r i a t e p r i m e r s , di-
g e s t e d w i t h MboI g a v e 2 0 2 - and 17-bp f r a g m e n t s in n o n -
A F - 5 0 8 alleles and r e m a i n e d u n c l e a v e d in the m u t a t e d al-
lele. A s a P C R artifact, h e t e r o d u p l e x e s w e r e f o r m e d be-
t w e e n n o r m a l and A F - 5 0 8 alleles (B, lanes 1, 2, u p p e r
band).
T h e s l o w e r m o b i l i t y o f h e t e r o d u p l e x e s on p o l y a c r y -
l a m i d e gels has b e e n u s e d to i d e n t i f y h e t e r o z y g o u s indi-

The G-551D mutation creates an Mbol restriction site. Using the

same primers as above, the 425-bp amplification product from the
normal allele remains undigested when treated with MboI, whereas
the mutant allele produces two fragments of 243 and 182-bp, re-
spectively.
For detection of N-1303K a 290-bp segment was amplified. The
sample digested with BstN1 remains intact when the mutation is
present while normal alleles are cleaved to 266-bp and 24-bp frag-
ments.
Primer sequences:
F-5" > A A T G T T C A A G G G A C T C C A < 3"
R-5" > C A C T C C A C T G T T C A T A G G G A T C C A G < 3"
Analysis of the G-542X mutation was performed by amplification
of a 295-bp fragment followed by digestion with BstNl, which
yields a 101-bp and a 194-bp fragment in both normal and positive
samples. The 194-bp fragment remains uncleaved in the mutant al-
Fig. lA, B Restriction enzymatic detection of R-553X and AF-
lele but is trimmed to 171-bp in the absence of the mutation.
508 mutations in a Chilean CF patient. Polymerase chain reaction
Primer sequences: (PCR) amplification products cleaved with specific restriction en-
F-5" > G C A G A G A A A G A C A T T A T A G T T C T T < 3" zymes were fractionated on a 12% polyacrylamide gel. A DNA of a
R-5" > G C A C A G A T T C T G A T G A A C C A T A T T < 3" normal male control (lanes 1 and 2) and a CF patient (lanes 3 and
4) obtained by use of primers described in Materials and methods
to detect R-553X alleles and incubated with (+) and without (-) re-
Results striction enzyme HindlI. B The PCR product of exon 10 from
DNA or the same CF patient (lanes 1 and 2) and normal control
(lanes 3 and 4) obtained as described in Materials and methods to
S c r e e n i n g o f the patients for A F - 5 0 8 , G - 5 5 1 D , G - 5 4 2 X , detect F-508 alleles incubated with (+) or without (-) restriction
R - 5 5 3 X and N - 1 3 0 3 K m u t a t i o n s i d e n t i f i e d the g e n o t y p e s enzyme Mbol. A heteroduplex upper band (lanes 1 and 2) indi-
s h o w n in T a b l e 1. O f 36 patients a n a l y z e d , 20 w e r e f o u n d cates AF-508 heterozygosity

Table 2 Mutation anaylsis of CF chromosomes in Chilean patients
Mutation No. positive Percent of all
chromosomes CF chromosomes tested
(n = 72)
AF-508 21 29.2
G-542X 0 0
G-551D 0 0
R-553X 3 4.2
N-1303K 0 0
Other 48 66.6
viduals bearing AF-508 and AI-507 deletions (Rommens
et al. 1990; Nelson et al. 1991). The AF-508 mutation was
detected in all the alleles where the heteroduplex was
formed, which suggests that the infrequent AI-507 dele-
tion is not present in the sample.
The frequency of chromosomes bearing the mutations
under study is shown in Table 2. The AF-508 and R-553X
mutations account for 33% of the altered chromosomes.
The remaining chromosomes (66.6%) are still uncharac-
terized. According to the recessive character of the disor-
der and the clinical features of the patients, these chromo-
somes must carry other mutations distinct from the five
analyzed.
Discussion
The Chilean population arose predominantly from a mix-
ture of Caucasians and Amerindians. It is expected that
most, if not all, CF genes in Chileans come from Euro-
pean immigrants, mainly from Spain.
The most common mutation detected in Caucasian CF
patients is AF-508, but its frequency varies significantly
between countries and ethnic groups. The AF-508 dele-
tion is present in 5 0 % - 8 8 % of CF chromosomes in the
different populations of western Europe and in 3 0 % - 3 4 %
of CF chromosomes among some ethnic groups of the
Middle East (Tsui 1992; McIntosh and Cutting 1992).
D N A screening for five of the most common CF muta-
tions demonstrates that two of these (AF-508 and R-
553X) account for 33.3% of the altered chromosomes in
the Chilean sample. Although the CF group under study
was small, statistical analysis (z test for proportions) sup-
ports the observation that there is a reduced frequency of
the AF-508 mutation (29.2%) compared with that ob-
served in northwest European (67%, P < 0.0001) or in
Spanish populations (55%, P < 0.0001), ( E W G C F G 1990;
Estivill et al. 1989; The Cystic Fibrosis Genetic Analysis
Consortium 1990). Only 5% of the patients analyzed were
homozygous for this mutation (n -- 36).
The present percent Amerindian admixture in the Chilean
population is close to 30%, as determined by genetic mark-
ers, such as haptoglobins, Rh factor and ABO. According
to the same markers, our CF sample shows a higher Cau-
casian component than the general Chilean population
(Aspillaga et al. 1993; Cifuentes et al. 1988). The low fre-
293
quency observed for AF-508 suggests therefore that racial
admixture alone does not account for the incidence of this
mutation in the group.
The cde Rh(-) haplotype, absent in the Araucanians
was found in 25% of the independent haplotypes under
study (n = 135). Its frequency is 0.38 in Europeans and
0.22 in the Chilean population. The cDE haplotype fre-
quency has been estimated to be 0.38 in Araucanians,
0.17 in Europeans and 0.23 in Chileans. Our sample (n =
135) showed a frequency of 0.15 for this haplotype. When
the AB0 blood system was analyzed the observed fre-
quency for the 0 allele was 0.71 (n = 96). Its frequency
has been estimated as 0.89 in Araucanians, 0.66 in Euro-
peans and 0.77 in the Chilean population. The frequency
for these three independent alleles lies between the values
estimated for the Chilean and the European populations.
We found 3 out of 72 chromosomes bearing the infre-
quent R-553X mutation. This mutation, present in 1.5%
of the CF chromosomes analyzed worldwide (Hamosh et
al. 1991), seems to be more common in black than in Cau-
casian American CF individuals (Cutting et al. 1990;
Ober et al. 1992). Typing of blood samples from our CF
patients and their families showed a higher occurrence for
the cDe (Rh) haptotype than that estimated for the Chilean
population (7.4% vs 2.55%, P < 0.019; ascertained for
135 independent genes or haplotypes calculated from ge-
nealogies). This cDe haplotype is quite frequent in Afri-
can natives (Cavalli-Sforza and Bodmer 1971) and in a
Chilean population with African ancestors (Acufia et al.
1988). Even though the families were not aware of black
ancestries, our findings of both the infrequent R-553X
mutation and the Rh haplotype point to some African
genotype present in our patients.
Chile is one of the Latin American countries with the
highest life expectancy; however, respiratory ailments are
the second cause of death in children (Anuario de De-
mografia 1991). The mean age of CF diagnosis is 2 years,
which is the same average age of death of patients. Life
expectancy for survivors is 7 years on average (Tagle
1988). It is highly probable that CF is underdiagnosed,
masked by other bronchial and lung diseases.
These data, although preliminary, also strongly suggest
the existence in Chilean patients of some CF mutations
different from the five common types analyzed. This large
group of uncharacterized CF mutations deserves further
study. Likewise, in order to account properly for the real
incidence of CF in Chile, it will be neccessary to deter-
mine the prevalent types of mutations in the population.
Acknowledgements We with to thank Dr. Pilar Carvallo for her
generous advice, Dr. David Holmes and Dr. Catherine Connelly
for critical review of the manuscript, Dr. Carlos Valenzuela for sta-
tistical analysis of the data and Dr. Francisco Sepfilveda (ACRF,
Cambridge, UK) for providing us with the PCR primers. We are
grateful to the Corporaci6n Nacional para la Fibrosis Quistica for
their work with the CF patients and their families in obtaining
samples. This work was supported by a grant from FONDECYT.
Proy. 91-1106 Chile.
294
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