Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Asians with chronic pulmonary disease: a pilot study.
ABSTRACT Little is known about the relationship between cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in Asian patients and severe asthma or idiopathic bronchiectasis. We investigated this potential relationship in the Singaporean Chinese.
Twenty patients with chronic pulmonary disease, 14 with severe asthma and 6 with idiopathic bronchiectasis, were screened for CFTR mutations by direct gene sequencing. The frequencies of identified putative mutations were compared against 40 unaffected controls and 96 unselected population samples.
Three missense mutations (I125T, I556V, and Q1352H) and 1 splice site variant (intron 8 12TG5T) were identified in a total of 10 patients, representing a combined mutant/variant allele frequency of 0.25. These alleles were also observed in the controls, but at a significantly lower allele frequency of 0.09 (P<0.01). Furthermore, the I125T mutation was significantly associated with the idiopathic bronchiectasis sub-group (P<0.05).
The significantly higher frequency of CFTR mutations among patients with chronic pulmonary disease compared with unaffected controls suggests that these mutations may increase risk for disease. The association of I125T with idiopathic bronchiectasis alone suggests that different mutations predispose to different disease.
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ABSTRACT: Cystic fibrosis transmembrane conductance regulator (CFTR) is an asthma susceptibility gene. Individuals heterozygous for CFTR gene mutation may develop obstructive pulmonary disease like bronchial asthma. To find out the association of S549N and IVS8-5T variants of the CFTR gene with bronchial asthma and its severity and to assess the combinational effect of S549N and IVS8-5T variants on severity of disease. Included were 250 clinically diagnosed bronchial asthma cases aged 5 months to 15 years and 250 age- and sex-matched controls. All cases were further categorized into four different categories as per Global Initiative for Asthma criteria (GINA) guidelines: mild intermittent (83), mild persistent (96), moderate persistent (52), and severe persistent (19). Screening for S549N and 5T variants was done using the polymerase chain reaction-restriction fragment length polymorphism method. The proportion of IVS8-5T variant was found significantly higher in cases (10.8%) as compared with controls (2.4%) (p=0.001); however, no significant difference in the proportion of S549N was observed among cases (2.0%) and controls (0.8%) (p=0.447). Individuals mutant for IVS8-5T variant had increased risk for persistent asthma (p=0.000). We conclude that IVS8-5T variant is associated with bronchial asthma and can also increase severity of the disease.Genetic Testing and Molecular Biomarkers 04/2012; 16(8):884-91. · 1.44 Impact Factor
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ABSTRACT: Several lines of evidence suggest that in Caucasian populations, mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene are associated with susceptibility to lung disease caused by nontuberculous mycobacteria (NTM). However, there is little data available in Asian populations, in which the prevalence of CF is very low. Therefore, we investigated this potential relationship in a Korean population. Sixty patients who fulfilled the diagnostic criteria for NTM lung disease were screened for genetic alterations in the CFTR gene by whole-exon resequencing. For all identified CFTR gene variants, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) genotyping was performed. Genotype and haplotype data were compared between 360 patients with NTM lung disease and 446 healthy controls. Among 13 CFTR genetic variants that were found by whole-exon resequencing, Q1352H showed a significantly higher frequency in NTM patients than in controls, giving an odds ratio (OR) of 4.27 (95% confidence interval (CI), 1.43-12.78). A haplotype with Q1352H showed the strongest association with the disease, with an OR of 3.73 (95% CI, 1.50-9.25). Furthermore, all Q1352H alleles were associated with the V allele of the V470M variant. Our results suggest that CFTR gene variants may increase susceptibility to NTM lung disease in the Korean population. Q1352H appears to be strongly related to NTM lung disease susceptibility in the Korean population.Journal of Human Genetics advance online publication, 21 March 2013; doi:10.1038/jhg.2013.19.Journal of Human Genetics 03/2013; · 2.37 Impact Factor
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ABSTRACT: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene accounts for an autosomal recessive condition called cystic fibrosis (CF). In the Indian subcontinent, CF and its related diseases are under-diagnosed by the medical community due to the poor knowledge of the disease and its confounding diagnosis, and also due to the poor medical facilities available for these patients, thus causing an increased infant mortality rate with a low life expectancy in general. The aim of the study was to document the spectrum and distribution of CFTR mutations in controls, asthma and chronic pancreatitis cases of Northen India. A total of 800 subjects including 400 controls, 250 asthma cases and150 chronic pancreatitis cases were analyzed for 6 mutations (F508del, G542X, G551D, R117H, W1282X, and S549N) and IVS8 Tn polymorphism. Out of 800 subjects, 18% [(Asthma - 24% (n=250), CP - 29.33% (n=150) cases and controls - 9.3% (n=400)] were positive for heterozygous mutation, 0.8% of the (n=250) asthmatic cases (n=250) were homozygous for IVS8 T5 polymorphism while no subjects were found positive for W1282X mutation. T5 polymorphism was more common in asthmatic cases while F508del mutation in chronic pancreatitis cases. The carrier frequency of F508del, G542X, G551D, R117H, S549N and T5 were 0.015, 0.025, 0.02, 0.005, 0.005, and 0.022 respectively. The cumulative carrier frequency was 0.093. CFTR mutations were underestimated in Indian population. The present study will serve in establishment of genetic screening and prenatal setup for Indian population.Gene 01/2014; · 2.20 Impact Factor
Cystic fibrosis transmembrane conductance regulator (CFTR) gene
mutations in Asians with chronic pulmonary disease: A pilot studyB
Nicola S.P. Ngiama,b,*, Samuel S. Chonga, Lynette P.C. Sheka,b, Denise L.M. Goha,b,
K.C. Ongc, S.Y. Chngb, G.H. Yeoa, Daniel Y.T. Goha,b
aDepartment of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, 5, Lower Kent Ridge Road, Singapore 119074, Singapore
bChildren’s Medical Institute, National University Hospital, 5, Lower Kent Ridge Road, Singapore 119074, Singapore
cKC Ong Chest and Medical Clinic, 3, Mount Elizabeth, #12-03, Mount Elizabeth Medical Centre, Singapore 228510, Singapore
Received 14 December 2005; received in revised form 19 February 2006; accepted 19 February 2006
Available online 6 March 2006
Background: Little is known about the relationship between cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in
Asian patients and severe asthma or idiopathic bronchiectasis. We investigated this potential relationship in the Singaporean Chinese.
Methods: Twenty patients with chronic pulmonary disease, 14 with severe asthma and 6 with idiopathic bronchiectasis, were screened for
CFTR mutations by direct gene sequencing. The frequencies of identified putative mutations were compared against 40 unaffected controls
and 96 unselected population samples.
Results: Three missense mutations (I125T, I556V, and Q1352H) and 1 splice site variant (intron 8 12TG5T) were identified in a total of 10
patients, representing a combined mutant/variant allele frequency of 0.25. These alleles were also observed in the controls, but at a
significantly lower allele frequency of 0.09 (P<0.01). Furthermore, the I125T mutation was significantly associated with the idiopathic
bronchiectasis sub-group (P<0.05).
Conclusions: The significantly higher frequency of CFTR mutations among patients with chronic pulmonary disease compared with
unaffected controls suggests that these mutations may increase risk for disease. The association of I125Twith idiopathic bronchiectasis alone
suggests that different mutations predispose to different disease.
D 2006 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
Keywords: CFTR mutations; Bronchiectasis; Asthma; Asians
The CFTR gene is located on the long arm of
chromosome 7 and consists of 230 kb and contains 27
coding exons. There are more than 1400 CFTR sequence
variations identified in the cystic fibrosis (CF) mutation
database (http://www.genet.sickkids.on.ca/cftr/). The types
and distributions of mutations vary significantly among
different populations [1–3]. The majority of mutations have
been identified in European and North American popula-
tions but the spectrum of mutations and genetic polymor-
phism has not been well described in Chinese and Indians.
Even less is known in Malays.
The association between CFTR mutations and asthma is
controversial. Mennie et al.  did not find any association
between the CFTR gene mutations and asthma in a British
population. Looking at specific mutations, the link between
DF508 heterozygosity and CFTR mutations with asthma has
also been conflicting. Some authors  suggested that
DF508 heterozygosity was associated with an increased
susceptibility to asthma in a Danish population while
Schroeder et al.  suggested that obligate DF508 carriers
are protected from asthma. There is some evidence that
1569-1993/$ - see front matter D 2006 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
iData was presented at the 39th Singapore–Malaysia Congress of
Medicine, 30 June – 3 July, 2005 in Singapore for the Young Investigator’s
* Corresponding author. National University Hospital, Children’s Medical
Institute, 5 Lower Kent Ridge Road, Main Building Level 4, Singapore
119074, Singapore. Tel.: +65 67724420; fax: +65 67797486.
E-mail address: email@example.com (N.S.P. Ngiam).
Journal of Cystic Fibrosis 5 (2006) 159 – 164
CFTR gene mutations may be associated with other
respiratory diseases. A group of investigators in Italy has
identified 5 different CFTR mutations and 3 rare DNA
polymorphisms in a series of 16 patients with diffuse bron-
chiectasis . Tzetis et al  showed that CFTR mutations
were seen in 26.3% of patients with disseminated bronchi-
ectasis of unknown origin. Several other studies have sug-
gested an increased prevalence of CFTR mutation in adult
disseminated bronchiectasis [9,10]. Casals et al.  suggest
that heterozygosity for CFTR mutations has pathogenic
consequences which contribute to the development of bron-
chiectasis in adults. However, these findings have recently
been refuted in 2 studies that did not find CFTR mutations to
play a major role in the pathogenesis of adult bronchiectasis
[12,13]. In addition, Marchand et al.  found an increased
frequency of CFTR mutations in a small group of patients
(n=21) with allergic bronchopulmonary aspergillosis.
There is little data available on the types and distribution
of CFTR mutations in the Asian populations [15–17] and
there are no studies looking at its role in chronic pulmonary
disease (excluding CF). Asian patients with congenital
bilateral absence of vas deferens (CBAVD) have been
investigated and in Japanese patients with CBAVD,
missense mutations including Q1352H were detected .
In Taiwanese patients with CBAVD, 5 novel mutations that
did not overlap with those found in Caucasian populations
were identified . In Asian patients with CF, less is
known. The most prevalent CFTR mutation in Taiwanese is
1898+5G>T with a 30% frequency . Other mutations
have been identified in a small number of patients of
Chinese origin . Hence, it is the aim of this study to
investigate if there is an association between CFTR gene
mutations and chronic pulmonary disease (e.g. idiopathic
bronchiectasis and severe bronchial asthma) in the Chinese
Singaporean population. Our second aim was to determine
the frequency of CFTR gene mutations in the same
2.1. Study design
This is a case–control study conducted from 2002 to
2004 in the 3 general hospitals of the National Healthcare
Group. The study was approved by the institution’s review
board and informed consent was taken prior to enrolling
each individual in the study.
Cases were defined as individuals with severe asthma or
idiopathic bronchiectasis for which no underlying cause had
been identified. The criteria used for diagnosing severe
asthma were according to the Global Initiative for Asthma
(GINA) guidelines. The criterion used for diagnosing
idiopathic bronchiectasis was a chronic cough (3 months
in a 2 year duration) productive of purulent sputum. These
patients may or may not have had CT evidence of
bronchiectasis and/or clubbing. The underlying cause in
these patients could not be found. Cases were excluded if
they had an abnormal sweat conductivity (> 60 mmol/L),
classical symptoms of CF, chronic obstructive pulmonary
disease, immunodeficiency, primary ciliary dyskinesia or a
history of bronchopulmonary dysplasia.
Controls were individuals with no history of chronic
respiratory symptoms or pulmonary disease. There was no
known family history of cystic fibrosis in all controls. We
also analyzed a sample of unselected Chinese cord bloods to
estimate the population frequency of putative mutations we
identified in our patients.
2.2. Sweat testing
Sweat testing was performed as a screen for CF. This is a
well-established method used for the diagnosis of CF.
Sweating was stimulated by pilocarpine iontophoresis using
the Webster sweat inducer, the sample collected in a
microbore tubing (Wescor Macroduct, Wescor Inc., Logan,
USA) and the conductivity measured using the Wescor
‘‘Sweat-Chek’’ conductivity analyser. Any sweat conduc-
tivity result greater than or equal to 60 mmol/L (equivalent
sodium chloride, NaCl) was considered positive. This
method is comparable to the Gibson-Cooke (QPIT) method
and is also recommended by The National Committee for
Clinical Laboratory Standards, USA 1994 and endorsed by
the Cystic Fibrosis Foundation of the USA.
2.3. Molecular methods
2.3.1. DNA extraction
Genomic DNAwas extracted from anti-coagulated blood
using conventional methods. DNA was stored at 4 -C until
2.3.2. CFTR mutation screening
Patient DNA samples were screened for mutations within
the CFTR gene by direct gene sequencing using PCR
primers as described by Wong et al. . The CFTR gene is
located on chromosome 7q, and consists of 27 exons, with a
protein product of 1480 amino acids and a molecular mass
of 168,138 Da. It was amplified in 27 separate fragments,
then sequenced using the Big Dye terminator cycle
sequencing kit (Applied Biosystems), and analyzed on an
ABI 3100 Genetic Analyzer (Applied Biosystems). Results
were analyzed with the aid of the Sequence Navigator
software (Applied Biosystems). The control samples were
screened using the same method for the four mutations that
were detected in the patients.
2.4. Statistical analysis
Differences between proportions were compared using
the Chi-square test or the Fischer’s Exact Test with the SPSS
12.0 statistical program. All values were based on two-sided
N.S.P. Ngiam et al. / Journal of Cystic Fibrosis 5 (2006) 159–164
comparisons and values of less than 0.05 were considered to
be statistically significant. P-values were not corrected for
Twenty patients (11 males and 9 females) of Chinese
ethnicity were included in this study. The age range was 21
to 76 years (median=51.5 years). 6 patients had bronchi-
ectasis and 14 had severe asthma. The normal control
group consisted of 40 unrelated individuals of the same
ethnicity who were free of chronic respiratory symptoms
Three missense mutations (I125T in exon 4, I556V in
exon 11, and Q1352H in exon 22) and one splice site variant
(intron 8 12TG5T) were identified among the patients. The
normal controls and population samples were subsequently
screened for the presence of these four variant alleles. The
distributions of the mutations in the patient, normal control,
and population groups are shown in Tables 1 and 2.
Overall, 10 (50%) patients had at least one CFTR
mutation compared to 7 (17.5%) in the controls (Table 1,
P<0.01). The variant allele frequency in the affected group
was 25% compared to 8.75% in the normal control group
and 14.2% in the population sample (Table 2).
On subgroup analysis, the I125T mutation was found to
be significantly associated with idiopathic bronchiectasis but
not severe asthma. Of the 6 patients with idiopathic
bronchiectasis, 2 (33.3%) were heterozygous for I125T,
compared to 1 in 40 normal controls (2.5%) and 2 in 96
unselected population samples (2.1%) (Table 1). In marked
contrast, I125Twas not observed in the severe asthma group.
The I125T mutant allele frequency was 16.7% compared to
1.3% in the normal controls (P<0.05) and an estimated
population frequency of 1% (P<0.02) (Table 2). One other
patient was heterozygous for the 12TG5T polymorphism,
which did not differ in genotype or allele frequency from the
normal controls or population group.
higher in the severe asthma patients, where 2 of 14 patients
normal controls (2.5%) and an estimated population hetero-
zygote frequency of 4.2% (4 in 95) (Table 1). However, the
genotype and allele frequency differences did not reach
statistical significance. Similarly, 3 severe asthma patients
were heterozygous for the I556V mutation (21.4%), com-
pared to 10% (4 of 40) in the normal controls, and an
estimated population frequency of 12.5% (12 of 96).
Furthermore, 2 severe asthma patients were heterozygous
for the 12TG5T polymorphism (14.3%) compared to 1 of 40
normal controls (2.5%) and an estimated population frequen-
cy of 9.7% (9 in 93). These observations indicate a trend
towards higher incidence of these alleles in the affected
the estimated allele frequency in the population, suggesting
the possibility of an effect of these mutations on increased
risk for developing lung disease.
Frequency of CFTR gene variants in the Singaporean Chinese
VariationGenotype Unselected population
CFTR gene variant allele frequency comparisons between patient and population and normal control groups
VariationAllele Unselected population
aP value for disease vs. unselected population.
bP value for disease vs. healthy controls.
* Statistically significant (P<0.05).
N.S.P. Ngiam et al. / Journal of Cystic Fibrosis 5 (2006) 159–164
CFTR gene mutations have been found to be associated
not only with classical CF but also with other CF-like
diseases such as bronchiectasis, chronic pancreatitis and
congenital bilateral absence of the vas deferens (CBAVD).
The heterogeneity of the CF phenotype may be due to the
nature of the mutations itself, and/or the effects of
environmental factors. Our study identified four CFTR
mutations in the population and patients, with only the
I125T mutation showing a significant association with
The distribution of CF mutations seen in the Singaporean
Chinese population is different from that seen in the
Caucasian population. In our patients, we did not find any
of the 10 most common disease-causing mutations reported
in the Caucasians. These are R117H (exon 4), 621UVG>T
(intron 4), F508del (exon 10), 1717-1 G>A (intron 10),
G542X (exon 11), G551D (exon 11), R553X (exon 11),
R1162X (exon 19), W1282X (exon 20) and N1303K (exon
21). This may have implications in the testing process for
local patients suspected to have diseases related to mutations
in the CFTR gene. The process either will need to involve
sequencing the entire CFTR gene in all patients or testing
with a panel of the most common mutations found in our
population. As the spectrum of mutations identified in our
study is small, the second option is a possible cost-effective
In this study, 50% of patients with idiopathic bronchi-
ectasis and severe asthma had at least one CFTR mutation or
variant. A higher frequency of CFTR mutations in patients
with disseminated bronchiectasis, asthma and chronic
obstructive pulmonary disease was also found in other
studies which showed a frequency of between 26% and 40%
Of the 4 mutations or variants that we found, only the
I125T mutation was significant, showing an association
with idiopathic bronchiectasis. Interestingly, the I125T
mutation was first described in a Chinese woman and
reported by Mittre in the CF Genetic Analysis Consortium
. However, the phenotype of the patient it was found in
was not described. While this mutation occurred in a higher
proportion of our patients with idiopathic bronchiectasis, a
disease-causing role cannot be proven until functional
studies on the protein product are performed. This missense
mutation, a TYC transition at nucleotide 506 in exon 4,
results in a non-conservative IleYThr substitution at amino
acid 125, which is located in the second transmembrane
domain of CFTR.
Although the higher proportion of the Q1352H mutation
in our patient population was not statistically significant, it
is likely that this mutation has a role to play in disease
causation. In a study in a Korean population, it was found
that the heterozygote frequency of Q1352H was signifi-
cantly higher in patients with bronchiectasis and chronic
pancreatitis. Q1352H was found on molecular studies to be
a significant mutation. It is located in the sequence called
the ‘‘linker peptide’’ of the CFTR. This peptide is essential
for the integrity of the protein and for regulation of ATP
hydrolysis. The change of the last glutamine into histidine
(Q1352H) resulted in defects in protein expression and
affected gating properties of single channel kinetics .
This mutation seems to be an Asian mutation as it has been
identified in Japanese individuals with CF  as well as
those with CBAVD.  Q1352H was also recently
detected in a Korean patient with alcoholic chronic
pancreatitis although it was not thought to predispose to
the disease . Larger studies will need to be done in the
Singaporean population to determine the frequency of this
The first description of I556V was made in a male
French patient having atypical CF who was also compound
heterozygous for another mutation, R31C in exon 2 . He
had asthma-like bronchopathy, chronic diarrhea since
childhood, along with an abnormal sweat chloride test.
One of his children inherited his I556V mutation, and had
chronic bronchitis suggestive of CF, although the authors
indicated that the diagnosis was not clear. In the Korean
study, I556V was shown to reduce the current density in the
whole-cell chloride current by reducing the open probability
of the channel . In our population, there was no
statistical difference between the distribution in our controls
and our patients. This is could be the effect of a lack of
power or could disprove a disease-causing role of this
It is known that polymorphisms in IVS8 Tnaffect the
RNA splicing of exon 9 and that the IVS8 T5variation
reduces splice acceptor efficiency. Therefore, this variation
reduces the proper RNA and protein synthesis and seems
to be related to CF and CF-related diseases like CBAVD
. In addition, when IVS8 T5is combined with other
variations such as an increased number of IVS8 (TG)mand
the V470 variant, CFTR activity reduces and there is a
higher disease penetrance . Three alleles (T5, T7 and
T9) can be found at this polymorphism locus . These
alleles determine the length of a DNA sequence of
thymines in intron 8 which then determine the presence
of exon 9 in the CFTR mRNA . Exon 9 encodes part
of the functionally important first nucleotide-binding
domain of the CFTR. Shorter stretches of thymine residues
results in a higher proportion of CFTR transcripts lacking
exon 9 sequences. This translates to CFTR proteins that do
not mature, resulting in reduced apical chloride channel
activity . The IVS8-5T has been found to have
incomplete penetrance and this is due to the number of
TG repeats adjacent to 5T. Individuals with 5T adjacent to
either 12 or 13 TG repeats were substantially more likely
to exhibit a disease phenotype . The 12TG5T variant
was found in patients with asthma and bronchiectasis in
our study. This may be a contributing factor towards
disease expression in view of the functional importance of
N.S.P. Ngiam et al. / Journal of Cystic Fibrosis 5 (2006) 159–164
Fifty percent of our patients with asthma had either a
mutation or the 12TG5T variant. The mutations were the
I556V and the Q1352H mutations. However, this was not
statistically different from the controls. To our knowledge,
this is the first study reporting CFTR mutations in Asian
asthmatic patients. Analysis of larger cohorts of Asian
asthmatics would be necessary to resolve the conflicting
conclusions of the role of CFTR mutations in asthma from
Each subgroup in this study had few patients and this
may be the reason why the differences in allele frequencies
did not reach statistical significance. However, this pilot
study has yielded sufficiently promising results to warrant
further follow-up studies to better delineate the spectrum of
CFTR mutations in our population. Future plans for this
study would be to screen a large group of patients and
controls. Functional molecular studies would be helpful to
detect if the amino acid changes in the CFTR protein results
in significant changes in chloride channel function.
In summary, the types and distribution of CFTR
mutations in the Singaporean Chinese population is different
from that in the European and North American population.
There is a higher incidence of the I125T alleles in patients
with idiopathic bronchiectasis. Screening practices for
CFTR mutations in patients with disease may need to
change in light of these findings.
This work was supported by grants from the National
University of Singapore Academic Research Fund (R178-
000-075-112) and the National Medical Research Council
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