Transcript Slide 1

ASSOCIATION OF INTERLEUKIN-13 (IL-13) GENE POLYMORPHISMS WITH
IL-13 PRODUCTION IN MALAYSIAN ASTHMATICS: A PILOT STUDY
Tan, L.K.#, Raj, V.L.*, Liam, C.K.*, Naidu, R.+, Radhakrishnan, A.K.#
# International Medical University, Kuala Lumpur, Malaysia *University Malaya, Kuala Lumpur, Malaysia;
+Monash University Malaysia, Selangor, Malaysia
Table 2: Results of the Hardy-Weinberg equilibrium (HWE) test
Total subjects
INTRODUCTION
IL-13 SNPs
Asthma is one of the most common respiratory disorders encountered in both children and adults.
Clinically, it is characterised by intermittent and reversible airway obstruction, bronchohyperresponsiveness (BHR) and airway inflammation1. Asthma is currently a worldwide problem,
with over 300 million people worldwide suffer from asthma2. The development of asthma appears to
be determined by the interaction between host susceptibility and a variety of environmental
exposures3. High serum immunoglobulin E (IgE) levels have been reported to be correlated with the
clinical expression of allergy and asthma and a strong genetic component has been shown to
contribute to this association4. Genetic studies in several populations have identified a region on
chromosome 5q31-q33 that contains the asthma susceptibility gene in several populations5,6,7,8. This
region contains a cluster of pro-inflammatory cytokines genes that play an important role in immune
regulation. Two members of this gene cluster, interleukin (IL)-4 and IL-13 have been shown to have a
role in the pathogenesis of asthma8,9. These cytokines, produced by T-helper-2 (TH2) cells induce
class-switching of B-lymphocytes to produce immunoglobulin E (IgE) class of antibodies. Several
single nucleotide polymorphisms (SNPs) of the human IL-13 gene have been reported [7,8]. Two of
these SNPs (-1111, 4257), are reported to have the strongest relationship with the incidence of
asthma in a Dutch population study8. This is a pilot study conducted on the Malaysian population to
investigate the allelic frequency of two SNPs (-1111, 4257) in the human IL-13 gene and its impact on
IL-13 production. This study can provide valuable insight into the overall mechanisms that cause
susceptibility to asthma. The outcome of this study may provide a basis for future studies and
contribute to early detection and diagnosis of asthma, as well as development of more effective
therapeutic approaches.
MATERIALS AND METHODS
χ2
p-value
-1111C>T
1.132
0.287
4257G>A
0.955
0.328
Table 2: Genotype data obtained from RFLP and sequencing was first tested with HWE
test before further genetic analysis to ensure that the genotype distributions have no
statistically significant departures from the HWE. P-values for both SNPs were >0.05.
Genotype distributions of both SNPs in all subjects were in HWE.
Table 3: The IL-13 SNP allele frequencies for asthmatic patients and control subjects.
Allele frequency
Frequencies
Asthmatic
patients
(n=87)
Control subjects
(n=94)
C
91 (52.3%)
125 (66.5%)
-1111
4257
T
83 (47.7%)
63 (33.5%)
G
95 (54.6%)
105 (55.9%)
A
79 (45.4%)
7.561
0.006
1
0.057
0.811
1
IL-13 Production
(pg/mL)
IL-13 Production
Lymphocyte Culture
(72 hours)
Mann-Whitney
test
Asthmatic patients
(mean ± standard deviation)
Control subjects
(mean ± standard deviation)
p-value
CC
345.41 ± 117.71
222.63 ± 93.30
<0.001
CT
252.96 ± 119.40
208.31 ± 107.64
0.089
TT
295.92 ± 95.70
158.05 ± 65.40
0.001
GG
264.16 ± 93.50
209.44 ± 91.54
0.068
GA
285.62 ± 93.50
215.25 ± 103.46
0.011
AA
300.41 ± 116.75
182.26 ± 92.72
0.008
DNA Extraction
-1111C>T
Quantification of IL-13
production by ELISA
Restriction Fragment Length
Polymorphism (RFLP)
DF*
Table 4: The relationship between the SNPs in the human IL-13 gene and IL-13 production
Genotype
Polymerase Chain Reaction
(PCR)
p-value
Table 3: A significant association between allele frequencies in the promoter region (-1111)
were found with asthmatic patients and control subjects (p=0.006). The mutant T-allele
may have an effect on the development of asthma in Malaysian asthmatics. These results
matched previous studies done in the Dutch7,8 and African American populations9.
Recruitment of asthmatics and control subjects
(5 ml blood)
Allele Frequencies
83 (44.1%)
Chi-square test
4257G>A
DNA sequencing
DISCUSSION
RESULTS AND DISCUSSION
 Allele frequencies of the Malaysian population for the -1111C wild-type allele and the 4257G
wild-type allele of the IL-13 gene were 0.665 and 0.558 respectively in control subjects and 0.523
and 0.546 respectively in asthmatics.
Table 1: Racial distribution of asthmatic patients and control subjects recruited
Malay
Chinese
Indian
Total
Asthmatic patients
29
31
27
87
Control subjects
30
31
33
94
1
2
3
PCR Product
4
5
6
bp
1
2
3
PCR Product
CC
TT
CT
GG
4
AA
5
 It was hypothesised that a higher frequency of mutant alleles of SNPs in IL-13 gene would be
found in asthmatic patients compared to control subjects. The findings for SNP at promoter
region -1111 supported this hypothesis with statistically significant (p=0.006) results. Although
the results for SNP at position 4257 also supported this hypothesis but the association observed
was not statistically significant (p=0.811).
6
bp
SNP at site -1111 resulted in patients carrying homozygous wild-type CC and homozygous
mutant AA alleles had higher IL-13 production. The results also showed that the 4257A mutant
allele is associated with significantly higher IL-13 production in asthmatics compared with
control subjects.
GA
REFERENCES
372
343
(a) Hpy99I (-1111)
236
210
178
(b) NlaIV (4257)
FIGURE-1: The restriction enzyme Hpy99I recognises the SNP at site -1111C>T; NlaIV
recognises the SNP at site 4257G>A. In both gels, Lanes 1 and 6 represent 100 bp DNA ladder
as molecular marker. Lane 2 represent undigested PCR products. Lane 3 represents RFLP
patterns seen when only the respective wild-type alleles are present. Lane 4 shows RFLP
patterns when only the respective homozygous mutant alleles are present. Lane 5 shows the
restriction patterns observed when respective heterozygous alleles are present.
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