Samples were denatured by 30 cycles of 30 s at 94°C, annealed for 30 s at 58°C, and extended for 30 s at 72°C, using the thermal cycler. A 152-bp fragment containing the +49 A/G polymorphism in exon 1 of CTLA-4 was amplified. The forward primer was designed with a single-base mismatch for the last nucleotide, which corresponded to the + 47 position, to introduce a base change in the sequence of the PCR product. This substitution created a BstEII restriction site in the A allele. Amplified products were incubated at 60°C for 2 h using 5 U BstEII per reaction. Digested products underwent electrophoresis on a 3.5% agarose gel. Buy allegra online so The digested A allele gave a fragment of 130 bp, and the G allele gave an intact 152-bp fragment (Fig 1).
Statistical analysis was performed using statistical software (SPSS, version 8.0; SPSS; Chicago, IL). The x2 test and t test were used when appropriate.
CTLA-4 Promoter (—318 C/T) Polymorphism
No deviation from the Hardy-Weinberg equilibrium was observed in any group examined (asthma group, x2 = 0.458 and p = 0.795; control group, X2 = 1.168 and p = 0.558). The distribution of genotypic frequencies was not significantly different for the asthma group and the control group (p = 0.690; Table 1). When the asthma group was divided into patients with atopic asthma and nona-topic asthma, the distribution of genotypic frequencies did not differ among the atopic asthma group, the nonatopic asthma group, and the control group (p = 0.311; Table 2). The relationships between the CTLA-4 promoter (—318 C/T) polymorphism and asthma severity were studied. Sixty-one patients were classified as having mild-to-moderate asthma (mild intermittent, 17 patients; mild persistent, 31 patients; moderate persistent, 13 patients), and 27 patients had severe asthma.
Figure 1. CTLA-4 exon 1 and promoter genotypes by PCR-RFLP. The GG (152 bp), AG (152 bp, 130 bp), AA (130 bp), CC (226 bp), CT (226 bp, 130 bp, 96 bp), and Tt (130 bp, 96 bp) genotypes are shown.
Table 1—CTLA-4 Promoter (—318 C/T) Polymorphism in Asthmatic Patients and Healthy Control Subjects
|Groups||Genotypes CC CT TT|
|Asthma patients (n = 88)||70 (79.5)||16(18.2)||2 (2.3)|
|Healthy control subjects||67 (77.9)||15 (7.4)||4(4.7)|
|(n = 86)|
Table 2—CTLA-4 Promoter (—318 C/T) Polymorphism According to Atopy
|Asthma Severity||CC||CT||TT||C Allele||T Allele|
|Mild to moderate (n = 61)||53 (86.9)||7(11.5)||1 (1.6)||113(92.6)||9 (7.4)|
|Severe (n = 27)||17 (63.0)||9 (33.3)||1 (3.7)||43 (79.6)||11 (20.4)|