A case–control study was conducted in Makassar, Indonesia, from June to December 2025.
A total of 80 pregnant women were included, consisting of:
40 subjects with asthma during pregnancy (case group) 40 non-asthmatic pregnant women (control group)
Subjects were recruited from Wirahusada Medical Center, Indonesia.
Inclusion criteria:
Pregnant women diagnosed with asthma based on clinical and GINA criteria Willing to participate and provide informed consent
Exclusion criteria:
Chronic pulmonary diseases other than asthma Severe systemic illness
Asthma control was classified according to Global Initiative for Asthma (GINA) criteria into:
Controlled asthma Uncontrolled asthma
All patients subsequently followed the study flow, underwent genetic testing, and had their asthma control assessed. The overall research process up to data analysis is illustrated in
Genomic DNA was extracted from peripheral blood samples using a standard Chelex-based method. Approximately 3 mL of peripheral venous blood was collected from the cubital vein into EDTA-containing vacutainer tubes and stored at −20 °C until processing.
Prior to extraction, frozen samples were thawed in a 37 °C water bath. A total of 200 μL of whole blood was mixed with 800 μL of saponin solution and incubated at 4 °C for 5 minutes. The mixture was then centrifuged at 10,000 rpm for 10 minutes at room temperature. The supernatant was discarded, and the pellet was washed once with 500 μL of phosphate-buffered saline (PBS), followed by centrifugation at 4,000 rpm for 5 minutes. This washing step was repeated three times.
Subsequently, 150 μL of distilled water (ddH 2O) and 20% Chelex solution were added to the pellet. The mixture was heated in boiling water (100 °C) for 10 minutes and then centrifuged at 10,000 rpm for 10 minutes. The supernatant containing genomic DNA was transferred into a new microtube and used as a template for polymerase chain reaction (PCR). PCR amplification was performed using two pairs of primers targeting β2-adrenergic receptor (β2-AR) gene polymorphisms at codons 16 (Arg16Gly) and 27 (Gln27Glu).
The primer sequences were as follows:
Forward (5′-CGCCTTCTTGCTGGCACCCAAT-3′)
Reverse (5′-CCAACTACTTCATCACTTCACTGG-3′)
Forward (5′-GAATGAGGCTTCCAGGCGTC-3′)
Reverse (5′-GGCCCATGACCAGATCAGCA-3′)
Each PCR reaction mixture contained approximately 100 ng of genomic DNA template, PCR buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl), 1.5 mM MgCl 2, 200 μM dNTPs, 1.25 U Taq DNA polymerase, and 10 pmol of each primer in a final reaction volume adjusted with nuclease-free water.
PCR amplification was carried out in a thermal cycler with the following conditions: initial denaturation at 94 °C for 2 minutes, followed by 30 cycles of denaturation at 94 °C for 30 seconds, primer annealing at 55 °C for Arg16Gly polymorphism and 60 °C for Gln27Glu polymorphism (each for 30 seconds), and extension at 72 °C for 1 minute. A final extension step was performed at 72 °C for 5 minutes.
The PCR products were purified using a commercial purification kit (Qiagen, Germany) according to the manufacturer’s instructions. DNA sequencing was performed using an automated sequencer (ABI Prism, USA). Sequencing reactions were carried out in a unidirectional manner, using conditions similar to PCR amplification, consisting of 30 cycles of denaturation at 96 °C, annealing at 50 °C, and extension at 60 °C.
Following sequencing, the products were subjected to polyacrylamide gel electrophoresis and analyzed using the automated sequencer system. The resulting gel images were processed using dedicated software to generate electropherograms, which were subsequently interpreted to determine genotype variations.
Data were analyzed using statistical software. Associations were evaluated using chi-square tests. Odds ratios (OR) with 95% confidence intervals (CI) were calculated. A p-value <0.05 was considered statistically significant.