Supplementary methods
Genotyping
Genotyping of the GSTP1, GSTM1, and C3 gene polymorphisms was
performed as previously described1,2. We genotyped 21 SNPs in 15 genes (Table S1) by
the allele-specific amplification (ASA) using either the TaqMan probe (TaqMan-ASA)3
or SYBR Green detection. For the TaqMan-ASA method, 2  Platinum qPCR
SuperMix-UDG (Invitrogen, Carlsbad, CA, USA) was used as master mix, whereas 2 
Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) was used for ASA with
SYBR Green detection according to the manufacturer’s instructions. The primer and
TaqMan probe sequences are shown in Table S2. The samples were analyzed using an
ABI PRISM 7000 Sequence Detector System (Applied Biosystems, Foster City, CA,
USA) and Chromo4 Real-Time System (Bio-Rad, Hercules, CA, USA). The
thermoprofile was 50ºC for 2 min, 95ºC for 2 min, and 45 cycles of 95ºC for 15 s and
60ºC for 30 s (for TGFB1 -509C>T: 45 cycles of 95ºC for 15 s, 58ºC for 20 s, and 72ºC
for 30 s).
10 other SNPs in 8 genes (Table S1) were genotyped by the single nucleotide
primer extension reaction using SNaPshot Multiplex Kit (Applied Biosystems)
according to the manufacturer’s instructions with slight modification. Multiplex PCR
amplification was performed with 8 sets of primers (listed in Table S3a) using TaKaRa
Ex Taq HS (TaKaRa Bio, Otsu, Japan) on a GeneAmp PCR System 9700 (Applied
Biosystems). The amplification condition consisted of initial denaturation of 95ºC for 3
min, 40 cycles at 95ºC for 20 sec, 60ºC for 30 sec, 72ºC for 1 min, and a final extension
at 72ºC for 5 min. Subsequently, the SNaPshot reaction was performed with the
SNaPshot primers (Table S3b) using the PCR products treated with shrimp alkaline
phosphatase (SAP; TaKaRa Bio) and exonuclease I (ExoI; New England Biolabs,
Beverly, MA, USA) to remove primers and unincorporated dNTPs. The SNaPshot
primers were designed to anneal adjacent to the SNP of interest and were modified by
the addition of non-homologous 5′-poly (dA) tails to alter the primer lengths. Following
the treatment with calf intestine alkaline phosphatase (CIAP; TaKaRa Bio) to remove
the 5’-phosphoryl group of unincorporated ddNTPs, the SNaPshot products were
analyzed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The resulting
data were analyzed with GeneMapper v3.5 Software (Applied Biosystems).
Genotyping of the STAT6 exon 1 GT repeat, NOS1 intron 2 GT repeat, IL12B
-6415CTCTAA>GC, and SOCS1 -1478delCA was performed by fragment analysis of
the PCR product. PCR amplifications were carried out using TaKaRa Ex Taq HS with
6-carboxyfluorescein (FAM)-labeled and non-labeled primer pairs (Table S4). The PCR
conditions were as follows: an initial denaturation of 95ºC for 3 min, followed by 35
cycles at 95ºC for 20 sec, 60ºC for 30 sec, and 72ºC for 30 sec, with a final extension at
72ºC for 10 min. The PCR products were electrophoresed on an ABI PRISM 3100
Genetic Analyzer as described earlier and the fragment lengths were determined using
GeneMapper v3.5 Software.
References
1.
2.
3.
Inoue, H., Mashimo, Y., Funamizu, M., Shimojo, N., Hasegawa, K., Hirota, T. et
al. Association study of the C3 gene with adult and childhood asthma. J Hum
Genet 53, 728-38 (2008).
Kamada, F., Mashimo, Y., Inoue, H., Shao, C., Hirota, T., Doi, S. et al. The
GSTP1 gene is a susceptibility gene for childhood asthma and the GSTM1 gene
is a modifier of the GSTP1 gene. Int Arch Allergy Immunol 144, 275-86 (2007).
Fujii, K., Matsubara, Y., Akanuma, J., Takahashi, K., Kure, S., Suzuki, Y. et al.
Mutation detection by TaqMan-allele specific amplification: application to
molecular diagnosis of glycogen storage disease type Ia and medium- chain
acyl-CoA dehydrogenase deficiency. Hum Mutat 15, 189-96 (2000).