Vitamin D-Related Gene Polymorphisms, Plasma 25-Hydroxy-Vitamin D, Cigarette Smoke and Non-Small Cell Lung Cancer (NSCLC) Risk


(g) (h) 
(i) (j) (k) (l) Figure S1. Cont. (m) (n) (o)



  Figure S1. Cont. 


Figure S1 .

 S1 Figure S1. Genotyping of the Vitamin D-related gene polymorphisms Polymorphism by PCR-RFLP. (a) Genotyping of the VDR rs10735810 (Fok1) Polymorphism by PCR-RFLP. Lanes 2 and 3 CC (265 bp); Lanes 1 and 5 TT (196 bp and 69 bp); Lanes 4, 6 and 7 CT (265 bp, 196 bp and 69 bp); (b) Genotyping of the VDR rs11568820 (Cdx2) Polymorphism by PCR-RFLP. Lanes 3 and 4 TT (135 bp); Lanes 1 and 5 CC (72 bp and 63 bp); Lane 2 TC (135 bp, 72 bp and 63 bp); (c) Genotyping of the VDR rs1544410 (Bsm1) Polymorphism by PCR-RFLP. Lanes 1, 2 and 3 GG (191 bp); Lanes 4 and 5 AA (115 and 76 bp); Lanes 6 and 7 GA (76, 115 and 191 bp); (d) Genotyping of the VDR rs7975232 (Apa1) Polymorphism by PCR-RFLP. Lanes 3 and 4 CC (745 bp); Lane 1 AA (528 and 217 bp); Lane 2 CA (745, 528 and 217 bp); (e) Genotyping of the VDR rs731236 (Taq1) Polymorphism by PCR-RFLP. Lanes 3 and 4 TT (494bp); Lane 1 CC (201 and 293 bp); Lane 2 TC (201, 293 and 494 bp); (f) Genotyping of the CYP24A1 rs6068816 Polymorphism by PCR-RFLP. Lane 2 CC (648 bp); Lane 1 TT (415 and 233 bp); Lane 3 CT (648, 415 and 233 bp); (g) Genotyping of the CYP24A1 rs2244719 Polymorphism by PCR-RFLP. Lanes 3 and 4 TT (574 bp); Lane 5 CC (390 and 184 bp); Lanes 1, 2 and 6 TC (574, 390 and 184 bp); (h) Genotyping of the CYP24A1 rs4809960 Polymorphism by PCR-RFLP. Lanes 1 and 2 TT (710 bp); Lanes 6, 7 and 8 CC (531 and 179 bp); Lanes 9; 3, 4 and 5 TC (710, 531 and 179 bp); (i) Genotyping of the CYP24A1 rs2762939 Polymorphism by PCR-RFLP. Lane 3 GG (560 bp); Lane 1 CC (470 and 90 bp); Lane 2 GC (560, 470 and 90 bp); (j) Genotyping of the CYP24A1 rs2181874 Polymorphism by PCR-RFLP. Lane 2 GG (735 bp); Lanes 1 and 4 AA (392 and 343 bp); Lane 3 GA (735, 392 and 343 bp); (k) Genotyping of the CYP24A1 rs2296241 Polymorphism by PCR-RFLP. Lanes 1 and 2 GG (732 bp); Lane 5 AA (518 and 214 bp); Lanes 3 and 4 GA (574, 390 and 184 bp); (l) Genotyping of the CYP27B1 rs10877012 Polymorphism by PCR-RFLP. Lanes 3, 4, 5, 6 GG (476 bp); Lanes 7 TT (310 and 166 bp); Lanes 1, 2 GT (476, 310 and 166 bp); (m) Genotyping of the CYP27B1 rs3782130 Polymorphism by PCR-RFLP. Lane 1 CC (620 bp); Lanes 2, 3, 5 and 6 GG (430 and 190 bp); Lane 4 CG (620, 430 and 190 bp); (n) Genotyping of the GC rs7041 Polymorphism by PCR-RFLP. Lanes 5, 6 and 7 TT (482 bp); Lanes 1 and 2 GG (298 and 184 bp); Lanes 3 and 4 TG (482, 298 and 184 bp); (o) Genotyping of the GC rs4588 Polymorphism by PCR-RFLP. Lane 3 CC (600 bp), Lane 2 AA (400 and 200 bp), Lanes 1 and 4 CA (600, 400 and 200 bp). 


Table S1 .

 S1 Characteristics of the single-nucleotide polymorphisms (SNPs) and primers, polymerase chain reaction (PCR) conditions used in the study. 


	SNP a Reference	Gene	Chr b	Position	Type	W/V c MAF d	Primer Sequence	PCR Conditions
	rs10735810	VDR (Fok1)	12: (46559162)	No block (Exon 4)	Missense	C/T	0.41	F:5′-AGCTGGCCCTGGCACTGACTCTGCTCT-3′ R:5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′	°C for 5 min (95 °C for 30 s, 61 °C for s, 72 °C for 30 s) ×40 cycles, 72 °C for min
	rs11568820	VDR (Cdx2)	12: (46588812)	BlockC2 (Intron 2)	Intron	T/C	0.49	F:5′-AGTAAACTAGGTCACAATAAAAACTTATTTCTT-3′ R:5′-AGTAAACTAGGTCACAGTAAAAACTTATTTCTT-3′	°C for 5 min (95 °C for 30 s, 56 °C for s, 72 °C for 30 s) ×40 cycles, 72 °C for min
	rs1544410	VDR (Bsm1)	12: (46526102)	Block B (Intron 2)	Intron	G/A	0.11	F:5′-CAGAGCATGGACAGGGAGCAA-3′ R:5′-GCAACTCCTCATGGCTGAGGTCTC-3′	°C for 5 min (95 °C for 30 s, 60 °C for s, 72 °C for 30 s) ×40 cycles, 72 °C for min
	rs7975232	VDR (Apa1)	12: (46525104)	Block B (Intron 8)	Intron	C/A	0.44	F:5′-GCTCTCAGCTGGGCCCCTCACTGCTCAAT-3′ R:5′-GCTCTCAGCTGGGCACCTCACTGCTCAAT-3′	°C for 5 min (95 °C for 30 s, 70 °C for s, 72 °C for 45 s) ×40 cycles, 72 °C for min
	rs731236	VDR (Taq1)	12: (46525024)	Block B (Exon 11)	Synonymous	T/C	0.13	F:5′-CAGAGCATGGACAGGGAGCAA-3′ R:5′-GCAACTCCTCATGGCTGAGGTCTC-3′	°C for 5 min (95 °C for 30 s, 55 °C for s, 72 °C for 45 s) ×40 cycles, 72 °C for min
	rs6068816	CYP24A1	20: (52214498)	Block B (Exon 6)	Synonymous	C/T	0.48	F:5′-ACTTCGTCAGTAACGGACGAGTGA-3′ R:5′-GAGTCGAGGTCATATCGTGAGTGA-3′	°C for 5 min (95 °C for 30 s, 60 °C for s, 72 °C for 45 s) ×40 cycles, 72 °C for min
	rs2244719	CYP24A1	20: (52216265)	Block C (Intron 4)	Intron	T/C	0.18	F:5′-ATAGGGTTTTTTTTCCTTCCATTTCATCA-3′ R: 5′-ATAGGGTTTTTTTTTCTTCCATTTCATCA-3′	°C for 5 min (95 °C for 30 s, 65 °C for s, 72 °C for 45 s) ×40 cycles, 72 °C for min
	rs4809960	CYP24A1	20: (52219480)	Block D (Intron 4)	Intron	T/C	0.11	F:5′-ACTTCGTCAGTAACGGACGGCAGG-3′ R:5′-GAGTCGAGGTCATATCGTGGCAGGT-3′	°C for 5 min (95 °C for 30 s, 60 °C for s, 72 °C for 45 s) ×40 cycles, 72 °C for min

Table S1 .

 S1 Cont. 


	SNP a Reference	Gene	Chr b	Position	Type	W/V c MAF d Primer Sequence	PCR Conditions
	rs2762939	CYP24A1	20: (52214658)	Block B (Intron 5)	Intron	G/C	0.40	F:5′-CTCTCTCTGCACCGGTCCTGGGAGCAATG-3′ R:5′-CTCTCTCTGCACCGCTCCTGGGAGCAATG-3′	95 °C for 5 min (95°C for 30 s, 52 °C for 30 s, 72 °C for 30 s) ×40 cycles, °C for 5 min
	rs2181874	CYP24A1	20: (52217885)	No Block (Intron 4)	Intron	G/A	0.15	F:5′-GTAACCGGGAGCTGCGGGAATGTATTCAG-3′ R:5′-GTAACCGGGAGCTGTGGGAATGTATTCAG-3′	95 °C for 5 min (95 °C for 30 s, °C for 30 s, 72 °C for 45 s) ×40 cycles, °C for 5 min
	rs2296241	CYP24A1	20: (52219626)	Block D (Exon 4)	Synonymous	G/A	0.49	F:5′-TTGAAGGTCTTGGCCGATTTTATGGGCAG-3′ R:5′-TTGAAGGTCTTGGCTGATTTTATGGGCAG-3′	95 °C for 5 min (95 °C for 30 s, °C for 30 s, 72 °C for 30 s) ×40 cycles, °C for 5 min
	rs10877012	CYP27B1	12: (56448352)	No block	downstream	G/T	0.41	F:5′-CTGGCACAGAGCCTCATAG-3′ R:5′-CGTATGCCTGTAGTGCCTT-3′	95 °C for 5 min (95 °C for 30 s, °C for 30 s, 72 °C for 30 s) ×35 cycles, °C for 5 min
	rs3782130	CYP27B1	12: (56448165)	Block A	downstream	C/G	0.39	F:5′-GGCCCTCTCTCTGTTGCCTAG-3′ R:5′-CAACACTTTGGGAGGCTAAGG-3′	95 °C for 5 min (95 °C for 30 s, °C for 30 s, 72 °C for 30 s) ×35 cycles, °C for 5 min
	rs7041	GC	4: (72837198)	Block A (Exon 11)	Missense	T/G	0.36	F:5′-AAATAATGAGCAAATGAAAGAAGAC-3′ R:5′-CAATAACAGCAAAGAAATGAGTAGA-3′	95 °C for 3min (95 °C for 30 s, 64 °C for 30 s, 72 °C for 30 s) ×35 cycles, °C for7 min
	rs4588	GC	4: (72837187)	Block A (Exon 11)	Missense	C/A	0.28	F:5′-AAATAATGAGCAAATGAAAGAAGAC-3′ R:5′-CAATAACAGCAAAGAAATGAGTAG-3′	95 °C for 3min (95 °C for 30 s, 64 °C for 30 s, 72 °C for 30 s) ×35 cycles, °C for 7 min

a http://hapmap.org; b Chr (Chromosome); c W/V (Wild/Variation); d MAF (Minor Allele Frequency) among controls.
Xiayu Wu, Jiaoni Cheng, Kaiyun Yang
Vitamin D-Related Gene Polymorphisms, Plasma 25-Hydroxy-Vitamin D, Cigarette Smoke and Non-Small Cell Lung Cancer (NSCLC) Risk