Impact of Cytokine Gene Polymorphisms on Risk and Treatment Outcomes of Aplastic Anemia

Journal: the Annals of Hematology

Yun-Gyoo Lee1, Inho Kim1, 4, 5, Jin Hee Kim2, 6, Ji-Yeon Bae4, Ji-Hyun Kwon1, Dong-Yeop Shin1, Jong-Eun Lee7, Eun Young Song3, Hyun Kyoung Kim3, Sung-Soo Yoon1, 4, Sung Sup Park3, Dong Soon Lee3, Kyou-Sup, Han3,Myoung Hee Park3, Yun-Chul Hong2, 6, Seonyang Park1, 4, 5, Byoung-Kook Kim1, 4

1Department of Internal Medicine, 2Preventive Medicine,3Laboratory Medicine, Seoul National University Hospital, Seoul, Korea

4Cancer Research Institute, 5Diagnostic DNA chip Center, Seoul National University College of Medicine, Seoul, Korea

6Institute of Environmental Medicine, Seoul National University Medical Research Center, Seoul, Korea

7DNA Link Inc., Seoul, Korea

Corresponding author

Inho Kim, M.D., Ph.D.,
Department of Internal Medicine

Seoul National University Hospital
101 Daehang-ro, Jongno-gu, Seoul 110-744, Korea

Tel: 82 2 2072 0834
Email:

Genotyping Method

The genotyping was screened using single base primer extension assay using ABI PRISM SNaPShot Multiplex kit (ABI, Foster City, CA, USA) according to manufacturer’s recommendation. Briefly, the genomic DNA flanking the interested single nucletide polymorphism (SNP) was amplified with PCR reaction with Forward and Reverse primer pairs and standard PCR reagents in 10μL reaction volume, containing 10ng of genomic DNA, 0.5pM of each oligonucleotide primer, 1μL of 10X PCR buffer, 250M dNTP (2.5mM each) and 0.25 unit i-StarTaq DNA Polymerase (5unit/µl) (iNtRON Biotechnology, Sungnam, Kyungki-Do, Korea). The PCR reactions were carried out as follows; 10 min at 95℃ for 1 cycle, and 35 cycles on 95℃ for 30s, Tm℃ for 1min, 72℃ for 1min followed by 1 cycle of 72℃ for 10mins. After amplification, the PCR products were treated with 1 unit each of shrimp alkaline phosphatase (SAP) (USB Corporation, Cleveland, OH, USA)and exonuclease I (USB Corporation, Cleveland, OH, USA) at 37℃ for 75 minutes and 72℃ for 15 minutes to purify the amplified products. One microliter of the purified amplification products were added to a SNaPshot Multiplex Ready reaction mixture containing 0.15pmols of genotyping primer for primer extension reaction. The primer extension reaction was carried out for 25cycles of 96℃ for 10 seconds, 50℃ for 5 seconds, and 60℃ for 30 seconds. The reaction products were treated with 1 unit of SAP at 37℃ for 1 hour and 72℃ for 15 minutes to remove excess fluorescent dye terminators. One microliter of the final reaction samples containing the extension products were added to 9 μL of Hi-Di formamide (ABI, Foster City, CA). The mixture was incubated at 95℃ for 5 min, followed by 5min on ice and then analyzed by electrophoresis in ABI Prism 3730xl DNA analyzer. Analysis was carried out using Genemapper software (version 4.0; Applied Biosystems). Table 1 shows the primer sets and Tm used for the SNaPshot assay.
Table 1. Primer sets and Tm for the SNaPshot assay.

Gene / SNP name (rs number) / Strand / Primer sequence / Tm / Additive
IFNG / -2353A/T (rs7139169) / Reverse / Forward / GCAGAAGACACGCGAATAG / 55 / -
Reverse / ATCCTCCTTAAAATTAATCTTAGATTCTC
Genotyping / GGTTTATACTTTTCTAAGAGTTCTG
-1616C/T (rs2069705) / Reverse / Forward / CAGTTTTACAGGTAAGGAGACTGAG / 55 / -
Reverse / TTTGCATTTCTACCTGTACTGTGTA
Genotyping / TATCTAGCTATATGATTGTGAGTTA
+874A/T (rs2430561) / Forward / Forward / ATATTCAGACATTCACAATTGATT / 60 / -
Reverse / TATTATACGAGCTTTAAAAGATAGTTCC
Genotyping / TTTATXCTTACAACACAAAATCAAATC
TNF / -1037C/T (rs1799724) / Forward / Forward / TAGGAGAATGTCCAGGGCTAT / 55 / -
Reverse / AGGCTCTTTCACTCCCTGG
Genotyping / GTCGAGTATGGGGACCCCCMNTTAA
-1031C/T (rs1799964) / Forward / Forward / cagagagcttcagggatatg / 55 / Betaine
Reverse / gtctcctgtaacccattcct
Genotyping / aaggagaagctgagaaga
-863C/A (rs1800630) / Forward / Forward / GGGAGAACAAAAGGATAAGG / 55 / Betaine
Reverse / TGAAGCTCTCACTTCTCAGG
Genotyping / AAGTCGAGTATGGGGACCCCC
-308G/A (rs1800629) / Forward / Forward / AGAAGGAAACAGACCACAGAC / 55 / Betaine
Reverse / GGGAAAGAATCATTCAACCA
Genotyping / TAGGTTTTGAGGGGCATG
TGFB / -590 C/T (rs1800469) / Forward / Forward / TCAGAGCTGACCCCAGCTAA / 65 / Betaine
Reverse / GGCCACCGTCCTCATCTC
Genotyping / CCTCCTGACCCTTCCATCC
P10L C/T (rs1800470) / Reverse / Forward / GCCCATCTAGGTTATTTCC / 60 / Betaine
Reverse / TGCCAGTCACTTCCTACC
Genotyping / AGCAGCGGTAGCAGCAGC
FAS / -670G/A (rs1800682) / Reverse / Forward / GCGATTTGGCTTAAGTTGTT / 60 / -
Reverse / GAGAAGTCAGGGTGAGGAAG
Genotyping / GCAACATGAGAGGCTCACAGACGTT