Genotyping of the VNTR polymorphism in the exon 4 of the L-SIGN gene and its negative association with the risk of SARS coronavirus infection
Lab/Group: He/Zhou Lab (Beijing Institute of Radiation Medicine)
Related Journal & Article Information
Journal: Nature Genetics
Article Title: Lack of support for an association between CLEC4M homozygosity and protection against SARS coronavirus infection
Introduction
A convincing association study includes at least appropriate sampling, accurate genotyping, and appropriate statistical analysis (Hattersley et al. 2005). Additionally, association studies require replication in independent populations (Lohmueller et al. 2003). We have followed these rules in a recent study to assess the association between the risk of severe acute respiratory syndrome (SARS) coronavirus infection and the variable number tandem repeat (VNTR) polymorphism in the exon 4 of the L-SIGN gene (for liver/lymph node-specific ICAM-3 grabbing nonintegrin, encoded by CLEC4M) (Zhi et al. 2007). In this study, the individuals of three independent case-control populations have been rigorously selected and all of them are unrelated adults (Zhang et al. 2005). The genotyping of the VNTR polymorphism was performed by separating PCR products on agarose gel. As for the statistical analysis, we have used widely accepted methods to evaluate Hardy-Weinberg equilibrium, compare allele or genotype frequencies, and assess the genetic association. By use of this protocol for an association study, we found that there is lack of support for a genetic association between the L-SIGN polymorphism and risk of SARS coronavirus infection, which was previously reported by Chan et al. (Chan et al. 2006; Zhi et al. 2007).
Materials
Reagents
1. EDTANa2 (analytical-pure, 10mM)
2. Tris-saturated phenol (pH 8.0)
3. Chloroform (analytical-pure)
4. Isoamyl alcohol (analytical-pure)
5. Proteinase K
6. Ex Taq DNA polymerase [Code: DRR01AM, supplied with EX Taq, 10×Ex Taq Buffer (Mg2+ Free), dNTP Mixture and MgCl2. TaKaRa Bio Inc.]
7. Regular agarose (LOT NO. 101645. Shanghai Yito, China)
Equipment
1. PCR System (GeneAmp® PCR system 9700, 96-well, Applied Biosystems)
2. Centrifuge (Eppendorf Centrifuge 5810R, Eppendorf)
3. Gel Documentation system (Gel DocTM XR 170-8170, Bio-Rad)
4. Spectrophotometer (SmartSpecTM Plus spectrophotometer, Bio-Rad)
5. Water system (PURELAB Ultra Genetic, ELGA LabWater)
6. Refrigerator (-20~4ºC, BCD-257WD, Panasonic)
7. Ultra-low temperature freezer (-80ºC, MDF-U32V, SANYO)
Procedure
A. Blood collection and DNA preparation
1. Rigorously select case-control samples. The case-control population contain unrelated adults recruited from outpatient clinics and hospitals.
2. Collect 5ml blood by standard venipuncture into a tube with 40 μl EDTANa2 (10 mM) for each participant.
3. Extract genomic DNA from 5 ml whole blood using standard phenol/chloroform method.
4. Determine DNA concentration using a spectrophotometer.
5. Dilute DNA samples to 10 ng/μl and distribute them to 96-well plates.
B. Polymorphism genotyping
6. Analyze the VNTR polymorphism by PCR in the case-control populations in a blind manner.
7. The PCR reaction was carried out at 94ºC for 2 min, 32 cycles of denaturing at 94ºC for 30 s, annealing at 56.5ºC for 30 s and extension at 72ºC for 40 s, and final extension at 72ºC for 10 min, using a forward primer 5’-CTTTTCTTCTTGGCCCAGTGT-3’ and a reverse primer 5’-CTCAGGCAGGGTTGGTAACTC-3’ in a reaction mixture of 15 μl containing 10~20 ng genomic DNA, 0.25 mM dNTPs, 1.5 mM MgCl2, 200 pM of each primer and 1 unit (U) Ex Taq DNA polymerase (TaKaRa, Otsu, Shiga).
8. Separate the amplified products by 2% agarose gel electrophoresis.
9. Determine the length of alleles using marker standards and express genotypes as the number-of-repeat combinations.
10. Validate the accuracy of genotyping data for the VNTR polymorphism by resequencing 15% masked, random samples of patients and controls.
C. Statistical analysis
11. The allele and genotype frequencies for the polymorphism can be determined by direct gene counting. The fitness to Hardy-Weinberg equilibrium can be tested by using the Markov chain method implemented in the GENEPOP software package (available at: http://wbiomed.curtin.edu.au/genepop/). Comparisons of sex distribution between patient and control groups can be performed by use of the Chi-square test. Differences of mean age between the groups can be analyzed by use of an unpaired t test. Comparison of allele or genotype frequencies can be done using the CLUMP program (available at: http://www.mds.qmw.ac.uk/statgen/dcurtis/software). Genetic association for heterozygote and homozygote genotype with the SARS risk can be assessed by use of logistic regression using SPSS software (version 9.0; SPSS Inc., Chicago, IL), and the odds ratio (OR) and 95% confidential interval (c.i.) can be calculated and adjusted for age and gender, where appropriate. An association can be considered significant at a P value of less than 0.05, and all statistical tests should be two-sided.
Troubleshooting
Critical Steps
Anticipated Results
References
1. Hattersley, A.T. et al. Lancet. 366, 1315-1323 (2005).
2. Lohmueller, K.E. et al. Nat. Genet. 33, 177-182 (2003).
3. Zhi, L, et al. Nat. Genet. In press (2007).
4. Zhang, H. et al. J. Infect. Dis. 192, 1355-1361 (2005).
5. Chan, V.S. et al. Nat. Genet. 38, 38-46 (2006).
Acknowledgements
We thank all the tested individuals, their families and collaborating clinicians for their participation. This study was supported in part by grants from the Chinese High-tech Program (2001AA224011 and 2002BA711A10), Medicine and Health Research Program (01Z018), Chinese National Science Fund for Creative Research Groups (30321003 and 30621063), Beijing Science & Technology NOVA program (2006A54) and Chinese State Key Basic Research Program (2006CB910803).
Keywords
L-SIGN, VNTR, Genotyping, Association study