Reply to: “CCL3L1 and HIV/AIDS susceptibility” and “Experimental aspects of copy number variant assays at CCL3L1” (original) (raw)

He et al. reply:

We read with interest the three correspondences1,2,3 that are directly or indirectly related to our previous publication in Nature Medicine4. Because of methodological concerns with their own C-C chemokine ligand-3–like-1 (CCL3L1) gene quantitative PCR (qPCR) assay, Urban et al.1 assume that similar limitations apply to the assay that we and others have used and, by extension, infer that all of the several reported associations for CCL3L1 copy number with HIV/AIDS suceptibility (Supplementary Table 1) are spurious. We tested the validity of this assumption. Notwithstanding their statement that they “assayed for CCL3L1 copy number using a previously described method” (described in ref. 5 here), Urban et al.1 made two major modifications to our qPCR assay, which we show adversely affect assay performance. Although they used the same probe sequences, instead of using tetramethylrhodamine (TAMRA) as the quencher, they used minor groove binder (MGB), a change that raises the melting temperature of the probe 8°C higher than the optimal temperature (Supplementary Table 2). Urban et al.1 also raise the concern that our assay is confounded, because they observed that CCL3L1 gene copy number (GCN) differed with the quality and the amount of input DNA. Our published protocol specifically recommended a fixed amount of high-quality input DNA between 2 and 10 ng5, and, consequently, we used qPCR standard curves comprising twofold dilutions from 25 to 0.78 ng. By contrast, Urban et al.1 used variable amounts of input DNA (up to 100 ng) and constructed standard curves of tenfold dilutions from 100 ng to 1 pg.

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References

  1. Urban, T.J. et al. Nat. Med. 15, 1110–1112 (2009).
    Article CAS PubMed PubMed Central Google Scholar
  2. Bhattacharya, T. et al. Nat. Med. 15, 1112–1115 (2009).
    Article CAS PubMed Google Scholar
  3. Field, S.F. et al. Nat. Med. 15, 1115–1117 (2009).
    Article CAS PubMed PubMed Central Google Scholar
  4. Ahuja, S.K. et al. Nat. Med. 14, 413–420 (2008).
    Article CAS PubMed PubMed Central Google Scholar
  5. Gonzalez, E. et al. Science 307, 1434–1440 (2005).
    Article CAS PubMed Google Scholar
  6. Townson, J.R., Barcellos, L.F. & Nibbs, R.J. Eur. J. Immunol. 32, 3016–3026 (2002).
    Article CAS PubMed Google Scholar
  7. Shostakovich-Koretskaya, L. et al. AIDS 23, 679–688 (2009).
    PubMed Google Scholar
  8. Dolan, M.J. et al. Nat. Immunol. 8, 1324–1336 (2007).
    Article CAS PubMed Google Scholar
  9. Guérin, S. et al. AIDS 14, 2788–2790 (2000).
    Article PubMed Google Scholar
  10. Yamashita, T.E. et al. AIDS 15, 735–746 (2001).
    Article CAS PubMed Google Scholar
  11. Valdez, H., Purvis, S.F., Lederman, M.M., Fillingame, M. & Zimmerman, P.A. J. Am. Med. Assoc. 282, 734 (1999).
    Article CAS Google Scholar
  12. Hendrickson, S.L. et al. J. Acquir. Immune Defic. Syndr. 48, 263–271 (2008).
    Article CAS PubMed PubMed Central Google Scholar
  13. Kasten, S. et al. Eur. J. Med. Res. 5, 323–328 (2000).
    CAS PubMed Google Scholar
  14. Colobran, R. et al. Genes Immun. 9, 279–288 (2008).
    Article CAS PubMed Google Scholar
  15. Perry, G.H. et al. Am. J. Hum. Genet. 82, 685–695 (2008).
    Article CAS PubMed PubMed Central Google Scholar
  16. Alkan, C. et al. Nat. Genet. advance online publication, doi: 10.1038/ng.437 (30 August 2009).
  17. Gornalusse, G. et al. PLoS Genet. 5, e1000359 (2009).
    Article PubMed PubMed Central Google Scholar
  18. Degenhardt, J.D. et al. PLoS Genet. 5, e1000346 (2009).
    Article PubMed PubMed Central Google Scholar

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Author notes

  1. Henry M. Jackson Foundation, Wilford Hall United States Air Force Medical Center, Lackland Air Force Base, Texas, USA.
  2. Infectious Disease Service, San Antonio Military Medical Center, Brooke Army Medical Center, Fort Sam Houston, Texas, USA.

Authors and Affiliations

  1. Veterans Administration Research Center for AIDS and HIV-1 Infection, South Texas Veterans Health Care System, San Antonio, Texas, USA
    Weijing He, Hemant Kulkarni, John Castiblanco, Una Aluyen, Robert Maldonado, Andrew Carrillo, Madeline Griffin, Amanda Lipsitt, Lisa Beachy, Robert A Clark, Matthew J Dolan, Matthew J Dolan & Sunil K Ahuja
  2. Department of Medicine, University of Texas Health Science Center, San Antonio, Texas, USA
    Weijing He, Hemant Kulkarni, John Castiblanco, Una Aluyen, Robert Maldonado, Andrew Carrillo, Madeline Griffin, Amanda Lipsitt, Lisa Beachy, Robert A Clark & Sunil K Ahuja
  3. Department of Pediatrics, University of California–San Diego School of Medicine, La Jolla, California, USA
    Chisato Shimizu & Jane C Burns
  4. Department of General Pediatrics and Pediatric Infectious Diseases, Dnepropetrovsk State Medical Academy, Dnepropetrovsk, Ukraine
    Ludmila Shostakovich-Koretskaya
  5. Laboratorio de Biología Celular y Retrovirus, Hospital de Pediatría 'J.P. Garrahan', Buenos Aires, Argentina
    Andrea Mangano & Luisa Sen
  6. Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, Scotland, UK
    Robert J B Nibbs
  7. AIDS Virus Research Unit, National Institute for Communicable Diseases/National Health Laboratory Services and University of the Witwatersrand, Johannesburg, South Africa
    Caroline T Tiemessen
  8. AIDS Clinical Research Unit, University of Miami, Miller School of Medicine, Miami, Florida, USA
    Hector Bolivar
  9. Departments of Pediatrics and Genome Sciences, University of Washington and Seattle Children's Hospital, Seattle, Washington, USA
    Michael J Bamshad

Authors

  1. Weijing He
  2. Hemant Kulkarni
  3. John Castiblanco
  4. Chisato Shimizu
  5. Una Aluyen
  6. Robert Maldonado
  7. Andrew Carrillo
  8. Madeline Griffin
  9. Amanda Lipsitt
  10. Lisa Beachy
  11. Ludmila Shostakovich-Koretskaya
  12. Andrea Mangano
  13. Luisa Sen
  14. Robert J B Nibbs
  15. Caroline T Tiemessen
  16. Hector Bolivar
  17. Michael J Bamshad
  18. Robert A Clark
  19. Jane C Burns
  20. Matthew J Dolan
  21. Sunil K Ahuja

Corresponding author

Correspondence toSunil K Ahuja.

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He, W., Kulkarni, H., Castiblanco, J. et al. Reply to: “CCL3L1 and HIV/AIDS susceptibility” and “Experimental aspects of copy number variant assays at _CCL3L1_”.Nat Med 15, 1117–1120 (2009). https://doi.org/10.1038/nm1009-1117

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