Skip to content
2000
  1. Home
  2. A-Z Publications
  3. Current HIV Research
  4. Volume 22, Issue 6
  5. Article
Volume 22, Issue 6
  • ISSN: 1570-162X
  • E-ISSN: 1873-4251

Abstract

Introduction

The C-C chemokine receptor type 5 (CCR5) is a major co-receptor for human immunodeficiency virus (HIV). Some individuals carry the CCR5 delta-32 genetic polymorphism. People with homozygous CCR5 delta-32 gene are nearly completely resistant to HIV-1 infection. High-resolution melting curve (HRM) analysis is a post-PCR technique utilized for identifying genetic variations in a quick, affordable, and closed-tube assay. The objective of this study was to develop an HRM assay for easy detection of delta-32 mutations.

Materials and Methods

DNA was extracted from peripheral blood mononuclear cells. HRM was performed to detect delta-32 mutation. The study investigated the impact of various factors, including annealing temperature, template concentration, touchdown PCR, additives, amplicon size, and program settings, on HRM Tm differentiation.

Results

It was expected that there would be a 4°C Tm difference between amplicons with and without delta-32 mutation, but the test showed a difference of only 2.3°C. In attempts to identify heterozygote delta-32 variants, a Tm difference of only 0.4°C could be achieved. Various modifications were applied, such as adjusting the template concentration, using touchdown PCR, and adding DMSO and glycerol. However, none of these changes helped to differentiate the Tm effectively, especially in delta-32 heterozygote samples.

Conclusion

The HRM test identified four samples with heterozygote mutations in each HIV-infected (8.89%) and control (5.72%) groups. More importantly, this study showed that identifying the delta-32 mutation of the CCR5 gene using HRM assay is not as straightforward as previously suggested in some literature, and it requires special setup conditions.

© 2024 Bentham Science Publishers
Loading

Article metrics loading...

/content/journals/chr/10.2174/011570162X326491240906064322
2024-12-01
2025-01-21

  • From This Site

    /content/journals/chr/10.2174/011570162X326491240906064322
    dcterms_title,dcterms_subject,pub_keyword
    -contentType:Contributor -contentType:Concept -contentType:Institution
    10
    5
Loading full text...

Full text loading...

References

  1. BergerE.A. MurphyP.M. FarberJ.M. Chemokine receptors as HIV-1 coreceptors: Roles in viral entry, tropism, and disease.Annu. Rev. Immunol.199917165770010.1146/annurev.immunol.17.1.65710358771
     [Google Scholar]
  2. AlkhatibG. BergerE.A. HIV coreceptors: From discovery and designation to new paradigms and promise.Eur. J. Med. Res.200712937538417933717
     [Google Scholar]
  3. BrelotA. ChakrabartiL.A. CCR5 revisited: How mechanisms of HIV entry govern AIDS pathogenesis.J. Mol. Biol.2018430172557258910.1016/j.jmb.2018.06.02729932942
     [Google Scholar]
  4. BlanpainC. LibertF. VassartG. ParmentierM. CCR5 and HIV Infection.Receptors Channels200281193110.3109/1060682021213512402506
     [Google Scholar]
  5. MichaelN.L. LouieL.G. RohrbaughA.L. SchultzK.A. DayhoffD.E. WangC.E. SheppardH.W. The role of CCR5 and CCR2 polymorphisms in HIV-1 transmission and disease progression.Nat. Med.19973101160116210.1038/nm1097‑11609334732
     [Google Scholar]
  6. Eugen-OlsenJ. IversenA.K.N. GarredP. KoppelhusU. PedersenC. BenfieldT.L. SorensenA.M. KatzensteinT. DickmeissE. GerstoftJ. SkinhøjP. SvejgaardA. NielsenJ.O. HofmannB. Heterozygosity for a deletion in the CKR-5 gene leads to prolonged AIDS-free survival and slower CD4 T-cell decline in a cohort of HIV-seropositive individuals.AIDS199711330531010.1097/00002030‑199703110‑000079147421
     [Google Scholar]
  7. LaurichesseJ.J. PersozA. TheodorouI. RouziouxC. DelfraissyJ.F. MeyerL. Improved virological response to highly active antiretroviral therapy in HIV-1-infected patients carrying the CCR5 Δ32 deletion.HIV Med.20078421321910.1111/j.1468‑1293.2007.00455.x17461848
     [Google Scholar]
  8. MarmorM. SheppardH.W. DonnellD. BozemanS. CelumC. BuchbinderS. KoblinB. SeageG.R.III Homozygous and heterozygous CCR5-Δ32 genotypes are associated with resistance to HIV infection.J. Acquir. Immune Defic. Syndr.200127547248110.1097/00126334‑200108150‑0000911511825
     [Google Scholar]
  9. LiuS. KongC. WuJ. YingH. ZhuH. Effect of CCR5-Δ32 heterozygosity on HIV-1 susceptibility: A meta-analysis.PLoS One201274e3502010.1371/journal.pone.003502022496885
     [Google Scholar]
  10. NiJ. WangD. WangS. The CCR5-Delta32 genetic polymorphism and HIV-1 infection susceptibility: A meta-analysis.Open Med. (Wars.)201813146747410.1515/med‑2018‑006230426084
     [Google Scholar]
  11. de Roda HusmanA.M. KootM. CornelissenM. KeetI.P. BrouwerM. BroersenS.M. BakkerM. RoosM.T. PrinsM. de WolfF. CoutinhoR.A. MiedemaF. GoudsmitJ. SchuitemakerH. Association between CCR5 genotype and the clinical course of HIV-1 infection.Ann. Intern. Med.19971271088289010.7326/0003‑4819‑127‑10‑199711150‑000049382366
     [Google Scholar]
  12. FrançaL.T.C. CarrilhoE. KistT.B.L. A review of DNA sequencing techniques.Q. Rev. Biophys.200235216920010.1017/S003358350200379712197303
     [Google Scholar]
  13. SlatkoB.E. GardnerA.F. AusubelF.M. Overview of next-generation sequencing technologies.Curr. Protoc. Mol. Biol.20181221e5910.1002/cpmb.5929851291
     [Google Scholar]
  14. WittwerC.T. High-resolution DNA melting analysis: advancements and limitations.Hum. Mutat.200930685785910.1002/humu.2095119479960
     [Google Scholar]
  15. ReedGH. KentJO. WittwerCT. High-resolution DNA melting analysis for simple and efficient molecular diagnostics.Pharmacogenomics20078659760810.2217/14622416.8.6.597
     [Google Scholar]
  16. MontgomeryJ.L. SanfordL.N. WittwerC.T. High-resolution DNA melting analysis in clinical research and diagnostics.Expert Rev. Mol. Diagn.201010221924010.1586/erm.09.8420214540
     [Google Scholar]
  17. KianiS.J. RamshiniM. Bokharaei-SalimF. DonyaviT. EshratiB. KhoshmirsafaM. GhorbaniS. TavakoliA. MonavariS.H. GhalejooghZ.Y. Abbasi-KolliM. High resolution melting curve analysis for rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants.Acta Virol.2023671919810.4149/av_2023_10936950889
     [Google Scholar]
  18. RamshiniM. Bokharaei-SalimF. DonyaviT. KhoshmirsafaM. GhorbaniS. KhatamiA. Single nucleotide polymorphism in toll-like receptor 3 gene as a potential risk factor for severe outcome of coronavirus disease 2019.Rev Res Med Microbiol2019
     [Google Scholar]
  19. LopalcoL. CCR5: From natural resistance to a new anti-HIV strategy.Viruses20102257460010.3390/v202057421994649
     [Google Scholar]
  20. VossenR.H.A.M. AtenE. RoosA. den DunnenJ.T. High-resolution melting analysis (HRMA)-more than just sequence variant screening.Hum. Mutat.200930686086610.1002/humu.2101919418555
     [Google Scholar]
  21. DonyaviT. Bokharaei-SalimF. NahandJ.S. GarshasbiS. EsghaeiM. SadeghiM. JamshidiS. KhanalihaK. Evaluation of CCR5-Δ32 mutation among individuals with high risk behaviors, neonates born to HIV-1 infected mothers, HIV-1 infected individuals, and healthy people in an Iranian population.J. Med. Virol.20209281158116410.1002/jmv.2565831854469
     [Google Scholar]
  22. NansenA. ChristensenJ.P. AndreasenS.Ø. BartholdyC. ChristensenJ.E. ThomsenA.R. The role of CC chemokine receptor 5 in antiviral immunity.Blood20029941237124510.1182/blood.V99.4.123711830471
     [Google Scholar]
  23. RasmussenH. WergeT. A closed-tube assay for genotyping of the 32-bp deletion polymorphism in the chemokine receptor 5 (CCR5) gene: Dissociation analysis of amplified fragments of DNA.Mol. Cell. Probes200721181110.1016/j.mcp.2006.05.00316879946
     [Google Scholar]
  24. NischalkeH.D. NattermannJ. LichterfeldM. WoitasR.P. RockstrohJ.K. SauerbruchT. SpenglerU. Rapid determination of the Δ32 deletion in the human CC-chemokine receptor 5 (CCR5) gene without DNA extraction by lightcycler real-time polymerase chain reaction.AIDS Res. Hum. Retroviruses200420775075410.1089/088922204152463415307921
     [Google Scholar]
  25. KorbieD.J. MattickJ.S. Touchdown PCR for increased specificity and sensitivity in PCR amplification.Nat. Protoc.2008391452145610.1038/nprot.2008.13318772872
     [Google Scholar]
  26. RouxK.H. Single-step PCR optimization using touchdown and stepdown PCR programming.Methods Mol Biol2002192313610.1385/1‑59259‑177‑9:031
     [Google Scholar]
  27. RahimiH. FarajollahiM.M. HosseiniA. Distribution of the mutated delta 32 allele of CCR5 co-receptor gene in Iranian population.Med. J. Islam. Repub. Iran20142814025694998
     [Google Scholar]
  28. KhorramdelazadH. HakimizadehE. HassanshahiG. RezayatiM. SendiH. ArababadiM.K. CCR5 Δ 32 mutation is not prevalent in Iranians with chronic HBV infection.J. Med. Virol.201385696496810.1002/jmv.2351023588722
     [Google Scholar]
  29. HeydarifardZ. TabarraeiA. MoradiA. Polymorphisms in CCR5Δ32 and risk of HIV-1 infection in the southeast of caspian sea, Iran.Dis. Markers2017201711510.1155/2017/419010729209099
     [Google Scholar]
/content/journals/chr/10.2174/011570162X326491240906064322
Loading
Detection Of CCR5 Delta-32 Mutation Using High-Resolution Melting Curve Analysis: Challenges and Facts
Current HIV Research 22, 368 (2024); https://doi.org/10.2174/011570162X326491240906064322
/content/journals/chr/10.2174/011570162X326491240906064322
/content/journals/chr/10.2174/011570162X326491240906064322
Loading

Data & Media loading...

Supplements

Supplementary material is available on the publisher's website along with the published article.

file format download Download

  • Article Type:     Research Article
Keyword(s): chemokine receptor R5 (CCR5); delta-32 deletion; genetic diversity; High-resolution melting curve analysis (HRM); human immunodeficiency virus (HIV); mutation
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test