AEGiS-15IAC: Development of Novel Test-System for Simultaneous Detection of del32 and m303 Mutations in CCR5 Gene Coding HIV-1 Entry Coreceptor.

15th International AIDS Conference

Bangkok, Thailand - July 11-16, 2004

Development of Novel Test-System for Simultaneous Detection of del32 and m303 Mutations in CCR5 Gene Coding HIV-1 Entry Coreceptor.

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. A10200)

Apryatin S, Nikolaeva I, Rakhmanaliev E, Sulimova G, Ruban S, Sizyakina L, Sidorovich I
Institute of Immunology RF Ministry of Health, Moscow, Russian Federation

BACKGROUND: The human gene for CCR5 chemokine receptor affects susceptibility to HIV infection. The mutations del32 and m303 in a homozygous provide complete resistance to HIV sexual transmission. The cases of resistance to HIV infection in the absence homozygous genotype were shown; some of them could be explained by trans-position of these mutations at heterozygous individuals carrying both alleles. Existing test-systems are capable to detect del32 and m303 mutations, but not relative position of those. The data on del32/m303 genotype should be taken in account for selecting study populations for HIV vaccine trials. In this work, we present novel test-system for simultaneous detection of del32 and m303 mutations in CCR5 gene coding HIV-1 entry coreceptor.

METHODS: Patients: HIV seropositive (n=107), Caucasian. HIV seronegative (n=247): Caucasian (n=188, including 9 HIV seronegative women from HIV discordant pairs), Asian (n=50), total of 354. Reference test-system: "Vecto-CCR5-amply-100" commercial kit ("Vector-Best", Russia).

RESULTS: We developed the test-system for the simultaneous detection of mutations del32 and m303. The original primers, flanking a part of CCR5 gene (fragment that includes possible sites of both mutations del32 and m303), were selected and used for PCR-PLRF. Amplicons were treated with Hind II endonuclease, which specifically recognizes the sequence coinciding with m303 mutation site. A set of products can be obtained depending on del32/m303 genotype. The developed test-system was assessed with representative panel (354 DNA samples) in comparison with commercial kit "Vecto-CCR5-amply-100", and results completely coincided.

CONCLUSIONS: The developed test-system allows quickly and effectively revealing both del32 and m303 mutations simultaneously, as well as distinguishing a trans-position (both mutations located in different gene copes) from cis-position (mutations located in one gene copy) for the case of m303/del32 genotype.

Keywords: AEGIS, HIV-1, Receptors, CCR5, HIV Infections, Mutation, HIV Seropositivity, Acquired Immunodeficiency Syndrome, Receptors, Chemokine, Genotype, Alleles, Human Development, Russia, Humans, Female, growth & development, genetics

040711
A10200