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Rapid Progression to AIDS in HIV + Individuals with a Structural Variant of the Chemokine Receptor CX 3 CR1.
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Rapid Progression to AIDS in HIV+ Individuals with a Structural Variant of the Chemokine Receptor CX3CR1 Faure, S., M. Laurence, D. Costagliola, C. Vaneensberghe, E. Genin, B. Autran, J.F. Delfraissy, D.H. McDermott, P.M. Murphy, P. Debre, I. Theodorou, and C. Combadiere. 2000. Rapid progression to AIDS in HIV+ Individuals with a structural variant of the chemokine receptor CX3CR1. Science 287:2274-2277.
CHEMOKINE RECEPTOR 5 (CCR5) • CCR5 has to some extent been a key prospect to fighting HIV infection. • The HIV virus uses this receptor as entry into macrophages and CD4 cells. • Mutations in CCR5 (CCR5 delta 32) have been known to decrease rate and progression of HIV-1 infections. • Predominantly found among Caucasian populations. • A frame shift is caused by the deletion of 32-bp within the coding region, which give CCR5 delta 32 its mutation. • Prevents the binding of HIV-1 to macrophages and CD4 cells, which is located on the cell surface of macrophages and CD4 cells. • Most patients were found to have the heterozygous mutation, the focus of many experiments went to heterozygous CCR5 HIV-1 infected patients (Caucasians).
Introduction to CX3CR1 • 7-membrane receptor that has a high attraction to fractalkine. • Fractalkine is a transmembrane molecule CX3CR1 specific chemokine that stimulates leukocytic adhesion and migration. • Functions as a mediator of migratory and adhesive functions of fractalkine. • Natural Killer cells have CX3CR1 expressed on their cell surface and use fractalkine as their adhesive and migratory inducing agent. • Functions as CCR5 pertaining to HIV-1 entry. • CX3CR1 it shows a limited interaction when testing HIV’s DNA envelops. • Fractalkine has the ability to block HIV from binding to CX3CR1.
Can CX3CR1 Regulate HIV Disease? • Observe the comparison between genetic variants and outcomes of the altered disease forming the rational.
Single stranded conformation polymorphism (SSCP) • Technique used to amplify base pair fragments. • The fragments being amplified would encode for the whole CX3CR1 receptor. • Technique includes the use of a primer for amplification. • Final results placed on a mutation detection gel that shows different types of mutation for CX3CR1.
Single Nucleotide Polymorphism (SNP) • Variation in DNA sequences. • These variations in the human genome can be used to help identify differences in humans being susceptible to, or protected from, a mass of diseases. • Comparisons in variations of the human genome is important for understanding how various biochemical shapes and cellular functions, and also sheds light on past human evolution. • The main use of SNPs, especially in this experiment, would be to find how individual genes contribute to certain diseases that have complex, multigene basis. • How researchers were able to discover how receptor CCR5 mutation can lead to resistance to HIV and AIDS.
An Example of SNP • http://www.nature.com/cgitaf/DynaPage.taf?file=/nature/journal/v409/n6822/full/409822a0_fs.html&content_filetype=pdf. Chakravarti, A. 2001. Single nucleotide polymorphisms: . . .to a future of genetic medicine. Nature 409: 822 – 823.
How Was Experiment Conducted? • 78 random French Caucasian's blood was used to screen the coding sequence for CX3CR1 mutations in these donors. • The method they used to screen the blood donors was via the single stranded conformation polymorphism (SSCP) technique. • There was a detection of 5 single nucleotide polymorphisms (SNPs) in the transmembrane domains of the receptor. • Four of these SNPs were nonsynonymous and 1 was synonymou.s
Synonymous and Nonsynonymous Substations • Studied in molecular sequence evolution. • Synonymous (silent) substitution is greatly invisible when dealing with natural selection. • Nonsynonymous (amino acid replacement) maybe shown to go under serious selection stress. • These two mutations contribute greatly to understanding the mechanism to DNA sequence evolution. • Observed in natural selection when genetic mutations in certain individual have the advantage to fight off disease. • Allows for scientist to observe these mutations as if a guide to showing possible, functional mutation. • Help guide scientists to figuring out which mutations in CX3CR1 could be involved in progression to disease
The Synonymous SNP Mutation • The synonymous SNP mutation. • This was the only mutation that consisted of a nonconservative exchanging from polar to nonpolar side chain on the 7th transmembrane domain. • This means that the mutation went from a side chain that was polar to a side chain that was nonpolar.
What Was Found From SNP? • SNPs positions T57A and V122I were only found once. • This means that they are not likely to be good representations of genetic variants. • V249I and T280 who had 25.7% and 13.5% presence among the 78 individuals who were infected. • Success in Caucasian populations.
Restriction Fragment Length Polymorphism-Based Method (RFLP) • Used as a genetic linkage map of the human genome. • Develop arbitrary single copies of DNA probes. • DNA probes are competent in exposing DNA sequence polymorphisms. • Each probe defines a place on the chromosome where a specific gene is located. • Amplify DNA segments in order to find V249I and T280M mutations.
Two SNPs (V249I and T280M) showed higher percentile presence were used for further study. • 565 individuals from four HIV cohorts. • Cohorts consisted of: • IMMUNOCO: patients who had intermediate progression. • ALT: patients whom were without symptom (asymptomatic) and had long-term progress. • SEROCO: patients whom had a known seroconversion date. • The three cohorts used RFLP method to identify the different mutations to CX3CR1 from their genomes.
Haplotype and Genotype • Haplotype as a group of alleles of different genes on a single chromosome that are so closely linked that are considered a single unit. • Genotype is a sequence of DNA that is used to make up who you are individually or as a group. • Genotypes are the DNA sequences passed to offspring.
The results from RFLP used (from the four cohorts) to identify the possible genotypes from the CX3CR1 receptor.
Six possible genotypes and, among them, the cohorts that had these genotypes • Genotypes separated into three haplotypes: V249 T280, I249 T280, and I249 M280. • Separated into allele presence among those haplotypes. • The number tallied by adding the genotypes that consisted of the alleles.
Genotype Separated Into Haplotypes and Distributed Among HIV-Infected Cohorts
Hardy Weinberg Expectations • Methods Weinberg used to identify the process of evolution. • Believed that evolution can not be met if seven conditions occur in a population. • Developed a mathematical equation for genotype frequencies in a population and also to track generation changes (Hardy Weinberg equilibrium equation; p2 + 2pq + q2 = 1). • p represents the frequency of the dominant allele. • q is the frequency of the recessive allele for a trait called by a pair of alleles (homozygous recessive). • Used to observe HIV-1 infected individuals who had the allele CX3CR1. • Was expected that 13 indivduals would be homozygous for T280 but, actually, it was 21 individuals (p<0.045 means 4.5%).
What Was Found From the Hardy-Weinberg Expectations? • There was no deviation among uninfected individuals. • Observed CCR5 wild-type among HIV-infected individuals. • CX3CR1-V249I, CCR5-Δ32, nor CCR2-64I experienced a significant digression from the Hardy-Weinberg expectation among HIV-infected and uninfected individuals. • Haplotype I249M280 may cause increase vulnerability to HIV infection, but more study must be done.
Observation of the Progression of HIV With This Haplotype I249 M280 • IMMUNOCO and ALT cohorts were used and odd ratio, 95% confidence interval (CI), and P-value were observed. • Observation were compared with the haplotype V249 T280. • The two CX3CR1 haplotypes were compared with CCR5 (wt and wt/∆32) who were highly frequent among ALT cohorts. • Found that indeed CX3CR1 I249 M280 is involved in HIV disease progression.
Haplotype I249 M280 had less number value among the CX3CR1 cohorts. • IMMUNO had more presence of 3+4+6 haplotype than ALT. • indicating a high progression among cohorts who possessed 3+4+6 haplotype of CX3CR1.
Results • CX3CR1─M280 homozygotes showed an increase progression to clinical AIDS • The same analysis found in Kaplan-Meiers Survival analysis on CD4 decline and progression that were found among heterozygotes and homozygotes wild-type. • CX3CR2—M280 homozygote individuals had a substantial drop in being AIDS free over a period of time.
Receptor Involved With Disease Progression, CX3CR1, Compared With CCR5 and CCR2 Genes • CXCR1—I249 M280 haplotype was not correlatedwith CCR5 and CCR2. • Allele M280 for the CCR5 wild type receptor showed a steep drop in the proportion of AIDS free patients in the same amount of time as CX3CR1—M280. • The CCR5 wt. M280 showed a drop as low as 0.25 in proportion of AIDS free individuals from SEROCO cohort. • CX3CR1—M280 had a proportion of AIDS free individual in ~80 months from SEROCO cohort.
Fractalkine Binding On CX3CR1 Haplotypes (V249 T280, I249 T280, and I249 T280).
Fractalkine Binding Compared With Primary Peripheral Blood Mononuclear Cells (PBMCs) • V249 T280 showed the highest number of specific binding as 125I-labled increased. • I249 T280 had the least number of specific binding as 125I-labled increased, which indicates that T280 has some effect on ligand recognition. • More work must be done to identify whether the effect on expression is due to distribution or stability of receptor protein, modulation of synthesis, or linkage with promoter alleles that
CX3CR1-1249 M280 Is Associated With HIV Disease Progression • There’s a limitation on coreceptor activity. • Accelerated progression maybe indicated from seroconvertor. • Factalkine may not be relevant to mutational analysis. • there’s also the possibility that receptor expression and fratalkine binding could compromise normal immune responses.
