F8 haplotype and inhibitor risk: results from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort.

Ancestral background, specifically African descent, confers higher risk for development of inhibitory antibodies to factor VIII (FVIII) in haemophilia A. It has been suggested that differences in the distribution of FVIII gene (F8) haplotypes, and mismatch between endogenous F8 haplotypes and those comprising products used for treatment could contribute to risk. Data from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort were used to determine the association between F8 haplotype 3 (H3) vs. haplotypes 1 and 2 (H1 + H2) and inhibitor risk among individuals of genetically determined African descent. Other variables known to affect inhibitor risk including type of F8 mutation and human leucocyte antigen (HLA) were included in the analysis. A second research question regarding risk related to mismatch in endogenous F8 haplotype and recombinant FVIII products used for treatment was addressed. Haplotype 3 was associated with higher inhibitor risk among those genetically identified (N = 49) as of African ancestry, but the association did not remain significant after adjustment for F8 mutation type and the HLA variables. Among subjects of all racial ancestries enrolled in HIGS who reported early use of recombinant products (N = 223), mismatch in endogenous haplotype and the FVIII proteins constituting the products used did not confer greater risk for inhibitor development. Haplotype 3 was not an independent predictor of inhibitor risk. Furthermore, our findings did not support a higher risk of inhibitors in the presence of a haplotype mismatch between the FVIII molecule infused and that of the individual.

Detecting AIDS restriction genes: from candidate genes to genome-wide association discovery.

The screening of common genetic polymorphisms among candidate genes for AIDS pathology in HIV exposed cohort populations has led to the description of 20 AIDS restriction genes (ARGs), variants that affect susceptibility to HIV infection or to AIDS progression. The combination of high-throughput genotyping platforms and the recent HapMap annotation of some 3 million human SNP variants has been developed for and applied to gene discovery in complex and multi-factorial diseases. Here, we explore novel computational approaches to ARG discovery which consider interacting analytical models, various genetic influences, and SNP-haplotype/LD structure in AIDS cohort populations to determine if these ARGs could have been discovered using an unbiased genome-wide association approach. The procedures were evaluated by tracking the performance of haplotypes and SNPs within ARG regions to detect genetic association in the same AIDS cohort populations in which the ARGs were originally discovered. The methodology captures the signals of multiple non-independent AIDS-genetic association tests of different disease stages and uses association signal strength (odds ratio or relative hazard), statistical significance (p-values), gene influence, internal replication, and haplotype structure together as a multi-facetted approach to identifying important genetic associations within a deluge of genotyping/test data. The complementary approaches perform rather well and predict the detection of a variety of undiscovered ARGs that affect different stages of HIV/AIDS pathogenesis using genome-wide association analyses.

Effect of a single amino acid change in MHC class I molecules on the rate of progression to AIDS.

BACKGROUND: From studies of genetic polymorphisms and the rate of progression from human immunodeficiency virus type 1 (HIV-1) infection to the acquired immunodeficiency syndrome (AIDS), it appears that the strongest susceptibility is conferred by the major-histocompatibility-complex (MHC) class I type HLA-B*35,Cw*04 allele. However, cytotoxic T-lymphocyte responses have been observed against HIV-1 epitopes presented by HLA-B*3501, the most common HLA-B*35 subtype. We examined subtypes of HLA-B*35 in five cohorts and analyzed the relation of structural differences between HLA-B*35 subtypes to the risk of progression to AIDS. METHODS: Genotyping of HLA class I loci was performed for 850 patients who seroconverted and had known dates of HIV-1 infection. Survival analyses with respect to the rate of progression to AIDS were performed to identify the effects of closely related HLA-B*35 subtypes with different peptide-binding specificities. RESULTS: HLA-B*35 subtypes were divided into two groups according to peptide-binding specificity: the HLA-B*35-PY group, which consists primarily of HLA-B*3501 and binds epitopes with proline in position 2 and tyrosine in position 9; and the more broadly reactive HLA-B*35-Px group, which also binds epitopes with proline in position 2 but can bind several different amino acids (not including tyrosine) in position 9. The influence of HLA-B*35 in accelerating progression to AIDS was completely attributable to HLA-B*35-Px alleles, some of which differ from HLA-B*35-PY alleles by only one amino acid residue. CONCLUSIONS: This analysis shows that, in patients with HIV-1 infection, a single amino acid change in HLA molecules has a substantial effect on the rate of progression to AIDS. The different consequences of HLA-B*35-PY and HLA-B*35-Px in terms of disease progression highlight the importance of the epitope specificities of closely related class I molecules in the immune defense against HIV-1.

Polygenic and multifactorial disease gene association in man: Lessons from AIDS.

In an age when the majority of monogenic human disease genes have been identified, a particular challenge for the coming generation of human geneticists will be resolving complex polygenic and multifactorial diseases. The tools of molecular and population genetic association have much potential as well as peril in uncovering small cryptic genetic effects in disease. We have used a candidate gene approach to identify eight distinct human loci with alleles that in different ways influence the outcome of exposure to HIV-1, the AIDS virus. The successes in these gene hunts have validated the approach and illustrate the strengths and limitations of association analysis in an actual case history. The integration of genetic associations, well-described clinical cohorts, extensive basic research on AIDS pathogenesis, and functional interpretation of gene connections to disease offers a formula for detecting such genes in complex human genetic phenotypes.

Influence of CCR5 promoter haplotypes on AIDS progression in African-Americans.

OBJECTIVES: To test the hypothesis that the CCR5 promoter variants in HIV-1-infected African-Americans affect the rate of progression to AIDS and to determine the extent of linkage disequilibrium between the CCR5P1 allele and the CCR5 59029A variant (referred to here as CCR5-2459A), both of which have been shown independently to accelerate AIDS progression in Caucasians. DESIGN: We used survival analysis to assess the effects of CCR5 promoter variants in HIV-1 seroincident Caucasians and African-Americans. SUBJECTS AND METHODS: Genotypes were determined for 806 Caucasians and 1067 African-Americans, which included 700 seroconverters, enrolled in four HIV/AIDS natural history cohort studies. These genotypes were used to determine linkage and haplotypes for CCR2 and CCR5 alleles. Survival analysis was used to assess the effect of CCR2, CCR5, and CCR5 promoter haplotypes on progression to AIDS in seroincident African-Americans. RESULTS: A survey of Caucasians and African-Americans demonstrated complete linkage disequilibrium between CCR5P1 and CCR5-2459A sites. The composite CCR5P1 haplotype (including the CCR5-2459A allele) is shown to be associated with rapid progression to AIDS endpoints in both African-American and Caucasian cohorts, but the effect is recessive in Caucasians and dominant in African-Americans. This is probably due to the presence of modulating genes or as yet unidentified polymorphisms that may differ between racial groups.

High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA.

The nucleocapsid (N) protein of mouse hepatitis virus (MHV) is the major virion structural protein. It associates with both viral genomic RNA and subgenomic mRNAs and has structural and non-structural roles in replication including viral RNA-dependent RNA transcription, genome replication, encapsidation and translation. These processes all involve RNA-protein interactions between the N protein and viral RNAs. To better understand the RNA-binding properties of this multifunctional protein, the N protein was expressed in Escherichia coli as a chimeric protein fused to glutathione-S-transferase (GST). Biochemical analyses of RNA-binding properties were performed on full-length and partial N protein segments to define the RNA-binding domain. The full-length N protein and the GST-N protein fusion product had similar binding activities with a dissociation constant (K(d)) of 14 nM when the MHV 5'-leader sequence was used as ligand. The smallest N protein fragment which retained RNA-binding activity was a 55 aa segment containing residues 177-231 which bound viral RNA with a K(d) of 32 nM. A consensus viral sequence recognized by the N protein was inferred from these studies; AAUCYAAAC was identified to be the potential minimum ligand for the N protein. Although the core UCYAA sequence is often tandemly repeated in viral genomes, ligands containing one or more repeats of UCYAA showed no difference in binding to the N protein. Together these data demonstrate a high-affinity, specific interaction between the N protein and a conserved RNA sequence present at the 5'-ends of MHV mRNA.

New class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS.

The genetics of resistance to infection by HIV-1 cohort consists of 200 slow and 75 rapid progressors to AIDS corresponding to the extremes of HIV disease outcome of 20,000 Caucasians of European descent. A comprehensive analysis of HLA class I and class II genes in this highly informative cohort has identified HLA alleles associated with fast or slow progression, including several not described previously. A quantitative analysis shows an overall HLA influence independent of and equal in magnitude (for the protective effect) to the effect of the CCR5-Delta32 mutation. Among HLA class I genes, A29 (p = 0.001) and B22 (p < 0.0001) are significantly associated with rapid progression, whereas B14 (p = 0.001) and C8 (p = 0.004) are significantly associated with nonprogression. The class I alleles B27, B57, C14 (protective), and C16, as well as B35 (susceptible), are also influential, but their effects are less robust. Influence of class II alleles was only observed for DR11. These results confirm the influence of the immune system on disease progression and may have implications on peptide-based vaccine development.

HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage.

A selective advantage against infectious disease associated with increased heterozygosity at the human major histocompatibility complex [human leukocyte antigen (HLA) class I and class II] is believed to play a major role in maintaining the extraordinary allelic diversity of these genes. Maximum HLA heterozygosity of class I loci (A, B, and C) delayed acquired immunodeficiency syndrome (AIDS) onset among patients infected with human immunodeficiency virus-type 1 (HIV-1), whereas individuals who were homozygous for one or more loci progressed rapidly to AIDS and death. The HLA class I alleles B*35 and Cw*04 were consistently associated with rapid development of AIDS-defining conditions in Caucasians. The extended survival of 28 to 40 percent of HIV-1-infected Caucasian patients who avoided AIDS for ten or more years can be attributed to their being fully heterozygous at HLA class I loci, to their lacking the AIDS-associated alleles B*35 and Cw*04, or to both.

Mouse hepatitis virus nucleocapsid protein as a translational effector of viral mRNAs.

The mouse hepatitis virus (MHV) nucleocapsid protein stimulated translation of a chimeric reporter mRNA containing an intact MHV 5'-untranslated region and the chloramphenicol acetyltransferase (CAT) coding region. The nucleocapsid protein binds specifically the tandemly repeated-UCYAA- of the MHV leader. This RNA sequence is the same as the intergenic motif found in the genome RNA. Preferential translation of viral mRNA in MHV infected cells is stimulated in part by this interaction and represents a specific, positive translational control mechanism employed by coronaviruses.

Genetic restriction of AIDS pathogenesis by an SDF-1 chemokine gene variant. ALIVE Study, Hemophilia Growth and Development Study (HGDS), Multicenter AIDS Cohort Study (MACS), Multicenter Hemophilia Cohort Study (MHCS), San Francisco City Cohort (SFCC)

Stromal-derived factor (SDF-1) is the principal ligand for CXCR4, a coreceptor with CD4 for T lymphocyte cell line-tropic human immunodeficiency virus-type 1 (HIV-1). A common polymorphism, SDF1-3'A, was identified in an evolutionarily conserved segment of the 3' untranslated region of the SDF-1 structural gene transcript. In the homozygous state, SDF1-3'A/3'A delays the onset of acquired immunodeficiency syndrome (AIDS), according to a genetic association analysis of 2857 patients enrolled in five AIDS cohort studies. The recessive protective effect of SDF1-3'A was increasingly pronounced in individuals infected with HIV-1 for longer periods, was twice as strong as the dominant genetic restriction of AIDS conferred by CCR5 and CCR2 chemokine receptor variants in these populations, and was complementary with these mutations in delaying the onset of AIDS.

Frequency of HLA allele-specific peptide motifs in HIV-1 proteins correlates with the allele's association with relative rates of disease progression after HIV-1 infection.

An HLA allele-specific cytotoxic T lymphocyte response is thought to influence the rate of disease progression in HIV-1-infected individuals. In a prior study of 139 HIV-1-infected homosexual men, we identified HLA class I alleles and observed an association of specific alleles with different relative hazards for progression to AIDS. Seeking an explanation for this association, we searched HIV-1 protein sequences to determine the number of peptides matching motifs defined by combinations of specific amino acids reported to bind 16 class I alleles. Analyzing complete sequences of 12 clade B HIV isolates, we determined the number of allele motifs that were conserved (occurring in all 12 isolates) and nonconserved (occurring in only one isolate), as well as the average number of allele motifs per isolate. We found significant correlations with an allele's association with disease progression for counts of conserved motifs in gag (R = 0.73; P = 0.002), pol (R = 0.58, P = 0.024), gp120 (R = 0.78, P = 0.00056), and total viral protein sequences (R = 0.67, P = 0.0058) and also for counts of nonconserved motifs in gag (R = 0.62, P = 0.013), pol (R = 0.74, P = 0.0017), gp41 (R = 0.52, P = 0.046), and total viral protein (R = 0.71, P = 0.0033). We also found significant correlations for the average number of motifs per isolate for gag, pol, gp120, and total viral protein. This study provides a plausible functional explanation for the observed association of different HLA alleles with variable rates of disease progression.

Modeling defective interfering virus therapy for AIDS: conditions for DIV survival.

The administration of a genetically engineered defective interfering virus (DIV) that interferes with HIV-1 replication has been proposed as a therapy for HIV-1 infection and AIDS. The proposed interfering virus, which is designed to superinfect HIV-1 infected cells, carries ribozymes that cleave conserved regions in HIV-1 RNA that code for the viral envelope protein. Thus DIV infection of HIV-1 infected cells should reduce or eliminate viral production by these cells. The success of this therapeutic strategy will depend both on the intercellular interaction of DIV and HIV-1, and on the overall dynamics of virus and T cells in the body. To study these dynamical issues, we have constructed a mathematical model of the interaction of HIV-1, DIV, and CD4+ cells in vivo. The results of both mathematical analysis and numerical simulation indicate that survival of the engineered DIV purely on a peripheral blood HIV-1 infection is unlikely. However, analytical results indicate that DIV might well survive on HIV-1 infected CD4+ cells in lymphoid organs such as lymph nodes and spleen, or on other HIV-1 infected cells in these organs.

Coronavirus translational regulation: leader affects mRNA efficiency.

Cells infected with the murine coronavirus, mouse hepatitis virus (MHV), show decreased host protein synthesis concomitant with an increase in viral protein synthesis. We examined the in vitro translation property of the conserved MHV 5'-leader RNA sequence by constructing chimeric mRNAs in which the 72-nt 5'-leader of M protein mRNA (A59 strain) was positioned upstream of the human alpha-globin coding region in a T7 expression vector. Synthetic 5'-capped transcripts of these mRNA constructs were translated in cell-free extracts prepared from uninfected and MHV-infected murine DBT cells. Nonviral mRNAs translated readily in both uninfected and infected cell-free extracts. By contrast, replacement of the human alpha-globin 5'-untranslated region (UR) with the MHV 5'-leader increased translation ca. three- to fourfold in cell-free extracts from MHV-infected cells versus translation in extracts from uninfected cells. Chimeric globin mRNA containing the reverse complementary sequence of the viral leader RNA in the 5'-UR showed no such increase in translation, indicating sequence specificity for the effect. A 13-nt region (-UCUAAUCCAAACA-) immediately proximal to the start codon was found to be important for the increased translation of the MHV leader-containing mRNAs. These data indicate that the apparent down-regulation of host translation is not primarily due to an inhibition of host translation but also involves a significant stimulation of viral translation in cis by a structural feature of the MHV 5'-leader RNA sequence in conjunction with a virus-specified or virus-induced factor.

Localization of the RNA-binding domain of mouse hepatitis virus nucleocapsid protein.

The 454-amino acid nucleocapsid (N) protein of mouse hepatitis virus (MHV) binds the leader RNA sequence located at the 5' ends of all plus-sense genomic and subgenomic viral mRNAs. Purified N protein was cleaved with formic acid to determine which domain interacts with the leader RNA sequence. Incubation at 42 degrees C resulted in partial cleavage into two fragments of M(r)s of approximately 32K and 37K and three fragments of 17K, 16K and 14K. Incubation at 56 degrees C resulted in complete cleavage yielding only the three lower molecular mass products. Both the 32K and 37K partial cleavage products and one of the complete cleavage products bind MHV leader RNA, suggesting that the central region of the N protein contains the RNA-binding domain. Monoclonal antibody mapping of the cleavage products confirmed that the MHV leader RNA binding domain is contained within the central 140-amino acid fragment, comprising amino acids 169 to 308. Analysis of the amino acids within this domain indicates no similarity to any previously described RNA-binding protein, suggesting that N protein may possess a unique RNA-binding motif.

A mechanism of immune escape by slow-replicating HIV strains.

Strains of human immunodeficiency virus (HIV) differ greatly in their ability to replicate in T4 cells. Fast-replicating strains are observed during the early and late stages of HIV infection, while slow-replicating strains prevail during the intermediate, latent, stage. The prevalence of slow-replicating strains has been attributed to these strains' ability to escape the immune response. However, how these strains are able to avoid being eliminated by an immune response for several years has not been explained. Recent experiments indicate that HIV may be selectively transferred from infected macrophages to T4 cells specific for HIV antigens. Thus, HIV may preferentially infect those T4 cells necessary for generating a protective immune response. To determine the conditions under which an HIV-specific immune response can be blocked, we developed a mathematical model incorporating the process of viral transfer from infected macrophages to HIV-specific T4 cells along with the known processes of macrophage-T4 interaction, immune stimulation, and viral infection of T4 cells and macrophages. Our model shows that the mechanism of viral transfer to HIV-specific T4 cells can allow slow-replicating strains of HIV to escape immune response under conditions in which an immune response occurs against fast-replicating strains. The model also suggests that in addition to slow replication, the ability to reduce or block T-cell activation may be an important characteristic of escape mutants.

Trace elements in the hair of healthy and malnourished children.

Pre-school age children from the metropolitan area of Guadalajara, Mexico, consisting of 47 malnourished children were evaluated for mineral status by hair samples and nutritional status by anthropometric measurements and haemoglobin levels. Hair minerals were determined by either atomic absorption spectroscopy or instrumental neutron activation analysis. Malnourished children had significantly lower hair zinc while the iron and copper levels were significantly higher. Hair concentrations of calcium, magnesium, and manganese were not significantly different between healthy and malnourished children. Sixty per cent of the malnourished children's haemoglobin values were either marginal or deficient while 45 per cent of the haematocrits readings were either marginal or deficient. Malnourished children in weight-for-age category were 81 per cent malnourished; by weight-for-height 55 per cent were malnourished and by height-for-age 59 per cent were malnourished. The overall nutritional status of malnourished children can be characterized as mild to severe malnutrition with a chronic stunting of growth. Hair zinc values were a good indicator of nutritional status.

Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription.

The interaction of the mouse hepatitis virus (MHV) nucleocapsid protein (N) and viral RNA was examined. Monoclonal antibody specific for N protein coimmunoprecipitated MHV genomic RNA as well as all six MHV subgenomic mRNAs found in MHV-infected cells. In contrast, monoclonal antibodies to the MHV E2 or E1 envelope glycoproteins, an anti-I-A monoclonal antibody, and serum samples from lupus patients did not immunoprecipitate the MHV mRNAs. Moreover, the anti-N monoclonal antibody did not coimmunoprecipitate vesicular stomatitis virus RNA or host cell RNA under conditions which immunoprecipitated all MHV RNAs. These data suggest a specific interaction between the N protein and the virus-specific mRNAs. Both the membrane-bound and cytosolic small MHV leader-specific RNAs of greater than 65 nucleotides long were immunoprecipitated only by anti-N monoclonal antibody. These data suggest that an N binding site is present within the leader RNA sequences at a site at least 65 nucleotides from the 5' end of genomic RNA and all six subgenomic mRNAs. The larger leader-containing RNAs originating from mRNA 1 and mRNA 6, as well as the MHV negative-stranded RNA, were also immunoprecipitated by the anti-N monoclonal antibody. These data indicate that the MHV N protein is associated with MHV-specific RNAs and RNA intermediates and may play an important functional role during MHV transcription and replication.

Punctate intranodal gold deposits simulating microcalcifications on mammograms.

Some axillary lymph nodes are frequently seen on mammograms. Rarely, such nodes will demonstrate opacities that appear to be multiple punctate calcific deposits. The literature is of little guidance as to the meaning of this appearance. One report indicates that breast cancers that are manifested as extensive punctate calcifications very rarely occur in this pattern in metastatically involved axillary lymph nodes. The authors suggest that such punctate densities are much more often indicative of intranodal gold deposits and that they occur almost invariably in patients with rheumatoid arthritis who have undergone prolonged chrysotherapy. This observation was confirmed in one patient and suspected in three others with similar clinical histories.

Phosphorylation of the mouse hepatitis virus nucleocapsid protein.

Analysis of the radiolabeled tryptic peptides derived from the nucleocapsid proteins of two serotypes of mouse hepatitis virus showed each to have a small number of unique peptides; however, two biologically distinct variants of the JHM strain appeared identical. Analysis of [32P]-labeled nucleocapsid-derived peptides showed that phosphorylation occurs at only a few sites and that all three viruses differed in the sites of phosphorylation. No differences in the sites of phosphorylation were found between the nucleocapsid proteins derived from purified virions and the membranes or the cytosol of infected cells, suggesting that post-translational phosphorylation plays no role in the regulation of viral assembly. These data show unequivocal evidence that the nucleocapsid proteins of mouse hepatitis virus strains differ in the sites of phosphorylation.