Effects of the number of markers per haplotype and clustering of haplotypes on the accuracy of QTL mapping and prediction of genomic breeding values

The aim of this paper was to compare the effect of haplotype definition on the precision of QTL-mapping and on the accuracy of predicted genomic breeding values. In a multiple QTL model using identity-by-descent (IBD) probabilities between haplotypes, various haplotype definitions were tested i.e. including 2, 6, 12 or 20 marker alleles and clustering base haplotypes related with an IBD probability of > 0.55, 0.75 or 0.95. Simulated data contained 1100 animals with known genotypes and phenotypes and 1000 animals with known genotypes and unknown phenotypes. Genomes comprising 3 Morgan were simulated and contained 74 polymorphic QTL and 383 polymorphic SNP markers with an average r² value of 0.14 between adjacent markers. The total number of haplotypes decreased up to 50% when the window size was increased from two to 20 markers and decreased by at least 50% when haplotypes related with an IBD probability of > 0.55 instead of > 0.95 were clustered. An intermediate window size led to more precise QTL mapping. Window size and clustering had a limited effect on the accuracy of predicted total breeding values, ranging from 0.79 to 0.81. Our conclusion is that different optimal window sizes should be used in QTL-mapping versus genome-wide breeding value prediction.     

Comparison of the mutagenic activity of the benzene metabolites, hydroquinone and para-benzoquinone in the supF forward mutation assay: a role for minor DNA adducts formed from hydroquinone in benzene mutagenicity

Benzene, a ubiquitous environmental pollutant and occupational hazardous chemical, is a recognised human leukaemogen and rodent carcinogen. The mechanism by which benzene exerts its carcinogenic effects is to date unknown but it is considered that mutations induced by benzene-DNA adducts may play a role. The benzene metabolite, para-benzoquinone (p-BQ) following reaction in vitro with DNA, forms four major adducts, which include two adducts on 2'-deoxyguanosine 3'-monophosphate (dGp). Reaction of DNA with the benzene metabolite hydroquinone (HQ) results in only one major DNA adduct, which corresponds to one of the dGp adducts formed following reaction with p-BQ. The mutagenicity of the adducts formed from these two benzene metabolites was investigated using the supF forward mutation assay. Metabolite-treated plasmid (pSP189) containing the supF gene was replicated in human Ad293 cells before being screened in indicator bacteria. Treatment with 5-20 mM p-BQ gave a 12 to 40-fold increase in mutation rate compared to 5-20 mM HQ treatment, a result reflected in the level of DNA modification observed (8 to 26-fold increase compared to HQ treatment). Treatment with p-BQ gave equal numbers of GC --> TA transversions and GC --> AT transitions, whereas treatment with HQ gave predominantly GC-->AT transitions. The spectra of mutations achieved for the two individual treatments were shown to be significantly different (P = 0.004). A combination of both treatments also resulted in a high level of GC --> AT transitions and a synergistic increase in the number of multiple mutations, which again predominated as GC --> AT transitions. Sites of mutational hotspots were observed for both individual treatments and one mutational hotspot was observed in the multiple mutations for the combined treatment. These results suggest that the dGp adducts formed from benzene metabolite treatment may play an important role in the mutagenicity and myelotoxicity of benzene.     

The effect of high-density lipoproteins on the formation of lipid/protein conjugates during in vitro oxidation of low-density lipoprotein

High-density lipoprotein (HDL) incubated with low-density lipoprotein (LDL) under oxidising conditions has previously been reported to decrease the accumulation of lipid peroxides on LDL and to diminish the biological effects of LDL, which would have been present had it been oxidatively modified in the absence of HDL. Thus far direct evidence that oxidative modification of LDL is diminished by HDL has, however, been lacking. We used electrospray ionisation mass spectrometry (ESI-MS) to detect 4-hydroxy-2-nonenal (HNE)-modified histidine residues of tryptic fragments of LDL which had been subject to Cu(2+) induced oxidation both in the presence and absence of human or avian HDL. HNE-modified angiotensin II was introduced into the incubation mixture as an internal standard and to check that HDL did not interfere in the detection of HNE-modified peptides non-specifically. Our results confirmed earlier reports that HNE modification of histidine occurs during the oxidation of LDL and for the first time revealed a marked attenuation of the process in the presence of human HDL with no effect on the detection of HNE-modified angiotensin II by ESI-MS. Avian HDL, which lacks the anti-oxidative enzyme paraoxonase, did not affect the formation of apo B adducts. Our findings therefore suggest that covalent linkage of lipid peroxidation products to LDL protein as well as the accumulation of lipid peroxides on LDL is diminished in the presence of HDL containing paraoxonase.     

Proteomic analysis of chronic lymphocytic leukemia subtypes with mutated or unmutated Ig V(H) genes

Chronic lymphocytic leukemia (CLL) is a common hematopoietic malignant disease with variable outcome. CLL has been divided into distinct groups based on whether somatic hypermutation has occurred in the variable region of the immunoglobulin heavy-chain locus or alternatively if the cells express higher levels of the CD38 protein. We have analyzed the proteome of 12 cases of CLL (six mutated (M-CLL) and six unmutated (UM-CLL) immunoglobulin heavy-chain loci; seven CD38-negative and five CD38-positive) using two-dimensional electrophoresis and mass spectrometry. Statistical evaluation using principal component analysis indicated significant differences in patterns of protein expression between the cases with and without somatic mutation. Specific proteins indicated by principal component analysis as varying between the prognostic groups were characterized using mass spectrometry. The levels of F-actin-capping protein beta subunit, 14-3-3 beta protein, and laminin-binding protein precursor were significantly increased in M-CLL relative to UM-CLL. In addition, primary sequence data from tandem mass spectrometry showed that nucleophosmin was present as several protein spots in M-CLL but was not detected in UM-CLL samples, suggesting that several post-translationally modified forms of nucleophosmin vary between these two sample groups. No specific differences were found between CD38-positive and -negative patient samples using the same approach. The results presented show that proteomic analysis can complement other approaches in identifying proteins that may have potential value in the biological and diagnostic distinction between important clinical subtypes of CLL.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

Mutation spectra induced by alpha-acetoxytamoxifen-DNA adducts in human DNA repair proficient and deficient (xeroderma pigmentosum complementation group A) cells

Tamoxifen, a breast cancer drug, has recently been approved for the chemoprevention of this disease. However, tamoxifen causes hepatic carcinomas in rats through a genotoxic mechanism and increases the risk of endometrial tumors in women. DNA adducts have been detected at low levels in human endometrium, and there is much interest in determining whether DNA damage plays a role in tamoxifen-induced endometrial carcinogenesis. This study investigates the mutagenicity of tamoxifen DNA adducts formed by alpha-acetoxytamoxifen, a reactive ester producing the major DNA adduct formed in livers of tamoxifen-treated rats. pSP189 plasmid DNA containing the supF gene was treated with alpha-acetoxytamoxifen and adduct levels (0.5-8.0 adducts per plasmid) determined by (32)P-postlabeling. Adducted plasmids were transfected into nucleotide excision repair proficient (GM00637) or deficient (GM04429, XPA) human fibroblasts. After replication, plasmids were recovered and screened in indicator bacteria. Relative mutation frequencies increased with the adduct level, with 1.3-3.6-fold higher numbers of mutations in the XP cells compared to the GM00637 cells, indicating that NER plays a significant role in the removal of these particular tamoxifen DNA adducts. The majority of sequence alterations (91-96%) occurred at GC base pairs, as did mutation hotspots, although the type and position of mutations was cell-specific. In both cell lines, as the adduct level increased, the proportion of GC --> AT transitions decreased and GC --> TA transversions, mutations known to arise from the major tamoxifen adducts, increased. Given the high mutagenicity of dG-N(2)-tamoxifen adducts, if not excised, they may potentially contribute to the initiation of endometrial cancer in women.