Anna Minoprio

Heart Rate as a Predictor of Mortality: The MATISS Project

OBJECTIVES This study sought to verify the independent role of heart rate in the prediction of all-cause, cardiovascular, and noncardiovascular mortality in a low-risk male population. METHODS In an Italian population-based observational study, heart rate was measured in 2533 men, aged 40 to 69 years, between 1984 and 1993. Data on cardiovascular risk factors were collected according to standardized procedures. Vital status was updated to December 1997. RESULTS Of 2533 men followed up (representing 24,457 person-years), 393 men died. Age-adjusted death rates for 5 heart rate levels showed increasing trends. The adjusted hazard rate ratios for each heart rate increment were 1.52 (95% confidence interval [CI] = 1.29, 1.78) for all-cause mortality, 1.63 (95% CI = 1.26, 2.10) for cardiovascular mortality, and 1.47 (95% CI = 1.19, 1.80) for noncardiovascular mortality. Relative risks between extreme levels were more than 2-fold for all endpoints considered. CONCLUSIONS Heart rate is an independent predictor of cardiovascular, noncardiovascular, and total mortality in this Italian middle-aged male population.

MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay†

MUTYH‐associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8‐hydroxyguanine (8‐oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh−/− mouse defective cells. Several parameters, including accumulation of 8‐oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell‐based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild‐type protein. Our cell‐based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity. Hum Mutat 30:1–8, 2009.

Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis

The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G>T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition.

Genotype-phenotype analysis of S326C OGG1 polymorphism: a risk factor for oxidative pathologies.

8-Oxoguanine DNA glycosylase (OGG) activity was measured by an in vitro assay in lymphocytes of healthy volunteers genotyped for various OGG1 polymorphisms. Only homozygous carriers of the polymorphic C326 allele showed a significantly lower OGG activity compared to the homozygous S326 genotype. The purified S326C OGG1 showed a decreased ability to complete the repair synthesis step in a base excision repair reaction reconstituted in vitro. The propensity of this variant to dimerize as well as its catalytic impairment were shown to be enhanced under oxidizing conditions. Mass spectrometry revealed that the extra cysteine of the variant protein is involved in disulfide bonds compatible with significant conformational changes and/or dimerization. We propose that the S326C OGG1 catalytic impairment and its susceptibility to dimerization and disulfide bond formation in an oxidizing environment all concur to decrease repair capacity. Consequently, the C326 homozygous carriers may be at increased risk of oxidative pathologies.

Understanding the role of the Q338H MUTYH variant in oxidative damage repair

The MUTYH DNA–glycosylase is indirectly engaged in the repair of the miscoding 7,8-dihydro-8-oxo-2′-deoxyguanine (8-oxodG) lesion by removing adenine erroneously incorporated opposite the oxidized purine. Inherited biallelic mutations in the MUTYH gene are responsible for a recessive syndrome, the MUTYH-associated polyposis (MAP), which confers an increased risk of colorectal cancer. In this study, we functionally characterized the Q338H variant using recombinant proteins, as well as cell-based assays. This is a common variant among human colorectal cancer genes, which is generally considered, unrelated to the MAP phenotype but recently indicated as a low-penetrance allele. We demonstrate that the Q338H variant retains a wild-type DNA–glycosylase activity in vitro, but it shows a reduced ability to interact with the replication sensor RAD9:RAD1:HUS1 (9–1–1) complex. In comparison with Mutyh−/− mouse embryo fibroblasts expressing a wild-type MUTYH cDNA, the expression of Q338H variant was associated with increased levels of DNA 8-oxodG, hypersensitivity to oxidant and accumulation of the population in the S phase of the cell cycle. Thus, an inefficient interaction of MUTYH with the 9–1–1 complex leads to a repair-defective phenotype, indicating that a proper communication between MUTYH enzymatic function and the S phase checkpoint is needed for effective repair of oxidative damage.

Loss of MUTYH function in human cells leads to accumulation of oxidative damage and genetic instability

The DNA glycosylase MUTYH (mutY homolog (Escherichia coli)) counteracts the mutagenic effects of 8-oxo-7,8-dihydroguanine (8-oxodG) by removing adenine (A) misincorporated opposite the oxidized purine. Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated adenomatous polyposis (MAP). Here we designed new tools to investigate the biochemical defects and biological consequences associated with different MUTYH mutations in human cells. To identify phenotype(s) associated with MUTYH mutations, lymphoblastoid cell lines (LCLs) were derived from seven MAP patients harboring missense as well as truncating mutations in MUTYH. These included homozygous p.Arg245His, p.Gly264TrpfsX7 or compound heterozygous variants (p.Gly396Asp/Arg245Cys, p.Gly396Asp/Tyr179Cys, p.Gly396Asp/Glu410GlyfsX43, p.Gly264TrpfsX7/Ala385ProfsX23 and p.Gly264TrpfsX7/Glu480del). DNA glycosylase assays of MAP LCL extracts confirmed that all these variants were defective in removing A from an 8-oxoG:A DNA substrate, but retained wild-type OGG1 activity. As a consequence of this defect, MAP LCLs accumulated DNA 8-oxodG in their genome and exhibited a fourfold increase in spontaneous mutagenesis at the PIG-A gene compared with LCLs from healthy donors. They were also hypermutable by KBrO3—a source of DNA 8-oxodG—indicating that the relatively modest spontaneous mutator phenotype associated with MUTYH loss can be significantly enhanced by conditions of oxidative stress. These observations identify accumulation of DNA 8-oxodG and a mutator phenotype as likely contributors to the pathogenesis of MUTYH variants.

Influence of defects on the electrophoretic, thermodynamic and dielectric properties of a 21 base pair DNA in solution.

The thermodynamic and dielectric properties of a 21 base pair DNA have been evaluated and compared with those of samples with some defects. In particular, fragments in which the absence of a phosphate group (nick) or of two nucleotides (gap) causes chain interruptions were studied. Measurements of ultraviolet absorption as a function of temperature at different oligomer concentrations and at various ionic strengths were performed. Dielectric spectroscopy at radiofrequencies (1 MHz-1 GHz) was applied on aqueous solutions of the duplexes at 5 degrees C, where the solutes are thermally stable. Dielectric dispersions with 30-40 MHz characteristic frequencies were defined. The results of melting experiments indicate a thermal destabilization of the oligomers containing the defects. Electrophoretic data and the dielectric results show that the conformations of the nicked and control samples are very similar, while the oligomer with a gap is more compact with a different charge distribution at the ends.

Formation and Repair of Mismatches Containing Ribonucleotides and Oxidized Bases at Repeated DNA Sequences.

Background: Whether ribonucleotide and base excision repair (RER and BER) interfere during repair synthesis events is unknown. Results: Complex mispairs containing ribonucleotides and oxidized bases are formed by polymerase β and processed by RER and BER enzymes. Conclusion: Complex lesions modify the efficiency of DNA/RNA repair systems. Significance: This work explains how complex mispairs can compromise BER and RER. The cellular pool of ribonucleotide triphosphates (rNTPs) is higher than that of deoxyribonucleotide triphosphates. To ensure genome stability, DNA polymerases must discriminate against rNTPs and incorporated ribonucleotides must be removed by ribonucleotide excision repair (RER). We investigated DNA polymerase β (POL β) capacity to incorporate ribonucleotides into trinucleotide repeated DNA sequences and the efficiency of base excision repair (BER) and RER enzymes (OGG1, MUTYH, and RNase H2) when presented with an incorrect sugar and an oxidized base. POL β incorporated rAMP and rCMP opposite 7,8-dihydro-8-oxoguanine (8-oxodG) and extended both mispairs. In addition, POL β was able to insert and elongate an oxidized rGMP when paired with dA. We show that RNase H2 always preserves the capacity to remove a single ribonucleotide when paired to an oxidized base or to incise an oxidized ribonucleotide in a DNA duplex. In contrast, BER activity is affected by the presence of a ribonucleotide opposite an 8-oxodG. In particular, MUTYH activity on 8-oxodG:rA mispairs is fully inhibited, although its binding capacity is retained. This results in the reduction of RNase H2 incision capability of this substrate. Thus complex mispairs formed by an oxidized base and a ribonucleotide can compromise BER and RER in repeated sequences.

Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

Abstract DNA trinucleotide repeat (TNR) expansion underlies several neurodegenerative disorders including Huntingtons disease (HD). Accumulation of oxidized DNA bases and their inefficient processing by base excision repair (BER) are among the factors suggested to contribute to TNR expansion. In this study, we have examined whether oxidation of the purine dNTPs in the dNTP pool provides a source of DNA damage that promotes TNR expansion. We demonstrate that during BER of 8-oxoguanine (8-oxodG) in TNR sequences, DNA polymerase β (POL β) can incorporate 8-oxodGMP with the formation of 8-oxodG:C and 8-oxodG:A mispairs. Their processing by the OGG1 and MUTYH DNA glycosylases generates closely spaced incisions on opposite DNA strands that are permissive for TNR expansion. Evidence in HD model R6/2 mice indicates that these DNA glycosylases are present in brain areas affected by neurodegeneration. Consistent with prevailing oxidative stress, the same brain areas contained increased DNA 8-oxodG levels and expression of the p53-inducible ribonucleotide reductase. Our in vitro and in vivo data support a model where an oxidized dNTPs pool together with aberrant BER processing contribute to TNR expansion in non-replicating cells.

EFFECT OF THYMINE DIMER INTRODUCTION IN A 21 BASE PAIR OLIGONUCLEOTIDE

Abstract— It is well known that the pyrimidine dimers are the main damage produced by UV radiation on the DNA structure. However, while studies on the photoproduct structure have been carried out extensively, uncertainties still exist on the implication that a single damaging event has on the overall conformation. In particular, the extension of the damage influence on the polynucleotide chain is a matter of debate. This problem is especially important to understanding some steps of the repair mechanisms. In this study we performed a chemical‐physical characterization of 21 base pair oligonucleotides containing a single thymine dimer in one strand. Thermodynamic parameters were determined by means of thermal denaturation experiments, and static fluorescence measurements were performed to unequivocally define the primary structure‐conformation relationship in this specific case. We used hydroxyl radicals, produced by means of γ‐irradiation of the sample solution, to detect fine structure changes. Our data show that the introduction of a single thymine dimer might cause only a slight distortion of the helix geometry, as judged by the evaluation of the enthalpic and the entropic terms and by the small changes observed in the binding of ethidium bromide to DNA. The modifications in the sugar phosphate backbone subsequent to the damaging event are especially evident, near the thymine dimer, toward the 5′‐end direction in the strand containing the dimer.