Kenneth B. Beckman

Oxidative Decay of DNA

The study of DNA oxidation has progressed from an exploratory phase, during which its basic biochemistry was established, into a field branching out into numerous areas. Early on, radiation biologists discovered that radiolysis of water generates oxygen free radicals, which are responsible for many of the consequences of irradiating living things. The characterization of radiation-induced oxidative DNA lesions, and the connection between radiation and cancer, caused a surge of interest in DNA oxidation per se and raised the possibility of DNA damage from biological oxidants. Nucleic acid biochemists, cancer biologists, and toxicologists then set out to ask key questions: “how much oxidative DNA damage is there, how does it get there, how and when is it removed, and what are the consequences?” A proliferation of techniques has resulted in the confirmation of the early hypotheses and also delivered some surprises. In this minireview, we have outlined some of the most interesting recent results. Extensive reviews on DNA oxidation published elsewhere have discussed the earlier work in detail (1– 5). A companion minireview by Henle and Linn (6) covers in depth the biochemistry of DNA oxidation.

Germ-line epigenetic modification of the murine A vy allele by nutritional supplementation

Environmental effects on phenotype can be mediated by epigenetic modifications. The epigenetic state of the murine Avy allele is highly variable, and determines phenotypic effects that vary in a mosaic spectrum that can be shifted by in utero exposure to methyl donor supplementation. We have asked if methyl donor supplementation affects the germ-line epigenetic state of the Avy allele. We find that the somatic epigenetic state of Avy is affected by in utero methyl donor supplementation only when the allele is paternally contributed. Exposure to methyl donor supplementation during midgestation shifts Avy phenotypes not only in the mice exposed as fetuses, but in their offspring. This finding indicates that methyl donors can change the epigenetic state of the Avy allele in the germ line, and that the altered state is retained through the epigenetic resetting that takes place in gametogenesis and embryogenesis. Thus a mothers diet may have an enduring influence on succeeding generations, independent of later changes in diet. Although other reports have suggested such heritable epigenetic changes, this study demonstrates that a specific mammalian gene can be subjected to germ-line epigenetic change.

Genome-wide association study identifies multiple susceptibility loci for pulmonary fibrosis

We performed a genome-wide association study of non-Hispanic, white individuals with fibrotic idiopathic interstitial pneumonias (IIPs; n = 1,616) and controls (n = 4,683), with follow-up replication analyses in 876 cases and 1,890 controls. We confirmed association with TERT at 5p15, MUC5B at 11p15 and the 3q26 region near TERC, and we identified seven newly associated loci (Pmeta = 2.4 × 10−8 to 1.1 × 10−19), including FAM13A (4q22), DSP (6p24), OBFC1 (10q24), ATP11A (13q34), DPP9 (19p13) and chromosomal regions 7q22 and 15q14-15. Our results suggest that genes involved in host defense, cell-cell adhesion and DNA repair contribute to risk of fibrotic IIPs.

Characterizing the genetic basis of transcriptome diversity through RNA-sequencing of 922 individuals

Understanding the consequences of regulatory variation in the human genome remains a major challenge, with important implications for understanding gene regulation and interpreting the many disease-risk variants that fall outside of protein-coding regions. Here, we provide a direct window into the regulatory consequences of genetic variation by sequencing RNA from 922 genotyped individuals. We present a comprehensive description of the distribution of regulatory variation--by the specific expression phenotypes altered, the properties of affected genes, and the genomic characteristics of regulatory variants. We detect variants influencing expression of over ten thousand genes, and through the enhanced resolution offered by RNA-sequencing, for the first time we identify thousands of variants associated with specific phenotypes including splicing and allelic expression. Evaluating the effects of both long-range intra-chromosomal and trans (cross-chromosomal) regulation, we observe modularity in the regulatory network, with three-dimensional chromosomal configuration playing a particular role in regulatory modules within each chromosome. We also observe a significant depletion of regulatory variants affecting central and critical genes, along with a trend of reduced effect sizes as variant frequency increases, providing evidence that purifying selection and buffering have limited the deleterious impact of regulatory variation on the cell. Further, generalizing beyond observed variants, we have analyzed the genomic properties of variants associated with expression and splicing and developed a Bayesian model to predict regulatory consequences of genetic variants, applicable to the interpretation of individual genomes and disease studies. Together, these results represent a critical step toward characterizing the complete landscape of human regulatory variation.

Mitochondrial Aging: Open Questionsa

ABSTRACT: Interest in the role of mitochondria in aging has intensified in recent years. This focus on mitochondria originated in part from the free radical theory of aging, which argues that oxidative damage plays a key role in degenerative senescence. Among the numerous mechanisms known to generate oxidants, leakage of the superoxide anion and hydrogen peroxide from the mitochondrial electron transport chain are of particular interest, due to the correlation between species‐specific metabolic rate (“rate of living”) and life span. Phenomenological studies of mitochondrial function long ago noted a decline in mitochondrial function with age, and on‐going research continues to add to this body of knowledge. The extranuclear somatic mutation theory of aging proposes that the accumulation of mutations in the mitochondrial genome may be responsible in part for the mitochondrial phenomenology of aging. Recent studies of mitochondrial DNA (mtDNA) deletions have shown that they increase with age in humans and other mammals. Currently, there exist numerous important and fundamental questions surrounding mitochondria and aging. Among these are (1) How important are mitochondrial oxidants in determining overall cellular oxidative stress? (2) What are the mechanisms of mitochondrial oxidant generation? (3) How are lesions and mutations in mtDNA formed? (4) How important are mtDNA lesions and mutations in causing mitochondrial dysfunction? (5) How are mitochondria regulated, and how does this regulation change during aging? (6) What are the dynamics of mitochondrial turnover? (7) What is the relationship between mitochondrial damage and lipofuscinogenesis? (8) What are the relationships among mitochondria, apopotosis, and aging? and (9) How can mitochondrial function (ATP generation and the establishment of a membrane potential) and dysfunction (oxidant generation) be modulated and degenerative senescence thereby treated?

Resistance Exercise Reverses Aging in Human Skeletal Muscle

Human aging is associated with skeletal muscle atrophy and functional impairment (sarcopenia). Multiple lines of evidence suggest that mitochondrial dysfunction is a major contributor to sarcopenia. We evaluated whether healthy aging was associated with a transcriptional profile reflecting mitochondrial impairment and whether resistance exercise could reverse this signature to that approximating a younger physiological age. Skeletal muscle biopsies from healthy older (N = 25) and younger (N = 26) adult men and women were compared using gene expression profiling, and a subset of these were related to measurements of muscle strength. 14 of the older adults had muscle samples taken before and after a six-month resistance exercise-training program. Before exercise training, older adults were 59% weaker than younger, but after six months of training in older adults, strength improved significantly (P<0.001) such that they were only 38% lower than young adults. As a consequence of age, we found 596 genes differentially expressed using a false discovery rate cut-off of 5%. Prior to the exercise training, the transcriptome profile showed a dramatic enrichment of genes associated with mitochondrial function with age. However, following exercise training the transcriptional signature of aging was markedly reversed back to that of younger levels for most genes that were affected by both age and exercise. We conclude that healthy older adults show evidence of mitochondrial impairment and muscle weakness, but that this can be partially reversed at the phenotypic level, and substantially reversed at the transcriptome level, following six months of resistance exercise training.

Endogenous oxidative damage of mtDNA.

Almost a decade ago, based on analytical measurements of the oxidative DNA adduct 8-oxo-deoxyguanosine (oxo8dG), it was reported that mitochondrial DNA suffers greater endogenous oxidative damage than nuclear DNA. The subsequent discovery that somatic deletions of mitochondrial DNA occur in humans, and that they do so to the greatest extent in metabolically active tissues, strengthened the hypothesis that mitochondrial DNA is particularly susceptible to endogenous oxidative attack. This hypothesis was (and is) appealing for a number of reasons. Nevertheless, solid direct support for the hypothesis is lacking. Since the initial measurements, attempts to repeat the observation of greater oxidation of mitochondrial DNA have resulted in a range of measurements that spans over four orders of magnitude. Moreover, this range includes values that are as low as published values for nuclear DNA. In the last 2 years or so, it has become apparent that the quantification of oxidative DNA adducts is prone to artifactual oxidation. We have reported that the analysis of small quantities of DNA may be particularly susceptible to such interference. Because yields of mitochondrial DNA are generally low, a systematic artifact associated with low quantities of DNA may have elevated the apparent level of adduct oxo8dG in mitochondrial DNA relative to nuclear DNA in some studies. Whatever the cause for the experimental variation, the huge disparity between published measurements of oxidative damage makes it impossible to conclude that mitochondrial DNA suffers greater oxidation than nuclear DNA. Despite the present confusion, however, there are reasons to hypothesize that this is indeed the case. We briefly describe methods being developed by a number of workers that are likely to surmount current obstacles and allow the hypothesis to be tested definitively.

AR intragenic deletions linked to androgen receptor splice variant expression and activity in models of prostate cancer progression

Reactivation of the androgen receptor (AR) during androgen depletion therapy (ADT) underlies castration-resistant prostate cancer (CRPCa). Alternative splicing of the AR gene and synthesis of constitutively active COOH-terminally truncated AR variants lacking the AR ligand-binding domain has emerged as an important mechanism of ADT resistance in CRPCa. In a previous study, we demonstrated that altered AR splicing in CRPCa 22Rv1 cells was linked to a 35-kb intragenic tandem duplication of AR exon 3 and flanking sequences. In this study, we demonstrate that complex patterns of AR gene copy number imbalances occur in PCa cell lines, xenografts and clinical specimens. To investigate whether these copy number imbalances reflect AR gene rearrangements that could be linked to splicing disruptions, we carried out a detailed analysis of AR gene structure in the LuCaP 86.2 and CWR-R1 models of CRPCa. By deletion-spanning PCR, we discovered a 8579-bp deletion of AR exons 5, 6 and 7 in the LuCaP 86.2 xenograft, which provides a rational explanation for synthesis of the truncated AR v567es AR variant in this model. Similarly, targeted resequencing of the AR gene in CWR-R1 cells led to the discovery of a 48-kb deletion in AR intron 1. This intragenic deletion marked a specific CWR-R1 cell population with enhanced expression of the truncated AR-V7/AR3 variant, a high level of androgen-independent AR transcriptional activity and rapid androgen independent growth. Together, these data demonstrate that structural alterations in the AR gene are linked to stable gain-of-function splicing alterations in CRPCa.

Paraoxonase Polymorphisms, Haplotypes, and Enzyme Activity in Latino Mothers and Newborns

Recent studies have demonstrated widespread pesticide exposures in pregnant women and in children. Plasma paraoxonase 1 (PON1) plays an important role in detoxification of various organophosphates. The goals of this study were to examine in the Center for Health Assessment of Mothers and Children of Salinas (CHAMACOS) birth cohort of Latina mothers and their newborns living in the Salinas Valley, California, the frequencies of five PON1 polymorphisms in the coding region (192QR and 55LM) and the promoter region (−162AG, −909CG, and −108CT) and to determine their associations with PON1 plasma levels [phenylacetate arylesterase (AREase)] and enzyme activities of paraoxonase (POase) and chlorpyrifos oxonase (CPOase). Additionally, we report results of PON1 linkage analysis and estimate the predictive value of haplotypes for PON1 plasma levels. We found that PON1−909, PON1−108, and PON1192 had an equal frequency (0.5) of both alleles, whereas PON1−162 and PON155 had lower variant allele frequencies (0.2). Nearly complete linkage disequilibrium was observed among coding and promoter polymorphisms (p < 0.001), except PON1192 and PON1−162 (p > 0.4). Children’s PON1 plasma levels (AREase ranged from 4.3 to 110.7 U/mL) were 4-fold lower than their mothers’ (19.8 to 281.4 U/mL). POase and CPOase activities were approximately 3-fold lower in newborns than in mothers. The genetic contribution to PON1 enzyme variability was higher in newborns (R2 = 25.1% by genotype and 26.3% by haplotype) than in mothers (R2 = 8.1 and 8.8%, respectively). However, haplotypes and genotypes were comparable in predicting PON1 plasma levels in mothers and newborns. Most of the newborn children and some pregnant women in this Latino cohort may have elevated susceptibility to organophosphate toxicity because of their PON1192 genotype and low PON1 plasma levels.

Comparing genetic ancestry and self-described race in African Americans born in the United States and in Africa

Genetic association studies can be used to identify factors that may contribute to disparities in disease evident across different racial and ethnic populations. However, such studies may not account for potential confounding if study populations are genetically heterogeneous. Racial and ethnic classifications have been used as proxies for genetic relatedness. We investigated genetic admixture and developed a questionnaire to explore variables used in constructing racial identity in two cohorts: 50 African Americans and 40 Nigerians. Genetic ancestry was determined by genotyping 107 ancestry informative markers. Ancestry estimates calculated with maximum likelihood estimation were compared with population stratification detected with principal components analysis. Ancestry was approximately 95% west African, 4% European, and 1% Native American in the Nigerian cohort and 83% west African, 15% European, and 2% Native American in the African American cohort. Therefore, self-identification as African American agreed well with inferred west African ancestry. However, the cohorts differed significantly in mean percentage west African and European ancestries (P < 0.0001) and in the variance for individual ancestry (P ≤ 0.01). Among African Americans, no set of questionnaire items effectively estimated degree of west African ancestry, and self-report of a high degree of African ancestry in a three-generation family tree did not accurately predict degree of African ancestry. Our findings suggest that self-reported race and ancestry can predict ancestral clusters but do not reveal the extent of admixture. Genetic classifications of ancestry may provide a more objective and accurate method of defining homogenous populations for the investigation of specific population-disease associations. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1329–38)