John K. Wiencke

DNA methylation arrays as surrogate measures of cell mixture distribution

BackgroundThere has been a long-standing need in biomedical research for a method that quantifies the normally mixed composition of leukocytes beyond what is possible by simple histological or flow cytometric assessments. The latter is restricted by the labile nature of protein epitopes, requirements for cell processing, and timely cell analysis. In a diverse array of diseases and following numerous immune-toxic exposures, leukocyte composition will critically inform the underlying immuno-biology to most chronic medical conditions. Emerging research demonstrates that DNA methylation is responsible for cellular differentiation, and when measured in whole peripheral blood, serves to distinguish cancer cases from controls.ResultsHere we present a method, similar to regression calibration, for inferring changes in the distribution of white blood cells between different subpopulations (e.g. cases and controls) using DNA methylation signatures, in combination with a previously obtained external validation set consisting of signatures from purified leukocyte samples. We validate the fundamental idea in a cell mixture reconstruction experiment, then demonstrate our method on DNA methylation data sets from several studies, including data from a Head and Neck Squamous Cell Carcinoma (HNSCC) study and an ovarian cancer study. Our method produces results consistent with prior biological findings, thereby validating the approach.ConclusionsOur method, in combination with an appropriate external validation set, promises new opportunities for large-scale immunological studies of both disease states and noxious exposures.

Aging and environmental exposures alter tissue-specific DNA methylation dependent upon CPG island context

Epigenetic control of gene transcription is critical for normal human development and cellular differentiation. While alterations of epigenetic marks such as DNA methylation have been linked to cancers and many other human diseases, interindividual epigenetic variations in normal tissues due to aging, environmental factors, or innate susceptibility are poorly characterized. The plasticity, tissue-specific nature, and variability of gene expression are related to epigenomic states that vary across individuals. Thus, population-based investigations are needed to further our understanding of the fundamental dynamics of normal individual epigenomes. We analyzed 217 non-pathologic human tissues from 10 anatomic sites at 1,413 autosomal CpG loci associated with 773 genes to investigate tissue-specific differences in DNA methylation and to discern how aging and exposures contribute to normal variation in methylation. Methylation profile classes derived from unsupervised modeling were significantly associated with age (P<0.0001) and were significant predictors of tissue origin (P<0.0001). In solid tissues (n = 119) we found striking, highly significant CpG island–dependent correlations between age and methylation; loci in CpG islands gained methylation with age, loci not in CpG islands lost methylation with age (P<0.001), and this pattern was consistent across tissues and in an analysis of blood-derived DNA. Our data clearly demonstrate age- and exposure-related differences in tissue-specific methylation and significant age-associated methylation patterns which are CpG island context-dependent. This work provides novel insight into the role of aging and the environment in susceptibility to diseases such as cancer and critically informs the field of epigenomics by providing evidence of epigenetic dysregulation by age-related methylation alterations. Collectively we reveal key issues to consider both in the construction of reference and disease-related epigenomes and in the interpretation of potentially pathologically important alterations.

Glioma Groups Based on 1p/19q, IDH, and TERT Promoter Mutations in Tumors

BACKGROUND The prediction of clinical behavior, response to therapy, and outcome of infiltrative glioma is challenging. On the basis of previous studies of tumor biology, we defined five glioma molecular groups with the use of three alterations: mutations in the TERT promoter, mutations in IDH, and codeletion of chromosome arms 1p and 19q (1p/19q codeletion). We tested the hypothesis that within groups based on these features, tumors would have similar clinical variables, acquired somatic alterations, and germline variants. METHODS We scored tumors as negative or positive for each of these markers in 1087 gliomas and compared acquired alterations and patient characteristics among the five primary molecular groups. Using 11,590 controls, we assessed associations between these groups and known glioma germline variants. RESULTS Among 615 grade II or III gliomas, 29% had all three alterations (i.e., were triple-positive), 5% had TERT and IDH mutations, 45% had only IDH mutations, 7% were triple-negative, and 10% had only TERT mutations; 5% had other combinations. Among 472 grade IV gliomas, less than 1% were triple-positive, 2% had TERT and IDH mutations, 7% had only IDH mutations, 17% were triple-negative, and 74% had only TERT mutations. The mean age at diagnosis was lowest (37 years) among patients who had gliomas with only IDH mutations and was highest (59 years) among patients who had gliomas with only TERT mutations. The molecular groups were independently associated with overall survival among patients with grade II or III gliomas but not among patients with grade IV gliomas. The molecular groups were associated with specific germline variants. CONCLUSIONS Gliomas were classified into five principal groups on the basis of three tumor markers. The groups had different ages at onset, overall survival, and associations with germline variants, which implies that they are characterized by distinct mechanisms of pathogenesis. (Funded by the National Institutes of Health and others.).

Variants in the CDKN2B and RTEL1 regions are associated with high-grade glioma susceptibility.

The causes of glioblastoma and other gliomas remain obscure. To discover new candidate genes influencing glioma susceptibility, we conducted a principal component–adjusted genome-wide association study (GWAS) of 275,895 autosomal variants among 692 adult high-grade glioma cases (622 from the San Francisco Adult Glioma Study (AGS) and 70 from the Cancer Genome Atlas (TCGA)) and 3,992 controls (602 from AGS and 3,390 from Illumina iControlDB (iControls)). For replication, we analyzed the 13 SNPs with P < 10−6 using independent data from 176 high-grade glioma cases and 174 controls from the Mayo Clinic. On 9p21, rs1412829 near CDKN2B had discovery P = 3.4 × 10−8, replication P = 0.0038 and combined P = 1.85 × 10−10. On 20q13.3, rs6010620 intronic to RTEL1 had discovery P = 1.5 × 10−7, replication P = 0.00035 and combined P = 3.40 × 10−9. For both SNPs, the direction of association was the same in discovery and replication phases.

Human Lymphocytes Exposed to Low Doses of Ionizing Radiations Become Refractory to High Doses of Radiation as Well as to Chemical Mutagens that Induce Double-strand Breaks in DNA

Human lymphocytes exposed to low doses of ionizing radiation from incorporated tritiated thymidine or from X-rays become less susceptible to the induction of chromatid breaks by high doses of X-rays. This response can be induced by 0.01 Gy (1 rad) of X-rays, and has been attributed to the induction of a repair mechanism that causes the restitution of X-ray-induced chromosome breaks. Because the major lesions responsible for the induction of chromosome breakage are double-strand breaks in DNA, attempts have been made to see if the repair mechanism can affect various types of clastogenic lesions induced in DNA by chemical mutagens and carcinogens. When cells exposed to 0.01 Gy of X-rays or to low doses of tritiated thymidine were subsequently challenged with high doses of tritiated thymidine or bleomycin, which can induce double-strand breaks in DNA, or mitomycin C, which can induce cross-links in DNA, approximately half as many chromatid breaks were induced as expected. When, on the other hand, the cells were challenged with the alkylating agent methyl methanesulfonate (MMS), which can produce single-strand breaks in DNA, approximately twice as much damage was found as was induced by MMS alone. The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive response that follows exposure to low doses of alkylating agents.

The landscape of recombination in African Americans

Recombination, together with mutation, gives rise to genetic variation in populations. Here we leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and we identify about 2,500 recombination hotspots that are active in people of West African ancestry but nearly inactive in Europeans. The probability of a crossover at these hotspots is almost fully controlled by the alleles an individual carries at PRDM9 (P value < 10−245). We identify a 17-base-pair DNA sequence motif that is enriched in these hotspots, and is an excellent match to the predicted binding target of PRDM9 alleles common in West Africans and rare in Europeans. Sites of this motif are predicted to be risk loci for disease-causing genomic rearrangements in individuals carrying these alleles. More generally, this map provides a resource for research in human genetic variation and evolution.

DNA Methylation, Isocitrate Dehydrogenase Mutation, and Survival in Glioma

BACKGROUND Although much is known about molecular and chromosomal characteristics that distinguish glioma histological subtypes, DNA methylation patterns of gliomas and their association with other tumor features such as mutation of isocitrate dehydrogenase (IDH) genes have only recently begun to be investigated. METHODS DNA methylation of glioblastomas, astrocytomas, oligodendrogliomas, oligoastrocytomas, ependymomas, and pilocytic astrocytomas (n = 131) from the Brain Tumor Research Center at the University of California San Francisco, as well as nontumor brain tissues (n = 7), was assessed with the Illumina GoldenGate methylation array. Methylation data were subjected to recursively partitioned mixture modeling (RPMM) to derive methylation classes. Differential DNA methylation between tumor and nontumor was also assessed. The association between methylation class and IDH mutation (IDH1 and IDH2) was tested using univariate and multivariable analysis for tumors (n = 95) with available substrate for sequencing. Survival of glioma patients carrying mutant IDH (n = 57) was compared with patients carrying wild-type IDH (n = 38) using a multivariable Cox proportional hazards model and Kaplan-Meier analysis. All statistical tests were two-sided. RESULTS We observed a statistically significant association between RPMM methylation class and glioma histological subtype (P < 2.2 × 10(-16)). Compared with nontumor brain tissues, across glioma tumor histological subtypes, the differential methylation ratios of CpG loci were statistically significantly different (permutation P < .0001). Methylation class was strongly associated with IDH mutation in gliomas (P = 3.0 × 10(-16)). Compared with glioma patients whose tumors harbored wild-type IDH, patients whose tumors harbored mutant IDH showed statistically significantly improved survival (hazard ratio of death = 0.27, 95% confidence interval = 0.10 to 0.72). CONCLUSION The homogeneity of methylation classes for gliomas with IDH mutation, despite their histological diversity, suggests that IDH mutation is associated with a distinct DNA methylation phenotype and an altered metabolic profile in glioma.

Ethnic variation in the prevalence of a common NAD(P)H quinone oxidoreductase polymorphism and its implications for anti-cancer chemotherapy.

The NAD(P)H quinone oxidoreductase (NQO1:EC 1.6.99.2) is an important biotransformation enzyme system that is also known to metabolize important novel chemotherapeutic compounds. The gene that codes for this enzyme has recently been found to be polymorphic in humans. Here, we describe the ethnic distribution of the polymorphism and note that this may have implications for anti-tumour drug development and use.

Induction of the adaptive response by X-rays is dependent on radiation intensity

Human lymphocytes pretreated with low (0.01 Gy) but not high (0.5 Gy) doses of X-rays become somewhat refractory to the induction of chromatid deletions by subsequent exposure to high (1.5 Gy) doses of X-rays (i.e. the yield of chromatid deletions is less than the sum of the yields induced by the pre-exposure and the subsequent challenge doses). This adaptive response can also be induced by pretreating the cells with very low, or even high, concentrations of tritiated thymidine. Because high concentrations of tritiated thymidine result in high doses of radiation that are delivered at very low dose-rates (i.e. less than 0.01 Gy/min), the lack of adaptation following high pre-treatment doses of X-rays could be attributed to their higher dose-rates. To test the effect of X-ray intensity on the induction of the adaptive response, lymphocytes were irradiated with 0.5 Gy of X-rays at 0.005-0.5 Gy/min at 28-30 h of culture, and then irradiated with 1.5 Gy at 48 h. Chromatid deletions were measured 6 h later. The results show that 0.5 Gy of X-rays given at low dose-rates (0.005 or 0.01 Gy/min), but not at high dose-rates (0.1, 0.2, or 0.5 Gy/min), are capable of inducing the adaptive response. Furthermore, experiments in which a male subjects cells exposed to 0.5 Gy given at 0.005 Gy/min were cocultivated with a female subjects cells irradiated with 0.5 Gy at 0.5 Gy/min showed that cells exposed to radiation at low and high intensity progress to metaphase equally and, therefore, that the lack of an adaptive response at high dose-rates cannot be attributed to selection of radioresistant cells. Although the induction of the adaptive response at higher X-ray doses occurs at low radiation intensity, there seems to be a minimum dose required for this effect; e.g., 0.01-Gy pretreatments induced the adaptive response when given at 0.2 Gy/min, but not at 0.005 Gy/min. Thus, the adaptive response is dependent both on the total dose of the pretreatment and on the rate at which the dose is given.

The transcription factor GABP selectively binds and activates the mutant TERT promoter in cancer

A mutant promoters partner in crime Telomerase is an enzyme that maintains the ends of chromosomes. TERT, the gene coding for the enzymes catalytic subunit, is not expressed in healthy somatic cells, but its expression is reactivated in the majority of human cancers. The resultant high levels of telomerase help cancer cells survive and multiply. Recurrent mutations in the promoter region of TERT are associated with high telomerase levels in multiple cancer types. Bell et al. show that a specific transcription factor called GABP is selectively recruited to the mutant form of the TERT promoter, which activates TERT gene expression Science, this issue p. 1036 Cancer-associated mutations in the promoter of the telomerase gene allow increased activation by transcription factor binding. Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, which are fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBMs), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identified the functional consequence of these mutations in GBMs to be recruitment of the multimeric GA-binding protein (GABP) transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native E26 transformation-specific motifs critically cooperate with these mutations to activate TERT, probably by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.