30 research outputs found
Differential predictors for alcohol use in adolescents as a function of familial risk
Abstract: Traditional models of future alcohol use in adolescents have used variable-centered approaches, predicting alcohol use from a set of variables across entire samples or populations. Following the proposition that predictive factors may vary in adolescents as a function of family history, we used a two-pronged approach by first defining clusters of familial risk, followed by prediction analyses within each cluster. Thus, for the first time in adolescents, we tested whether adolescents with a family history of drug abuse exhibit a set of predictors different from adolescents without a family history. We apply this approach to a genetic risk score and individual differences in personality, cognition, behavior (risk-taking and discounting) substance use behavior at age 14, life events, and functional brain imaging, to predict scores on the alcohol use disorders identification test (AUDIT) at age 14 and 16 in a sample of adolescents (N = 1659 at baseline, N = 1327 at follow-up) from the IMAGEN cohort, a longitudinal community-based cohort of adolescents. In the absence of familial risk (n = 616), individual differences in baseline drinking, personality measures (extraversion, negative thinking), discounting behaviors, life events, and ventral striatal activation during reward anticipation were significantly associated with future AUDIT scores, while the overall model explained 22% of the variance in future AUDIT. In the presence of familial risk (n = 711), drinking behavior at age 14, personality measures (extraversion, impulsivity), behavioral risk-taking, and life events were significantly associated with future AUDIT scores, explaining 20.1% of the overall variance. Results suggest that individual differences in personality, cognition, life events, brain function, and drinking behavior contribute differentially to the prediction of future alcohol misuse. This approach may inform more individualized preventive interventions
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Differential predictors for alcohol use in adolescents as a function of familial risk
Abstract: Traditional models of future alcohol use in adolescents have used variable-centered approaches, predicting alcohol use from a set of variables across entire samples or populations. Following the proposition that predictive factors may vary in adolescents as a function of family history, we used a two-pronged approach by first defining clusters of familial risk, followed by prediction analyses within each cluster. Thus, for the first time in adolescents, we tested whether adolescents with a family history of drug abuse exhibit a set of predictors different from adolescents without a family history. We apply this approach to a genetic risk score and individual differences in personality, cognition, behavior (risk-taking and discounting) substance use behavior at age 14, life events, and functional brain imaging, to predict scores on the alcohol use disorders identification test (AUDIT) at age 14 and 16 in a sample of adolescents (N = 1659 at baseline, N = 1327 at follow-up) from the IMAGEN cohort, a longitudinal community-based cohort of adolescents. In the absence of familial risk (n = 616), individual differences in baseline drinking, personality measures (extraversion, negative thinking), discounting behaviors, life events, and ventral striatal activation during reward anticipation were significantly associated with future AUDIT scores, while the overall model explained 22% of the variance in future AUDIT. In the presence of familial risk (n = 711), drinking behavior at age 14, personality measures (extraversion, impulsivity), behavioral risk-taking, and life events were significantly associated with future AUDIT scores, explaining 20.1% of the overall variance. Results suggest that individual differences in personality, cognition, life events, brain function, and drinking behavior contribute differentially to the prediction of future alcohol misuse. This approach may inform more individualized preventive interventions
Differential Comparative Genomic Hybridization Analysis of Normal and Glycosyl Phosphatidyl Inositol Deficient Clones in Paroxysmal Noctorunal Hemoglobinuria.
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High-Resolution Genomic Scan for Cryptic Chromosomal Lesions in MDS and AML
Abstract
Cytogenetic analysis is of eminent importance for the diagnosis and prognosis of hematologic malignancies. Due to limitations of traditional karyotyping, novel technologies which improve resolution and sensitivity are under development. In array-based comparative genomic hybridization (A-CGH), differentially labeled test and reference DNA samples are hybridized to genomic microarrays. Differences in sequence copy number between the samples are reflected in a shift of the fluorescent intensity. The resolution of A-CGH is limited solely by the number of clones; it is theoretically possible to achieve linear coverage of the chromosomes. The principle of the CGH techniques allows for detection of unbalanced chromosomal changes of the whole genome. These types of genomic aberrations are most common in MDS, but may exist and further subclassify malignancies with defined balanced translocations. In MDS, depending on the study, 40–60% of patients have a normal or non-informative karyotype by traditional methods. It is likely that this number may be reduced if the resolution and sensitivity level is increased. Additionally, diagnosis of patients with known chromosomal abnormalities can be further refined.
We first applied A-CGH to the analysis of normal marrow (N=8) to establish whether it will detect chromosomal defects that may acquired and are compatible with normal hematopoiesis. Moreover, defects may be present in healthy elderly. We utilized arrays of up to 2621 clones with a maxium coverage of 1Mb (Vysis, Spectral Genomics). The results were verified by a dye-swap protocol on two arrays per sample. Four controls showed a normal array profile or only changes in clones previously identified as having a polymorphic copy number within the human genome. The remaining controls had changes including a loss of material on 6p (N=1), loss of 6p and 8q material (N=1) and a gain of 4p and loss of 9p sequences (N=1). These changes may reflect unidentified polymorphisms. In contrast, one control had gains of multiple contiguous clones on chromosomes 9, 15 and 22. We also studied the marrow of patients with advanced MDS (N=43) using A-CGH and traditional cytogenetics. The cohort included patients with known singular lesions (N=7) and complex karyotypes (N=1). The remaining patients had either normal or non-informative cytogenetics. For a del 5q patient and a trisomy 21 patient, A-CGH verified the karyotype without identifying further lesions, in a second del 5q patient was a gain of material on 19p, and a monosomy X patient had a gain of 1p36 by CGH. In 3 cases with partially clonal defects, A-CGH did not detect the abnormality. A normal genomic composition was confirmed in a patient with noninformative (N=1) and normal (N=1) karyotypes. Losses of material on 2q and 3q and gains of material on 22q and the 11p telomeric region were identified in a patient with normal cytogenetics, while another "normal" had gains on 2p, 14q and 21q. Additionally, one normal karyotype had loss of chromosome 16 material and one had loss of 6p sequences. This pilot study demonstrates the utility of A-CGH analysis to study chromosomal aberrations in MDS. A-CGH allows for the detection of cytogenetically undetected abnormalities. Analysis of a large number of samples may allow for the detection of consensus defects or global genomic instability with clinical implications
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DNA Repair Gene Expression in CD34+ Bone Marrow Cells in MDS Patients with Mutiple Chromosomal Abnormalities by Array-Based Comparative Genomic Hybridization (A-CGH)
Abstract
Based on the high rate of chromosomal defects in MDS, inherent chromosomal instability (CIN) has been hypothesized as a key pathophysiologic factor of clonal evolution. Predisposition to DNA damage may be primarily due to acquired/inherited weakness in DNA repair machinery; such insufficiency may become manifest after a long latency following cumulative exposure to genotoxic agents. Subsequent changes in chromosomal structure and stepwise acquisition of neoplastic features could lead to leukemic progression. Previously, a number of allelic polymorphisms in DNA repair genes were observed. These variants may lead to altered expression of corresponding proteins. Conversely, if DNA damage is a primary defect, upregulation of specific DNA repair enzymes may be compensatory. Irrespective of the initial pathogenetic defect, we theorized that broad analysis of DNA repair machinery in MDS may point towards specific lesions that could be a subject of more targeted studies. Therefore, we examined levels of DNA repair enzymes using gene expression arrays. For proper comparisons, CD34 cells from 10 MDS patients (4 RA, 6 RAEB/RAEBt) and healthy controls were used. Expression array results were confirmed by Taqman PCR. Reference expression was established by pooling RNA from 12 controls. For more targeted analysis, A-CGH based genomic scan was used to better assess the extent of DNA damage in patients. The expression of 22 out of 113 DNA repair genes tested was detectable at levels >1,5X background; 2-level normalization of gene expression was performed according to variation of mRNA input (housekeeping gene-ACTB) and inter-assay variation in the signal intensity (biotinylated artificial sequence -BAS2C). Our combined standard sample was validated against individual controls; signals <1,5X pooled expression were obtained. Using expression levels of normal CD34 cells as a reference we found that 19 genes were upregulated in concordant fashion. The most dramatically increased genes included APEX, ATM, XRCC1, XRCC5 and MPG. This finding favors the theory that overexpression of the DNA repair machinery is a compensatory event to cope with a primarily increased level of DNA damage. When we subgrouped MDS patients according to FAB criteria, the expression of DNA repair genes (e.g., CIB1, ERCC1, SUMO1) increased with the malignant progression. For further analysis we have defined CIN phenotype by the presence of large or multiple small defects as determined by A-CGH. When patients with CIN vs. those with normal karyotype were compared, we found that chromosomal damage was not accompanied by a higher expression of DNA repair genes. MPG was most dramatically upregulated in all MDS patients. This gene involved in excision of methylated bases can induce single stranded breaks (SSB) and increase sensitivity to alkylating agents. Our finding suggest that either increased purine methylation induces a compensatory mechanism (MPG upregulation) or that overactivity of MPG itself results in increased base excision. Alternatively, overexpression of MPG may lead to SSB especially because downstream genes (e.g. XRCC3 or DNA ligase III) were not accordingly upregulated. In conclusion, our studies form a basis for further analysis of clinical phenotypes associated with upregulation of specific DNA repair genes and may indicate possible therapeutic targets in molecularly defined subtypes of MDS
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High-Resolution Genomic Arrays Facilitate Detection of Novel Cryptic Chromosomal Lesions in MDS
Abstract
The evolution of abnormal hematopoietic clones characterized by acquired chromosomal abnormalities is the central event in the pathogenesis of MDS. Defective chromosomes have significant clinical implications in the management of MDS and suggest the presence of an inherent chromosomal instability. As karyotypic lesions are not found in all MDS patients, it is possible that in some the dysplastic clone may evolve without a chromosomal defect or, more likely, the resolution of routine metaphase cytogenetics is not sufficient to detect smaller lesions; in many instances lack of growth precludes the analysis.
Array-based comparative genomic hybridization (A-CGH) allows for a high-resolution genomic scan that circumvents some of the limitations associated with the use of conventional cytogenetics. We hypothesized that high-resolution genomic analysis of genetic gains and losses by A-CGH may detect cryptic lesions, particularly in patients with negative/non-informative cytogenetics that may be of clinical/scientific significance. We examined bone marrow cells from 39 MDS patients (18 RA/RARS, 11 RAEB-t, 6 CMML and 4 secondary AML) and 11 controls using a 2632 BAC microarray and CGH. Dye swapping on duplicate arrays assured reproducibility of the CGH results, confirmed globally by a high resolution 50K SNP microarray in 4 patients and by microsatellite analysis in others.
By traditional cytogenetics 19 patients had chromosomal lesions, 18 were normal and 2 tests non-informative. When A-CGH was applied, a normal karyotype was found in only 15% of patients in comparison to 46% by metaphase cytogenetics. Of note is that both cases with uninformative cytogenetics showed an abnormal CGH result and in several patients (N=11) with an abnormal karyotype additional lesions were found. Karyotypic results were confirmed in 7 cases; discordant analysis may be due to a lower proportion of dysplastic cells in marrow.
Irrespective of the genomic area affected, when we studied the raw number of lesions more advanced forms of MDS (RAEB-t/AML) were evenly distributed between patients subdivided on sheer number of lesions (0, 1–17, >17). Many hotspots of genomic instability shared between patients were identified. For example, 1p26.3, 10q26 and 4p16 lesions were found in 2 or more patients. Interestingly, these regions contain genes of potential pathologic significance, including tubulin gamma complex associated protein 2 (TUBGCR2) and histone stem-loop binding protein (SLBP). Cryptic lesions on chromosome 7 (e.g. 7p21, 7q31) were identified in 5 patients with normal cytogenetics. These patients suffered from severe cytopenias, consistent with the prognosis of monosomy 7 and highlighting a consensus defect on chromosome 7. Certain chromosomes were rarely or never affected, implying that a more targeted array might be designed for clinical use.
A-CGH Cytogenetics Unsuccessful Normal Abnormal Unsuccessful (N=2) 0 0 2 Normal (N=18) 0 3 15 Abnormal (n=19) 0 3 16
In summary, our study highlights the superior level of resolution of A-CGH as compared to metaphase analysis in the diagnosis of MDS. A prospective analysis is underway to determine the prognostic value of CGH-detected lesions and their pathophysiologic significance
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High-Density Genomic Scan with 50K SNP Arrays Reveals Existence of Cryptic Chromosomal Lesions and Germ Line Allelic Polymorphism That Might Determine Predisposition to MDS
Abstract
Chromosomal damage is a hallmark of MDS. Inability to detect abnormal karyotype in a portion of MDS patients is consistent with the theory that large lesions may represent the extreme of possible DNA damage. Predisposition to MDS may be due to inherited defects; possibly, genes coding for detoxifying enzymes, DNA repair genes and immunogenetic factors could be involved and the risk can be multifactorial. To date studies dealing with complex pathogenesis of MDS have been based on empiric approaches allowing for very limited insights into the possibly complex genetic traits and DNA changes during evolution of the disease. Gene array technology facilitates detailed genomic analysis. We have applied 50K SNP Affymetrix arrays to the analysis of the MDS genome. The goal of our study was to establish the feasibility of this technology to study 2 separate aspects: i) to detect acquired cryptic chromosomal damage, ii) to investigate germ line allelic variants in MDS that could constitute predisposition factors. Our MDS cohort included 22 patients 15 RA/RS, 7 RAEB; 11 with typical cytogenetic abnormalities and 11 normal karyotypes. Using a threshold value 6 -log10(pval) we confirmed previously known changes in 8/11 patients. The remaining 3 showed multiple scattered lesions throughout affected chromosomes; false negatives were likely due to dilution by non-clonal cells. However, using a high density SNP scan, novel previously cryptic lesions contiguous over various chromosomal portions were found in 5/11 patients with normal metaphase cytogenetics and some patients with previously established defects. Chromosomes 5, 7 and 8 showed a higher number of smaller defects consistent with the pathogenesis of MDS; deletions within 7p14, 7p12.2 and 7q21.3 were found in 4 patients without obvious mononosomy 7. Similarly, deletions of various size within 5q12.3–35.5 were seen in 4/15 patients with low-grade MDS. Analysis of such defects within phenotypically defined subsets of patients may reveal possible consensus lesions or commonly affected genes.
We also investigated the presence of putatively MDS-specific genotypes. Overrepresentation of otherwise rare SNP may suggest existence of cryptic changes involving adjacent portions of the genome. Globally SNP array allowed for classification of 55953 SNP in 22 patients. 28% were heterozygous, while 36% were homozygous for either variant. Complex analysis of such a large number of genotypes may allow for determination of potential associations between genetic makeup and clinical sub-entities. Based on the allelic frequencies of individual SNP in MDS group and controls, we have identified significantly overrepresented SNPs occurring in either homo- or heterozygous form. For example, FOXL1 SNP occurring at a frequency of 2.4% in homo- and heterozygote form in normals was found in 25% of patients, and 10% of patients were homozygous for a Semaphorin SNP present in only 0.015% of controls. Similarly, PARP variant showed an allelic frequency of 2.7 vs. 14% in controls and patients, respectively. In general, our study show the potential value of high density SNP arrays in precise analysis of clonal genomic lesions and/loss heterozygozity as well as complex genotypic profiles that may potentially contribute to inherited predisposition traits