Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

Progressive fibrosis of the interstitium is the dominant final pathway in renal destruction in native and transplanted kidneys. Over time, the continuum of molecular events following immunological and nonimmunological insults lead to interstitial fibrosis and tubular atrophy and culminate in kidney failure. We hypothesize that these insults trigger changes in DNA methylation (DNAm) patterns, which in turn could exacerbate injury and slow down the regeneration processes, leading to fibrosis development and graft dysfunction. Herein, we analyzed biopsy samples from kidney allografts collected 24 months posttransplantation and used an integrative multi-omics approach to understand the underlying molecular mechanisms. The role of DNAm and microRNAs on the graft gene expression was evaluated. Enrichment analyses of differentially methylated CpG sites were performed using GenomeRunner. CpGs were strongly enriched in regions that were variably methylated among tissues, implying high tissue specificity in their regulatory impact. Corresponding to this methylation pattern, gene expression data were related to immune response (activated state) and nephrogenesis (inhibited state). Preimplantation biopsies showed similar DNAm patterns to normal allograft biopsies at 2 years posttransplantation. Our findings demonstrate for the first time a relationship among epigenetic modifications and development of interstitial fibrosis, graft function, and inter-individual variation on long-term outcomes.

L1 penalized continuation ratio models for ordinal response prediction using high-dimensional datasets.

Health status and outcomes are frequently measured on an ordinal scale. For high-throughput genomic datasets, the common approach to analyzing ordinal response data has been to break the problem into one or more dichotomous response analyses. This dichotomous response approach does not make use of all available data and therefore leads to loss of power and increases the number of type I errors. Herein we describe an innovative frequentist approach that combines two statistical techniques, L(1) penalization and continuation ratio models, for modeling an ordinal response using gene expression microarray data. We conducted a simulation study to assess the performance of two computational approaches and two model selection criteria for fitting frequentist L(1) penalized continuation ratio models. Moreover, we empirically compared the approaches using three application datasets, each of which seeks to classify an ordinal class using microarray gene expression data as the predictor variables. We conclude that the L(1) penalized constrained continuation ratio model is a useful approach for modeling an ordinal response for datasets where the number of covariates (p) exceeds the sample size (n) and the decision of whether to use Akaike Information Criterion (AIC) or Bayesian Information Criterion (BIC) for selecting the final model should depend upon the similarities between the pathologies underlying the disease states to be classified.

High-throughput DNA methylation datasets for evaluating false discovery rate methodologies.

When analyzing high-throughput genomic data, the multiple comparison problem is most often addressed through estimation of the false discovery rate (FDR), using methods such as the Benjamini & Hochberg, Benjamini & Yekutieli, the q-value method, or in controlling the family-wise error rate (FWER) using Holm's step down method. To date, research studies that have compared various FDR/FWER methodologies have made use of limited simulation studies and/or have applied the methods to one or more microarray gene expression dataset(s). However, for microarray datasets the veracity of each null hypothesis tested is unknown so that an objective evaluation of performance cannot be rendered for application data. Due to the role of methylation in X-chromosome inactivation, we postulate that high-throughput methylation datasets may provide an appropriate forum for assessing the performance of commonly used FDR methodologies. These datasets preserve the complex correlation structure between probes, offering an advantage over simulated datasets. Using several methylation datasets, commonly used FDR methods including the q-value, Benjamini & Hochberg, and Benjamini & Yekutieli procedures as well as Holm's step down method were applied to identify CpG sites that are differentially methylated when comparing healthy males to healthy females. The methods were compared with respect to their ability to identify CpG sites located on sex chromosomes as significant, by reporting the sensitivity, specificity, and observed FDR. These datasets are useful for characterizing the performance of multiple comparison procedures, and may find further utility in other tasks such as comparing variable selection capabilities of classification methods and evaluating the performance of meta-analytic methods for microarray data.

MicroRNA profiles in allograft tissues and paired urines associate with chronic allograft dysfunction with IF/TA.

Despite the advances in immunosuppression, renal allograft attrition over time remains unabated due to chronic allograft dysfunction (CAD) with interstitial fibrosis (IF) and tubular atrophy (TA). We aimed to evaluate microRNA (miRNA) signatures in CAD with IF/TA and appraise correlation with paired urine samples and potential utility in prospective evaluation of graft function. MiRNA signatures were established between CAD with IF/TA versus normal allografts by microarray. Validation of the microarray results and prospective evaluation of urine samples was performed using real-time quantitative-PCR (RT-qPCR). Fifty-six miRNAs were identified in samples with CAD-IF/TA. Five miRNAs were selected for further validation based on array fold change, p-value and in silico predicted mRNA targets. We confirmed the differential expression of these five miRNAs by RT-qPCR using an independent set of samples. Differential expression was detected for miR-142-3p, miR-204, miR-107 and miR-211 (p < 0.001) and miR-32 (p < 0.05). Furthermore, differential expression of miR-142-3p (p < 0.01), miR-204 (p < 0.01) and miR-211 (p < 0.05) was also observed between patient groups in urine samples. A characteristic miRNA signature for IF/TA that correlates with paired urine samples was identified. These results support the potential use of miRNAs as noninvasive markers of IF/TA and for monitoring graft function.

Ordinal response prediction using bootstrap aggregation, with application to a high-throughput methylation data set.

Many investigators conducting translational research are performing high-throughput genomic experiments and then developing multigenic classifiers using the resulting high-dimensional data set. In a large number of applications, the class to be predicted may be inherently ordinal. Examples of ordinal outcomes include tumor-node-metastasis (TNM) stage (I, II, III, IV); drug toxicity evaluated as none, mild, moderate, or severe; and response to treatment classified as complete response, partial response, stable disease, or progressive disease. While one can apply nominal response classification methods to ordinal response data, in doing so some information is lost that may improve the predictive performance of the classifier. This study examined the effectiveness of alternative ordinal splitting functions combined with bootstrap aggregation for classifying an ordinal response. We demonstrate that the ordinal impurity and ordered twoing methods have desirable properties for classifying ordinal response data and both perform well in comparison to other previously described methods. Developing a multigenic classifier is a common goal for microarray studies, and therefore application of the ordinal ensemble methods is demonstrated on a high-throughput methylation data set.

Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a cancer and leukemia group B study.

The FLT3 gene is mutated by an internal tandem duplication (ITD) in 20-25% of adults with acute myeloid leukemia (AML). We studied 82 adults <60 years of age with primary AML and normal cytogenetics, who received uniform high-dose therapy and found FLT3 ITD in 23 (28%) patients. When the 23 FLT3 ITD+ cases were compared with the 59 cases with wild-type (WT) FLT3, disease-free survival (DFS) was inferior (P = 0.03), yet overall survival (OS) was not different (P = 0.14). However, 8 (35%) of 23 FLT3 ITD/+ cases also lacked a FLT3 WT allele (FLT3(ITD-R)) as determined by PCR and loss of heterozygosity. Thus, three genotypic groups were identified: normal FLT3(WT/WT), heterozygous FLT3(ITD/WT), and hemizygous FLT3(ITD/-). DFS and OS were significantly inferior for patients with FLT3(ITD/-) (P = 0.0017 and P = 0.0014, respectively). Although DFS and OS for FLT3(WT/WT) and FLT3(ITD/WT) groups did not differ (P = 0.32 and P = 0.98, respectively), OS of the FLT3(ITD/-) group was worse than the FLT3(WT/WT) (P = 0.0005) and FLT3(ITD/WT) (P = 0.008) groups. We propose a model in which FLT3(ITD/-) represents a dominant positive, gain-of-function mutation providing AML cells with a greater growth advantage compared with cells having the FLT3(WT/WT) or FLT3(ITD/WT) genotypes. In conclusion, we have identified the FLT3(ITD/-) genotype as an adverse prognostic factor in de novo AML with normal cytogenetics. A poor prognosis of the relatively young FLT3(ITD/-) adults (median age, 37 years), despite treatment with current dose-intensive regimens, suggests that new treatment modalities, such as therapy with a FLT3 tyrosine kinase inhibitor, are clearly needed for this group of patients.

Comparison of cytogenetic and molecular genetic detection of t(8;21) and inv(16) in a prospective series of adults with de novo acute myeloid leukemia: a Cancer and Leukemia Group B Study.

PURPOSE: To prospectively compare cytogenetics and reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22), aberrations characteristic of core-binding factor (CBF) acute myeloid leukemia (AML), in 284 adults newly diagnosed with primary AML. PATIENTS AND METHODS: Cytogenetic analyses were performed at local laboratories, with results reviewed centrally. RT-PCR for AML1/ETO and CBFbeta/MYH11 was performed centrally. RESULTS: CBF AML was ultimately identified in 48 patients: 21 had t(8;21) or its variant and AML1/ETO, and 27 had inv(16)/t(16;16), CBFbeta/MYH11, or both. Initial cytogenetic and RT-PCR analyses correctly classified 95.7% and 96.1% of patients, respectively (P =.83). Initial cytogenetic results were considered to be false-negative in three AML1/ETO-positive patients with unique variants of t(8;21), and in three CBFbeta/MYH11-positive patients with, respectively, an isolated +22; del(16)(q22),+22; and a normal karyotype. The latter three patients were later confirmed to have inv(16)/t(16;16) cytogenetically. Only one of 124 patients reported initially as cytogenetically normal was ultimately RT-PCR-positive. There was no false-positive cytogenetic result. Initial RT-PCR was falsely negative in two patients with inv(16) and falsely positive for AML1/ETO in two and for CBFbeta/MYH11 in another two patients. Two patients with del(16)(q22) were found to be CBFbeta/MYH11-negative. M4Eo marrow morphology was a good predictor of the presence of inv(16)/t(16;16). CONCLUSION: Patients with t(8;21) or inv(16) can be successfully identified in prospective multi-institutional clinical trials. Both cytogenetics and RT-PCR detect most such patients, although each method has limitations. RT-PCR is required when the cytogenetic study fails; it is also required to determine whether patients with suspected variants of t(8;21), del(16)(q22), or +22 represent CBF AML. RT-PCR should not replace cytogenetics and should not be used as the only diagnostic test for detection of CBF AML because of the possibility of obtaining false-positive or false-negative results.

Quantification of CBFbeta/MYH11 fusion transcript by real time RT-PCR in patients with INV(16) acute myeloid leukemia.

Amplification of the CBFbeta/MYH11 fusion transcript by a qualitative reverse transcription-polymerase chain reaction (RT-PCR) has been used to detect minimal residual disease (MRD) and assess the risk for disease relapse in inv(16)(p13q22) acute myeloid leukemia (AML). This strategy has, however, produced conflicting results and because of an uncertain predictive value, its use in the clinical setting cannot be recommended. The objective of the current study was to evaluate if quantification by Real Time RT-PCR could be useful to determine levels of CBFbeta/MYH11 fusion transcripts predictive of clinical outcome in inv(16)(p13q22) AML at diagnosis or during remission. Bone marrow (BM) samples from 16 patients with inv(16) AML enrolled on a German multicenter trial (AML HD93) were analyzed for levels of CBFbeta/MYH11 fusion transcripts by Real Time RT-PCR at diagnosis (n= 14), during remission (n= 10) and at relapse (n=6). The CBFbeta/MYH11 transcript copy number in each sample was normalized to copies of an internal control housekeeping transcript (ie 18S). The copy number measured at diagnosis or relapse were 3 to 4 log higher that those measured during remission, following completion of induction treatment. A high CBFbeta/MYH11 transcript copy number at diagnosis had a significant correlation with a high percentage of BM blasts (Spearman's coefficient = -0.66; P= 0.03), and a borderline correlation with a short complete remission (CR) duration (Spearman's coefficient = -0.51; P= 0.07). No difference in levels of CBFbeta/MYH11 fusion transcripts measured during intensification therapy was found between patients destined to relapse and those who continued in CCR (P= 0.75). Following completion of the entire chemotherapy program, patients that during CR showed a CBFbeta/MYH11 fusion transcript copy number >10 had a significantly shorter CR duration (P= 0.002) and higher risk for disease relapse (P= 0.05) than patients with a CBFbeta/MYH11 fusion transcript copy number <10. The results of the current study, therefore, suggest that it is possible to determine in remission samples a threshold of CBFbeta/MYH11 transcript copy number above which relapse occurs and below which continuous CR is likely.

Depsipeptide (FR 901228) promotes histone acetylation, gene transcription, apoptosis and its activity is enhanced by DNA methyltransferase inhibitors in AML1/ETO-positive leukemic cells.

In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting histone deacetylase (HDAC) and silencing AML1target genes important for hematopoietic differentiation. We hypothesized that depsipeptide (FR901228), a novel HDAC inhibitor evaluated in ongoing clinical trials, restores gene transcription and cell differentiation in AML1/ETO-positive cells. A dose-dependent increase in H3 and H4 histone acetylation was noted in depsipeptide-treated AML1/ETO-positive Kasumi-1 cells and blasts from a patient with t(8;21) AML. Consistent with this biological effect, we also showed a dose-dependent increase in cytotoxicity, expression of IL-3, here used as read-out for silenced AML1-target genes, upregulation of CD11b with other morphologic changes suggestive of partial cell differentiation in Kasumi-1 cells. Some of these biologic effects were also attained in other myeloid leukemia cell lines, suggesting that depsipeptide has differentiation and cytotoxic activity in AML cells, regardless of the underlying genomic abnormality. Notably, the activity of depsipeptide was enhanced by 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor (DNMT). These two agents in combination resulted in enhanced histone acetylation, IL-3 expression, and cytotoxicity, suggesting HDAC and DNMT activities as a potential dual target in future therapeutic strategies for AML1/ETO and other molecular subgroups of AML.

Kidney grafts from HCV-positive donors: advantages and disadvantages.

The Organ Procurement and Transplantation Network database (2001-2006) was reviewed for kidney transplant (KT) recipients, to evaluate the effects of use of grafts from donors positive for hepatitis C virus (HCV) on recipient outcome. Data for 76,787 de novo adult KT recipients were included in the analysis. Serologic tests revealed HCV positivity in 6.25% of cadaver kidneys and 2.97% of living-donor kidneys. Median follow-up in patients still alive was 36 months. At multivariable Cox regression analysis in recipients of cadaver kidney, HCV serostatus was significantly associated with overall and graft survival (both P < .001), with a hazard ratio for HCV-positive patients of 1.43 for overall survival and 1.48 for graft survival. Similar results were obtained for living-donor kidney recipients. Recipients of HCV-positive organs tended to be male and African American and to have a shorter waiting time. Infection was the most commonly reported cause of death in recipients of organs from HCV-positive donors. In patients willing to accept HCV-positive grafts (929 [25.6%]), waiting time was significantly shortened (P < .001). However, this benefit was offset by decreased patient survival (P < .001) and graft survival (P = .007).

Monoamine oxidase A and childhood adversity as risk factors for conduct disorder in females.

BACKGROUND: Recent studies among males have reported a genotype-environment interaction (GxE) in which low-activity alleles at the monoamine oxidase A (MAOA) locus conferred greater sensitivity to the effects of childhood adversity on risk for conduct disorder (CD). So far, few studies of females have controlled for gene-environment correlation or used females heterozygous for this X-linked gene. METHOD: Logistic regression analysis of a sample of 721 females ages 8-17 years from the longitudinal Virginia Twin Study of Adolescent Behavioral Development (VTSABD) assessed the additive effects of MAOA genotypes on risk for CD, together with the main effect of childhood adversity and parental antisocial personality disorder (ASP), as well as the interaction of MAOA with childhood adversity on risk for CD. RESULTS: A significant main effect of genotype on risk for CD was detected, where low-activity MAOA imparted the greatest risk to CD in girls while controlling for the significant effects of maternal ASP and childhood adversity. Significant GxE with weak effect was detected when environmental exposure was untransformed, indicating a higher sensitivity to childhood adversity in the presence of the high-activity MAOA allele. The interaction was no longer statistically significant after applying a ridit transformation to reflect the sample sizes exposed at each level of childhood adversity. CONCLUSIONS: The main effect of MAOA on risk for CD in females, its absence in males and directional difference of interaction is suggestive of genotype-sex interaction. As the effect of GxE on risk for CD was weak, its inclusion is not justified.

A disattenuated correlation estimate when variables are measured with error: illustration estimating cross-platform correlations.

Previous cross-platform reproducibility studies have compared consistency of intensities as well as consistency of fold changes across different platforms using Pearson's correlation coefficient. In this study, we propose the use of measurement error models for estimating gene-specific correlations. Additionally, gene-specific reliability estimates are shown to be useful in prioritizing clones for sequence verification rather than selecting clones using a simple random sample. The proposed 'disattenuated' correlation may prove useful in a wide variety of studies when both X and Y are measured with error, such as in confirmation studies of microarray gene expression values, wherein more reliable laboratory assays such as real-time polymerase chain reaction are used.

Somatic treatment of catatonia.

OBJECTIVE: The authors reviewed the recent literature regarding the treatment of catatonia as a syndrome of multiple etiologies. Given the historical and clinical association of catatonia with schizophrenia, the authors' examined the assumption that the first-line treatment of catatonia is antipsychotic medication. METHODS: Articles published between January 1, 1985 and December 31, 1994 were located using the Paperchase medical literature search system. Additionally, references from those identified articles were examined for possible inclusion in this review. To be included in this review, articles had to be written in English and report specific symptoms of catatonia to determine, retrospectively, if DSM-IV criteria for catatonia were met. RESULTS: Seventy publications met inclusion criteria and reported on a total of 178 patients and included 270 separate treatment episodes. Most of the articles were case-reports, although a few case-series were identified. Multiple causes of catatonia were identified in these reports. The most commonly reported treatment for catatonia was with benzodiazepines which were effective in 70 percent of the cases, with lorazepam demonstrating the highest frequency of use and a 79 percent complete response rate. Electroconvulsive therapy (ECT) was also efficacious (85%) and was more likely to provide a positive outcome in cases of malignant catatonia. Antipsychotics demonstrated poor efficacy. CONCLUSIONS: Catatonia is a nonspecific syndrome with multiple etiologies. Treatment of catatonia should be based on the underlying cause when it is identifiable. Lorazepam appears to offer a safe, effective first-line treatment of catatonia. ECT should be considered when rapid resolution is necessary (e.g., malignant catatonia) or when an initial lorazepam trial fails.

Polymorphisms in cytokines and growth factor genes and their association with acute rejection and recurrence of hepatitis C virus disease in liver transplantation.

Acute rejection (AR) and recurrence of hepatitis C virus (HCV) infection are complications after liver transplantation (LTx). Genetic factors play a role in cytokine production as a consequence of polymorphisms within cytokine genes. Our goal was to identify genetic factors that might be associated with AR and recurrence of HCV in liver transplant recipients (LTxRs). We studied 77 Caucasian LTxRs and 100 Caucasian healthy individuals. We studied single-nucleotide polymorphisms (SNPs) in tumor necrosis factor-alpha[TNF-alpha, interleukin-6 (IL-6), IL-10, transforming growth factor-beta1, and angiotensin-converting enzyme genes by SNaPSHOT trade mark Multiplex assay. SNPs were classified as high producers (HP), intermediate producers (IP), or low producers (LP), and their association with AR and recurrence of HCV were studied. The frequency of TNF-alpha IP and HP genotypes was significantly higher in LTxRs with AR in comparison to patients without AR (TNF-alpha HP -238: 63 vs 20%, p < 0.001; TNF-alpha HP -308: 47.4 vs 20%, p = 0.02). The frequency of IL-6 IP and HP genotypes was higher in patients with AR episodes, but the difference was not statistically significant (p = 0.14). However, when we analyzed the simultaneous presence of pro-inflammatory genotypes in the same patient, we found a significant difference between patients with and without AR, respectively (42.1 vs 14.6%, p = 0.012). Moreover, the frequency of the IL-10 LP genotype was higher in LTx patients with AR (p = 0.001) compared to patients without AR. There was an association between pro-inflammatory genotypes and HCV recurrence. Our data suggest that cytokine gene polymorphisms might play a role in AR and HCV recurrence in LTxRs.

BAALC, the human member of a novel mammalian neuroectoderm gene lineage, is implicated in hematopoiesis and acute leukemia.

The molecular basis of human leukemia is heterogeneous. Cytogenetic findings are increasingly associated with molecular abnormalities, some of which are being understood at the functional level. Specific therapies can be developed based on such knowledge. To search for new genes in the acute leukemias, we performed a representational difference analysis. We describe a human gene in chromosome 8q22.3, BAALC (brain and acute leukemia, cytoplasmic), that is highly conserved among mammals but evidently absent from lower organisms. We characterized BAALC on the genomic level and investigated its expression pattern in human and mouse, as well as its complex splicing behavior. In vitro studies of the protein showing its subcellular localization suggest a function in the cytoskeleton network. Two isoforms are specifically expressed in neuroectoderm-derived tissues, but not in tumors or cancer cell lines of nonneural tissue origin. We show that blasts from a subset of patients with acute leukemia greatly overexpress eight different BAALC transcripts, resulting in five protein isoforms. Among patients with acute myeloid leukemia, those overexpressing BAALC show distinctly poor prognosis, pointing to a key role of the BAALC products in leukemia. Our data suggest that BAALC is a gene implicated in both neuroectodermal and hematopoietic cell functions.