Differential DNA methylation patterns of small B-cell lymphoma subclasses with different clinical behavior.

Non-Hodgkin's lymphoma (NHL) is a group of malignancies of the immune system with variable clinical behaviors and diverse molecular features. Despite the progress made in classification of NHLs based on classical methods, molecular classifications are a work in progress. Toward this goal, we used an array-based technique called differential methylation hybridization (DMH) to study small B-cell lymphoma (SBCL) subtypes. A total of 43 genomic DMH experiments were performed. From these results, several statistical methods were used to generate a set of differentially methylated genes for further validation. Methylation of LHX2, POU3F3, HOXC10, NRP2, PRKCE, RAMP, MLLT2, NKX6.1, LRP1B and ARF4 was validated in cell lines and patient samples and demonstrated subtype-related preferential methylation patterns. For LHX2 and LRP1B, bisulfite sequencing, real-time reverse transcriptase-polymerase chain reaction and induction of gene expression following treatment with the demethylating agent, 5'-aza-2'-deoxycytidine, were confirmed. This new epigenetic information is helping to define molecular portraits of distinct subtypes of SBCL that are not recognized by current classification systems and provides valuable potential insights into the biology of these tumors.

Microarray image spot segmentation using the method of projections.

Microarray technology is increasingly used as a means of high throughput analysis of human, non-human and plant genomes. Manual methods of array production using this technology lead to many inherent problems in the microarray image produced. The density of the spots in the images produced is very high, such that neighboring spots can overlap. The image background is often not uniform, containing noise that is often difficult to distinguish from actual spots. In this research, a projections-based approach is investigated for spot segmentation in paired radio probe microarray images. An important aspect of spot segmentation is the capability to perform corresponding spot-to-spot comparisons between the paired images. Experimental results are presented for spot segmentation in isolated and paired microarray images.

Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays.

It is now clear that aberrant DNA methylation observed in cancer cells is not restricted to a few CpG islands, but affects multiple loci. When this epigenetic event occurs at the 5'-end of the regulatory region of genes, it is frequently associated with transcriptional silencing. To investigate further this widespread event in the tumor genome, we developed a novel microarray containing 7776 short GC-rich tags tethered to glass slide surfaces. This DNA chip was used to study 17 paired tissues of breast tumors and normal controls. Amplicons, representing differential pools of methylated DNA fragments between tumors and normal controls, were cohybridized to the microarray panel. Hypermethylation of multiple CpG island loci was then detected in a two-color fluorescence system. Approximately 1% (on average, 83 loci) of these CpG islands examined were hypermethylated in this patient group. Hierarchical clustering segregated these tumors based on their methylation profiles and identified a group of CpG island loci that corresponds to the hormone-receptor status of breast cancer. This observation was independently confirmed by examining a single locus, the promoter of the human glypican 3 gene, which was predominately hypermethylated in the hormone receptor-negative tumors. Our findings support the notion that hypermethylation of critical CpG island loci influences cancer development and produces distinct epigenetic signatures for particular tumor subtypes.

Structural analysis of TCRalpha and beta chains from human T-Cell clones specific for small nuclear ribonucleoprotein polypeptides Sm-D, Sm-B and U1-70 kDa: TCR complementarity determining region 3 usage appears highly conserved.

Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are systemic autoimmune diseases that are characterized by the presence of autoantibodies reactive with U small nuclear RNP (snRNP) autoantigens. Both B and T cells are important in the pathogenesis of the disease, and T- and B-cell immunity against snRNP polypeptides have been shown to be linked in vivo. Currently, several alternative hypotheses for the pathogenesis of these diseases have been proposed. These include loss of tolerance, modified self-antigens, molecular mimicry and nondirected immune activation. To help distinguish between the various models of disease pathogenesis, we have characterized the T-cell receptor (TCR) CDR3 from a large panel of well-characterized human T-cell clones and lines specific for individual snRNP polypeptides. The results presented here reveal highly restricted TCR usage across patients by the snRNP-reactive T cells based on the deduced amino acid sequence of the CDR3 loop. These data support the hypothesis that T-cell responses against self antigens in SLE and MCTD are antigen driven and that there are a limited number of T-cell epitopes present on the snRNP autoantigens.

An information system for improving clinical laboratory outcomes.

Laboratories performing clinical molecular diagnostic and cytogenetic testing require improved information systems to address their specialized data processing needs. We developed an application that automates result reporting, documents quality assurance information, and tracks specimens. While similar functionality was implemented in both the molecular diagnostic and cytogenetic modules, we present an outcome assessment of the cytogenetic laboratory's use of the system since it maintained a relatively constant number of personnel, test procedures, and samples over a three-year period. Upon implementation, significant reductions occurred in the time taken from receipt of sample to the release of the final report by 44% (P < 0.001) and 48% (P < 0.001) for peripheral blood and bone marrow samples, respectively. The number of cases processed per technologist increased by 26% (P = 0.017). We attribute these gains in quality improvement to the automation of clerical tasks and decision support provided by the information system.

CpG island arrays: an application toward deciphering epigenetic signatures of breast cancer.

CpG island hypermethylation is a frequent epigenetic event in cancer. We have recently developed an array-based method, called differential methylation hybridization (DMH), allowing for a genome-wide screening of CpG island hypermethylation in breast cancer cell lines (T. H-M. Huang et al., Hum. Mol. Genet., 8: 459-470, 1999). In the present study, DMH was applied to screen 28 paired primary breast tumor and normal samples and to determine whether patterns of specific epigenetic alterations correlate with pathological parameters in the patients analyzed. Amplicons, representing a pool of methylated CpG DNA derived from these samples, were used as hybridization probes in an array panel containing 1104 CpG island tags. Close to 9% of these tags exhibited extensive hypermethylation in the majority of breast tumors relative to their normal controls, whereas others had little or no detectable changes. Pattern analysis in a subset of CpG island tags revealed that CpG island hypermethylation is associated with histological grades of breast tumors. Poorly differentiated tumors appeared to exhibit more hypermethylated CpG islands than their moderately or well-differentiated counterparts (P = 0.041). This early finding lays the groundwork for a population-based DMH study and demonstrates the need to develop a database for examining large-scale methylation data and for associating specific epigenetic signatures with clinical parameters in breast cancer.

Analysis of T cell receptors specific for U1-70kD small nuclear ribonucleoprotein autoantigen: the alpha chain complementarity determining region three is highly conserved among connective tissue disease patients.

The U1-70kD autoantigen is a major target of B cell responses in patients with connective tissue diseases (CTD). T cell responses are important in the pathogenesis of CTD, however little is known about autoantigen-specific T cells in these diseases. We have recently proven that U1-70kD-reactive human T cells exist. To further characterize these autoreactive T cells, U1-70kD-reactive T cell clones have been generated from patients with CTD using either a recombinant fusion protein or synthetic peptides spanning the U1-70kD polypeptide. T cell receptors (TCR) isolated from the U1-70kD-reactive T cell clones were sequenced and the third complementarity-determining region (CDR3) compared to determine if a common motif was present. mAb blocking of antigen-induced proliferation was done to determine the HLA restriction element used in recognition of the U1-70kD autoantigen by T cells. The results presented here indicate that TCRAV CDR3 usage is highly restricted among U1-70kD autoantigen-specific human T cells clones derived from CTD patients with distinctive structural features. Furthermore, the recognition of the U1-70kD autoantigen occurs in the context of HLA-DR.

Data-driven homologue matching for chromosome identification.

Karyotyping involves the visualization and classification of chromosomes into standard classes. In "normal" human metaphase spreads, chromosomes occur in homologous pairs for the autosomal classes 1-22, and X chromosome for females. Many existing approaches for performing automated human chromosome image analysis presuppose cell normalcy, containing 46 chromosomes within a metaphase spread with two chromosomes per class. This is an acceptable assumption for routine automated chromosome image analysis. However, many genetic abnormalities are directly linked to structural or numerical aberrations of chromosomes within the metaphase spread. Thus, two chromosomes per class cannot be assumed for anomaly analysis. This paper presents the development of image analysis techniques which are extendible to detecting numerical aberrations evolving from structural abnormalities. Specifically, an approach to identifying "normal" chromosomes from selected class(es) within a metaphase spread is presented. Chromosome assignment to a specific class is initially based on neural networks, followed by banding pattern and centromeric index criteria checking, and concluding with homologue matching. Experimental results are presented comparing neural networks as the sole classifier to our homologue matcher for identifying class 17 within normal and abnormal metaphase spreads.

Integrating molecular diagnostic and flow cytometric reporting for improved longitudinal monitoring of HIV patients.

Studies have shown that monitoring HIV-infected patients undergoing antiretroviral therapy is best represented by combined measurement of plasma HIV-1 RNA and CD4+ T-lymphocytes [1]. This pilot study at the University of Missouri-Columbia integrates molecular diagnostic and flow cytometric data reporting to provide current and historical HIV-1 RNA levels and CD4+ T-cell counts. The development of a single database for storage and retrieval of these values facilitates composite report generation that includes longitudinal HIV-1 RNA levels and CD4+ T-cell counts for all patients. Results are displayed in tables and plotted graphically within a web browser. This method of data presentation converts individual data points to more useful medical information and could provide clinicians with decision support for improved monitoring of HIV patients undergoing antiretroviral therapy.

Homologue matching using the Choquet integral.

Automated Giemsa-banded chromosome image research has been largely restricted to classification schemes associated with isolated chromosomes within metaphase spreads. In normal human metaphase spreads, there are 46 chromosomes occurring in homologous pairs for the autosomal classes, 1-22, and X chromosome for females. For optimizing automated human chromosome image analysis, many existing techniques assume cell normalcy. With many genetic abnormalities directly linked to structural and numerical aberrations of chromosomes within the metaphase spread, the two chromosome per class assumption may not be appropriate for anomaly analysis. At the University of Missouri, a data-driven homologue matching approach has been developed to identify all normal chromosomes within a metaphase spread from a selected class. Chromosome assignment to a specific class is initially based on neural networks, followed by banding pattern and centromeric index criteria checking, and concluding with homologue matching utilizing a density profile-based classifier, a shape profile-based classifier, and a binary band profile-based classifier. Based on preliminary results for the profile-based classifiers assigning chromosome 17, the Choquet integral is presented as an extension to the homologue matching approach. Experimental results are presented comparing the extended homologue matching approach to the transportation algorithm for identifying chromosome 21 within normal metaphase spreads.

The effects of image manipulation on automated karyotyping.

Karyotyping is one of the standard tools for human genetic investigations. Karyotyping involves the classification and interpretation of chromosomes found in a metaphase spread. As part of the automated karyotyping process, image manipulation is required for appropriately positioning metaphase spread chromosomes in their corresponding karyotype. Image manipulation, specifically image rotation, reorganizes the grey-level information within chromosomes to facilitate analysis. Statistical tests are performed to compare features related to banding pattern and length between unmanipulated chromosomes and corresponding rotated chromosomes. Based on experimental results, reorganizing the grey-level information yields statistically different chromosome features. Depending on the purpose of chromosome image analysis, the interpretation process of karyotyping could be impaired with chromosome feature distortion due to image rotation.

Central nervous system manifestations of human ehrlichiosis.

Since 1989, we have confirmed the diagnosis of human ehrlichiosis in 57 patients. Although routine radiological studies of the central nervous system (CNS) or analyses of cerebrospinal fluid (CSF) samples were not done for these patients, primary care physicians detected symptoms or signs that prompted them to perform such studies. CSF samples were examined for 15 of the 57 patients. Findings in eight of the 15 CSF samples were abnormal, and the most common abnormalities were lymphocytic pleocytosis and elevated protein levels. A search of the English-language literature revealed 21 additional cases in which CSF examinations were performed; in 13 of these cases, CSF findings were abnormal. The most common clinical finding that predicted CSF abnormalities was a change in mental status. A total of 14 patients underwent computerized tomographic studies, and none of these studies showed abnormalities. Four (19%) of the 21 patients with CNS manifestations of ehrlichiosis and abnormal CSF findings died.

Effects of altered prenatal hormonal environment on expression of autoimmune disease in NZB/NZW mice.

F1 hybrid New Zealand Black (NZB) x New Zealand White (NZM) (NZB/NZW) mice spontaneously develop an autoimmune disease analogous to systemic lupus erythematosus (SLE). Testosterone experts a powerful suppressive effect on this disorder in adult NZB/NZW mice. A series of experiments was designed to determine if disease would also be suppressed by exposing fetal NZB/NZW mice to increased testosterone. A model was developed in which NZB dams carrying NZB/NZW fetuses were treated with testosterone in a dose adequate to masculinize the external genitalia in female fetuses. NZB/NZW mice that were derived from testosterone-treated dams and control NZB/NZW offspring were followed in a longevity study and had serial assays to assess development of SLE. Additional experiments were carried out to measure lymphocyte subsets and responses to mitogens. Results were compared with F1 hybrid offspring of C57BL/6 dams crossed with DBA/2 males, which are not autoimmune and do not develop SLE. Spleen cells from these groups were tested for Thy 1.2, CD4, CD8, and IgM receptors, and for responses to the mitogens Concanavalin A (ConA) and lipopolysaccharide. Control male NZB/NZW fetuses had unexpectedly high serum estradiol, which decreased significantly with maternal testosterone treatment. The testosterone-exposed male NZB/NZW fetuses developed into adults that lived longer than male NZB/NZW controls. Testosterone treatment of the dam was associated with elevated terminal anti-DNA levels but did not alter markers of renal diseases in adult NZB/NZW mice of either sex. Testosterone-exposed NZB/NZW females had altered T-lymphocyte subsets and testosterone-exposed males had increased response to ConA compared to controls. In male NZB/NZW fetuses whose mothers were administered testosterone, the naturally high level of circulating estradiol observed in untreated male fetuses was decreased significantly. This decrease was associated with an increase in longevity. This unique observation has important implications for fetal exposure to endocrine disruptors in the environment.

Apoptosis of gamma/delta T cells in human ehrlichiosis.

Expansion of activated T cells expressing the T-cell receptor (TCR) gamma/delta, CD45RO, and HLA-DR antigens is a prominent feature of acute infection with Ehrlichia chaffeensis in humans. The fate of these activated cells and the resolution of the gamma/delta T-cell response with return to the usual alpha/beta T-cell populations in this disease are not clearly understood. At a morphologic level, apoptotic cells are present in the peripheral blood during the acute and resolution phases of the infection. Simple culture of density gradient-separated lymphocytes from the blood of patients with acute ehrlichiosis produced cell death rapidly in the media compared to alpha/beta T cells. This loss of viability after incubation was apparently mediated by apoptosis, based on flow cytometric and morphologic analyses. The results suggest that most primed (CD45RO+) and activated (HLA-DR+) gamma/delta T cells in acute ehrlichiosis might be subject to removal from the body by programmed or apoptotic cell death.

A centromere attribute integration approach to centromere identification.

Automated and nonautomated approaches to chromosome classification involves assessing several chromosome attributes. The centromere is an important attribute which provides insight to other features such as chromosome orientation and the banding pattern sequence. Improving the ability to identify the centromere will enhance feature determination and analysis. Techniques to identify the centromere attempt to isolate specific centromere attributes. The centromere can be characterized as possessing the following properties: 1) usually the narrowest region in the chromosome image, 2) usually located in a region containing extreme concavities along the chromosome contour, and 3) usually located in a region of uniform dark grey-level. A centromere attribute integration approach for automated centromere identification has been developed which has a correct identification rate of 93.5% on a diversified data set. This approach determines and evaluates centromere candidates based on quantified centromere attributes. Centromere attribute integration incorporates other commonly used techniques for centromere identification. Some of the techniques integrated into the experimental algorithm include evaluating chromosome curvature, analyzing the shape profile, and inspecting the width profile.

Centromere attribute integration based chromosome polarity assignment.

Automated karyotyping involves evaluating quantified chromosome attributes for proper classification. Chromosome attributes derived from the banding pattern require the correct chromosome polarity for correct banding sequence interpretation. Chromosome polarity is defined in terms of determining the short and long arms of the chromosome using the centromere as the reference point for measuring the chromosome length on both sides of the centromere. In addition to banding sequence interpretation, polarity is used in the chromosome orientation for chromosome repositioning from the metaphase spread to the karyotype. Automated polarity determination is often not performed for classifying chromosomes in the metaphase spread image. Polarity may be determined user interactively, by the system, or not at all. In order to reduce the computational complexity of evaluating banding sequence features using both chromosome ends as reference points, there is a need to improve chromosome polarity determination in automated karyotyping. A centromere attribute integration approach has been developed at the University of Missouri-Columbia which performs correct chromosome polarity assessment at a rate comparable to other studies of 96.1% on a diversified data set.