Automatic quantitation of multiple sclerosis lesions on MR images

A two-pronged approach for the automatic quantitation of multiple sclerosis (MS) lesions on magnetic resonance (MR) images has been developed. This method includes the design and use of a pulse sequence for improved lesion-to-tissue contrast (LTC) and seeks to identify and minimize the sources of false lesion classifications in segmented images. The new pulse sequence, referred to as AFFIRMATIVE (Attenuation of Fluid by Fast Inversion Recovery with MAgnetization Transfer Imaging with Variable Echoes), improves the LTC, relative to spin-echo images, by combining Fluid-Attenuated Inversion Recovery (FLAIR) and Magnetization Transfer Contrast (MTC). In addition to acquiring fast FLAIR/MTC images, the AFFIRMATIVE sequence simultaneously acquires fast spin-echo (FSE) images for spatial registration of images, which is necessary for accurate lesion quantitation. Flow has been found to be a primary source of false lesion classifications. Therefore, an imaging protocol and reconstruction methods are developed to generate "flow images" which depict both coherent (vascular) and incoherent (CSF) flow. An automatic technique is designed for the removal of extra-meningeal tissues, since these are known to be sources of false lesion classifications. A retrospective, three-dimensional (3D) registration algorithm is implemented to correct for patient movement which may have occurred between AFFIRMATIVE and flow imaging scans. Following application of these pre-processing steps, images are segmented into white matter, gray matter, cerebrospinal fluid, and MS lesions based on AFFIRMATIVE and flow images using an automatic algorithm. All algorithms are seamlessly integrated into a single MR image analysis software package. Lesion quantitation has been performed on images from 15 patient volunteers. The total processing time is less than two hours per patient on a SPARCstation 20. The automated nature of this approach should provide an objective means of monitoring the progression, stabilization, and/or regression of MS lesions in large-scale, multi-center clinical trials. ^

NOVEL PHANTOMS AND POST-PROCESSING FOR DIFFUSION SPECTRUM IMAGING

High Angular Resolution Diffusion Imaging (HARDI) techniques, including Diffusion Spectrum Imaging (DSI), have been proposed to resolve crossing and other complex fiber architecture in the human brain white matter. In these methods, directional information of diffusion is inferred from the peaks in the orientation distribution function (ODF). Extensive studies using histology on macaque brain, cat cerebellum, rat hippocampus and optic tracts, and bovine tongue are qualitatively in agreement with the DSI-derived ODFs and tractography. However, there are only two studies in the literature which validated the DSI results using physical phantoms and both these studies were not performed on a clinical MRI scanner. Also, the limited studies which optimized DSI in a clinical setting, did not involve a comparison against physical phantoms. Finally, there is lack of consensus on the necessary pre- and post-processing steps in DSI; and ground truth diffusion fiber phantoms are not yet standardized. Therefore, the aims of this dissertation were to design and construct novel diffusion phantoms, employ post-processing techniques in order to systematically validate and optimize (DSI)-derived fiber ODFs in the crossing regions on a clinical 3T MR scanner, and develop user-friendly software for DSI data reconstruction and analysis. Phantoms with a fixed crossing fiber configuration of two crossing fibers at 90° and 45° respectively along with a phantom with three crossing fibers at 60°, using novel hollow plastic capillaries and novel placeholders, were constructed. T2-weighted MRI results on these phantoms demonstrated high SNR, homogeneous signal, and absence of air bubbles. Also, a technique to deconvolve the response function of an individual peak from the overall ODF was implemented, in addition to other DSI post-processing steps. This technique greatly improved the angular resolution of the otherwise unresolvable peaks in a crossing fiber ODF. The effects of DSI acquisition parameters and SNR on the resultant angular accuracy of DSI on the clinical scanner were studied and quantified using the developed phantoms. With a high angular direction sampling and reasonable levels of SNR, quantification of a crossing region in the 90°, 45° and 60° phantoms resulted in a successful detection of angular information with mean ± SD of 86.93°±2.65°, 44.61°±1.6° and 60.03°±2.21° respectively, while simultaneously enhancing the ODFs in regions containing single fibers. For the applicability of these validated methodologies in DSI, improvement in ODFs and fiber tracking from known crossing fiber regions in normal human subjects were demonstrated; and an in-house software package in MATLAB which streamlines the data reconstruction and post-processing for DSI, with easy to use graphical user interface was developed. In conclusion, the phantoms developed in this dissertation offer a means of providing ground truth for validation of reconstruction and tractography algorithms of various diffusion models (including DSI). Also, the deconvolution methodology (when applied as an additional DSI post-processing step) significantly improved the angular accuracy of the ODFs obtained from DSI, and should be applicable to ODFs obtained from the other high angular resolution diffusion imaging techniques.

Functional Analysis of Drosophila Integrator Complex in snRNA 3' End Processing

Uridine-rich small nuclear RNAs (U snRNAs) play essential roles in eukaryotic gene expression by facilitating the removal of introns from mRNA precursors and the processing of the replication-dependent histone pre-mRNAs. Formation of the 3’ end of these snRNAs is carried out by a poorly characterized, twelve-membered protein complex named Integrator Complex. In the effort to understand Integrator Complex function in the formation of the snRNA 3’ end, we performed a functional RNAi screen in Drosophila S2 cells to identify protein factors required for snRNA 3’ end formation. This screen was conducted by using a fluorescence-based reporter that elicits GFP expression in response to a deficiency in snRNA processing. Besides scoring the known Integrator subunits, we identified Asunder and CG4785 as additional core members of the Integrator Complex. Additionally, we also found a conserved requirement for Cyclin C and Cdk8 in both fly and human snRNA 3’ end processing. We have further demonstrated that the kinase activity of Cdk8 is critical for snRNA 3’ end processing and is likely to function independent of its well-documented function within the Mediator Cdk8 module. Taken together, this work functionally defines the Drosophila Integrator Complex and demonstrates a novel function for Cyclin C/Cdk8 in snRNA 3’ end formation. This thesis work has also characterized an important functional interaction mediated by a microdomain within Integrator subunit 12 (IntS12) and IntS1 that is required for the activity of the Integrator Complex in processing the snRNA 3’ end. Through the development of a reporter-based functional RNAi-rescue assay in Drosophila S2 cells, we analyzed domains within IntS12 required for snRNA 3’ end formation. This analysis unexpectedly revealed that an N-terminal 30 amino acid region and not the highly conserved central PHD finger domain, is required for snRNA 3’ end cleavage. The IntS12 microdomain (1-45) functions autonomously, and is sufficient to interact and stabilize the putative scaffold protein IntS1. Our findings provide more details of the Integrator Complex for understanding the molecular mechanism of snRNA 3’ end processing. Moreover, these results lay the foundation for future studies of the complex through the identification of a novel functional domain within one subunit and the identification of additional subunits.

Autoregulation of TRA-2 pre -mRNA splicing

One of the most elegant and tightly regulated mechanisms for control of gene expression is alternative pre-mRNA splicing. Despite the importance of regulated splicing in a variety of biological processes relatively little is understood about the mechanisms by which specific alternative splice choices are made and regulated. The transformer-2 (tra-2) gene encodes a splicing regulator that controls the use of alternative splicing pathways in the sex determination cascade of D. melanogaster and is particularly interesting because it directs the splicing of several distinct pre-mRNAs in different manners. The tra-2 protein positively regulates the splicing of both doublesex (dsx) and fruitless (fru) pre-mRNAs. Additionally tra-2 controls exuperantia (exu) by directing the choices between splicing and cleavage/polyadenylation and autoregulates the tra-2 pre-mRNA processing by repressing the removal of a specific intron (called M1). The goal of this study is to identify the molecular mechanisms by which TRA-2 protein affects the alternative splicing of pre-mRNA deriving from the tra-2 gene itself.^ The autoregulation of M1 splicing plays a key role in regulation of the relative levels of two functionally distinct TRA-2 protein isoforms expressed in the male germline. We have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative pre-mRNAs produced in D. virilis testes suggests that the germline-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster.^ To identify elements necessary for regulation of tra-2 M1 splicing, we mutagenized evolutionarily conserved sequences within the tra-2 M1 intron and flanking exons. Constructs containing these mutations were used to generate transgenic fly lines that have been tested for their ability to carry out autoregulation. These transgenic fly experiments elucidated several elements that are necessary for setting up a context under which tissue-specific regulation of M1 splicing can occur. These elements include a suboptimal 3$\sp\prime$ splice site, an element that has been conserved between D. virilis and D. melanogaster, and an element that resembles the 3$\sp\prime$ portion of a dsx repeat and other splicing enhancers.^ Although important contextual features of the tra-2 M1 intron have been delineated in the transgenic fly experiments, the specific RNA sequences that interact directly with the TRA-2 protein were not identified. Using Drosophila nuclear extracts from Schneider cells, we have shown that recombinant TRA-2 protein represses M1 splicing in vitro. UV crosslinking analysis suggests that the TRA-2 protein binds to several different sites within and near the M1 intron. ^

The Pre-entry Program at UTMSH: Effect on Academic Performance of First-Year Medical Students

Introduction: The Pre-Entry Program at The University of Texas Medical School at Houston is presented to assist entering students who are judged to be at risk for academic difficulty. It requires a significant commitment of time on the part of faculty, staff and students. The effectiveness of this program needs to be evaluated. [See PDF for complete abstract]

Evaluating the prevalence, content and readability of complementary and alternative medicine (CAM) web pages on the internet.

Complementary and alternative medicine (CAM) use is growing rapidly. As CAM is relatively unregulated, it is important to evaluate the type and availability of CAM information. The goal of this study is to deter-mine the prevalence, content and readability of online CAM information based on searches for arthritis, diabetes and fibromyalgia using four common search engines. Fifty-eight of 599 web pages retrieved by a "condition search" (9.6%) were CAM-oriented. Of 216 CAM pages found by the "condition" and "condition + herbs" searches, 78% were authored by commercial organizations, whose pur-pose involved commerce 69% of the time and 52.3% had no references. Although 98% of the CAM information was intended for consumers, the mean read-ability was at grade level 11. We conclude that consumers searching the web for health information are likely to encounter consumer-oriented CAM advertising, which is difficult to read and is not supported by the conventional literature.

FUNCTIONS OF DEADENYLATION FACTORS IN MRNA DECAY AND MRNA PROCESSING BODY FORMATION

Most newly synthesized messenger RNAs possess a 5’ cap and a 3’ poly(A) tail. The process of poly(A) tail shortening, also termed deadenylation, is important for post-transcriptional gene regulation, because deadenylation not only leads to mRNA translational inhibition but also is the first step of major mRNA degradation. Translationally inhibited mRNAs can be stored and/or degraded in dynamic cytoplasmic foci termed mRNA processing bodies, or P bodies, which are conserved in eukaryotes. To shed new light on the mechanisms of P body formation and P body functions, I focused on the link between deadenylation factors and P bodies. I found that the two major deadenylation complexes, Pan3-Pan2 and Ccr4-Caf1, can both be enriched in P bodies. The deadenylase activity of the Ccr4-Caf1 complex is prerequisite for P body formation. Pan3, but not the deadenylase Pan2, is essential for P body formation. While the C-terminal domain of Pan3 is important for interaction with Pan2, Pan3 N-terminal domain is important for Pan3 to form cytoplasmic foci colocalizing with P bodies and to promote mRNA decay. Interestingly, Pan3 N-terminal domain may be phosphorylated to regulate Pan3 localization and functions. Aside from the functions of the two deadenylation complexes in P bodies, I also studied all reported human P body proteins as a whole using bioinformatics. This effort not only has generated a comprehensive picture of the functions of and interactions among human P body proteins, but also has predicted proteins that may regulate P body formation and/or functions. In summary, my study has established a direct link between mRNA deadenylation and P body formation and has also led to new hypotheses to guide future research on how P body dynamics are controlled.

Application of Signal Advance Technology to Electrophysiology

Medical instrumentation used in diagnosis and treatment relies on the accurate detection and processing of various physiological events and signals. While signal detection technology has improved greatly in recent years, there remain inherent delays in signal detection/ processing. These delays may have significant negative clinical consequences during various pathophysiological events. Reducing or eliminating such delays would increase the ability to provide successful early intervention in certain disorders thereby increasing the efficacy of treatment. In recent years, a physical phenomenon referred to as Negative Group Delay (NGD), demonstrated in simple electronic circuits, has been shown to temporally advance the detection of analog waveforms. Specifically, the output is temporally advanced relative to the input, as the time delay through the circuit is negative. The circuit output precedes the complete detection of the input signal. This process is referred to as signal advance (SA) detection. An SA circuit model incorporating NGD was designed, developed and tested. It imparts a constant temporal signal advance over a pre-specified spectral range in which the output is almost identical to the input signal (i.e., it has minimal distortion). Certain human patho-electrophysiological events are good candidates for the application of temporally-advanced waveform detection. SA technology has potential in early arrhythmia and epileptic seizure detection and intervention. Demonstrating reliable and consistent temporally advanced detection of electrophysiological waveforms may enable intervention with a pathological event (much) earlier than previously possible. SA detection could also be used to improve the performance of neural computer interfaces, neurotherapy applications, radiation therapy and imaging. In this study, the performance of a single-stage SA circuit model on a variety of constructed input signals, and human ECGs is investigated. The data obtained is used to quantify and characterize the temporal advances and circuit gain, as well as distortions in the output waveforms relative to their inputs. This project combines elements of physics, engineering, signal processing, statistics and electrophysiology. Its success has important consequences for the development of novel interventional methodologies in cardiology and neurophysiology as well as significant potential in a broader range of both biomedical and non-biomedical areas of application.

Diffusion tensor imaging of cocaine-treated rodents.

Studies in cocaine-dependent human subjects have shown differences in white matter on diffusion tensor imaging (DTI) compared with non-drug-using controls. It is not known whether the differences in fractional anisotropy (FA) seen on DTI in white matter regions of cocaine-dependent humans result from a pre-existing predilection for drug use or purely from cocaine abuse. To study the effect of cocaine on brain white matter, DTI was performed on 24 rats after continuous infusion of cocaine or saline for 4 weeks, followed by brain histology. Voxel-based morphometry analysis showed an 18% FA decrease in the splenium of the corpus callosum (CC) in cocaine-treated animals relative to saline controls. On histology, significant increase in neurofilament expression (125%) and decrease in myelin basic protein (40%) were observed in the same region in cocaine-treated animals. This study supports the hypothesis that chronic cocaine use alters white matter integrity in human CC. Unlike humans, where the FA in the genu differed between cocaine users and non-users, the splenium was affected in rats. These differences between rodent and human findings could be due to several factors that include differences in the brain structure and function between species and/or the dose, timing, and duration of cocaine administration.

Coordinated changes in mRNA turnover, translation, and RNA processing bodies in bronchial epithelial cells following inflammatory stimulation.

Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.

NOVEL MECHANISMS OF ANTIGEN PROCESSING THAT ENHANCE BCG VACCINE EFFICACY

Mycobacterium tuberculosis, the causative agent of tuberculosis, is the most lethal single infectious agent afflicting man today causing 2 million deaths per year. The World Health Organization recommends a vaccine as the best option to prevent this disease. The current vaccine, BCG, has a variable efficacy and does not protect adults. It is known that BCG vaccine becomes sequestered in special phagosome compartments of macrophages that do not fuse with lysosomes. Since lysosome fusion is necessary for peptide production and T cell priming leading to protective TH1 immunity, we hypothesized that vaccine efficacy is reduced and occurs perhaps due to non-lysosome dependent mechanisms. We therefore proposed an in depth analysis of phagosome environment, and its proteome to unravel mechanisms of antigen processing and presentation. We initially discovered that three mechanisms of pH regulation including vacuolar proton ATPase, phagocyte oxidase and superoxide dismutase (SOD) secretion from BCG vaccine affect antigen processing within phagosomes. These studies led to the discovery that a mutant of BCG vaccine which lacked SOD was a better vaccine. Subsequently, the proteomic analysis of vaccine phagosomes led to the discovery of novel protease (γ-secretase) enriched on BCG vaccine phagosomes. We then demonstrated that these proteases generated a peptide from the BCG vaccine which was presented through the MHC-II pathway to T cells and induced a TH1 response. The specificity of antigen production from γ-secretase was confirmed through siRNA knockdown of the components of the protease namely, nicastrin, presenilin and APH, which led to a decrease in antigen presentation. We therefore conclude that, even though BCG phagosomes are sequestered and do not fuse with lysosomes to generate peptide antigens, there are complex and novel in situ mechanisms within phagosomes that are capable of generating an immune response. We conclude that TH1 immunity to BCG vaccine arises mostly due to non-lysosome dependent immune mechanisms of macrophages and dendritic cells.

The processing of human pro-tumor necrosis factor

Human pro-TNF-$\alpha$ is a 26 kd type II transmembrane protein, and it is the precursor of 17 kd mature TNF. Pro-TNF release mature from its extracellular domain by proteolytic cleavage between resideu Ava ($-$1) and Val (+1). Both forms of TNF are biologically active and the native form of mature TNF is a bell-shaped trimer. The structure of pro-TNF was studied both in intact cell system and in an in vitro translation system by chemical crosslinking. We found that human pro-TNF protein exist as a trimer in intact cells (LPS-induced THP-1 cells and TNF cDNA transfected COS-3 cells) and this trimeric structure is assembled intracellularly, possibly in the ER. By analysis several deletion mutants, we observed a correlation between expression of pro-TNF cytotoxicity in a juxtacrine fashion and detection of the trimer, suggesting the trimeric structure is very important for its biologic activity. With a series of deletion mutants in the linking domain, we found that the small deletion did not block the cleavage and large deletion did regardless of the presence or absence of the native cleavage site, suggesting that the length of the residues between the plasma membrane and the base of the trimer determines the rate of the cleavage, possibly by blocking the accessibility of the cleavage enzyme to its action site. Our data also suggest that the native cleavage site is not sufficient for the release of mature TNF and alternative cleavage site(s) exists. ^

The unstable (CTG)$\sb{\rm n}$ trinucleotide repeat associated with myotonic dystrophy: What it does and where it came from

Myotonic dystrophy (DM), an autosomal dominant disorder mapping to human chromosome 19q13.3, is the most common neuromuscular disease in human adults.^ Following the identification of the mutation underlying the DM phenotype, an unstable (CTG)$\sb{n}$ trinucleotide repeat in the 3$\prime$ untranslated region (UTR) of a gene encoding a ser/thr protein kinase named DM protein kinase (DMPK), the study was targeted at two questions: (1) the identification of the disease-causing mechanism(s) of the unstable repeat, and at a more basic level, (2) the identification of the origin and the mechanism(s) involved in repeat instability. The first goal was to identify the pathophysiological mechanisms of the (CTG)$\sb{n}$ repeat.^ The normal repeat is transcribed but not translated; therefore, initial studies centered on the effect on RNA transcript levels. The vast majority of DM affecteds are heterozygous for the mutant expansion, so that the normal allele interferes with the analysis of the mutant allele. A quantitative allele-specific RT-PCR procedure was developed and applied to a spectrum of patient tissue samples and cell lines. Equal levels of unprocessed pre-mRNA were determined for the wild type (+) and disease (DM) alleles in skeletal muscle and cell lines of heterozygous DM patients, indicating that any nucleosome binding has no effect at the level of transcriptional initiation and transcription of the mutant DMPK locus. In contrast, processed mRNA levels from the DM allele were reduced relative to the + allele as the size of the expansion increased. The unstable repeat, therefore, impairs post-transcriptional processing of DM allele transcripts. This phenomenon has profound effects on overall DMPK locus steady-state transcript levels in cells missing a wild type allele and does not appear to be mediated by imprinting, decreased mRNA stability, generation of aberrant splice forms, or absence of polyadenylation of the mutant allele.^ In Caucasian DM subjects, the unstable repeat is in complete linkage disequlibrium with a single haplotype composed of nine alleles within and flanking DMPK over a physical distance of 30 kb. A detailed haplotype analysis of the DM region was conducted on a Nigerian (Yoruba) DM family, the only indigenous sub-Saharan DM case reported to date. Each affected member of this family had an expanded (CTG)$\sb{n}$ repeat in one of their DMPK alleles. However, unlike all other DM populations studied thus far, disassociation of the (CTG)$\sb{n}$ repeat expansion from other alleles of the putative predisposing haplotype was found. Thus, the expanded (CTG)$\sb{n}$ repeat in this family was the result of an independent mutational event. Consequently, the origin of DM is unlikely the result of a single mutational event, and the hypothesis that a single ancestral haplotype predisposes to repeat expansion is not compelling. (Abstract shortened by UMI.) ^

Fibrillins: Sequence, phylogeny and carboxy terminal domain proteolytic processing

Fibrillin-1 and -2 are large secreted glycoproteins that are known to be components of extracellular matrix microfibrils located in the vasculature, basement membrane and various connective tissues. These microfibrils are often associated with a superstructure known as the elastic fiber. During the development of elastic tissues, fibrillin microfibrils precede the appearance of elastin and may provide a scaffolding for the deposition and crosslinking of elastin. Using RT/PCR, we cloned and sequenced 3.85Kbp of the FBN2 gene. Five differences were found between our contig sequence and that published by Zhang et al. (1995). Like many extracellular matrix proteins, the fibrillins are modular proteins. We compared analogous domains of the two fibrillins and also members of the latent TGF-$\beta$ binding protein (LTBP) family to determine their phylogenetic relationship. We found that the two families are homologous. LTBP-2 is the most similar to the fibrillin family while FBN-1 is the most similar to the LTBP family. The fibrillin-1 carboxy terminal domain is proteolytically processed. Two eukaryotic protein expression systems, baculoviral and CHO-K1, were developed to examine the proteolytic processing of the carboxy terminal domain of the fibrillin-1 protein. Both expression systems successfully processed the domain and both processed a mutant less efficiently. In the CHO-K1 cells, processing occurred intracellularly. ^

Analysis of primary Wilms tumors for WT1 RNA processing alterations

Wilms tumor (WT) is an embryonal renal tumor with a heterogeneous genetic etiology that serves as a valuable model for studying tumorigenesis. Biallelic inactivation of the tumor suppressor gene WT1, a zinc-finger transcriptional regulator located at 11p13, is critical for the development of some Wilms tumors. Interestingly, WT1 genomic analysis has demonstrated mutations in less than 20% of WT cases. This suggests either other genes play a more major role in Wilms tumorigenesis or WT1 is functionally altered by mechanisms other than DNA mutation. Previous observations in rat and in WT xenograft cell lines have suggested that abnormal WT1 RNA processing (exon 6 RNA editing and aberrant exon 2 splicing, respectively) is a potential mechanism of altering WT1 function in the absence of a WT1 DNA mutation. However, the role of this abnormal RNA processing has not previously been assessed in primary Wilms tumors. ^ To test the hypothesis that abnormal WT1 RNA processing is a mechanism of WT1alteration during tumor development, WT1 RNA from 85 primary tumors was analyzed using reverse transcription and polymerase chain reaction amplification (RT-PCR). Although no evidence for WT1 RNA editing was observed, variable levels (5% to 50%) of aberrant WT1 exon 2 splicing were detected for 11 tumors in the absence of a detectable WT1 DNA mutation. Also, alteration of normal WT1 alternative splicing, observed as RNA isoform loss, was detected in five tumors with no apparent WT1 genomic alteration, although no consistent pattern of RNA isoform loss was detected. This abnormal WT1 splicing, detected by either loss of exon 2 from some of the transcripts or loss of RNA isoforms, is statistically correlated with relapse (p = 0.005). These studies demonstrate that abnormal WT1 RNA processing is not a common mechanism of abrogating normal WT1 function in primary tumors. However, in those cases in which abnormal WTI splicing is present, these data indicate that it may serve as a useful prognostic marker for relapse in WT patients. ^