Susan M. Sell

Whole genome microarray analysis of gene expression in Prader–Willi syndrome

Prader–Willi syndrome (PWS) is caused by loss of function of paternally expressed genes in the 15q11‐q13 region and a paucity of data exists on transcriptome variation. To further characterize genetic alterations in this classic obesity syndrome using whole genome microarrays to analyze gene expression, microarray and quantitative RT‐PCR analysis were performed using RNA isolated from lymphoblastoid cells from PWS male subjects (four with 15q11‐q13 deletion and three with UPD) and three age and cognition matched nonsyndromic comparison males. Of more than 47,000 probes examined in the microarray, 23,383 were detectable and 323 had significantly different expression in the PWS lymphoblastoid cells relative to comparison cells, 14 of which were related to neurodevelopment and function. As expected, there was no evidence of expression of paternally expressed genes from the 15q11‐q13 region (e.g., SNRPN) in the PWS cells. Alterations in expression of serotonin receptor genes (e.g., HTR2B) and genes involved in eating behavior and obesity (ADIPOR2, MC2R, HCRT, OXTR) were noted. Other genes of interest with reduced expression in PWS subjects included STAR (a key regulator of steroid synthesis) and SAG (an arrestin family member which desensitizes G‐protein‐coupled receptors). Quantitative RT‐PCR for SAG, OXTR, STAR, HCRT, and HTR2B using RNA isolated from their lymphoblastoid cells and available brain tissue (frontal cortex) from separate individuals with PWS and control subjects and normalized to GAPD gene expression levels validated our microarray gene expression data. Our analysis identified previously unappreciated changes in gene expression which may contribute to the clinical manifestations seen in PWS.

Molecular Analysis of Chromosome 7q21.3 in Uterine Leiomyoma: Analysis using Markers with Linkage to Insulin Resistance

Recent sibling-pair linkage analyses have indicated possible linkage of noninsulin dependent diabetes mellitus (NIDDM) with a number of markers on the long arm of chromosome 7. A coincidental and recent discovery is that specific genetic anomalies identified on chromosome 7 in uterine leiomyoma tumor cells in many cases correspond, cytogenetically, to the same region where genetic linkage to insulin resistance has been identified. In the present study, 15 closely spaced microsatellite markers were used to finely map deletion breakpoints and to test for allelic loss of 7q markers in 12 uterine leiomyoma tumor samples with cytogenetically defined deletions. Of the 9 informative tumor samples, three exhibited breakpoints in the same region where genetic linkage to insulin resistance has been identified (between PON and UT901). Because breakpoints in neoplasias often occur within or adjacent to expressed sequences, these breakpoints may provide a molecular tool to aid in the identification of candidate genes for insulin resistance.

Personalized online information search and visualization

BackgroundThe rapid growth of online publications such as the Medline and other sources raises the questions how to get the relevant information efficiently. It is important, for a bench scientist, e.g., to monitor related publications constantly. It is also important, for a clinician, e.g., to access the patient records anywhere and anytime. Although time-consuming, this kind of searching procedure is usually similar and simple. Likely, it involves a search engine and a visualization interface. Different words or combination reflects different research topics. The objective of this study is to automate this tedious procedure by recording those words/terms in a database and online sources, and use the information for an automated search and retrieval. The retrieved information will be available anytime and anywhere through a secure web server.ResultsWe developed such a database that stored searching terms, journals and et al., and implement a piece of software for searching the medical subject heading-indexed sources such as the Medline and other online sources automatically. The returned information were stored locally, as is, on a server and visible through a Web-based interface. The search was performed daily or otherwise scheduled and the users logon to the website anytime without typing any words. The system has potentials to retrieve similarly from non-medical subject heading-indexed literature or a privileged information source such as a clinical information system. The issues such as security, presentation and visualization of the retrieved information were thus addressed. One of the presentation issues such as wireless access was also experimented. A user survey showed that the personalized online searches saved time and increased and relevancy. Handheld devices could also be used to access the stored information but less satisfactory.ConclusionThe Web-searching software or similar system has potential to be an efficient tool for both bench scientists and clinicians for their daily information needs.

Allelic Loss Analysis by Capillary Electrophoresis: An Accurate, Automated Method for Detection of Deletions in Solid Tumors

In this report, we describe a fast and accurate capillary electrophoresis, PCR-based method for detecting loss of allelic heterozygosity in solid tumor samples. This automated method requires small sample sizes, and data can be obtained in less than 15 min. The method is particularly powerful for uncovering deletions in tumor sample preparations containing significant normal tissue contamination.

V(D)J recombinase precursors and coding structure of signal sequence directed rearrangement

Abstract In order to increase our understanding of the complex process of immunoglobulin and T-cell receptor signal sequence directed rearrangements in vertebrates, an evolutionary approach was designed to (1) assess structural motifs important for rearrangement, (2) discuss evidence for precursors of recombinase candidates and (3) characterize a putative example of a recombinase mediated translocation in a human tumor of non-hematologic origin. Genomic reorganization occurs as a normal, developmentally regulated process in a variety of organisms. In the vertebrates, recombination signal sequence directed V(D)J rearrangement operates during the development of both T- and B-cells to generate mature T-cell receptor and immunoglobulin variable region genes, respectively. It is argued that this type of signal sequence directed rearrangement event probably evolved from a primitive rearrangement system, and it is proposed that novel rearranging gene families exist in other terminally differentiating tissues.

An improved assay for genotyping the common Alu insertion in the tissue-type plasminogen activation gene PLAT.

An improved assay for genotyping the common Alu insertion in the tissue-type plasminogen activator (PLAT) locus is described in this report. The assay is a valuable asset to clinical researchers interested in exploring disease associations with this allele. The automation and improved accuracy will facilitate future population-based studies, as well as clinical screening.

Jets : a modification to speed flexible oligonucleotide array construction

We have come a long way since the days of Northern blots. With the completion, or near completion, of multiple genome sequencing projects, DNA microarray technology is emerging as an extremely powerful and fundamental tool that can be used to explore genetic and molecular pathways associated with disease and other cellular processes.1,2 Translation of this information to the clinical arena will result in improvements in patient diagnostics and treatments.3,4,5 The application of array technology to biological questions is quickly transforming scientific paradigm, from one that formerly centered on the analysis of a relatively few biological measurements to one that supports the simultaneous exploration of thousands of events. While array technology is a powerful tool for both researchers and clinicians, its utility is hindered by the expense of, and difficulties associated with, custom modification of the arrays. In a recent article published in Nature Biotechnology, Hughes et al describe the adaptation of ink-jet technology for the in situ synthesis of small quantities of unique 60-mer oligonucleotides directly onto glass slides.6 This ‘second generation’ ink-jet oligonucleotide synthesizer is based on the approach described by Blanchard et al.7 It will greatly facilitate array modification by eliminating the time necessary for large-scale cDNA or oligonucleotide synthesis, reorganization of microtiter dishes, re-spotting of the DNA elements, or the generation of new photolithographic masks. With this new application of ink-jet technology, small quantities of oligonucleotides, specified by the user, can be synthesized directly onto the array. This permits a rapid customization that can meet the widely varying needs of investigators.