Biomedical subjects

David C Ward

Publications and source records attributed to David C Ward.

10 recordsLinked to original sources

PubMed2006

Dimensional assessment of X-bearing and Y-bearing haploid and disomic human sperm with the use of fluorescence in situ hybridization and objective morphometry.

OBJECTIVE: To explore the dimensional attributes of haploid and disomic X-bearing and Y-bearing spermatozoa. DESIGN: Morphometric evaluation of more than 2,000 X-bearing and Y-bearing spermatozoa after identification of the genotype with fluorescence in situ hybridization. SETTING: Academic clinical and research andrology laboratory. MAIN OUTCOME MEASURE(S): Sperm head area, perimeter, long axis, short axis, shape factor, elliptical form factor (long axis/short axis), and tail length. RESULT(S): We found no differences in dimensions or dimensional distributions between X-bearing and Y-bearing spermatozoa, whether in the native or the decondensed state, or in oligozoospermic or normozoospermic men. There were inconsistent differences and a 70% overlap in the dimensions of haploid and disomic spermatozoa. The other 30% of sperm with disomic nuclei were either smaller or larger compared to haploid spermatozoa. CONCLUSION(S): There are no differences, or distinguishing characteristics, in dimensions or dimensional distributions between X-bearing and Y-bearing spermatozoa. Dimensional attributes do not discriminate between dysomic and haploid spermatozoa.

Cell Shape↗

PubMed2006

Thin-film technology for direct visual detection of nucleic acid sequences: applications in clinical research.

Certain optical conditions permit the unaided eye to detect thickness changes on surfaces on the order of 20 A, which are of similar dimensions to monomolecular interactions between proteins or hybridization of complementary nucleic acid sequences. Such detection exploits specific interference of reflected white light, wherein thickness changes are perceived as surface color changes. This technology, termed thin-film detection, allows for the visualization of subattomole amounts of nucleic acid targets, even in complex clinical samples. Thin-film technology has been applied to a broad range of clinically relevant indications, including the detection of pathogenic bacterial and viral nucleic acid sequences and the discrimination of sequence variations in human genes causally related to susceptibility or severity of disease.

Base Sequence↗

PubMed2005

Simultaneous detection of microsatellite repeats and SNPs in the macrophage migration inhibitory factor (MIF) gene by thin-film biosensor chips and application to rural field studies.

Microsatellite repeat and single nucleotide polymorphisms (SNPs) are abundant sources of genetic variation, but existing methodologies cannot simultaneously detect these variants in a facile or inexpensive way. We describe herein a thin-film biosensor chip based on an allele-discriminating oligonucleotide array that enables genotyping for both microsatellite repeats and SNPs in a single analysis. We validated this methodology for the functionally polymorphic -794 CATT(5-8) repeat and -173 G/C SNP present in the promoter of the human gene for macrophage migration inhibitory factor (MIF). In a comparison of 30 samples collected at a rural hospital in Zambia, we observed a 100% concordance for both the CATT repeat and G/C SNP between the biosensor methodology and the conventional capillary electrophoresis. The biosensor chips are low in cost and once printed, they are robust and require no instrumentation for analysis. When combined with multiple displacement amplification, this methodology can be utilized in primitive settings for the genotyping of nanogram quantities of DNA present in blood, dried and stored on filter paper samples. We applied this methodology to a field study of MIF genotype in children with malaria, and provide first evidence for a potential association between MIF alleles and malaria infection. We also present data supporting significant population stratification of the low- versus high-expression forms of MIF that may bear on the role of this gene in infectious diseases.

Biosensing Techniques↗

PubMed2005

Serum protein markers for early detection of ovarian cancer.

Early diagnosis of epithelial ovarian cancer (EOC) would significantly decrease the morbidity and mortality from this disease but is difficult in the absence of physical symptoms. Here, we report a blood test, based on the simultaneous quantization of four analytes (leptin, prolactin, osteopontin, and insulin-like growth factor-II), that can discriminate between disease-free and EOC patients, including patients diagnosed with stage I and II disease, with high efficiency (95%). Microarray analysis was used initially to determine the levels of 169 proteins in serum from 28 healthy women, 18 women newly diagnosed with EOC, and 40 women with recurrent disease. Evaluation of proteins that showed significant differences in expression between controls and cancer patients by ELISA assays yielded the four analytes. These four proteins then were evaluated in a blind cross-validation study by using an additional 106 healthy females and 100 patients with EOC (24 stage I/II and 76 stage III/IV). Upon sample decoding, the results were analyzed by using three different classification algorithms and a binary code methodology. The four-analyte test was further validated in a blind binary code study by using 40 additional serum samples from normal and EOC cancer patients. No single protein could completely distinguish the cancer group from the healthy controls. However, the combination of the four analytes exhibited the following: sensitivity 95%, positive predictive value (PPV) 95%, specificity 95%, and negative predictive value (NPV) 94%, a considerable improvement on current methodology.

Aged↗

PubMed2004

Peptide conjugates for chromosomal gene targeting by triplex-forming oligonucleotides.

Triplex-forming oligonucleotides (TFOs) are DNA-binding molecules, which offer the potential to selectively modulate gene expression. However, the biological activity of TFOs as potential antigene compounds has been limited by cellular uptake. Here, we investigate the effect of cell-penetrating peptides on the biological activity of TFOs as measured in an assay for gene-targeted mutagenesis. Using the transport peptide derived from the third helix of the homeodomain of antennapedia (Antp), we tested TFO-peptide conjugates compared with unmodified TFOs. TFOs covalently linked to Antp resulted in a 20-fold increase in mutation frequency when compared with 'naked' oligonucleotides. There was no increase above background in mutation frequency when Antp by itself was added to the cells or when Antp was linked to mixed or scrambled sequence control oligonucleotides. In addition, the TFO-peptide conjugates increased the mutation frequency of the target gene, and not the control gene, in a dose-responsive manner. Confocal microscopy using labeled oligonucleotides indicated increased cellular uptake of TFOs when linked to Antp, consistent with the gene-targeting data. These results suggest that peptide conjugation may enhance intranuclear delivery of reagents designed to bind to chromosomal DNA.

Animals↗

PubMed2004

Candidate locus for Gilles de la Tourette syndrome/obsessive compulsive disorder/chronic tic disorder at 18q22.

Gilles de la Tourette syndrome (GTS), obsessive compulsive disorder (OCD), and chronic tic disorder (CTD) are chronic, potentially debilitating neuropsychiatric disorders that often cluster in families. Comorbidity data and family and linkage studies support the hypothesis that these phenotypes, in some cases, share a common etiology. Studies of chromosomal abnormalities associated with this phenotypic spectrum further show that GTS, OCD, and CTD may represent alternate manifestations of a shared genetic condition. We report on a 14-year-old girl with severe OCD and a t(2;18)(p12;q22) translocation. The patient's chromosome 18 breakpoint localizes to the same chromosomal band as two previously reported rearrangements associated with GTS, OCD, and CTD, and fine maps to a genomic position approximately 5 Mb from these rearrangements. The clustering of these three breakpoints within a relatively small genetic interval suggests that 18q22 is a promising region for containing a gene or genes of etiologic importance in the development of the GTS/OCD/CTD phenotypic spectrum.

Adolescent↗

PubMed2003

Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips.

Single-nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation and provide excellent markers to identify genetic factors contributing to complex disease susceptibility. A rapid, sensitive, and inexpensive assay is important for large-scale SNP scoring. Here we report the development of a multiplex SNP detection system using silicon chips coated to create a thin-film optical biosensor. Allele-discriminating, aldehyde-labeled oligonucleotides are arrayed and covalently attached to a hydrazinederivatized chip surface. Target sequences (e.g., PCR amplicons) then are hybridized in the presence of a mixture of biotinylated detector probes, one for each SNP, and a thermostable DNA ligase. After a stringent wash (0.01 M NaOH), ligation of biotinylated detector probes to perfectly matched capture oligomers is visualized as a color change on the chip surface (gold to blue/purple) after brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. Testing of PCR fragments is completed in 30-40 min. Up to several hundred SNPs can be assayed on a 36-mm2 chip, and SNP scoring can be done by eye or with a simple digital-camera system. This assay is extremely robust, exhibits high sensitivity and specificity, and is format-flexible and economical. In studies of mutations associated with risk for venous thrombosis and genotyping/haplotyping of African-American samples, we document high-fidelity analysis with 0 misassignments in 500 assays performed in duplicate.

Biosensing Techniques↗

PubMed2003

[Numerical chromosome anomalies in human sperm at low sperm concentration].

INTRODUCTION: With the assisted reproduction techniques the natural selection of sperm is bypassed on different levels. AIM: The aim of the present work is to determine the frequencies of sperm numerical chromosome aberrations in infertile men with low sperm count and to examine the relationship between the spermatogram parameters (sperm count and motility) and the aneuploidy and diploidy frequencies. METHODS: 32 men with low sperm count were investigated. Semen analysis was performed according to the WHO criteria. Disomy and diploidy frequencies were detected with fluorescence in situ hibridization using 17, X and Y centromeric probes. 200,969 sperm were scored, with the mean of 6272 cells in each subjects. The rate of numerical chromosome anomalies were also estimated using the detected disomy and the diploidy frequencies. RESULTS: Mean sperm concentration of the 32 men was 18.2 million/ml (SD: +/- 8.43, range: 8.0-45.5), mean motility 49.4% (+/- 9.32, 30-69.2). The X/Y ratio was 1.07. The mean frequencies of sex chromosome disomy, chromosome 17 disomy and diploidy were 0.36%, 0.16% and 0.56%, respectively. The most frequent disomy was XY disomy (0.14). Sex chromosome disomy frequencies were higher in oligospermic samples (0.37% vs. 0.32%, OR = 1.18, 95% CI = 1.01-1.39, p < 0.001). With special regard to XY disomy (0.08% vs. 0.17%, OR = 1.99, 95% CI = 1.48-2.67, p < 0.001). In subjects with oligoasthenozoospermia the diploidy frequency increased (0.96%, OR = 2.39, 95% CI = 2.13-2.69, p < 0.001), mostly due to the elevated rate of diploid sperm of meiosis II. origin (XX or YY diploids). Estimated frequency of the numerical chromosome anomalies was 8.3 +/- 5.3 in the study population. Neither the sperm count, nor the sperm motility showed correlation with the detected frequency of the chromosome aberrations. CONCLUSIONS: In oligospermic patients there is a risk for elevated frequency of sperm with sex chromosome aneuploidy, especially the XY disomy. Furthermore, the diploidy frequency is increased in oligozooastenospermic samples. Nevertheless, classical parameters of semen analysis (sperm count and motility) do not correlate with the frequency of numerical chromosomal anomalies. The risk can be determined using fluorescens in situ hybridization on sperm.

Chromosome Aberrations↗

PubMed2003

Epigenetic abnormalities associated with a chromosome 18(q21-q22) inversion and a Gilles de la Tourette syndrome phenotype.

Gilles de la Tourette syndrome (GTS) is a potentially debilitating neuropsychiatric disorder defined by the presence of both vocal and motor tics. Despite evidence that this and a related phenotypic spectrum, including chronic tics (CT) and Obsessive Compulsive Disorder (OCD), are genetically mediated, no gene involved in disease etiology has been identified. Chromosomal abnormalities have long been proposed to play a causative role in isolated cases of GTS spectrum phenomena, but confirmation of this hypothesis has yet to be forthcoming. We describe an i(18q21.1-q22.2) inversion in a patient with CT and OCD. We have fine mapped the telomeric aspect of the rearrangement to within 1 Mb of a previously reported 18q22 breakpoint that cosegregated in a family with GTS and related phenotypes. A comprehensive characterization of this genomic interval led to the identification of two transcripts, neither of which was found to be structurally disrupted. Analysis of the epigenetic characteristics of the region demonstrated a significant increase in replication asynchrony in the patient compared to controls, with the inverted chromosome showing delayed replication timing across at least a 500-kb interval. These findings are consistent with long-range functional dysregulation of one or more genes in the region. Our data support a link between chromosomal aberrations and epigenetic mechanisms in GTS and suggest that the study of the functional consequences of balanced chromosomal rearrangements is warranted in patients with phenotypes of interest, irrespective of the findings regarding structurally disrupted transcripts.

Child↗

PubMed2003

Detection of chromosomal instability in paired breast surgery and ductal lavage specimens by interphase fluorescence in situ hybridization.

PURPOSE: Ductal lavage is a new modality for collecting exfoliated breast cells with the goal of detecting early neoplasia. The purpose of our study was to evaluate the correlation between cancer-associated abnormalities in breast lesions and exfoliated breast cells collected by ductal lavage. EXPERIMENTAL DESIGN: We performed histopathologic, cytologic, and molecular cytogenetic analyses on 39 paired cases of surgically excised breast lesions and ductal lavage specimens collected immediately before surgery. RESULTS: Abnormal cytology was detected in 7 of 15 (47%) of the evaluable lavages collected from malignant cases, versus 4 of 19 (21%) of the evaluable lavages harvested from benign cases for a sensitivity and specificity of 47 and 79%, respectively. Interphase fluorescence in situ hybridization analysis of all evaluable lavages revealed numeric changes on chromosomes 1, 8, 11, and/or 17 in 10 of 14 (71%) specimens from malignant cases versus 2 of 18 (11%) from benign cases for a sensitivity and specificity of 71 and 89%, respectively. CONCLUSIONS: Our study demonstrates that cytologic and genetic abnormalities associated with breast cancer progression can be detected in ductal lavage cells collected from women with in situ and invasive breast cancer and suggests that fluorescence in situ hybridization may have superior sensitivity and specificity compared with conventional cytology.

Breast↗