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Biomedical subjects

Robert A Welch

Publications and source records attributed to Robert A Welch.

7 recordsLinked to original sources
PubMed2006

Operator experience and sample quality in genetic amniocentesis.

OBJECTIVE: We sought to relate the frequency of maternal cell contamination in amniotic fluid samples that were submitted to a single laboratory for cytogenetic analysis to the experience and training of the physician who performed the amniocentesis. STUDY DESIGN: We reviewed the database of a single cytogenetics laboratory to compare the number of amniocenteses that were performed annually per physician to the rate of maternal cell contamination in genetic amniocentesis samples. Only samples that resulted in a 46 XY male karyotype were studied so that maternal cell contamination could be identified as having occurred when the karyotype revealed > or = 1 cell with 2 X chromosomes. Samples were categorized as being submitted by a physician who submitted > or = 50 or more samples annually versus < 50 samples to this laboratory. The frequency of maternal cell contamination was compared with annual operator volume with 2 x 2 tables and analyzed by chi-squared testing. RESULTS: Between 2000 and 2004, the laboratory received 6332 mid-trimester amniotic fluid samples that generated a male karyotype result. Fourteen of 2081 samples (0.67%) that were submitted by physicians who submitted < 50 samples grew > or = 1 46 XX cells, compared with 8 of 4251 samples (0.19%; chi-squared, 9.47; degrees of freedom, 1; P = .0021). CONCLUSION: Maternal cell contamination occurs more frequently in genetic amniocentesis samples that are obtained by physicians who perform < 50 genetic amniocenteses annually.

Amniocentesis↗
PubMed2005

Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance.

BACKGROUND: Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1-200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR Identifiler panel) and N = 49 SNP (TaqMan) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs. RESULTS: The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates. CONCLUSION: The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR Identifiler panel from wgaDNA equivalent to that of gDNA.

Biotechnology↗
PubMed2005

Association of multiple DRD2 polymorphisms with anorexia nervosa.

To investigate whether the dopaminergic system plays a role in the etiology of anorexia nervosa (AN) via the dopamine D2 receptor, we investigated association and transmission disequilibrium at seven single-nucleotide polymorphisms (SNPs) spanning about 75 kbp of the gene DRD2. We studied 191 probands with a DSM-IV diagnosis of AN, 457 parents and affected relatives with a DSM-IV eating disorder diagnosis, and 98 unrelated, female, normal weight controls. The -141 C/- insertion/deletion (-141 Indel), previously shown to affect DRD2 transcription efficiency, and multiple exon seven polymorphisms, one of which has previously been shown to affect DRD2 transcript stability, exhibited statistically significant association with diagnosis in haplotype transmission disequilibrium and in haplotype case : control analyses. Significant linkage disequilibrium between the -141 Indel and two exon seven SNPs (939Y and 957Y) was observed over a distance of >50 kbp in the AN probands but not in the controls. Genetically transmitted variation in D2 dopamine receptor expression mediated by functional polymorphisms affecting transcription and translation efficiency may play a role in vulnerability to AN.

Adolescent↗
PubMed2005

Effects of electron-beam irradiation on whole genome amplification.

Electron-beam (E-beam) irradiation, currently being used to sterilize mail addressed to selected ZIP codes in the United States, has significant negative effects on the genomic integrity of DNA extracted from buccal-cell washes. We investigated the yield, composition, and genotyping performance of whole genome amplified DNA (wgaDNA) derived from 24 matched samples of E-beam-irradiated and nonirradiated genomic DNA (gDNA) as a model for the effects of degraded gDNA on the performance of whole genome amplification. gDNA was amplified using the Multiple Displacement Amplification method. Three methods of DNA quantification analysis were used to estimate the yield and composition of wgaDNA, and 65 short tandem repeat and single nucleotide polymorphism genotyping assays were used to evaluate the genotyping performance of irradiated and nonirradiated gDNA and wgaDNA. Compared with wgaDNA derived from nonirradiated gDNA, wgaDNA derived from irradiated gDNA exhibited a significantly reduced yield of wgaDNA and significantly reduced short tandem repeat and single nucleotide polymorphism genotyping completion and concordance rates (P < 0.0001). Increasing the amount of irradiated gDNA input into whole genome amplification improved genotyping performance of wgaDNA but not to the level of wgaDNA derived from nonirradiated gDNA. Multiple Displacement Amplification wgaDNA derived from E-beam-irradiated gDNA is not suitable for genotyping analysis.

DNA↗
PubMed2004

Single nucleotide polymorphism genotyping by two colour melting curve analysis using the MGB Eclipse Probe System in challenging sequence environment.

Probe and primer design for single nucleotide polymorphism (SNP) detection can be very challenging for A-T DNA-rich targets, requiring long sequences with lower specificity and stability, while G-C-rich DNA targets present limited design options to lower GC-content sequences only. We have developed the MGB Eclipse Probe System, which is composed of the following elements: MGB Eclipse probes and primers, specially developed software for the design of probes and primers, a unique set of modified bases and a Microsoft Excel macro for automated genotyping, which ably solves, in large part, this challenge. Fluorogenic MGB Eclipse probes are modified oligonucleotides containing covalently attached duplex-stabilising dihydrocyclopyrroloindole tripeptide (DPI3), the MGB ligand (MGB is a trademark of Epoch Biosciences, Bothell, WA), which has the combined properties of allowing the use of short sequences and providing great mismatch discrimination. The MGB moiety prevents probe degradation during polymerase chain reaction (PCR), allowing the researcher to use real time data; alternatively, hybridisation can be accurately measured by a post-PCR two-colour melt curve analysis. Using MGB Eclipse probes and primers containing modified bases further enhances the analysis of difficult SNP targets. G- or C-rich sequences can be refractory to analysis due to Hoogsteen base pairing. Substitution of normal G with Epoch's modified G prevents Hoogsteen base pairing, allowing both superior PCR and probe-based analysis of GC-rich targets. The use of modified A and T bases allows better stabilisation by significantly increasing the Tm of the oligonucleotides. Modified A creates A-T base pairs that have a stability slightly lower than a G-C base pair, and modified T creates T-A base pairs that have a stability about 30 per cent higher than the unmodified base pair. Together, the modified bases permit the use of short probes, providing good mismatch discrimination and primers that allow PCR of refractory targets. The combination of MGB Eclipse probes and primers enriched with the MGB ligand and modified bases has allowed the analysis of refractory SNPs, where other methods have failed.

Base Sequence↗
PubMed2003

Specific roles of the obstetrician-gynecologist.

Obstetrician-gynecologists can play a major role in the prevention, identification, and treatment of substance abuse in women. Maintaining cognizance of abuse potential and consistently using office-screening techniques will identify patients who need assistance. Employing brief interventions at frequent office visits is an effective means of treating many of these patients. Those who do not respond to brief interventions may benefit from referral to an addictionologist. The US Supreme Court recently verified the Fourth Amendment rights of pregnant women by deciding that consent must be obtained to perform drug screening in pregnancy. Obstetrician-gynecologists will need to stay abreast of the changes that occur in their state laws as a result of this decision.

Attitude of Health Personnel↗