Biomedical subjects

Michele Caggana

Publications and source records attributed to Michele Caggana.

8 recordsLinked to original sources

PubMed2005

Development and characterization of on-chip biopolymer membranes.

In lab-on-a-chip applications, filtration is currently performed prior to sample loading or through pre-cast membranes adhered to the substrate. These membranes cannot be patterned to micrometer resolution, and their adhesion may be incompatible with the fabrication process or may introduce contaminants. We have developed an on-chip separation process using a biocompatible polymer that can be patterned and has controllable molecular rejection properties. We spun cast cellulose acetate (CA) membranes directly onto silicon wafers. Characterization of the molecular flux across the membrane showed that molecular weight and charge are major factors contributing to the membranes' rejection characteristics. Altering casting conditions such as polymer concentration in the casting solution and the quenching-bath composition and/or temperature allowed control of the molecular weight cut-off (MWCO). Three MWCOs; 300, 350, and 700 Da have been achieved for non-linear molecules. Molecular shape is also very important as much higher molecular weight single-stranded DNA was electrophoresed across the membranes while heme with a similar negative charge density was rejected. This was due to DNA's small molecular cross section. This is an important result because heme inhibits polymerase chain reactions (PCR) reducing the detection and characterization of DNA from blood samples.

Base Sequence↗

PubMed2005

Developing a sustainable process to provide quality control materials for genetic testing.

PURPOSE: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community. METHODS: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps. RESULTS: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step. CONCLUSIONS: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

Centers for Disease Control and Prevention, U.S.↗

PubMed2004

Development of a real-time polymerase chain reaction-based method for the measurement of relative allelic expression and identification of CYP2A13 alleles with decreased expression in human lung.

CYP2A13 is a human cytochrome P450 monooxygenase that is efficient in the metabolic activation of tobacco-specific nitrosamines. Sequence variations that affect CYP2A13 expression may contribute to interindividual differences in susceptibility to tobacco-related tumorigenesis. The aim of this study was to identify any impact of CYP2A13 single-nucleotide polymorphisms (SNPs) on CYP2A13 expression in human lung. Expression levels of CYP2A13 mRNA in normal lung displayed significant interindividual variation (>50-fold). Preliminary sequence analysis of CYP2A13 RNA-polymerase chain reaction (PCR) products suggested that a 7520C > G variation, located in the 3'-untranslated region, could be associated with low transcript abundance. Subsequently, we developed a method for the measurement of relative allelic expression, by taking advantage of the capability for melting-curve analysis in real-time PCR. Quantitative analyses using this method indicated that transcripts from the 7520G-containing alleles were >10-fold less abundant than those from the 7520C-containing alleles in 14 of 16 samples examined. The frequencies of the 7520C > G variation in anonymous White, African American, Hispanic, and Asian newborns from New York State were found to be 5.2, 26.8, 17.7, and 4.3%, respectively. The 7520C > G SNP was previously known to be present in both CYP2A13(*)1H and (*)3 alleles. However, analyses of SNP distribution indicated that, in 15 of the 16 heterozygous DNA samples, the 7520C > G SNP belonged to new CYP2A13(*)1 haplotypes. These findings provide a basis for further studies that associate CYP2A13 haplotypes with incidences of smoking-related lung tumors and for studies on the mechanisms of the low-expression phenotype of the 7520G-containing allele.

Adult↗

PubMed2004

Development of a rare cell fractionation device: application for cancer detection.

Isolating rare cells from biological fluids including whole blood or bone marrow is an interesting biological problem. Characterization of a few metastatic cells from cancer patients for further study is desirable for prognosis/diagnosis. Traditional methods have not proven adequate, due to the compositional complexity of blood, with its large numbers of cell types. To separate individual cells based on their mechanical characteristics, we have developed a series of massively parallel microfabricated sieving device. These devices were constructed with four successively narrower regions of channels numbering approximately 1800 per region. As cells traversed the device, they encountered each region and stopped at a gap width that prohibited passage due to their size. Cultured neuroblastoma cells, when mixed with whole blood and applied to the device, were retained in the 10-microm-wide by 20-microm-deep channels. All other cells migrated to the output. A derivative of the same device was utilized to characterize migration of whole blood. Adult white blood cells were retained at the 2.5-microm-wide by 5-microm-deep channels, while red blood cells passed through these channels. Devices designed to capture rare cells in peripheral circulation for downstream analysis will provide an important tool for diagnosis and treatment.

Cell Separation↗

PubMed2003

Single nucleotide polymorphisms of the human cyp2a13 gene: evidence for a null allele.

Human CYP2A13 is believed to be important in the metabolic activation of tobacco-specific nitrosamines in the respiratory tract; therefore, genetic polymorphisms of the CYP2A13 gene may be associated with interindividual differences in the risks of tobacco-related tumorigenesis. Our earlier studies identified a frequent single nucleotide polymorphism in CYP2A13 exon 5, Arg257Cys, which led to an approximate 50% decrease in metabolic activities. In the present study, three additional coding region mutations (Arg25Gln, Arg101Stop, and Asp158Glu) and several mutations in the introns and flanking regions were identified in a Chinese patient population. Of particular interest is the Arg101Stop mutation, which was due to a C > T change in exon 2. Thus, individuals homozygous for this nonsense mutation would not have a functional CYP2A13 protein and, therefore, might have reduced sensitivity to xenobiotic toxicity resulting from CYP2A13-mediated metabolic activation in the respiratory tract. The frequencies of the coding region mutations were further examined using random samples of white, black, Hispanic, and Asian newborns from New York. The frequency of the Arg25Gln mutation in Asian newborns (9.6%) was very similar to that found in the Chinese population (10.9%). On the other hand, the Arg101Stop mutation was not detected in 136 newborn samples examined (23 white, 21 black, 19 Hispanic, and 73 Asian), suggesting that this mutation may be unique for the Chinese patient population. Haplotype analysis indicated that the Arg25Gln and Arg257Cys mutations are parts of a common haplotype. However, an additional haplotype that consists of the 25Gln but not the 257Cys allele was also identified.

Alleles↗

PubMed2002

DNA banking for epidemiologic studies: a review of current practices.

To study genetic risk factors for common diseases, researchers have begun collecting DNA specimens in large epidemiologic studies and surveys. However, little information is available to guide researchers in selecting the most appropriate specimens. In an effort to gather the best information for the selection of specimens for these studies, we convened a meeting of scientists engaged in DNA banking for large epidemiologic studies. In this discussion, we review the information presented at that meeting in the context of recent published information. Factors to be considered in choosing the appropriate specimens for epidemiologic studies include quality and quantity of DNA, convenience of collection and storage, cost, and ability to accommodate future needs for genotyping. We focus on four types of specimens that are stored in these banks: (1) whole blood preserved as dried blood spots; (2) whole blood from which genomic DNA is isolated, (3) immortalized lymphocytes from whole blood or separated lymphocytes, prepared immediately or subsequent to cryopreservation; and (4) buccal epithelial cells. Each of the specimens discussed is useful for epidemiologic studies according to specific needs, which we enumerate in our conclusions.

Biological Specimen Banks↗

PubMed2002

Genetic polymorphisms of the human CYP2A13 gene: identification of single-nucleotide polymorphisms and functional characterization of an Arg257Cys variant.

Human cytochrome P450 2A13 (CYP2A13), which is highly efficient in the metabolic activation of a major tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), may play important roles in xenobiotic toxicity and tobacco-related tumorigenesis in the respiratory tract. The aim of this study was to identify any genetic polymorphisms of the CYP2A13 gene, which may alter the metabolic capacities of the enzyme. Polymerase chain reaction (PCR) single-strand conformational polymorphism analysis was used to identify single-nucleotide polymorphisms (SNPs) in all of the exons and at the exon-intron boundaries, and PCR-restriction fragment length polymorphism analysis and DNA sequencing were used to determine the frequencies of the newly identified variant alleles in the four major ethnic groups. Blood spot DNA from more than 100 individuals was used for these analyses. Seven variant alleles were found, but only one SNP was detected in the coding region, in exon 5, leading to an Arg257Cys amino acid change. The frequencies of the Arg257Cys allele in white, black, Hispanic, and Asian individuals are 1.9%, 14.4%, 5.8%, and 7.7%, respectively. Functional analysis of the variant protein was performed following its heterologous expression. The Arg257Cys variant was 37 to 56% less active than the wild-type Arg-257 protein toward all substrates tested. With NNK, Cys-257 had higher K(m) and lower V(max) values than did Arg-257, with a >2-fold decrease in catalytic efficiency. The Arg257Cys mutation could provide some protection against xenobiotic toxicity in the respiratory tract to individuals who are homozygous for the Cys-257 allele.

Amino Acid Substitution↗

PubMedDate not supplied

Characterization of beta-globin haplotypes using blood spots from a population-based cohort of newborns with homozygous HbS.

PURPOSE: A population-based cohort from three state newborn screening programs was used to describe beta-globin gene cluster variation. METHODS: Blood spots from newborns homozygous for HbS were genotyped for five restriction fragment length polymorphisms (RFLPs) to construct beta-globin haplotypes. Haplotype distributions were compared by race/ethnicity and sex. Expected heterozygosities were calculated and compared with observed heterozygosities. RESULTS: Haplotype distributions did not differ between sexes for either blacks or Hispanics. Neither racial/ethnic group deviated from Hardy-Weinberg equilibrium; however, Hispanics had higher heterozygosity at two RFLPs compared with blacks. CONCLUSION: The differences between populations probably reflect recent migration and admixture rather than selection.

Anemia, Sickle Cell↗