Prenatal diagnosis of tetrasomy 18p using multiplex fluorescent PCR and comparison with a variety of techniques

We report genetic characterization of isochromosome 18p using a combination of cytogenetic and molecular genetic methods, including multiplex fluorescent PCR. The patient was referred for chorionic villus sampling (CVS) due to advanced maternal age and maternal anxiety. The placental karyotype was 47,XX,+mar, with the marker having the appearance of a small supernumerary isochromosome. Because differentiating between isochromosomes and other structural rearrangements is normally very difficult, a variety of genetic tests including fluorescence in situ hybridization (FISH), PCR, and multiplex fluorescent PCR were undertaken to determine chromosomal origin and copy number and, thus, allow accurate diagnosis of the corresponding syndrome. FISH determined that the marker chromosome contained chromosome 18 material. PCR of a variety of short tandem repeats (STRs) confirmed that there was at least one extra copy of the maternal 18p material. However, neither FISH nor PCR could accurately determine copy number. Multiplex fluorescent PCR (MF-PCR) of STRs simultaneously determined that: (1) the marker included 18p material; (2) the marker was maternal in origin; (3) allele copy number indicated tetrasomy; and (4) contamination of the sample could be ruled out. Results were also rapid with accurate diagnosis of the syndrome tetrasomy 18p possible within 5 hours.

The molecular genetics of breast cancer: The contribution of comparative genomic hybridization

Comparative genomic hybridization (CGH) has been the technique of choice over the last 10 years for mapping DNA copy number changes in human tumors. Here we review the literature to demonstrate how CGH has contributed to the comprehension of molecular aspects of breast tumorigenesis. At least two distinct molecular pathways of breast cancer have been characterized that show a strong correlation with histological grade. It seems that grade I invasive ductal carcinomas (IDCs) arise from well-differentiated ductal carcinoma in situ (DCIS), whereas grade III IDCs come from poorly differentiated DCIS. In addition, dedifferentiation from a low- to a high-grade breast cancer has proven an unlikely phenomenon. CGH has been instrumental in dissecting distinct molecular pathways toward breast malignancy and in establishing a direct relationship between genotype and clinical pathological features. (C) 2005 Elsevier GrnbH. All rights reserved.

EGFR amplification and lack of activating mutations in metaplastic breast carcinomas

Metaplastic breast carcinomas are reported to harbour epidermal growth factor receptor (EGFR) overexpression in up to 80% of the cases, but EGFR gene amplification is the underlying genetic mechanism in around one-third of these. In this study, EGFR gene amplification as defined by chromogenic in situ hybridization and protein overexpression was examined in a cohort of 47 metaplastic breast carcinomas. Furthermore, the presence of activating EGFR mutations in exons 18, 19, 20, and 21 was investigated. Thirty-two cases showed EGFR overexpression and of these, 11 (34%) harboured EGFR gene amplification. In addition, EGFR amplification showed a statistically significant association with EGFR overexpression (p < 0.0094) and was restricted to carcinomas with homologous metaplasia. Ten cases, five with and five without EGFR amplification, were subjected to microarray-based CGH, which demonstrated that EGFR copy number gain may occur by amplification of a discrete genomic region or by gains of the short arm of chromosome 7 with a breakpoint near the EGFR gene locus, the minimal region of amplification mapping to EGFR, LANCL2, and SECOG. No activating EGFR mutations were identified, suggesting that this is unlikely to be a common alternative underlying genetic mechanism for EGFR expression in metaplastic breast carcinomas. Given that metaplastic breast carcinomas are resistant to conventional chemotherapy or hormone therapy regimens and that tumours with EGFR amplification are reported to be sensitive to EGFR tyrosine kinase inhibitors, these findings indicate that further studies are warranted to explore EGFR tyrosine kinase inhibitors as potential therapeutic agents for metaplastic breast carcinomas harbouring amplification of 7p11.2. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Effect of sequence variation in Plasmodium falciparum Histidine-Rich protein 2 on binding of specific monoclonal antibodies: Implications for rapid diagnostic tests for malaria

This Article Right arrow Full Text Right arrow Full Text (PDF) Right arrow Supplemental material Right arrow Alert me when this article is cited Right arrow Alert me if a correction is posted Services Right arrow Similar articles in this journal Right arrow Similar articles in PubMed Right arrow Alert me to new issues of the journal Right arrow Download to citation manager Right arrow Reprints and Permissions Right arrow Copyright Information Right arrow Books from ASM Press Right arrow MicrobeWorld Citing Articles Right arrow Citing Articles via HighWire Right arrow Citing Articles via Google Scholar Google Scholar Right arrow Articles by Lee, N. Right arrow Articles by McCarthy, J. Right arrow Search for Related Content PubMed Right arrow PubMed Citation Right arrow Articles by Lee, N. Right arrow Articles by McCarthy, J. Right arrow Pubmed/NCBI databases * Substance via MeSH Previous Article | Next Article Journal of Clinical Microbiology, August 2006, p. 2773-2778, Vol. 44, No. 8 0095-1137/06/$08.00+0 doi:10.1128/JCM.02557-05 Copyright © 2006, American Society for Microbiology. All Rights Reserved. Effect of Sequence Variation in Plasmodium falciparum Histidine- Rich Protein 2 on Binding of Specific Monoclonal Antibodies: Implications for Rapid Diagnostic Tests for Malaria{dagger} Nelson Lee,1,2 Joanne Baker,2 Kathy T. Andrews,1 Michelle L. Gatton,1,3 David Bell,4 Qin Cheng,2,3 and James McCarthy1* Australian Centre for International and Tropical Health and Nutrition, Queensland Institute of Medical Research and School of Population Health, University of Queensland, Queensland, Australia,1 Department of Drug Resistance and Diagnostics, Australian Army Malaria Institute, Brisbane, Australia,2 Malaria Drug Resistance and Chemotherapy, Queensland Institute of Medical Research, Queensland, Australia,3 World Health Organization, Regional Office for the Western Pacific, Manila, Philippines4 Received 8 December 2005/ Returned for modification 23 February 2006/ Accepted 26 May 2006 The ability to accurately diagnose malaria infections, particularly in settings where laboratory facilities are not well developed, is of key importance in the control of this disease. Rapid diagnostic tests (RDTs) offer great potential to address this need. Reports of significant variation in the field performance of RDTs based on the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) (PfHRP2) and of significant sequence polymorphism in PfHRP2 led us to evaluate the binding of four HRP2-specific monoclonal antibodies (MABs) to parasite proteins from geographically distinct P. falciparum isolates, define the epitopes recognized by these MABs, and relate the copy number of the epitopes to MAB reactivity. We observed a significant difference in the reactivity of the same MAB to different isolates and between different MABs tested with single isolates. When the target epitopes of three of the MABs were determined and mapped onto the peptide sequences of the field isolates, significant variability in the frequency of these epitopes was observed. These findings support the role of sequence variation as an explanation for variations in the performance of HRP2-based RDTs and point toward possible approaches to improve their diagnostic sensitivities

Microarray-based comparative genomic hybridisation of breast cancer patients receiving neoadjuvant chemotherapy

We analysed the molecular genetic profiles of breast cancer samples before and after neoadjuvant chemotherapy with combination doxorubicin and cyclophosphamide (AC). DNA was obtained from microdissected frozen breast core biopsies from 44 patients before chemotherapy. Additional samples were obtained before the second course of chemotherapy (D21) and after the completion of the treatment (surgical specimens) in 17 and 21 patients, respectively. Microarray-based comparative genome hybridisation was performed using a platform containing approx5800 bacterial artificial chromosome clones (genome-wide resolution: 0.9 Mb). Analysis of the 44 pretreatment biopsies revealed that losses of 4p, 4q, 5q, 12q13.11–12q13.12, 17p11.2 and 17q11.2; and gains of 1p, 2p, 7q, 9p, 11q, 19p and 19q were significantly associated with oestrogen receptor negativity. 16q21–q22.1 losses were associated with lobular and 8q24 gains with ductal types. Losses of 5q33.3–q4 and 18p11.31 and gains of 6p25.1–p25.2 and Xp11.4 were associated with HER2 amplification. No correlations between DNA copy number changes and clinical response to AC were found. Microarray-based comparative genome hybridisation analysis of matched pretreatment and D21 biopsies failed to identify statistically significant differences, whereas a comparison between matched pretreatment and surgical samples revealed a statistically significant acquired copy number gain on 11p15.2–11p15.5. The modest chemotherapy-driven genomic changes, despite profound loss of cell numbers, suggest that there is little therapeutic selection of resistant non-modal cell lineages.

Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA

Aim: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. Methods: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. Results: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 104/ mL, 6.3 × 102/mL and 1.6 × 103/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 109/mL, 2.08 × 106/mL and 4.40 × 107/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 105/ mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples, Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. Conclusion: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA. © 2006 The WJG Press. All rights reserved.

Modulation of higher-plant NAD(H)-dependent glutamate dehydrogenase activity in transgenic tobacco via alteration of beta subunit levels

Glutamate dehydrogenase (GDH; EC 1.4.1.2-1.4.1.4) catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate. In vascular plants the in vivo direction(s) of the GDH reaction and hence the physiological role(s) of this enzyme remain obscure. A phylogenetic analysis identified two clearly separated groups of higher-plant GDH genes encoding either the alpha- or beta-subunit of the GDH holoenzyme. To help clarify the physiological role(s) of GDH, tobacco (Nicotiana tabacum L.) was transformed with either an antisense or sense copy of a beta-subunit gene, and transgenic plants recovered with between 0.5- and 34-times normal leaf GDH activity. This large modulation of GDH activity (shown to be via alteration of beta-subunit levels) had little effect on leaf ammonium or the leaf free amino acid pool, except that a large increase in GDH activity was associated with a significant decrease in leaf Asp (similar to 51%, P=0.0045). Similarly, plant growth and development were not affected, suggesting that a large modulation of GDH beta-subunit titre does not affect plant viability under the ideal growing conditions employed. Reduction of GDH activity and protein levels in an antisense line was associated with a large increase in transcripts of a beta-subunit gene, suggesting that the reduction in beta-subunit levels might have been due to translational inhibition. In another experiment designed to detect post-translational up-regulation of GDH activity, GDH over-expressing plants were subjected to prolonged dark-stress. GDH activity increased, but this was found to be due more likely to resistance of the GDH protein to stress-induced proteolysis, rather than to post-translational up-regulation.