Bernhard G Zimmermann

Publications (14)55.68 Total impact

• Article: Cell-free DNA in maternal plasma: has the size-distribution puzzle been solved?

  Sinuhe Hahn, Bernhard G Zimmermann
  Clinical Chemistry 08/2010; 56(8):1210-1. · 7.91 Impact Factor
• Source

  Available from: PubMed Central

  Article: Prenatal diagnosis of fetal aneuploidies: post-genomic developments.

  Sinuhe Hahn, Laird G Jackson, Bernhard G Zimmermann
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  ABSTRACT: Prenatal diagnosis of fetal aneuploidies and chromosomal anomalies is likely to undergo a profound change in the near future. On the one hand this is mediated by new technical developments, such as chromosomal microarrays, which allow a much more precise delineation of minute sub-microscopic chromosomal aberrancies than the classical G-band karyotype. This will be of particular interest when investigating pregnancies at risk of unexplained development delay, intellectual disability or certain forms of autism. On the other hand, great strides have been made in the non-invasive determination of fetal genetic traits, largely through the analysis of cell-free fetal nucleic acids. It is hoped that, with the assistance of cutting-edge tools such as digital PCR or next generation sequencing, the long elusive goal of non-invasive prenatal diagnosis for fetal aneuploidies can finally be attained.
  Genome Medicine 01/2010; 2(8):50.
• Article: Detection of increased amounts of cell-free fetal DNA with short PCR amplicons.

  Aleksandra Sikora, Bernhard G Zimmermann, Corinne Rusterholz, Daniella Birri, Varaprasad Kolla, Olav Lapaire, Irene Hoesli, Vivian Kiefer, Laird Jackson, Sinuhe Hahn
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  ABSTRACT: A digital PCR approach has recently been suggested to detect greater amounts of cell-free fetal DNA in maternal plasma than conventional real-time quantitative PCR (qPCR). Because the digital qPCR approach uses shorter PCR amplicons than the real-time qPCR assay, we investigated whether a real-time qPCR assay appropriately modified for such short amplicons would improve the detection of cell-free fetal DNA. We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this "short" assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp). Qualitatively, both assays detected male cell-free fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT real-time qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons. The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy.
  Clinical Chemistry 11/2009; 56(1):136-8. · 7.91 Impact Factor
• Source

  Available from: Sinuhe Hahn

  Article: Digital PCR: a powerful new tool for noninvasive prenatal diagnosis?

  Bernhard G Zimmermann, Simon Grill, Wolfgang Holzgreve, Xiao Yan Zhong, Laird G Jackson, Sinuhe Hahn
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  ABSTRACT: Recent reports have indicated that digital PCR may be useful for the noninvasive detection of fetal aneuploidies by the analysis of cell-free DNA and RNA in maternal plasma or serum. In this review we provide an insight into the underlying technology and its previous application in the determination of the allelic frequencies of oncogenic alterations in cancer specimens. We also provide an indication of how this new technology may prove useful for the detection of fetal aneuploidies and single gene Mendelian disorders.
  Prenatal Diagnosis 11/2008; 28(12):1087-93. · 2.11 Impact Factor
• Source

  Available from: PubMed Central

  Article: Electrochemical detection of low-copy number salivary RNA based on specific signal amplification with a hairpin probe.

  Fang Wei, Jianghua Wang, Wei Liao, Bernhard G Zimmermann, David T Wong, Chih-Ming Ho
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  ABSTRACT: We developed a technique for electrochemical detection of salivary mRNA employing a hairpin probe (HP). Steric hindrance (SH) suppresses unspecific signal and generates a signal-on amplification process for target detection. The stem-loop configuration brings the reporter end of the probe into close proximity with the surface and makes it unavailable for binding with the mediator. Target binding opens the hairpin structure of the probe, and the mediator can then bind to the accessible reporter. Horseradish peroxidase is utilized to generate electrochemical signal. This signal-on process is characterized by a low basal signal, a strong positive readout and a large dynamic range. The SH is controlled via hairpin design and electrical field. By applying electric field control to HPs, the limit of detection of RNA is about 0.4 fM, which is 10 000-fold more sensitive than conventional linear probes. Endogenous Interleukin-8 mRNA is detected with the HP, and good correlation with the quantitative PCR technique is obtained. The resultant process allows a simple setup and by reducing the number of steps it is suited for the point-of-care detection of specific nucleic acid sequences from complex body fluids such as saliva.
  Nucleic Acids Research 07/2008; 36(11):e65. · 8.03 Impact Factor
• Article: Salivary mRNA targets for cancer diagnostics.

  Bernhard G Zimmermann, David T Wong
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  ABSTRACT: Head and neck squamous cell carcinoma (HNSCC) affects almost 1 million people worldwide per year. Despite therapeutic advances the overall survival rate remains low because diagnosis often occurs only at advanced stages with poor prognosis. Like in most cancers, the implementation of an early detection scheme would have a positive impact on this disease. Similarly, as oral cancer has a very high recurrence rate, the early identification of recurrence or second primary tumors is an important challenge. HNSCC detection is currently based on expert clinical examination of the upper aerodigestive tract and histologic analysis of suspicious areas, but it may be undetectable in hidden sites, and unfortunately visual screening for oral lesions is an often neglected part of dental healthcare. Our group is actively pursuing the assembly of a toolbox for the molecular analysis of oral fluid. Here we present our current status utilizing the salivary transcriptome for oral cancer diagnostics.
  Oral Oncology 06/2008; 44(5):425-9. · 2.86 Impact Factor
• Source

  Available from: Guido Krupp

  Article: Exon-level expression profiling: a comprehensive transcriptome analysis of oral fluids.

  Zhanzhi Hu, Bernhard G Zimmermann, Hui Zhou, Jianghua Wang, Bradley S Henson, Weixia Yu, David Elashoff, Guido Krupp, David T Wong
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  ABSTRACT: The application of global gene expression profiling to saliva samples is hampered by the presence of partially fragmented and degraded RNAs that are difficult to amplify and detect with the prevailing technologies. Moreover, the often limited volume of saliva samples is a challenge to quantitative PCR (qPCR) validation of multiple candidates. The aim of this study was to provide proof-of-concept data on the combination of a universal mRNA-amplification method with exon arrays for candidate selection and a multiplex preamplification method for easy validation. We used a universal mRNA-specific linear-amplification strategy in combination with Affymetrix Exon Arrays to amplify salivary RNA from 18 healthy individuals on the nanogram scale. Multiple selected candidates were preamplified in one multiplex reverse transcription PCR reaction, cleaned up enzymatically, and validated by qPCR. We defined a salivary exon core transcriptome (SECT) containing 851 transcripts of genes that have highly similar expression profiles in healthy individuals. A subset of the SECT transcripts was verified by qPCR analysis. Informatics analysis of the SECT revealed several functional clusters and sequence motifs. Sex-specific salivary exon biomarkers were identified and validated in tests with samples from healthy individuals. It is feasible to use samples containing fragmented RNAs to conduct high-resolution expression profiling with coverage of the entire transcriptome and to validate multiple targets from limited amounts of sample.
  Clinical Chemistry 06/2008; 54(5):824-32. · 7.91 Impact Factor
• Article: Real-time quantitative PCR for the detection of fetal aneuploidies.

  Bernhard G Zimmermann, Lech Dudarewicz
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  ABSTRACT: In prenatal analysis, one of the major concerns is the detection of fetal aneuploidies. Several molecular methods have been described recently for the rapid analysis of amniotic fluid and chorionic villi. Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) of short tandem repeats are already implemented in prenatal laboratories and permit the evaluation of the major chromosomal aberrations within 24 h. However, both methods have their disadvantages. The advent of real-time PCR has revolutionized the measurement of nucleic acid copy numbers in recent years. Quantitative PCR, a term that was only 10 years ago considered to be an oxymoron, is now widely accepted. We demonstrated previously the feasibility of detection of trisomy 21 by real-time PCR, and here we describe the modified test that permits simultaneous analysis of trisomies 18 and 21. This approach has been demonstrated in a recent large-scale analysis, and it is presented in detail in this chapter.
  Methods in molecular biology (Clifton, N.J.) 02/2008; 444:95-109.
• Article: Quantification of circulatory fetal DNA in the plasma of pregnant women.

  Bernhard G Zimmermann, Deborah G Maddocks, Neil D Avent
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  ABSTRACT: The analysis of cell-free fetal DNA in the circulation of the pregnant woman plays the pivotal role in noninvasive prenatal research. Here, we describe an improved method for the quantification of male DNA, which is a valuable research tool for the quantification of fetal DNA. The quantification of fetal DNA serves two main purposes. First, the levels may indicate certain pregnancy-related disorders such as preeclampsia even before onset of the disease; thus, the quantification may serve as a marker for early detection. Second, extraction and enrichment strategies of the fetal DNA compartment are important factors in the development and implementation of clinical tests, such as detection of fetal sex, Rhesus D status, point mutations, and aneuploidies. In this context, the quantification of fetal DNA is an important tool for the evaluation of protocols.
  Methods in molecular biology (Clifton, N.J.) 02/2008; 444:219-29.
• Article: Genomic targets in saliva.

  Bernhard G Zimmermann, Noh Jin Park, David T Wong
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  ABSTRACT: Saliva, the most accessible and noninvasive biofluid of our body, harbors a wide spectrum of biological analytes informative for clinical diagnostic applications. While proteomic constituents are a logical first choice as salivary diagnostic analytes, genomic targets have emerged as highly informative and discriminatory. This awareness, coupled with the ability to harness genomic information by high-throughput technology platforms such as genome-wide microarrays, ideally positions salivary genomic targets for exploring the value of saliva for detection of specific disease states and augmenting the diagnostic and discriminatory value of the saliva proteome for clinical applications. Buccal cells and saliva have been used as sources of genomic DNA for a variety of clinical and forensic applications. For discovery of disease targets in saliva, the recent realization that there is a transcriptome in saliva presented an additional target for oral diagnostics. All healthy subjects evaluated have approximately 3,000 different mRNA molecules in their saliva. Almost 200 of these salivary mRNAs are present in all subjects. Exploration of the clinical utility of the salivary transcriptome in oral cancer subjects shows that four salivary mRNAs (OAZ, SAT, IL8, and IL1b) collectively have a discriminatory power of 91% sensitivity and specificity for oral cancer detection. Data are also now in place to validate the presence of unique diagnostic panels of salivary mRNAs in subjects with Sjögren's disease.
  Annals of the New York Academy of Sciences 04/2007; 1098:184-91. · 3.15 Impact Factor
• Article: Characterization of salivary RNA by cDNA library analysis.

  Noh Jin Park, Xiaofeng Zhou, Tianwei Yu, Brigitta M N Brinkman, Bernhard G Zimmermann, Visswanathan Palanisamy, David T Wong
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  ABSTRACT: Oral fluid (saliva) meets the demands for a noninvasive and accessible diagnostic medium. Recent reports by our group and others described the presence and use of human RNA in saliva as a diagnostic or forensic tool, including the use for oral cancer detection. To gain insights into the integrity of salivary RNA, we examined in detail the integrity of salivary RNA by generating a cDNA library from pooled supernatant saliva of 10 healthy donors. From a library with a primary library titer of 1.3 x 10(6) cfu/mL of which 95% of the clones had inserts, we successfully sequenced 117 random colonies containing recombinant clones. BLAST search results indicated that all of these clones contained sequences of human origin. Most of the salivary RNAs appeared to be endonucleolytically cleaved at random positions as indicated by comparisons to respective full length parental RNAs from the Genbank. Twelve of the insert sequences matched to the normal salivary core transcriptome sequences, which are highly abundant mRNAs present in healthy individuals. This study provides an in-depth molecular analysis of the saliva transcriptome and should be a useful resource for future basic and translational studies of RNA in human saliva. In addition, this paper presents unequivocal evidence for the presence of RNA in saliva as determined by the use of diverse techniques such as reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), in vitro translation, and the construction of a salivary cDNA library.
  Archives of Oral Biology 02/2007; 52(1):30-5. · 1.60 Impact Factor
• Article: Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma.

  Bernhard G Zimmermann, Wolfgang Holzgreve, Neil Avent, Sinuhe Hahn
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  ABSTRACT: DNA of fetal origin is present in the plasma of pregnant women. The quantitative measurement of circulatory fetal DNA (cfDNA) by real-time quantitative PCR (qPCR) has been applied to investigate a possible correlation between increased levels and pregnancy-related disorders. However, as the levels of cfDNA are close to the detection limit (LOD) of the method used, the measurements may not be reliable. This is also problematic for the evaluation of preanalytical steps, such as DNA extraction and cfDNA enrichment by size separation. We optimized a protocol for the qPCR analysis of the multi-copy sequence DYS14 on the Y chromosome. This was compared with an established assay for the single-copy SRY gene. Probit regression analysis showed that the limit of detection (LOD) of the DYS14 assay, (0.4 genome equivalents (GE)) and limit of quantification (LOQ) were 10-fold lower in comparison to SRY (4 GE). The levels of cfDNA obtained from the first trimester of pregnancy could be quantified with high precision by the DYS14 assay (CV below 25%) as opposed to the SRY measurements (26-140%). Additionally, fetal sex was correctly determined in all instances. The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy can be measured reliably, targeting the DYS14 that is present in multiple copies per Y chromosome.
  Annals of the New York Academy of Sciences 10/2006; 1075:347-9. · 3.15 Impact Factor
• Article: Improvement of methods for the isolation of cell-free fetal DNA from maternal plasma: comparison of a manual and an automated method.

  Dorothy J Huang, Bernhard G Zimmermann, Wolfgang Holzgreve, Sinuhe Hahn
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  ABSTRACT: The low amount of cell-free fetal DNA present in the maternal circulation poses significant challenges to its use in future diagnostic applications, and ways of increasing the yield of this potential marker extracted from maternal plasma are constantly being explored. In this study, we compared two methods of DNA extraction, a manual and an automated method. Our analysis revealed that although the manual method yielded overall more total cell-free DNA, the automated system yielded higher quantities of cell-free DNA of fetal origin. Furthermore, the DNA isolated using the automated system appeared to be of greater purity than that isolated by the manual method, with fewer inhibitors to downstream real-time PCR reactions.
  Annals of the New York Academy of Sciences 10/2006; 1075:308-12. · 3.15 Impact Factor
• Article: Use of an automated method improves the yield and quality of cell-free fetal DNA extracted from maternal plasma.

  Dorothy J Huang, Bernhard G Zimmermann, Wolfgang Holzgreve, Sinuhe Hahn
  Clinical Chemistry 01/2006; 51(12):2419-20. · 7.91 Impact Factor