Heinz Ulrich G Weier

Biospecimen Reporting for Improved Study Quality (BRISQ)

Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues, it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality (BRISQ) recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The BRISQ guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.

A novel neuron-enriched homolog of the erythrocyte membrane cytoskeletal protein 4.1.

We report the molecular cloning and characterization of 4.1N, a novel neuronal homolog of the erythrocyte membrane cytoskeletal protein 4.1 (4.1R). The 879 amino acid protein shares 70, 36, and 46% identity with 4.1R in the defined membrane-binding, spectrin-actin–binding, and C-terminal domains, respectively. 4.1N is expressed in almost all central and peripheral neurons of the body and is detected in embryonic neurons at the earliest stage of postmitotic differentiation. Like 4.1R, 4.1N has multiple splice forms as evidenced by PCR and Western analysis. Whereas the predominant 4.1N isoform identified in brain is ∼135 kDa, a smaller 100 kDa isoform is enriched in peripheral tissues. Immunohistochemical studies using a polyclonal 4.1N antibody revealed several patterns of neuronal staining, with localizations in the neuronal cell body, dendrites, and axons. In certain neuronal locations, including the granule cell layers of the cerebellum and dentate gyrus, a distinct punctate-staining pattern was observed consistent with a synaptic localization. In primary hippocampal cultures, mouse 4.1N is enriched at the discrete sites of synaptic contact, colocalizing with the postsynaptic density protein of 95 kDa (a postsynaptic marker) and glutamate receptor type 1 (an excitatory postsynaptic marker). By analogy with the roles of 4.1R in red blood cells, 4.1N may function to confer stability and plasticity to the neuronal membrane via interactions with multiple binding partners, including the spectrin-actin–based cytoskeleton, integral membrane channels and receptors, and membrane-associated guanylate kinases.

Preimplantation genetic analysis of translocations: case-specific probes for interphase cell analysis

Abstract Carriers of balanced translocations show an increased risk of infertility and spontaneous abortions, because of errors in gametogenesis, and constitute a significant fraction of patients seeking assisted reproduction. The objective of this study was to design approaches for preimplantation diagnosis of chromosome translocations and to apply such techniques to the selection of chromosomally normal or balanced embryos prior to their transfer to the mother’s womb. Three slightly different approaches were assessed by means of chromosome-specific, non-isotopically labeled DNA probes and an assay based on fluorescence in situ hybridization- to score and characterize chromosomes in single blastomeres biopsied from embryos on their third day of development. The three approaches were used for preimplantation genetic diagnosis involving four couples who had enrolled in our IVF program and in which one of the partners was a carrier of one of the following translocations: 46,XX,t(12;20)(p13.1;q13.3), 46,XY,t(3;4) (p24;p15), 45,XY,der(14;15)(10q;10q), and 46,XY,t(6;11) (p22.1;p15.3). A total of 33 embryos were analyzed, of which 25 (75.8%) were found to be either unbalanced or otherwise chromosomally abnormal. Only a single embryo could be transferred to patients A and D, whereas three embryos were transferred to patient B in a total of two IVF cycles. Transfer of two embryos to patient C resulted in an ongoing pregnancy. Re-analysis of non-transferred embryos with additional probes confirmed the initial results in 95% (20/21) of the cases. In conclusion, case-specific translocation tests can be applied to any translocation carrier for the selection of normal or chromosomally balanced embryos prior to embryo transfer. This is expected significantly to increase the success rates in IVF cycles of translocation carriers, while preventing the spontaneous abortion or birth of abnormal offspring.

Clinical applications of fluorescence in situ hybridization

We review here the application of fluorescence in situ hybridization with chromosome-specific probes to chromosome classification and to detection of changes in chromosome number or structure associated with genetic disease. Information is presented on probe types that are available for disease detection. We discuss the application of these probes to detection of numerical aberrations important for prenatal diagnosis and to detection and characterization of numerical and structural aberrations in metaphase spreads and in interphase nuclei to facilitate tumor diagnosis.

Two-color hybridization with high complexity chromosome-specific probes and a degenerate alpha satellite probe DNA allows unambiguous discrimination between symmetrical and asymmetrical translocations

This report describes a fluorescence in situ hybridization approach to chromosome staining that facilitates detection of structural aberrations and allows discrimination between dicentric chromosomes and symmetrically translocated chromosomes. In this approach, selected whole chromosomes are stained in one color by hybridization with composite probes whose elements have DNA sequence homology along the length of the target chromosomes. In addition, all chromosomes are counterstained with a DNA specific dye so that structural aberrations between target and non-target chromosomes are clearly visible. Discrimination between dicentric chromosomes and symmetrical translocations is accomplished by hybridization with a second probe that is homologous to DNA sequences found in the centromeric region of all chromosomes. The centromeric marker is visualized in a different color, so that the number of centromeres per aberrant chromosome can be rapidly determined in the microscope by changing excitation and fluorescence filters.

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

SummaryProbe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.