Barbara J. Trask

DNA duplication associated with Charcot-Marie-Tooth disease type 1A

Charcot-Marie-tooth disease type 1A (CMT1A) was localized by genetic mapping to a 3 cM interval on human chromosome 17p. DNA markers within this interval revealed a duplication that is completely linked and associated with CMT1A. The duplication was demonstrated in affected individuals by the presence of three alleles at a highly polymorphic locus, by dosage differences at RFLP alleles, and by two-color fluorescence in situ hybridization. Pulsed-field gel electrophoresis of genomic DNA from patients of different ethnic origins showed a novel SacII fragment of 500 kb associated with CMT1A. A severely affected CMT1A offspring from a mating between two affected individuals was demonstrated to have this duplication present on each chromosome 17. We have demonstrated that failure to recognize the molecular duplication can lead to misinterpretation of marker genotypes for affected individuals, identification of false recombinants, and incorrect localization of the disease locus.

The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A.

Charcot–Marie-–ooth disease type 1A (CMT1A) is an autosomal dominant peripheral neuropathy associated with a large DNA duplication on the short arm of human chromosome 17. The trembler (Tr) mouse serves as a model for CMT1A because of phenotypic similarities and because the Tr locus maps to mouse chromosome 11 in a region of conserved synteny with human chromosome 17. Recently, the peripheral myelin gene Pmp–22 was found to carry a point mutation in Tr mice. We have isolated cDNA and genomic clones for human PM–22P. The gene maps to human chromosome 17p11.2–17p12, is expressed at high levels in peripheral nervous tissue and is duplicated, but not disrupted, in CMT1A patients. Thus, we suggest that a gene dosage effect involving PMP–22 is at least partially responsible for the demyelinating neuropathy seen in CMT1A.

Fluorescence in situ hybridization: applications in cytogenetics and gene mapping

Unique sequences, chromosomal subregions, or entire genomes can be specifically highlighted in metaphase or interphase cells by fluorescence in situ hybridization (FISH). This technique can be used to identify chromosomes, detect chromosomal abnormalities or determine the chromosomal location of specific sequences. FISH plays an increasingly important role in a variety of research areas, including cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping.

The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs.

The physical distance between DNA sequences in interphase nuclei was determined using eight cosmids containing fragments of the Chinese hamster genome that span 273 kb surrounding the dihydrofolate reductase (DHFR) gene. The distance between these sequences at the molecular level has been determined previously by restriction enzyme mapping (J.E. Looney and J.L. Hamlin, 1987, Mol. Cell Biol. 7: 569-577; C. Ma et al., 1988, Mol. Cell Biol. 8: 2316-2327). Fluorescence in situ hybridization was used to localize the DNA sequences in interphase nuclei of cells bearing only one copy of this genomic region. The distance between DNA sequences in interphase nuclei was correlated to molecular distance over a range of 25 to at least 250 kb. The observed relationship was such that genomic distance could be predicted to within 40 kb from interphase distance. The correct order of seven probes was derived from interphase distances measured for 19 pair-wise combinations of the probes. Measured distances between sequences approximately 200 kb apart indicate that the DNA is condensed 70- to 100-fold in hybridized nuclei relative to a linear DNA helix molecule. Cell lines with chromosome inversions were used to show that interphase distance increases with genomic distance in the 50-90 Mb range, but less steeply than in the 25-250 kb range.

Chapter 1 DNA Sequence Localization in Metaphase and Interphase Cells by Fluorescence in Situ Hybridization

Publisher Summary This chapter describes the in situ hybridization techniques used for labeling specific sequences in chromatin fixed to slides and in suspension and discusses the procedures used to label probes with biotin, digoxigenin, and aminoacetylfluorene (AAF). The simplest and most reproducible means of labeling DNA sequence probes is by nick translation. The chapter describes the techniques for one-color fluorescent detection of these probe labels along with the techniques used for the simultaneous detection of two probes (AAF and biotin or digoxigenin, and biotin) using two different fluorochromes. DNA can be chemically modified with AAF through a chemical reaction at the C-8 carbon of guanine by the carcinogen N -acetoxy-2- aminoacetylfluorene ( N -A-AAF). The AAF and biotin procedures are adapted to label nuclei in suspension for the quantitation of bound probe by flow cytometry or for analysis of nuclear organization by optical sectioning or confocal microscopy. The chapter reviews the procedure followed for suspension labeling. The procedures for DNA sequence localization in interphase and metaphase cells described in the chapter have a number of research applications.

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization

SummaryFluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluroescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluores-cence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase is consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.

Fluorescence in situ hybridization with DNA probes

Publisher Summary Fluorescence in situ DNA hybridization (FISH) is used to label fluorescently specific nucleic acid sequences in cells or chromosomes. The FISH procedure is used to reveal the location of these sequences and to quantify their copy number. The advantages of FISH over in situ hybridization with radioactively labeled probes include spatial resolution, convenience, and speed. FISH techniques approach the sensitivity of autoradiographic techniques. Several laboratories have reported the successful localization of the single-copy sites of specific sequences using probes of 2–5 kilobase pairs (kbp). Several schemes for probe modification and hybridization detection are described: biotinylated probe detected with fluorescently labeled avidin or antibiotin antibody, aminoacetylfluorene (AAF)-modified probe detected with an anti-AAF antibody, sulfonate-modified probe detected with an antisulfonate antibody, mercurated probe detected by a sulfhydryl group linked to either a fluorescent ligand or a hapten, which is detected with an antihapten antibody, and digoxigenin- labeled probes detected with an antidigoxigenin antibody. Biotin and digoxigenin moieties have been introduced in the probe through nick translation or random-primed polymerase reactions. Aminoacetylfluorene, sulfonate, and mercury have been introduced in the probe through simple chemical reactions.

A new system for high-resolution DNA sequence mapping in interphase pronuclei ☆

Cosmid clones containing human or hamster inserts have been hybridized in situ and localized with fluorescent reporter molecules in interphase nuclei (pronuclei) obtained after fusion of hamster eggs with either human or hamster sperm. Hamster egg cytoplasm processes the tightly packaged sperm DNA into large diffuse networks of chromatin fiber bundles, providing hybridization targets more extended than those available in somatic interphase cell nuclei. Pronuclear physical distances between hybridization signals were measured in micrometers and correlated to genomic distances determined by restriction fragment analyses, using cosmids from the Chinese hamster DHFR region and from the human Factor VIII/color vision pigment gene region (Xq28). The mean pronuclear distances between hybridization sites were about three times as large as those measured in somatic interphase cells for equivalent genomic distances. The relationship between physical and genomic distances was linear from less than 50 kb to at least 800 kb. The results show that physical distance in the sperm-egg system promises to extend the mapping range obtainable in somatic interphase nuclei below 50 kb and up to at least 800 kb.

The CCAAT/enhancer binding protein (C/EBPα) gene (CEBPA) maps to human chromosome 19q13.1 and the related nuclear factor NF-IL6 (C/EBPβ) gene (CEBPB) maps to human chromosome 20q13.1

The CEBPA gene encoding CCAAT/enhancer binding protein (C/EBP α ) has been mapped to human chromosome 19 and the CEBPB (formerly TCF5) gene encoding NF-IL6 (C/EBP β ) to human chromosome 20 by Southern blot analysis of Chinese hamster × human and mouse × human somatic cell hybrids. CEBPA has been further mapped to 19q13.1 between the loci GPI and TGFB using human × hamster somatic cell hybrids containing restricted fragments of human chromosome 19. This position was confirmed by fluorescence in situ hybridization. Furthermore, CEBPB has been mapped to 20q13.1 by fluorescence in situ hybridization.

Somatic cell hybrids, sequence-tagged sites, simple repeat polymorphisms, and yeast artificial chromosomes for physical and genetic mapping of proximal 17p

Somatic cell hybrids retaining the deleted chromosome 17 from 15 unrelated Smith-Magenis syndrome (SMS) [del(17)(p11.2p11.2)] patients were obtained by fusion of patient lymphoblasts with thymidine kinase-deficient rodent cell lines. Seventeen sequence-tagged sites (STSs) were developed from anonymous markers and cloned genes mapping to the short arm of chromosome 17. The STSs were used to determine the deletion status of these loci in these and four previously described human chromosome 17-retaining hybrids. Ten STSs were used to identify 28 yeast artificial chromosomes (YACs) from the St. Louis human genomic YAC library. Four of the 17 STSs identified simple repeat polymorphisms. The order and location of deletion breakpoints were confirmed and refined, and the regional assignment of several probes and cloned genes were determined. The cytogenetic band locations and relative order of six markers on 17p were established by fluorescence in situ hybridization mapping to metaphase chromosomes. The latter data confirmed and supplemented the somatic cell hybrid results. Most of the hybrids derived from [del(17)(p11.2p11.2)] patients demonstrated a similar pattern of deletion for the marker loci and were deleted for D17S446, D17S258, D17S29, D17S71, and D17S445. However, one of them demonstrated a unique pattern of deletion. This patient is deleted for several markers known to recognize a large DNA duplication associated with Charcot-Marie-Tooth (CMT) disease type 1A. These data suggest that the proximal junction of the CMT1A duplication is close to the distal breakpoint in [del(17)(p-11.2p11.2)] patients.