Prenatal diagnosis with repetitive in situ hybridization probes

We have used chromosome-specific repetitive sequences to detect the most common human aneuploidies prenatally. Together chromosome 21, 13, 18, X, and Y aneuploidy comprises 95% of the chromosome abnormalities that result in a high risk of abnormal phenotypes at birth. The X, Y, and 18 repetitive probes work reliably in multiple tissue types including directly examined and cultured amniocytes, chorionic villus cells, lymphocytes, and cultured fibroblasts. The probe that detects both chromosomes 13 and 21 routinely gives results in each cell type tested except directly studied amniocytes which can be interpreted in seven-ninths of the cases with protocol 1 and all tested samples with protocol 2. Our protocols diagnosed trisomy 21 in a 23-week fetus with low maternal serum AFP and a trisomy 18 in a direct chorionic villus sample 2 working days after the samples were obtained. Trisomy 21 also has been ruled out in a CVS karyotype first thought to be 47,XY,+21. These studies reflect the potential value of in situ hybridization to provide a more rapid, less expensive means to screen most at-risk fetal populations with less effort in first world cytogenetic laboratories, and to provide economical cytogenetic services in less developed countries. © 1992 Wiley-Liss, Inc.     

Oligonucleotide probes for the analysis of specific repetitive DNA sequences by fluorescence in situ hybridization.

Five non-isotopically labeled oligonucleotides have been designed and synthesized to facilitate the analysis of specific human and murine repetitive DNA sequences by fluorescence in situ hybridization (FISH). Three of the oligonucleotides contain alphoid DNA sequences; one hybridizes to the centromeres of all human and mouse chromosomes except the Y, while the other two are specific for human chromosomes 2 and 12, respectively. The fourth oligomer, containing sequences from the spacer region of a human 5S rDNA repeat, was used to confirm the map location of a approximately 100 copy 5S rDNA tandem repeat locus. The fifth oligomer, specific to the Alu family of repeats, generates a sharp R-banding pattern on human metaphase chromosomes, suitable for FISH karyotyping. These probes permit highly specific chromosome enumeration and aneuploidy detection with hybridization times as short as 30 minutes.     

A simplified protocol for fluorescence in situ hybridization with repetitive DNA probes and its use in clinical cytogenetics

A method for chromosome-specific staining and its use in clinical cytogenetics is described. This fluorescence in situ hybridization protocol for repetitive DNA probes results in yellow-green fluorescent signals on orange-red stained chromosomes. Special characteristics are its simplicity, the use of digoxigenin-11-dUTP for labeling, and the combination of high stringency criteria for hybridization and low stringency for washing. The method is particularly advantageous in cases with structurally abnormal extra chromosomes (ESACs), marker chromosomes of gonosomal origin, and chromosomal mosaicism. It may also facilitate the screening of cases for fragile X. The chromosome-specific staining can be done within 1 working-day.     

In situ hybridization with nonisotopic probes using different detection systems.

In order to evaluate a sensitive nonisotopic in situ hybridization method for routine work in pathology laboratories, we compared seven different detection systems, using digoxigenin- and biotin-labeled probes. The sensitivity of these methods was tested on four cases of cervical condyloma all known to be positive for HPV 6. Four of these methods gave satisfactory results without any background staining. The single biotin method and the single digoxigenin method were equally sensitive, while the two triple biotin methods, using mouse anti-biotin/anti-mouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase or mouse anti-biotin/alkaline phosphatase anti-mouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase as the detection systems, tremendously improved the sensitivity. The enhanced sensitivity of the nonisotopic in situ hybridization method make it useful in investigation of pathologic tissues.     

Prenatal diagnosis of Charcot-Marie-Tooth disease type 1a by multicolor in situ hybridization

Genetic heterogeneity within the most common genetic neuropathy, Charcot-Marie-Tooth disease (CMT) result in about 70% slow nerve conduction CMT1 and 30% normal nerve conduction CMT2. Autosomal dominant CMT1A on chromosome 17p11.2 represents about 70% of CMT1 cases and about 50% of all CMT cases. Three different size CMT1A duplications with variable flanking breakpoints were characterized by multicolor in situ hybridization and confirmed by pulsed field gel electrophoresis and quantitative polymerase chain reaction (PCR) amplification. These different size duplications result in the same CMT1A phenotype confirming that trisomy of a normal gene region results in CMT1A. the smallest duplication does not include the 409 locus used previously to screen for CMT1A duplications. Direct analysis of interphase nuclei from fetuses and at-risk patients by multicolor in situ hybridization to a commonly duplicated CMT1A probe is informative more often than polymorphic PCR analysis, faster than pulsed field gel electrophoresis (PFGE), and faster, more informative, and more reliable than restriction enzyme analysis. CMT1B restriction enzyme analysis of CMT pedigrees without CMT1A is expected to diagnose another 8% of at-risk CMT1 patients (total: 78%). © 1993 Wiley-Liss, Inc.     

Quantitative non-radioactive in situ hybridization of preproenkephalin mRNA with digoxigenin-labeled cRNA probes.

Non-radioactive detection of mRNA with in situ hybridization histochemistry has emerged as an important new technology for the study of gene expression. Quantitative in situ hybridization studies have generally relied upon counting of autoradiographic grains in the emulsion overlying cells containing hybridized, radioactively labeled probe. However, such high resolution studies require tedious grain counting over individual cells, frequently in addition to weeks of exposure to nuclear emulsion. The present report describes a quantitative, non-radioactive approach to the detection of a specific mRNA in the brain with the advantages of comparatively rapid tissue processing and computerized image analysis. The validity of this approach was tested by measuring the haloperidol-induced increase in the level of preproenkephalin mRNA in striatal sections of the rat brain using an RNA probe labeled with digoxigenin-11-UTP. Detection of probe hybridized to tissue sections was carried out enzymatically following complex formation with an antidigoxigenin-alkaline phosphatase conjugate. Using computerized image analysis, it was found that chronic treatment of rats with haloperidol resulted in a 50 +/- 6% increase in striatal neuronal optical density, a value in good agreement with previous studies using low-resolution radioactive methods, showing a 30-80% increase in striatal preproenkephalin mRNA hybridization signal.     

Detection ofLeishmania parasites by DNA in situ hybridization with non-radioactive probes

In situ hybridization techniques develop rapidly into diagnostic tools of considerable value for detection of viruses and bacteria. Here we report the application of this technique for the detection ofLeishmania parasites. Biotin-labelled total promastigote DNA was hybridized to culturedLeishmania parasites and to blood and impression smears of infected mice. In promastigotes kinetoplasts were strongly stained, nuclei somewhat more diffuse. In amastigotes both nuclear and kinetoplast DNA hybridized strongly. Amastigotes were easily detected in tissue of infected mice by their stable configuration of kinetoplast and nuclei. Cross-hybridization was observed betweenLeishmania donovani andL. tropica, but not between these two andL. braziliensis orTrypanosoma cruzi. A minor aspecific staining of host cell nuclei in the smears did not interfere with the detectability of the parasites.     

New localizations of VH sequences by in situ hybridization with biotinylated probes

The chromosomal localization of genes of three VH families (VH 1-3) was performed using in situ hybridization with biotinylated probes. Significantly strong signals were observed on chromosome 14, band 14q32, and on bands 16p11 and 15q11, although less frequently. Signal intensity and frequency were more important on chromosome 14 with all three probes, and on chromosome 16 with the VH2 and VH3 probes, while chromosome 15 was more marked than 16 with the VH1 probe. The localization of VH gene on chromosomes other than 14 suggests that several genes of the VH family had been simultaneously translocated in evolution and that the newly localized VH sequences may be pseudogenes.     

应用荧光原位杂交产前诊断未培养羊水细胞非整倍体

目的探讨荧光原位杂交(fluorescenceinsituhybridization,FISH)诊断未培养羊水细胞非整倍体的临床应用价值。方法对55例孕16～32周未培养羊水细胞进行FISH快速产前诊断,应用多色FISH对另4条染色体(X、Y、13号和18号)进行检测。以经母腹穿刺取胎血常规核型分析作为FISH检测结果对照。结果被检55例羊水未培养细胞均获得诊断结果,发现两例异常胎儿。1例为标准型21三体;另1例为21三体嵌合体。FISH检测与常规核型分析结果一致。结论FISH检测未培养羊水细胞非整倍体具有快速、简便、所用样本量少的优势,结果准确可靠,可达到产前诊断要求,有较大临床应用价值。     

Polymerase Chain reaction generated probes for fluorescence in situ hybridization

The extended use of Fish with centromeric probes in many cytogenetic laboratories is often impaired by the cost of this technique. Polymerase Chain Reaction (PCR) constitutes a simple way to generate and label such centromeric probes at low cost. Two types of human DNA source can be used: 1--Somatic hybrid cell lines containing a unique human chromosome. The specific amplification of the human subset of alphoid DNA is realised with a primer pair specific for the consensus region of human alpha satellite sequence. 2--Total Human DNA. This time, a primer pair specific for the alpha satellite DNA of the chromosome of interest must be designed. These probes, labelled during the PCR reaction by direct incorporation of modified dUTP, are actually widely used in our laboratory, alone or mixed with other probes (chromosome painting or locus specific probes).     

Prenatal in situ hybridization test for deleted steroid sulfatase gene

X-linked ichthyosis results from steroid sulfatase (STS) deficiency; 90% of affected patients have a complete deletion of the entire 146 kb STS gene on the distal X chromosome short arm (Xp22.3). In these families prenatal diagnosis and carrier testing can be completed in 2 days by hybridizing simultaneously 2 different cosmid probes labeled with fluorescein or Texas red and counterstaining interphase nuclear DNA with DAPI. An STS gene probe labeled with Texas red hybridizes specially to the steroid sulfatase gene on the X chromosome. A second flanking probe labeled with fluorescein hybridizes to both the normal Y chromosome and normal and STS deleted X chromosomes. In this fashion the interphase nuclei of normal males, affected males, normal females, and carrier females can be distinguished unambiguously. Because normal males and carrier females each show two yellow-green fluorescein spots and one Texas red STS spot, use of this test prenatally requires determining fetal sex independently with repetitive X and Y chromosome specific probes. This procedure can be used with lymphocytes, direct and cultured chorionic villus cells, direct and cultured amniocytes, and fibroblasts. Similar methods are anticipated to be useful for rapid diagnostic assessment of other aneuploid gene disorders. © 1993 Wiley-Liss, Inc.     

Non-isotopic in situ hybridization with digoxigenin and alkaline phosphatase labelled oligodeoxynucleotide probes. Applications in pituitary gland

We report the application of digoxigenin labelled oligonucleotide probes for the detection of hormonal messenger RNAs (mRNAs) in human pituitary adenomas. Positive signal for the appropriate mRNA was detected in tumours associated with Cushing's disease, acromegaly and hyperprolactinaemia, where immunoreactivity for adrenocorticotrophin (ACTH) growth hormone and prolactin had also been confirmed. In addition, we report the detection of proopiomelanocortin (POMC) mRNA in the rat pituitary gland using an oligodeoxynucleotide probe directly linked to alkaline phosphatase.     

Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections.

A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and 35S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.     

A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y-chromosome probes

Twenty-six laboratories used X and Y chromosome probes and the same procedures to process and examine 15,600 metaphases and 49,400 interphases from Phaseolus vulgaris-leucoagglutinin (PHA)-stimulated lymphocytes. In Part I, each laboratory scored 50 metaphases and 200 interphases from a normal male and a normal female from its own practice. In Part II, each laboratory scored 50 metaphases and 200 interphases on slides prepared by a central laboratory from a normal male and a normal female and three mixtures of cells from the male and female. In Part III, each laboratory scored 50 metaphases (in samples of 5, 10, 15, and 20) and 100 interphases (in samples of 5, 10, 15, 20, and 50) on new, coded slides of the same specimens used in Part II. Metaphases from male specimens were scored as 98–99% XY with no XX cells, and 97–98% of interphases were scored as XY with 0.04% XX cells. Metaphases from female specimens were scored as 96–97% XX with 0.03% XY cells, and 94–96% of interphases were scored as XX with 0.05% XY cells. Considering the data as a model for any probe used with fluorescence in situ hybridization (FISH), a statistical approach assessing the impact of analytical sensitivity on the numbers of observations required to assay for potential mosaicisms and chimerisms is discussed. The workload associated with processing slides and scoring 50 metaphases and 200 interphases using FISH averaged 27.1 and 28.6 minutes, respectively. This study indicates that multiple laboratories can test/develop guidelines for the rapid, efficacious, and cost-effective integration of FISH into clinical service. Am. J. Med. Genet. 76:318–326, 1998. © 1998 Wiley-Liss, Inc.     

In situ hybridization using biotinylated probes. An evaluation of different detection systems

In situ hybridization with biotin-labelled DNA probes is a powerful tool for the detection of viral sequences in infected tissues. However, sensitivity is low when compared to radiolabelled probes. In order to evaluate the impact on staining results of the detection system applied, we have tested various reagents. In our hands the sequential application of streptavidin and biotinylated alkaline phosphatase or development with a monoclonal anti-biotin antibody and the APAAP method gave consistently the best results. Furthermore, nitro blue tetrazolium/bromochloroindolyphosphate seems to be most suitable as a substrate for the alkaline phosphatase in this case. Use of other reagents, especially of a streptavidin-biotinylated alkaline phosphatase complex, resulted in a significantly lower staining intensity.     

Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis

The ability to visualize specific DNA sequences, on chromosomes and in nuclei, by fluorescence in situ hybridization (FISH) is fundamental to many aspects of genetics, genomics and cell biology. Probe selection is currently limited by the availability of DNA clones or the appropriate pool of DNA sequences for PCR amplification. Here, we show that liquid-phase probe pools from sequence capture technology can be adapted to generate fluorescently labelled pools of oligonucleotides that are very effective as repeat-free FISH probes in mammalian cells. As well as detection of small (15 kb) and larger (100 kb) specific loci in both cultured cells and tissue sections, we show that complex oligonucleotide pools can be used as probes to visualize features of nuclear organization. Using this approach, we dramatically reveal the disposition of exons around the outside of a chromosome territory core and away from the nuclear periphery.     

Aneuploidy assays on interphase nuclei by means of in situ hybridization with DNA probes

A method has been developed to detect chemically induced aneuploidyin interphase nuclei by means of in situ hybridization withchromosome-specific DNA probes. Lymphocyte cultures were treatedwith two known aneuploidy inducers, Benomyl and Griseofulvin.Two DNA fragments, QP23 and Y97, homologous to repetitive sequences,localized in the pericentromeric region of chromosome 9 andin the centromeric region of Y chromosome respectively, wereused as probes. Following autoradiography, grain clusters, revealingthe presence of the target chromosomes, were scored in restingnuclei. A marked increase in the frequency of cells with supernumeraryautoradiographic signals was observed with both probes at allconcentrations of the test compounds. The assay procedure appearedto be reproducible, sensitive and efficient in scoring largecell samples. It may therefore provide a useful tool for preliminaryscreening of potential aneuploidy inducers.     

Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization.

PURPOSE: This study was designed to evaluate the feasibility of using a targeted array-CGH strategy for prenatal diagnosis of genomic imbalances in a clinical setting of current pregnancies. METHODS: Women undergoing prenatal diagnosis were counseled and offered array-CGH (BCM V4.0) in addition to routine chromosome analysis. Array-CGH was performed with DNA directly from amniotic fluid cells with whole genome amplification, on chorionic villus samples with amplification as necessary, and on cultured cells without amplification. RESULTS: Ninety-eight pregnancies (56 amniotic fluid and 42 CVS specimens) were studied with complete concordance between karyotype and array results, including 5 positive cases with chromosomal abnormalities. There was complete concordance of array results for direct and cultured cell analysis in 57 cases tested by both methods. In 12 cases, the array detected copy number variation requiring testing of parental samples for optimal interpretation. Array-CGH results were available in an average of 6 and 16 days for direct and cultured cells, respectively. Patient acceptance of array-CGH testing was 74%. CONCLUSION: This study demonstrates the feasibility of using array-CGH for prenatal diagnosis, including reliance on direct analysis without culturing cells. Use of array-CGH should increase the detection of abnormalities relative to the risk, and is an option for an enhanced level of screening for chromosomal abnormalities in high risk pregnancies.