An Approach for Quantitative Assessment of Fluorescence In Situ Hybridization (FISH) Signals for Applied Human Molecular Cytogenetics
National Center of Mental Health, Russian Academy of Medical Sciences, Moscow, Russia (IYI,IVS,VVM,YBY), and Institute of Pediatrics and Children's Surgery, Russian Ministry of Health, Moscow, Russia (SGV)
Correspondence to: Y.B. Yurov, National Center of Mental Health, Russian Academy of Medical Sciences, Zagorodnoe sh.2, 119152, Moscow, Russia. E-mail: y_yurov{at}yahoo.com; i_yurov{at}mail.ru
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Summary |
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Key Words: quantitative FISH differentiation of FISH signals aneuploidy scoring low-level chromosomal mosaicism chromosome heteromorphism
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Introduction |
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Originally, quantitative analysis of fluorescent images was carried out for the improvement of routine cytogenetic tests (Pinkel et al. 1986). Subsequently, quantitative studies of FISH images were introduced to investigations of tissue-level gene expression, transcriptional activation, coexpression, and nuclear structure function. Despite increased interest in the development of the approaches to FISH signal quantitative assessment, there is as yet no standardized or commonly used protocol. Briefly, the main difficulties of such studies are the lack of stable reproducibility, the irregularity of the signal, and background autofluorescence (reviewed by Levsky and Singer 2003
). Therefore, development of adequate protocols for quantitative analysis of fluorescence images, especially for applied FISH studies, is an actual problem.
Here we present a relatively simple and rapid approach for the quantitative assessment of FISH signals based on the digital capturing of microscopic images and the intensity measuring of hybridization signals by Scion Image software originally developed for analyzing electrophoresis gels. We have carried out the tests for this technique studying aneuploidy and the low-level chromosomal mosaicism involving different human chromosomes in interphase nuclei of different tissues (chorionic villi, fetal skin, placenta, and neuronal cells of the adult brain). We have also investigated the efficiency of the approach in the differentiation of homologous chromosome heteromorphism by the quantitative analysis of alphoid DNA variation of chromosomes 13, 21, and X. In spite of many technical and theoretical limitations in the quantification of FISH signals, we were able to demonstrate the high reproducibility of the measurements after application of Scion Image software. We have concluded that this approach could be useful as an additional tool to visual microscopic analysis and to assist in correct FISH signal interpretation.
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Materials and Methods |
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The chorionic villi samples were obtained from the material of spontaneous abortions (510 weeks of gestation). These tissues were processed for FISH as follows: chorionic villi samples were washed in physiological solution three times. To clean the specimens of the rest of maternal deciduas and blood, samples were washed several times in 70% ethanol. They were then rinsed for 30 sec with 60% acetic acid and placed in solution of 60% acetic acid for 1520 min at room temperature and periodically mixed by inversion. Dispersed single-cell suspensions were fixed in a methanol-acetic acid (3:1) fixative mixture two times for 30 and 50 min. The cells were dropped onto wet slides and air dried at room temperature. Three slides with two drops of cell suspensions each were prepared for each sample.
Fetal skin, placenta, and samples of postmortem adult brain tissues were processed as described previously (Yurov et al. 2001a). Fifty milligrams of these samples were homogenized in 2 ml of phosphate-buffered saline (PBS), pH 7.3, with 0.1% Nonidet P-40, using a glass Teflon homogenizer. Samples were centrifuged at 1000 x g for 5 min followed by fixation of pellets in an ice-cold methanol-glacial acetic acid mixture (3:1), three times for 20 min. The resulting suspensions of nuclei were then dropped onto wet slides. The slides were allowed to dry overnight and were dehydrated by an ethanol series before use in FISH experiments. The quality of the slides with the fixed nuclei was estimated according to the absence of surrounding cytoplasm; another fixation step of cell suspensions was performed, if necessary.
Permission from the Ethics Committee of the National Center of Mental Health, Russian Academy of Medical Sciences was obtained. Written informed consent was obtained from the patients for whom molecular cytogenetic studies were carried out.
The set of DNA probes from the original collection developed at the Laboratory of Cytogenetics, National Center of Mental Health, Moscow, Russia, and including chromosome enumeration DNA probes specific to chromosomes 1, 7, 8, 9, 13, and 21; 14 and 22; 15, 16, 18, X, and Y was used (Yurov et al. 1996,2002
; Soloviev et al. 1998
). FISH studies were performed as described in detail previously (Soloviev et al. 1994
,1995
; Yurov et al. 1996
). For dual- and three-color hybridization, DNA probes were mixed in equal proportions (5 µl each probe at a concentration of 5 ng/µl for each probe). Labeled DNA probes were combined in the following order: (a) chromosome Yspecific probe (labeled by Cy3), chromosome Xspecific probe (labeled by fluorescein-FluorX), chromosome 1specific probe (labeled by biotin or AMCA); (b) chromosome 9specific probe (labeled by biotin) and chromosomes 13/21specific probe (labeled by Cy3); (c) chromosome 16specific probe (labeled by biotin) and chromosomes 14/22specific probe (labeled by Cy3); and (d) chromosome 15specific probe (labeled by biotin) and chromosome 18specific probe (labeled by Cy3). Hybridization was usually performed at 42C overnight, although clear hybridization signals were seen after 3060 min of hybridization. The slides were washed in 50% formamide, 2 x SSC at 4245C, three times for 2 min and rinsed in 0.12 x SSC for 5 min. Detection of biotin-labeled probes was performed as previously described (Pinkel et al. 1986
) by the use of a layer of fluorescein-avidin (Sigma; Moscow, Russia). Slides were mounted in antifade solution [0.2% p-phenylenediamine (Sigma) in 80% glycerol, 20 mM TRIS-HCl, pH 8.0], and 200 ng/ml DAPI (4',6-diamidino-2-phenylindole-2HCl).
For epifluorescence microscopy, a Leitz Orthoplan microscope (Leica Mikroskopie und Systeme; Wetzlar, Germany) equipped with a 100 W lamp was used with the following filter sets (Leica Mikroskopie und Systeme): A (No. 513,596) for DAPI fluorescence; I3 (No. 513,719) or GR (No. 513,821) for fluorescein isothiocyanate signals; GR (No. 513,821) for both fluorescein and cyanine signals; N2 (No. 513,609) for cyanine signals. All images were observed with the Plan-Neofluotar x40/1.30 or x63/1.300.60 oil immersion lenses.
The relative intensity of FISH signals was obtained by digital capturing of microscopic image by the monochrome CCD camera (Cohu 4910 series; Cohu Inc., San Diego, CA), LG-3 grayscale scientific PCI frame grabber (Scion Corporation; National Institutes of Health, Frederick, MD), and subsequent measuring of the intensity of hybridization signals by Scion Image Beta 4.0.2 (Scion Corporation) acquired from www.scioncorp.com (accessed 12/07/2001). The quantification of FISH signals from each digital image was processed by the macros supplied by the manufacturer. Numerical values of the signal relative intensity were compared with each other in the case of interphase FISH study. For the homologous chromosome differentiation, the ratio of the signal relative intensity from the digital image was obtained and compared with the value from another digital image. The reproducibility of the intensity measuring was assessed by several quantitative analyses (510 times) of the same interphase nucleus or metaphase spread. All interphase nuclei suspected to have one signal were subjected to quantitative assessment of FISH signals. Ten to 20 metaphase spreads were analyzed for each sample.
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Results |
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Differentiation of Homologous Chromosome by FISH with Chromosome-specific DNA Probes
The differentiation of homologous chromosomes was performed in a metaphase spread of peripheral blood lymphocytes of Down syndrome family members using an alphoid DNA probe for chromosomes 13 and 21. The technique was also applied for differentiation of homologous chromosome X in females without chromosome abnormalities being an initial stage of the X-chromosome inactivation patterns studied by FISH (described previously in detail in Yurov et al. 2001b) (Figures 1C and 1D and Figures 2A and 2B). The stable reproducibility of the FISH signal quantitative assessment was difficult to achieve because of the irregularity of the signal and background autofluorescence, particularly when comparing different slides. To solve this problem, we applied the comparison of the ratio of the relative intensity of the signals from different digital images. We tested the approach in the studies of chromosome-21 nondisjunction in 15 families with Down syndrome offspring (affected child, mother, and father). The technique allowed us to determine the paternal origin of the additional chromosome 21 in 12 families (80%)two paternal and 10 maternal; and in seven families (46.7%), it has allowed the determination of the meiosis stage of chromosome-21 nondisjunction (data not shown).
The differentiation of homologous chromosome X in females by FISH was found to be applicable to the molecular cytogenetic approach to the study of X-chromosome inactivation patterns. We studied 40 families (propositus and her mothers) with offspring affected by mental retardation not related to chromosome abnormality. An alphoid DNA probe specific for chromosome X was used. The example of quantitative assessment of variable chromosome X FISH signals used for differentiation of homologous chromosomes X is shown in Figure 1D and Figure 2A. The data show the high reproducibility of the measurement results, allowing the possibility of the differentiation of homologous chromosomes X by alphoid DNA heteromorphism. In 19 cases (47.5%), the approach of quantitative FISH signal assessment was found to be sufficiently efficient for the differentiation of homologous chromosomes X (data not shown).
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Discussion |
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It should be emphasized that in all the samples studied, over-position of signals was observed. As a result, the presence of a small but sufficient population of nuclei with one hybridization signal (instead of the two signals expected in a normal diploid cell) produces the problem of interpretation of the results. Additionally, the phenomenon of somatic pairing of homologous chromosomes observed with a higher frequency in brain tissues causes a misinterpretation of the aneuploidy FISH scoring. Therefore, the lack of an approach for signal discrimination could lead to misdiagnosis in the cases of low-level mosaicism. In the present paper, we show that digital quantification (rarely used in FISH analysis of aneuploidy) aids in avoiding the scoring of "pseudo-monosomy," the result of over-position (or somatic chromosome pairing), leading to more accurate study results. The application of the approach has allowed us to exclude or confirm monosomy in the samples studied. Our results show that a confidence interval in interphase FISH studies of aneuploidy could probably be significantly less than 95%. Therefore, on the basis of the data we have obtained, we conclude that this approach is efficient enough for studies of low-level chromosomal mosaicism in different tissues.
An additional application of the proposed approach is the differentiation of homologous chromosome parental origin. Homologous chromosomes in normal chromosomal complement usually have no morphological differences (with the exception of acrocentric chromosomes with variable short arms or heteromorphism of chromosomes 1, 9, and 16 after C-banding). However, variations of alpha satellite DNA present in the centromeric regions of all human chromosomes can be visualized by in situ hybridization with chromosome-specific alphoid DNA probes (Yurov et al. 1987,2001b
). In the present study, we analyzed the efficiency of the quantitative assessment of FISH signals for determining the parental origin of the additional chromosome 21 as well as differentiation of homologous chromosome X. The main difficulty of this study was that we had to compare the intensity of signals from the different slides. For more adequate comparative analysis, we proposed to use the ratio of relative intensity of FISH signals for chromosome 13 and 21 in the cases of chromosome 21 nondisjunction assays and chromosome-X signals in the case of X-chromosome inactivation study. We hypothesized that despite the variation of signal intensity from different slides, the ratio would not vary significantly, inasmuch as the ratio of the heterochromatin block size in homologous chromosomes is the same. The data obtained indicate that quantitative assessment of FISH signals is efficient up to 80% in the study of chromosome-21 nondisjunction. It should be emphasized that molecular cytogenetic studies are strongly recommended for more accurate cytogenetic diagnosis of trisomy 21, and therefore it would be quite helpful to determine the parental origin of the additional chromosome 21 and the meiosis stage of nondisjunction during a routine FISH analysis of a family with offspring affected by Down syndrome.
Initially, the molecular cytogenetic technique based on the identification of heteromorphism of homologous X chromosomes was found to sufficient for the study of X-chromosome inactivation patterns in Rett syndrome (Yurov et al. 2001b). However, the frequency of chromosome X with clearly visible heteromorphism after FISH with an alphoid DNA probe was found to be rather uncommon (in 8/33 individuals analyzed), probably due to the difficulty of comparative visual analysis of FISH signals. We propose the differentiation of active and inactive chromosome X with the quantitative assessment of FISH signals after application of a chromosome Xspecific alphoid DNA probe for improving significantly the efficiency of the molecular cytogenetic technique used in the study of X-chromosome inactivation. This assay is useful in X-chromosome inactivation studies in females with mosaic forms of the chromosome-X aneuploidy, because molecular genetic techniques are unable to precisely determine X-chromosome inactivation patterns in these cases. We have shown the application of the quantitative assessment of FISH signals for chromosome X to increase the efficiency of this technique up to 47.5%, based on the registration of chromosomal heteromorphism with low differences in alphoid DNA content between homologous chromosomes. Moreover, the results of initial molecular cytogenetic assays for X inactivation were in full agreement with the results of subsequent analysis of the same cases by means of a restriction/quantitative PCRbased assay (AR-assay), a commonly used technique for X-chromosome inactivation studies (Iourov et al. 2003
). We believe that this approach for the rapid quantitative assessment of FISH signals using Scion Image software will help in molecular cytogenetic studies of human chromosomes in different fields of research, including preimplantation, and pre- and postnatal diagnosis of aneuploidies by interphase FISH. This approach can be considered a highly efficient additional tool for use in studies of chromosome number variations in interphase nuclei, with a unique possibility for identification of aneuploidy with low-level chromosomal mosaicism.
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Acknowledgments |
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Footnotes |
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Received for publication May 29, 2004; accepted September 23, 2004
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Literature Cited |
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