Fiber Fluorescence In Situ Hybridization Research Articles

Concurrent activation of a novel putative transforming gene,myeov, and cyclin D1 in a subset of multiple myeloma cell lines with t(11;14)(q13;q32)

AbstractThrough the application of the NIH/3T3 tumorigenicity assay to DNA from a gastric carcinoma, we have identified a novel transforming gene, designated myeov (myeloma overexpressed gene in a subset of t[11;14]-positive multiple myelomas). Sequence analyses did not reveal any homology with sequences present in the GenBank, except the deduced protein structure predicts a transmembrane localization.Myeov was mapped to chromosome 11q13 and localized by DNA fiber fluorescence in situ hybridization (FISH) 360-kilobase (kb) centromeric of cyclin D1. In 3 of 7 multiple myeloma (MM) cell lines with a t(11;14)(q13;q32) and cyclin-D1 overexpression, Northern blot analysis revealed overexpression of myeov as well. In all 7 cell lines, the translocation breakpoint was mapped within the 360-kb region between myeov and cyclin D1. DNA fiber FISH with a contig of probes covering the constant region of the immunoglobulin heavy chain (IgH) revealed that exclusively in the 3myeov-overexpressing cell lines (KMS-12, KMS-21, and XG-5), either the 5′ Eμ enhancer or the most telomeric 3′ Eα enhancer was juxtaposed to myeov. Although cyclin D1overexpression represents a characteristic feature of all MM cell lines with t(11;14), our results demonstrate aberrant expression of a second putative oncogene in a subset of these cases, due to juxtaposition to IgH enhancers. The clinical relevance of this dual activation remains to be elucidated.

High Resolution Fiber-Fluorescence In Situ Hybridization.

High resolution fiber-Fluorescence in situ hybridization (FISH) is an advanced FISH technology that can effectively bridge the resolution gap between probe hybridizing on DNA molecules and chromosomal regions. Since various types of DNA and chromatin fibers can be generated reflecting different degrees of DNA/chromatin packaging status, fiber-FISH technology has been successfully used in diverse molecular cytogenetic/cytogenomic studies. Following a brief review of this technology, including its major development and increasing applications, typical protocols to generate DNA/chromatin fiber will be described, coupled with rationales, as well as technical tips. These released DNA/chromatin fibers are suitable for an array of cytogenetic/cytogenomic analyses.

Detection of Transgenes on DNA Fibers.

Fluorescence in situ hybridization (FISH) was developed for detecting specific DNA sequences directly on mitotic or meiotic chromosomes. However, the resolution of FISH on chromosomes is limited by condensed structure of chromatin, and it is difficult to differentiate two target sites close to each other. To overcome this issue, the objects was changed to stretched DNA fibers, and this fiber FISH technique has now been used for revealing genome structure at molecular level. Hybridization and detection procedures of fiber FISH are common with FISH on chromosomes. Therefore, application of fiber FISH is not difficult for the researchers of some experience in ordinary FISH. DNA fibers can be released from nuclei fixed on glass slides using a detergent. The DNA fibers were shred in FISH procedure, and the resultant fragments became small bead-like shape. This makes FISH signals on DNA fibers a series of dots. The size of DNA in the dot is estimated to be approximately 1 kb, it corresponding to the resolution of fiber FISH. This makes it possible to analyze structures of transgenes on DNA fibers in detail.

Characterization of two CENH3 genes and their roles in wheat evolution.

Wheat evolution is complex as a result of successive rounds of allopolyploidization and continuous selection during domestication. Diploid and tetraploid wheat species (Triticum spp.) were used as model systems in which to study the role of centromere-specific histone H3 variant (CENH3) in wheat evolution. We characterized two types of CENH3 genes, named αCENH3 and βCENH3, each of which has three slightly different copies derived from the AA, BB and DD genomes. Specific antibodies were raised against the two CENH3 proteins and were co-localized to centromeres with subtle differences. In most tetraploid wheat species, CENH3 genes are more highly expressed from the AA genome. In wild tetraploids, βCENH3 has a much lower expression level than αCENH3, while in cultivated tetraploids βCENH3 transcripts are enhanced to near αCENH3 levels. Comparison of the CENH3 proteins in wild and cultivated tetraploids revealed that the histone folding domain (HFD) of only βCENH3 is under positive selection, especially in the region responsible for targeting of CENH3 to the centromere. Taken together, positive selection of βCENH3 and its increased expression in tetraploid cultivars are indicative of adaptive evolution. Furthermore, the differences in localization between αCENH3 and βCENH3 observed using fiber fluorescence in situ hybridization (FISH) and immunodetection and in developmental phenotypes resulting from virus-reduced gene silencing imply their functional diversification during wheat evolution.

Tandem amplification of a chromosomal segment harboring 5-enolpyruvylshikimate-3-phosphate synthase locus confers glyphosate resistance in Kochia scoparia.

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.

Evolutionary dynamics of rDNA clusters in chromosomes of five clam species belonging to the family Veneridae (Mollusca, Bivalvia).

The chromosomal changes accompanying bivalve evolution are an area about which few reports have been published. To improve our understanding on chromosome evolution in Veneridae, ribosomal RNA gene clusters were mapped by fluorescent in situ hybridization (FISH) to chromosomes of five species of venerid clams (Venerupis corrugata, Ruditapes philippinarum, Ruditapes decussatus, Dosinia exoleta, and Venus verrucosa). The results were anchored to the most comprehensive molecular phylogenetic tree currently available for Veneridae. While a single major rDNA cluster was found in each of the five species, the number of 5S rDNA clusters showed high interspecies variation. Major rDNA was either subterminal to the short arms or intercalary to the long arms of metacentric or submetacentric chromosomes, whereas minor rDNA signals showed higher variability. Major and minor rDNAs map to different chromosome pairs in all species, but in R. decussatus one of the minor rDNA gene clusters and the major rDNA cluster were located in the same position on a single chromosome pair. This interspersion of both sequences was confirmed by fiber FISH. Telomeric signals appeared at both ends of every chromosome in all species. FISH mapping data are discussed in relation to the molecular phylogenetic trees currently available for Veneridae.

Characterization of Non-LTR Retrotransposable TRAS Elements in the Aphids Acyrthosiphon pisum and Myzus persicae (Aphididae, Hemiptera)

A non-LTR TRAS retrotransposon (identified as TRASAp1) has been amplified in the pea aphid Acyrthosiphon pisum and its presence has been assessed also in the peach potato aphid Myzus persicae. This TRAS element possesses 2 overlapping ORFs (a gag-ORF1 and a pol-ORF2 containing the reverse transcriptase and the endonuclease domains) that show a similarity ranging from 40% to 48% to proteins coded by other TRAS elements identified in insects (including the beetle Tribolium castaneum and the moth Bombyx mori). The study of the TRAS chromosomal insertion sites, performed by standard fluorescent in situ hybridization (FISH) and fiber FISH, showed that TRAS elements were located in a subtelomeric position, just before the telomeric (TTAGG) n repeats. In both the aphid species, TRAS elements were present at all termini of autosomes, but the 2 X chromosome telomeres show a clear-cut structural difference. Indeed, cromomycin A3 staining, together with FISH using a TRAS probe, revealed that TRAS signals only occur at the telomere opposite to the NOR-bearing one. Lastly, the analysis of the distribution of TRAS retrotransposons in a M. persicae strain possessing spontaneous fragmentations of the X chromosomes assessed that TRAS elements were not involved in the healing of de novo telomeres.

Localization of centromeric breaks in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) have very complex karyotypes that show all types of structural rearrangements. The most frequent aberrations are whole-arm translocations, which appear to have their breakpoints in centromeric or pericentromeric regions. We aimed to pinpoint the exact location of the breakpoints of these marker chromosomes with high-resolution cytogenetic and genetic analyses using microarray comparative genomic hybridization (CGH), multiplex ligation-dependent probe amplification (MLPA), and fiber fluorescence in situ hybridization (FISH). Among the seven cell lines in this study, six (84%) harbored one or more centromeric breakpoints or whole-arm translocations. In total, microarray CGH identified 163 breakpoints, 47 (29%) of which were in centromeric regions. Microarray CGH and MLPA results indicated that the translocation breakpoints were localized between the microarray oligonucleotide clones and MLPA probes closest to the centromere. High-resolution fiber-FISH revealed adjacent or minimally overlapping signals of probes that recognize the pericentromeric sequences of the two participating chromosomes. This indicates that whole chromosome arm translocation breakpoints occur within the pericentromeric chromatin and not the centromere core sequences.

Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells.

A de novo 22q11.22q11.23 interchromosomal tandem duplication in a boy with developmental delay, hyperactivity, and epilepsy

The recent development of high-throughput analysis for genomic copy numbers has enabled to identify microscopic chromosomal duplications that had never been recognized before. Microarray-based comparative genomic hybridization (aCGH) identified a de novo 2.1-Mb microduplication in the 22q11.22q11.23 region surrounded by low copy repeats (LCRs) LCR22E and LCR22H in a 5-year-old boy with developmental delay, hyperactivity, epilepsy, and distinctive facial features, which were within the wide range of the clinical manifestations of the patients with the same duplication pattern. Fiber-fluorescent in situ hybridization (FISH) analysis confirmed that the duplicated segments were aligned in a tandem configuration. Familial single nucleotide polymorphism (SNP) typing determined that the duplication was derived from paternal interchromosomal non-allelic homologous recombination (NAHR) during the first meiotic process of spermatogenesis. Although no patient with the deletions of the distal 22q11.2 has been reported as showing epilepsy, at least five patients including the presenting patient having the duplication between LCR22E and LCR22G showed epilepsy. Thus, the gain of the genomic copy number of this region may have epileptogenesis.

Comprehensive genetic analyses of PLP1 in patients with Pelizaeus–Merzbacher disease applied by array-CGH and fiber-FISH analyses identified new mutations and variable sizes of duplications

Pelizaeus–Merzbacher disease (PMD; MIM#312080) is a rare X-linked recessive neurodegenerative disorder. The main cause of PMD is alterations in the proteolipid protein 1 gene ( PLP1) on chromosome Xq22.2. Duplications and point mutations of PLP1 have been found in 70% and 10–25% of all patients with PMD, respectively, with a wide clinical spectrum. Since the underlining genomic abnormalities are heterogeneous in patients with PMD, clarification of the genotype-phenotype correlation is the object of this study. Comprehensive genetic analyses using microarray-based comparative genomic hybridization (aCGH) analysis and genomic sequencing were applied to fifteen unrelated male patients with a clinical diagnosis of PMD. Duplicated regions were further analyzed by fiber-fluorescence in situ hybridization (FISH) analysis. Four novel and one known nucleotide alterations were identified in five patients. Five microduplications including PLP1 were identified by aCGH analysis with the sizes ranging from 374 to 951-kb. The directions of five PLP1 duplications were further investigated by fiber-FISH analysis, and all showed tandem duplications. The common manifestations of the disease in patients with PLP1 mutations or duplications in this study were nystagmus in early infancy, dysmyelination revealed by magnetic resonance imaging (MRI), and auditory brain response abnormalities. Although the grades of dysmyelination estimated by MRI findings were well correlated to the clinical phenotypes of the patients, there is no correlation between the size of the duplications and the phenotypic severity.

Molecular Characterization of TMPRSS2-ERG Gene Fusion in the NCI-H660 Prostate Cancer Cell Line: A New Perspective for an Old Model

Recent studies have established that a significant fraction of prostate cancers harbor a signature gene fusion between the 5' region of androgen-regulated TMPRSS2 and an ETS family transcription factor, most commonly ERG. Studies on the molecular mechanisms and functional consequences of this important chromosomal rearrangement are currently limited to the VCaP cell line derived from a vertebral bone metastasis of a hormone-refractory prostate tumor. Here we report on the NCI-H660 cell line, derived from a metastasic site of an extrapulmonary small cell carcinoma arising from the prostate. NCI-H660 harbors TMPRSS2-ERG fusion with a homozygous intronic deletion between TMPRSS2 and ERG. We demonstrate this by real-time quantitative polymerase chain reaction, a two-stage dual-color interphase fluorescence in situ hybridization (FISH) assay testing for TMPRSS2 and ERG break-aparts, and single-nucleotide polymorphism oligonucleotide arrays. The deletion is consistent with the common intronic deletion found on chromosome 21q22.2-3 in human prostate cancer samples. We demonstrate the physical juxtaposition of TMPRSS2 and ERG on the DNA level by fiber FISH. The androgen receptor-negative NCI-H660 cell line expresses ERG in an androgen-independent fashion. This in vitro model system has the potential to provide important pathobiologic insights into TMPRSS2-ERG fusion prostate cancer.

Two novel translocation breakpoints upstream of SOX9 define borders of the proximal and distal breakpoint cluster region in campomelic dysplasia

The semilethal skeletal malformation syndrome campomelic dysplasia (CD) with or without XY sex reversal is caused by mutations within the SOX9 gene on 17q24.3 or by chromosomal aberrations (translocations, inversions or deletions) with breakpoints outside the SOX9 coding region. The previously published CD translocation breakpoints upstream of SOX9 fall into two clusters: a proximal cluster with breakpoints between 50-300 kb and a distal cluster with breakpoints between 899-932 kb. Here, we present clinical, cytogenetic and molecular data from two novel CD translocation cases. Case 1 with karyotype 46,XY,t(1;17)(q42.1;q24.3) has characteristic symptoms of CD, including mild tibial bowing, cryptorchidism and hypospadias. By standard fluorescence in situ hybridization (FISH) and by high-resolution fiber FISH, the 17q breakpoint was mapped 375 kb from SOX9, defining the centromeric border of the proximal breakpoint cluster region. Case 2 with karyotype 46,X,t(Y;17)(q11.2;q24.3) has the acampomelic form of CD and complete XY sex reversal. By FISH and somatic cell hybrid analysis, the 17q breakpoint was mapped 789 kb from SOX9, defining the telomeric border of the distal breakpoint cluster region. We discuss the structure of the 1 Mb cis-control region upstream of SOX9 and the correlation between the position of the 14 mapped translocation breakpoints with respect to disease severity and XY sex reversal.

Quantitative evaluation of partial deletions of the DAZ gene cluster

Partial deletions of the DAZ gene cluster are thought to cause spermatogenesis impairment. The presence of homologous copies of this gene in the Y chromosome does not allow PCR to be used for the identification of this abnormality. Hence, sequence family variants (SFV), following amplification of sY581, sY587 and sY586 and subsequent enzymatic digestion with Sau3A, DraI and TaqI, respectively, and the dual fiber fluorescence in situ hybridization (FISH) have been used to this aim. However, SFV is not always able to identify single DAZ gene copy deletions. We report a quantitative real-time PCR application to evaluate partial deletions of the DAZ gene cluster. To accomplish this, we designed a probe on exon 6 of the DAZ gene which is repeated 3 times in DAZ1, once in DAZ2 and DAZ3 and twice in DAZ4. Five normozoospermic healthy men (C1-C5) having 4 DAZ gene copies by SFV were selected. Fiber-FISH confirmed this outcome in C1-C4, but not in C5 who had an incomplete DAZ gene cluster. The men underwent then quantitative real-time PCR and C1 was arbitrarily selected as calibrator for the calculation of the DAZ gene signals because of the lowest variation in the threshold cycles. Real-time PCR identified 7.2+/-0.05 signals in C2-C4 and 5.4+/-0.05 signals in C5. The overall coefficient of variation was 1.4+/-0.2%. The loss of two signals in this subject may relate to a deletion of both DAZ2 and DAZ3 or of DAZ4 gene. Since SFV showed clearly the presence of DAZ2, it may be hypothesized that C5 lacks DAZ4. In conclusion, these data suggested that quantitative real-time PCR seems to be an effective and reproducible technique that can be used to study the DAZ gene cluster. In addition, the probe chosen for this approach may give indication on the DAZ gene copy deleted.

Visualization of episomal and integrated Epstein‐Barr virus DNA by fiber fluorescence in situ hybridization

For many Epstein-Barr virus (EBV)-associated malignancies, it is still a matter of controversy whether infected cells harbor episomal or chromosomally integrated EBV genomes or both. It is well established that the expression of EBV genes per se carries oncogenic potential, but the discrimination between episomal and integrated forms is of great relevance because integration events can contribute to the oncogenic properties of EBV, whereas host cells that exclusively harbor viral episomes may not carry the risks mediated by chromosomal integration. This notion prompted us to establish a reliable technique that not only allows to unequivocally discriminate episomal from integrated EBV DNA, but also provides detailed insights into the genomic organization of the virus. Here, we show that dynamic molecular combing of host cell DNA combined with fluorescence in situ hybridization (FISH) using EBV-specific DNA probes facilitate unambiguous discrimination of episomal from integrated viral DNA. Furthermore, the detection of highly elongated internal repeat 1 (IR1) sequences provides evidence that this method permits detection of major genomic alterations within the EBV genome. Thus, fiber FISH may also provide valuable insights into the genomic organization of viral genomes other than EBV.

In Situ Chromosomal Localization of rDNA Sites in “Safed Musli” Chlorophytum Ker-Gawl and Their Physical Measurement by Fiber FISH

Fluorescence In Situ Hybridization (FISH) technique has been applied on somatic chromosomes and extended DNA fibers in the medicinally important species of Chlorophytum to elucidate physical localization and measurement of the rDNA sites using two rRNA multigene families homologous to 45S and 5S rDNA. The two species of Chlorophytum, namely C. borivillianum and C. comosum, both with 2n = 28, reveal diversity for copy number and localization of rDNA sites. C. borivillianum is comprised of five 45S-rDNA sites:one each in the secondary constriction region of chromosomes 7, 8, 9; one in the subtelomeric region of the short arm of chromosome 2 and the telomeric region of the short arm of chromosome 12; and one 5S-rDNA site in the subtelomeric region of the long arm of chromosome 1. In C. comosum, there are three 45S-rDNA sites (one each in the short arm of chromosomes 12, 13, and 14) and two 5S-rDNA sites (in the secondary constriction regions of chromosomes 2 and 13). Fiber FISH analysis conducted on extended DNA fibers revealed variation in the size of continuous tandem strings for the two r-DNA families. Taking the standard value of native B DNA equivalent to 3.27 kb for 1 mum, it was estimated that the physical size of continuous DNA strings is of the order of approximately 90 kb, 180 kb, and 300 kb for 45S-rDNA and of the order of 60 kb, 150 kb for 5S-rDNA in C. comosum, grossly in correspondence to their respective physical sizes at metaphase.

Characterization of ancestral chromosome fusion points in the Indian muntjac deer.

Tandem fusion, a rare evolutionary chromosome rearrangement, has occurred extensively in muntjac karyotypic evolution, leading to an extreme fusion karyotype of 6/7 (female/male) chromosomes in the Indian muntjac. These fusion chromosomes contain numerous ancestral chromosomal break and fusion points. Here, we designed a composite polymerase chain reaction (PCR) strategy which recovered DNA fragments that contained telomere and muntjac satellite DNA sequence repeats. Nested PCR confirmed the specificity of the products. Two-color fluorescence in situ hybridization (FISH) with the repetitive sequences obtained and T2AG3 telomere probes showed co-localization of satellite and telomere sequences in Indian muntjac chromosomes. Adjacent telomere and muntjac satellite sequences were also seen by fiber FISH. These data lend support to the involvement of telomere and GC-rich satellite DNA sequences during muntjac chromosome fusions.

Molecular characterization of the recurrent unbalanced translocation der(1;7)(q10;p10)

AbstractAn unbalanced translocation der(1;7)(q10; p10) is a nonrandom chromosomal aberration commonly observed in myelodysplastic syndrome and acute myeloid leukemia. We molecularly analyzed the breakpoints of der(1;7)(q10;p10) by quantitative fluorescent in situ hybridization (FISH) analyses using centromeric satellite DNAs mapped to chromosomes 1 and 7 as probes. We found that the signal intensities of 2 centromere alphoid probes, D1Z7 on chromosome 1 and D7Z1 on chromosome 7, were almost invariably reduced on the derivative chromosome compared with those on their normal counterparts. These results suggest that this translocation results from the recombination between the 2 alphoids, which was further confirmed by fiber FISH experiments. Because the relative reduction in the intensities of D1Z7 and D7Z1 signals on the derivative chromosomes was highly variable among patients, it was estimated that the breakpoints in these patients were randomly distributed over several megabase pairs within each alphoid cluster except for its extreme end to the short arm. Our results provide a novel insight into the structural basis for generation of this translocation as well as its leukemogenic roles. (Blood. 2003;102:2597-2604)

Multiple sclerosis-associated virus-related pol sequences found both in multiple sclerosis and healthy donors are more frequently expressed in multiple sclerosis patients.

In the etiopathogenesis of multiple sclerosis (MS), both genetic and environmental factors play an important role. Among environmental factors, viral infections are most likely connected with the etiology of MS. There are many evidence suggesting possible involvement of retroviruses in the development of autoimmune diseases including MS. Multiple sclerosis-associated retrovirus (MSRV) seems to be the important candidate for viral etiology of MS. The aim of the study was to analyze MSRV pol sequences in patients with MS. As control, groups of myasthenia gravis, Parkinson's disease, and migraine patients, and healthy individuals have been studied. The MSRV pol sequences have been analyzed in RNA isolated from the serum and in DNA and RNA of peripheral blood lymphocytes from untreated MS patients and control groups. The MSRV pol sequences have been detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR technique, using specific oligonucleotide primers. In the serum RNA (cDNA), MSRV pol sequences have been identified in 31/32 MS patients. MSRV pol sequences were detected in serum cDNA of 9/17 myasthenia gravis patients, 7/16 Parkinson's disease patients, 10/21 migraine patients, and 13/27 healthy individuals. MSRV pol sequences were observed also in RNA from lymphocytes of all MS patients, 12/17 myasthenia gravis patients, 9/16 Parkinson's disease patients, 14/21 migraine patients, and 18/27 healthy donors. In the DNA from peripheral blood lymphocytes of all studied patients and healthy individuals, MSRV pol sequences have been found. The observed pattern of fiber-fluorescence in situ hybridization (FISH) signals suggests the presence of multiple copies of MSRV pol sequences, most likely tandemly dispersed in the genome. It can be concluded that MSRV pol sequences are endogenous, widespread in lymphocytes DNA, and transcribed into RNA of MS patients as well as of other studied patients and healthy individuals. However, more frequent expression of MSRV sequences detected in lymphocytes RNA (cDNA), as well as their presence in higher frequency in the serum of MS patients, may suggest the involvement of MSRV in the etiopathogenesis on MS.

Rapid Detection of IgH/BCL2 Rearrangement in Follicular Lymphoma by Interphase Fluorescence in Situ Hybridization with Bacterial Artificial Chromosome Probes

Follicular lymphomas (FLs) can be difficult to diagnose on aspirated specimens since the architectural pattern is not present. FLs characteristically have rearrangements in the IgH and BCL2 genes resulting from the reciprocal t(14;18) (q32; q21) translocation. Because of the dispersed distribution of breakpoints, fluorescence in situ hybridization (FISH) using genomic probes that span or flank the breakpoints is ideal for detecting this rearrangement in fine-needle aspiration (FNA) biopsies. To develop a set of probes, a bacterial artificial chromosome library was screened and the clones were mapped by fiber FISH. The probes were produced by the direct incorporation of fluorochrome-labeled nucleotides. The colocalization base FISH assay was applied to Cytospin preparations from FNA biopsies of lymph nodes from 26 patients with FL and 10 patients without FL. In those with FL, the percentage of cells with at least one IgH/BCL2 fusion signal ranged from 22% to 100% (mean, 63%), which was statistically significantly higher than that in FL-negative samples (mean, 2.7%). The probes demonstrated a significantly lower cutoff value (7%) in normal controls and effectively reduced the false-positive rate in FL-negative cases. These results were confirmed with fiber FISH assays on the same specimens. This interphase FISH assay is rapid and reliable for detecting rearrangements in the IGH/BCL2 gene, thereby aiding in the diagnosis of FL on FNA biopsy specimens.