Whole-chromosome Painting Probes Research Articles

Telomere Aberration Detection by PNA FISH Probe.

Telomere is a structure of the end cap of chromosomes. Telomere gets shorter as cell aging and progressing cell division. Shorter telomere may cause telomere fusion, thus inducing genomic instability. Telomere dysfunction can be visualized by PNA FISH probe against telomere repeat sequence (TTAGGG)n. PNA probes have higher hybridization affinity than DNA probes. The traditional FISH or modified FISH protocol can stain telomere relatively easier than whole-chromosome painting probes. This chapter introduces PNA telomere FISH protocol to visualize telomere signals on metaphase chromosomes.

Identification of satellited markers by microdissection and fluorescence in situ hybridization: a clinical case of isodicentric chromosome 22

BackgroundThe presence of small supernumerary marker chromosomes (sSMCs) in a karyotype leads to diagnostic questions because the resulting extra material may cause abnormal development depending on the origin of the duplication/triplication. Because SMCs are so small, their origin cannot be determined by conventional cytogenetic techniques, and new molecular cytogenetic methods are necessary. Here, we applied a target approach to chromosome rearrangement analysis by isolating a chromosome of interest via microdissection and using it in fluorescence in situ hybridization (FISH) as a probe in combination with whole-chromosome painting probes. This approach allows to identify origins of both the euchromatin and repeat-rich regions of a marker.Case presentationWe report a case of an adult male with congenital atresia of the rectum and anus, anotia, and atresia of the external auditory canal along with hearing loss. Karyotyping and FISH analysis with whole-chromosome painting probes of acrocentric chromosomes and the constructed microdissection library of the marker chromosome reliably identified an additional chromosome in some metaphases: mos 47,XY,+idic(22)(q11.2)[14]/46,XY [23].ConclusionWe propose to use whole-chromosome libraries and microdissected chromosomes in FISH to identify SMCs enriched with repeated sequences. We show that the methodology is successful in identifying the composition of a satellited marker chromosome.

Clinical Cytogenetics of the Dog: A Review.

Simple SummaryThe cytogenetic analysis of dogs is mainly focused on the diagnosis of disorders of sex development (DSD) and cancers. Unfortunately, the study of canine chromosomes is a challenging task due a high chromosome number (2n = 78) and the one-arm morphology of all autosomes. For years, the application of conventional cytogenetic techniques, Giemsa staining and G and DAPI (4′,6-diamidino-2-phenylindole) bandings, allowed the identification of sex chromosome aneuploidies and centric fusions. An advanced clinical cytogenetic diagnosis is also needed due to the fact that the dog is a valuable animal model in biomedical research. The application of hybridization methods, such as fluorescence in situ hybridization (FISH) and array comparative genome hybridization (aCGH), facilitated the detection of other chromosomal rearrangements. It can be foreseen that a wide use of modern molecular techniques (e.g., SNP microarray and next generation sequencing) will substantially extend the knowledge on canine chromosome mutations.The dog is an important companion animal and has been recognized as a model in biomedical research. Its karyotype is characterized by a high chromosome number (2n = 78) and by the presence of one-arm autosomes, which are mostly small in size. This makes the dog a difficult subject for cytogenetic studies. However, there are some chromosome abnormalities that can be easily identified, such as sex chromosome aneuploidies, XX/XY leukocyte chimerism, and centric fusions (Robertsonian translocations). Fluorescence in situ hybridization (FISH) with the use of whole-chromosome painting or locus-specific probes has improved our ability to identify and characterize chromosomal abnormalities, including reciprocal translocations. The evaluation of sex chromosome complement is an important diagnostic step in dogs with disorders of sex development (DSD). In such cases, FISH can detect the copy number variants (CNVs) associated with the DSD phenotype. Since cancers are frequently diagnosed in dogs, cytogenetic evaluation of tumors has also been undertaken and specific chromosome mutations for some cancers have been reported. However, the study of meiotic, gamete, and embryo chromosomes is not very advanced. Knowledge of canine genome organization and new molecular tools, such as aCGH (array comparative genome hybridization), SNP (single nucleotide polymorphism) microarray, and ddPCR (droplet digital PCR) allow the identification of chromosomal rearrangements. It is anticipated that the comprehensive use of chromosome banding, FISH, and molecular techniques will substantially improve the diagnosis of chromosome abnormalities in dogs.

Analysis of Chromosomal Damage Caused by Acetamiprid

Abstract Different chemicals can have genotoxic effects on the body, as confirmed by chromosome damage detection. Using conventional cytogenetic analysis and fluorescence in situ hybridization, we tested the extent of chromosome damage caused by the acetamiprid-based insecticide Mospilan 20SP on bovine peripheral blood lymphocytes at concentrations of, 2.5, 5, 25 and 50 µg.ml−1 after a 24 h incubation period. During the experiment, the presence of unstable aberrations—chromosomal and chromatid breaks and gaps—were detected by conventional cyto-genetic analysis. With increasing insecticide concentrations, we observed a statistically significant increase in chromosome damage frequency after 24 hours of exposure. Fluorescence in situ hybridization was used to detect stable structural aberrations; whole-chromosome painting probes for bovine chromosomes 1 and 7 (BTA 1 and BTA 7) were used for this purpose. As a result of exposure to the insecticide, neither BTA 1/BTA 7 translocations nor other types of translocations were observed.

Whole-chromosome paints in maize reveal rearrangements, nuclear domains, and chromosomal relationships

Whole-chromosome painting probes were developed for each of the 10 chromosomes of maize by producing amplifiable libraries of unique sequences of oligonucleotides that can generate labeled probes through transcription reactions. These paints allow identification of individual homologous chromosomes for many applications as demonstrated in somatic root tip metaphase cells, in the pachytene stage of meiosis, and in interphase nuclei. Several chromosomal aberrations were examined as proof of concept for study of various rearrangements using probes that cover the entire chromosome and that label diverse varieties. The relationship of the supernumerary B chromosome and the normal chromosomes was examined with the finding that there is no detectable homology between any of the normal A chromosomes and the B chromosome. Combined with other chromosome-labeling techniques, a complete set of whole-chromosome oligonucleotide paints lays the foundation for future studies of the structure, organization, and evolution of genomes.

A novel mosaic complex supernumerary marker chromosome in a girl with seizures: systematic characterization of the complex marker

Small supernumerary marker chromosomes (sSMC) are a heterogeneous group of chromosomes which are reported in variable phenotypes. They are chromosomal fragments or markers whose origins often cannot be determined by conventional cytogenetic methods alone and require molecular approaches. In general sSMC are equal in size or smaller than chromosome 20. The complex sSMC (CsSMC) arise from two different chromosomes. The aim of the present study is to identify and characterize the chromosomal abnormality in an 11year old girl with seizures, intellectual disability and developmental delay. Different methods like GTG banding, C-banding and NOR staining, spectral karyotyping (SKY), fluorescence in-situ hybridization (FISH) using whole chromosome paint probes (WCP) and bacterial artificial chromosome (BAC) clones were used. Also array CGH was done to check the gains and losses in the genome. The chromosomal analysis on the metaphases revealed a karyotype of mos 47,XX,+mar/46,XX. The marker was identified by SKY and aCGH, as a 33Mb duplication of the 3q region and a 38Mb duplication of the chromosome 9p region. This was confirmed by WCP FISH. Further fine mapping was done with 8 BAC clones from the 3q and 9p regions to confirm the marker chromosome. The marker showed the centromeric 9 region which was also confirmed by the BAC clones. The application of the combined cytogenetic methods like aCGH and FISH helped in the systematic characterization of the CsSMC. The accurate characterization of the CsSMC helps in increasing the knowledge of chromosomal origin, gene content, UPD and other imbalance in the genome.

Comparative analysis of individual chromosome involvement in micronuclei induced by mitomycin C and bleomycin in human leukocytes.

BackgroundMicronucleus (MN) assay is a well standardized approach for evaluation of clastogenic/aneugenic effects of mutagens. Fluorescence in situ hybridization (FISH) is successfully used to characterize the chromosomal content of MN. However, the relationships between nuclear positioning, length, and gene density of individual chromosomes and their involvement in MN induced by different mutagens have not been clearly defined.ResultsChromosomal content of MN was characterized in human leukocytes treated with mitomycin C (MMC) and bleomycin (BLM) by FISH using centromeric (cep) and whole-chromosome painting (wcp) probes. Involvement of chromosomes 8, 15 and 20 in MMC-induced and chromosomes 1, 9 and 16 in BLM-induced MN was studied, and correlated with chromosome size, gene density and interphase position. The results obtained were analyzed together with previous own data on the frequencies of inclusion of chromosomes 3, 4, 6, 7, 9, 16, 17, 18, and X in MMC-induced MN. It could be shown that MMC- and BLM-induced MN could contain material derived from all chromosomes investigated. Involvement of whole chromosomes 8, 15 and 20 in MMC-induced MN negatively correlated with gene density; however, analysis together with earlier studied chromosomes did not confirm this correlation. Inclusion of chromosomes 8, 15 and 20 in MMC-induced MN does not depend on their size and interphase position; the same result was found for the twelve overall analyzed chromosomes. In BLM-treated cells significant correlation between frequencies of involvement of chromosomes 1, 9 and 16 in MN and their size was found.ConclusionsOur results clearly revealed that BLM differs from MMC with respect to the distribution of induced chromosome damage and MN formation. Thus, DNA-damaging agents with diverse mechanism of action induce qualitatively different MN with regard to their chromosomal composition. Also this study demonstrates the utility of combined sequential application of cep and wcp probes for efficient detection of MN chromosomal content in terms of centric and acentric fragments.

The Ancestral Carnivore Karyotype As Substantiated by Comparative Chromosome Painting of Three Pinnipeds, the Walrus, the Steller Sea Lion and the Baikal Seal (Pinnipedia, Carnivora).

Karyotype evolution in Carnivora is thoroughly studied by classical and molecular cytogenetics and supplemented by reconstructions of Ancestral Carnivora Karyotype (ACK). However chromosome painting information from two pinniped families (Odobenidae and Otariidae) is noticeably missing. We report on the construction of the comparative chromosome map for species from each of the three pinniped families: the walrus (Odobenus rosmarus, Odobenidae–monotypic family), near threatened Steller sea lion (Eumetopias jubatus, Otariidae) and the endemic Baikal seal (Pusa sibirica, Phocidae) using combination of human, domestic dog and stone marten whole-chromosome painting probes. The earliest karyological studies of Pinnipedia showed that pinnipeds were characterized by a pronounced karyological conservatism that is confirmed here with species from Phocidae, Otariidae and Odobenidae sharing same low number of conserved human autosomal segments (32). Chromosome painting in Pinnipedia and comparison with non-pinniped carnivore karyotypes provide strong support for refined structure of ACK with 2n = 38. Constructed comparative chromosome maps show that pinniped karyotype evolution was characterized by few tandem fusions, seemingly absent inversions and slow rate of genome rearrangements (less then one rearrangement per 10 million years). Integrative comparative analyses with published chromosome painting of Phoca vitulina revealed common cytogenetic signature for Phoca/Pusa branch and supports Phocidae and Otaroidea (Otariidae/Odobenidae) as sister groups. We revealed rearrangements specific for walrus karyotype and found the chromosomal signature linking together families Otariidae and Odobenidae. The Steller sea lion karyotype is the most conserved among three studied species and differs from the ACK by single fusion. The study underlined the strikingly slow karyotype evolution of the Pinnipedia in general and the Otariidae in particular.

Single-cell analysis of aneuploidy events using yeast whole chromosome painting probes (WCPPs)

Aneuploidy is considered a widespread genetic variation in such cell populations as yeast strains, cell lines and cancer cells, and spontaneous changes in the chromosomal copy number may have implications for data interpretation. Thus, aneuploidy monitoring is essential during routine laboratory practice, especially while conducting biochemical and/or gene expression analyses. In the present study, we constructed a panel of whole chromosome painting probes (WCPPs) to monitor aneuploidy in a single yeast Saccharomyces cerevisiae cell. The WCPP-based system was validated using “normal” haploid and diploid cells, as well as disomic cells both with and without cell synchronisation. FISH that utilised WCPPs was combined with DNA cell cycle analysis (imaging cytometry) to provide a detailed analysis of signal variability during the cell cycle. Chromosome painting can be utilised to detect spontaneously formed disomic chromosomes and study aneuploidy-promoting conditions. For example, the frequency of disomic chromosomes was increased in cells lacking NAD+-dependent histone deacetylase Sir2p compared with wild-type cells (p<0.05). In conclusion, WCPPs may be considered to be a powerful molecular tool to identify individual genomic differences. Moreover, the WCPP-based system may be used at the single-cell level of analysis to supplement array-based techniques and high-throughput analyses at the population scale.

The Utility of Chromosome Microdissection in Clinical Cytogenetics: A New Reciprocal Translocation in Sheep

Local sheep breeders and scientists in Italy cooperate and conduct research on the genetic improvement of autochthonous genetic types (AGTs) by various approaches, including a cytogenetic breeding selection since 2011. The Laticauda sheep (Ovis aries, 2n = 54) breed is one of the AGTs reared in the Campania region (southern Italy). Performing cytogenetic analyses, we have detected and described a novel reciprocal translocation in a Laticauda sheep identified as 54,XX t(18;23)(q14;q26). Our data support recurring appeals that suggest the regular performance of cytogenetic analyses for monitoring genetic health of livestock species. In total, 5 cases of reciprocal translocations in sheep are known, including the new case. None of them has any phenotypic effect on the living offspring. However, affected animals are characterized by sterility or have a low fertility which can have an effect on breeding success and on economical balance. Presence and kind of the described novel chromosomal aberration were detected by performing CBA-banding and FISH mapping with telomeric probes. RBA-banding allowed the karyotyping of sheep chromosomes and the identification of aberrant chromosomes and regions involved in the new reciprocal translocation. Whole chromosome painting (WCP) probes received from equivalent chromosomes in cattle and the derivative sheep chromosome 18 confirmed the cytogenetic data. This way, our study underlined both the importance of WCP probes by chromosome microdissection and a new way to use WCP probes directly generated from derivative chromosomes.

Multicolor FISH methods in current clinical diagnostics

Multicolor FISH (mFISH) assays are currently indispensable for a precise description of derivative chromosomes. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole-chromosome painting probes in multiplex-FISH and spectral karyotyping. Since then, multiple approaches for chromosomal differentiation based on multicolor-FISH (MFISH) assays have been developed. Predominantly, they are applied to characterize marker or derivative chromosomes identified in conventional banding analysis. Since the introduction of array-based comparative genomic hybridization (aCGH), mFISH is also applied to verify and further delineate aCGH-detected aberrations. For the latter, it is important to consider the fact that aCGH cannot detect or characterize balanced rearrangements, which are important to be resolved in detail in infertility diagnostics. In addition, mFISH is necessary to distinguish different imbalanced situations detectable in aCGH; small supernumerary marker chromosomes have to be differentiated from insertions or unbalanced translocations. This review presents an overview on the available mFISH methods and their applications in pre- and post-natal clinical genetics.

Subchromosomal karyotype evolution in Equidae

Equidae is a small family which comprises horses, African and Asiatic asses, and zebras. Despite equids having diverged quite recently, their karyotypes underwent rapid evolution which resulted in extensive differences among chromosome complements in respective species. Comparative mapping using whole-chromosome painting probes delineated genome-wide chromosome homologies among extant equids, enabling us to trace chromosome rearrangements that occurred during evolution. In the present study, we performed subchromosomal comparative mapping among seven Equidae species, representing the whole family. Region-specific painting and bacterial artificial chromosome probes were used to determine the orientation of evolutionarily conserved segments with respect to centromere positions. This allowed assessment of the configuration of all fusions occurring during the evolution of Equidae, as well as revealing discrepancies in centromere location caused by centromere repositioning or inversions. Our results indicate that the prevailing type of fusion in Equidae is centric fusion. Tandem fusions of the type telomere-telomere occur almost exclusively in the karyotype of Hartmann's zebra and are characteristic of this species' evolution. We revealed inversions in segments homologous to horse chromosomes 3p/10p and 13 in zebras and confirmed inversions in segments 4/31 in African ass, 7 in horse and 8p/20 in zebras. Furthermore, our mapping results suggested that centromere repositioning events occurred in segments homologous to horse chromosomes 7, 8q, 10p and 19 in the African ass and an element homologous to horse chromosome 16 in Asiatic asses. Centromere repositioning in chromosome 1 resulted in three different chromosome types occurring in extant species. Heterozygosity of the centromere position of this chromosome was observed in the kiang. Other subtle changes in centromere position were described in several evolutionary conserved chromosomal segments, suggesting that tiny centromere repositioning or pericentric inversions are quite frequent in zebras and asses.

A novel t(3;12)(q21;p13) translocation in a patient with accelerated chronic myeloid leukemia after imatinib and nilotinib therapy.

The acquisition of secondary chromosomal aberrations in chronic myeloid leukemia (CML) patients with Philadelphia chromosome-positive (Ph+) karyotype signifies clonal evolution associated with the progression of the disease to its accelerated or blastic phase. Therefore, these aberrations have clinical and biological significance. T(3;12)(q26;p13), which is a recurrent chromosomal aberration observed in myeloid malignancies, is typically associated with dysplasia of megakaryocytes, multilineage involvement, short duration of any blastic phase, and extremely poor prognosis. We have identified a recurrent reciprocal translocation between chromosomes 3 and 12 with different breakpoint at bands 3q21 in the malignant cells from a 28-year-old man. The patient was initially diagnosed as having Ph+ CML in the chronic phase. The t(3;12)(q21;p13) translocation occurred 4 years after the patient was first diagnosed with CML while undergoing tyrosine kinase inhibitor therapy. We confirmed the t(3;12)(q21;p13) translocation via fluorescence in situ hybridization assay by using whole-chromosome paint probes for chromosomes 3 and 12. Our findings demonstrate that, similar to other recurrent translocations involving 3q26 such as t(3;3) and t(3;21), the t(3;12)(q21;p13) translocation is implicated not only in myelodysplastic syndrome and acute myeloid leukemia but also in the progression of CML. These findings extend the disease spectrum of this cytogenetic aberration.

Disorder of sexual development in a Yorkshire terrier (78, XY; SRY-positive)

A 9-month-old Yorkshire terrier was admitted to the clinic because of abnormal sexual behaviour and clitoral hypertrophy. External examination confirmed standard development of caudal genital organs: vagina, vulva and cervix uteri. Serum profile of gonadotropin hormones 17 β-estradiol (<10.0 pg.ml(-1)) and testosterone (9.1 ng.ml(-1)) revealed the presence of testicular tissue. A midline laparotomy was performed to detect the cranial parts of the genital system. Gonads resembling testicles, structures indicating epididymis and rudimentary deferent ducts were resected, along with adherent part of the uterus. Cytogenetic analysis showed a male chromosomal complement 78, XY in all metaphases of the studied Yorkshire terrier dog. The chromosomal constitution was confirmed by fluorescence in situ hybridisation (FISH) with whole-chromosome painting probes specific for chromosomes X and Y, as well as by polymerase chain reaction (PCR) amplification of the 271-bp Y-linked fragment of SRY (the sex-determining region on the Y chromosome) gene. Sequencing of the dog's SRY gene coding region did not reveal any mutation. To search for potential mutation in the SOX9 gene (Sry-box containing gene 9), which is considered to be one of the key genes involved in the sex determination process, the PCR fragments of exons 1, 2 and 3 originating from the canine patient were sequenced in order to compare with both male and female healthy control dogs. In the analysed regions of the SOX9 gene, no mutation was found.

Chromosomal Composition of Micronuclei in Human Leukocytes Exposed to Mitomycin C

Micronuclei (MN) can be induced by different mutagenic substances. Even though this has been known for decades, it is still not clear which genetic content, especially which chromosomes, these MN are constituted of and if there are any influences on this content by the MN-inducing substance. Also, the interphase position, size, and gene density of a chromosome could influence its involvement in MN formation. To study some of these questions, fluorescence in situ hybridization using centromeric and whole-chromosome painting probes for chromosomes 3, 4, 6, 7, 9, 16, 17, 18, and X was applied in mitomycin C (MMC)-induced MN in human leukocytes. The obtained results showed that material from all studied chromosomes was present in MN. Also, there was no correlation between interphase position, size, and gene density of the studied chromosomes and their migration in MN. Interestingly, material derived from chromosomes 9 and 16 was overrepresented in MMC-induced MN. Finally, further studies using substances other than MMC are necessary to clarify if the MN-inducing mutagen has an influence on the chromosomal content of the MN.

Identification of chromosomes and aberrations after exposure to pesticide in bovine lymphocytes

Tebuconazole belongs to the group of triazole fungicides. As a systemic foliar fungicide it was widely used in agriculture for crops protection, such as barley, wheat, peanuts and orchard fruits. Associations between exposure to pesticides and health outcomes in humans, including different kinds of cancer were reported by several authors. Effects in immune, hematological, nervous, endocrine and reproductive systems have also been described. Data about genotoxic effects of pesticides are rarely reported. We are interested in the detection of structural and numerical aberrations in bovine peripheral lymphocytes using fluorescence in situ hybridization (FISH) in vitro. Three whole chromosome painting probes (WCPs), BTA1 for the bovine chromosome 1 (red colour), BTA 5 for chromosome 5 (green colour) and BTA 7 for chromosome 7 (red colour), as well, were used in experiments. Aneuploidy and unstable structural aberrations were detected more precisely than those seen in conventional chromosome aberrations assays, but only on the marked chromosomes could be analysed.

Analysis Using Sperm-FISH of a Putative Interchromosomal Effect in Boars Carrying Reciprocal Translocations

The occurrence of interchromosomal effects (ICE) in reciprocal translocation carriers still remains contradictory in the human literature. We used the pig as an animal model to investigate whether the structure of the reciprocal translocations as well as the size and/or type of the chromosomes not involved in the rearrangement may influence the occurrence and the extent of ICE. Analyses of chromosomal sperm content by fluorescence in situ hybridization (FISH) using whole-chromosome painting probes for 7 chromosomes (1, 10, 11, 13, 18, X and Y) were carried out on sperm samples of 2 boars with normal semen parameters carrying different balanced reciprocal translocations: 38, XY, t(3;15)(q27;q13) or 38, XY, t(12;14)(q13;q21). One fertile boar with normal karyotype was also studied as a control. Aneuploidy rates for the 7 chromosomes were estimated by scoring 10,000 to 20,000 spermatozoa for each probe combination. No significant ICE was found except for chromosome 1 in the case of the t(3;15) translocation. Even if statistically significant, this ICE remained very weak and should have very little impact on the reproductive performance of the carrier boar. The size and/or type of chromosomes not involved in the translocation do not seem to have a major influence on the occurrence of ICE. The structure of the translocation could play a role, but complementary studies should be carried out to confirm this assumption.

Tracking genome organization in rodents by Zoo-FISH

The number of rodent species examined by modern comparative genomic approaches, particularly chromosome painting, is limited. The use of human whole-chromosome painting probes to detect regions of homology in the karyotypes of the rodent index species, the mouse and rat, has been hindered by the highly rearranged nature of their genomes. In contrast, recent studies have demonstrated that non-murid rodents display more conserved genomes, underscoring their suitability for comparative genomic and higher-order systematic studies. Here we provide the first comparative chromosome maps between human and representative rodents of three major rodent lineages Castoridae, Pedetidae and Dipodidae. A comprehensive analysis of these data and those published for Sciuridae show (1) that Castoridae, Pedetidae and Dipodidae form a monophyletic group, and (2) that the European beaver Castor fiber (Castoridae) and the birch mouse Sicista betulina (Dipodidae) are sister species to the exclusion of the springhare Pedetes capensis (Pedetidae), thus resolving an enduring trifurcation in rodent higher-level systematics. Our results together with published data on the Sciuridae allow the formulation of a putative rodent ancestral karyotype (2n = 50) that is thought to comprise the following 26 human chromosomal segments and/or segmental associations: HSA1pq, 1q/10p, 2pq, 2q, 3a, 3b/19p, 3c/21, 4b, 5, 6, 7a, 7b/16p, 8p/4a/8p, 8q, 9/11, 10q, 12a/22a, 12b/22b, 13, 14/15, 16q/19q, 17, 18, 20, X and Y. These findings provide insights into the likely composition of the ancestral rodent karyotype and an improved understanding of placental genome evolution.

Chromosome analysis of esophageal squamous cell carcinoma cell line KYSE 410-4 by repetitive multicolor fluorescence in situ hybridization

Chromosome aberrations are distinctive features of human malignant tumors. Analysis of chromosomal changes can illuminate the molecular mechanisms underlying the development and progression of cancer. To establish the technique of multicolor fluorescence in situ hybridization (M-FISH) for identifying chromosome aberrations in esophageal carcinoma cell line KYSE 410-4, four pools of 6-color whole-chromosome painting probes have been designed and hybridized on the same metaphase spread by four rounds of repetitive FISH. Repetitive 6-color M-FISH was successfully established and the cytogenetic abnormalities in KYSE 410-4 cells were characterized. Chromosome gains occurred at 2q, 3, 8, 17p, and X. An isochromosome 3q was visualized in the cell line, which might be one intermediate mechanism leading to 3p losses and/or 3q gains. Furthermore, 16 structural arrangements were detected, including four derivative chromosomes. The rearrangement of the centromeric regions accounted for approximately 44% of all rearrangements. The results added a more complete and accurate information of the genetic alterations to the classical cytogenetic description of KYSE 410-4 and provided a detailed cytogenetic background data for appropriate use of the cell line. The established 6-color M-FISH was useful for analyzing chromosomes in the whole genome of human tumors.

Premature ovarian failure in a patient with a complex chromosome rearrangement involving the critical region Xq24, characterized by analysis using fluorescence in situ hybridization by chromosome microdissection

To characterize a complex chromosome rearrangement previously detected by G-banding in peripheral blood lymphocytes as 46,X, inv(X)(p11;q2?), inv(4)(q?),ins(8)(q?) in a patient with primary amenorrhea. Case report. University faculty of medicine and hospital. A 16-year-old girl with primary amenorrhea. Microdissection of chromosomes labeled by fluorescence in situ hybridization and by reverse painting. Use of commercial whole-chromosome painting probes for the detection of the aberrant chromosomes 4, 8, and X. Fluorescence probes of the isolated derivative chromosomes are self-generated. The use of whole-chromosome painting probes allowed reliable identification of all chromosomes involved in the complex chromosome rearrangements. The DNA of those chromosomes was amplified and fluorescence labeled by polymerase chain reaction using degenerated oligonucleotide primers. These probes revealed breakpoints of the complex chromosome rearrangement by hybridization on normal and original chromosomes in 4q31.1, 8q24.1, Xp22.1, Xp11.4, and Xq24. We report on an Indian patient who has premature ovarian failure with primary amenorrhea as well as a hormone level increased for LH and FSH but decreased for TSH. She has a balanced complex translocation with three breakpoints in the X chromosome that were located by fluorescence in situ hybridization by chromosome microdissection, but no breakpoints localized in the critical regions for premature ovarian failure on the X chromosome. The breakpoint in Xq24 may be associated with the amenorrhea.