A new strategy for the detection of subtelomeric rearrangements

We present a new strategy for the detection of subtelomeric rearrangements. This approach is based on two hybridizations with different probe sets. The first set consists of microdissected subtelomeric probes (each 5-10 megabases in size) labeled combinatorially employing 7 different fluorochromes. With this set, subtelomeric interchromosomal exchanges can be detected in a 24-color experiment. The second set comprises a second generation of subtelomeric PAC-, P1- and BAC-clones. Probes for p- and q-arms are labeled with two different colors. This second set detects small deletions; in addition it provides regional information, so that translocated material identified by the first probe set can be assigned to the p- or q-arm of a chromosome. The test has been evaluated in a blind study on a series of subtle translocations and deletions.     

The use of repetitive DNA in cytogenetic studies of plant sex chromosomes

The structure of sex chromosomes in plants was analyzed by fluorescent in situ hybridization (FISH) with repetitive DNAs. FISH probes were successfully obtained from DNA libraries that were amplified from microdissected sex chromosomes. Some probes hybridized to the subtelomeric regions, where many kinds of repetitive DNAs are located with intrachromosomal similarity of their repeat units rather than interchromosomal similarity. For example, FISH with the subtelomeric repetitive sequence can easily show the location of the pseudoautosomal region (PAR) on the X chromosome of Silene latifolia. The other probes were localized on the interstitial region of the sex chromosomes. The interstitial region contains chloroplast DNAs or neighboring sequences of the internal telomeres, suggesting insertion or translocation occurred during differentiation of the sex chromosome. These data are very informative for understanding the structure of the plant sex chromosomes and their evolutionary process.     

Risk assessment and segregation analysis in a pericentric inversion inv6p23q25 carrier using FISH on decondensed sperm nuclei

Fluorescent in situ hybridization (FISH) in decondensed sperm nuclei has been used to determine the percentage of normal/balanced or unbalanced spermatozoa produced by an inv(6)(p23q25) carrier, and the possible interchromosomal effect (ICE) of the reorganized chromosomes on other chromosome pairs. A dual color FISH with specific subtelomeric probes for the 6p and 6q regions was performed to determine the segregation pattern of the inverted chromosome. ICE on chromosomes 18, X and Y was assessed using a triple color FISH assay. In the segregation analysis 10,049 spermatozoa were analyzed, and only 45.7% of them were normal/balanced. The high number of unbalanced gametes in our carrier could be the consequence of the large size of the inverted segment. This situation could facilitate the formation of an inversion loop, where formation of an odd number of chiasmata (usually one) result in the production of 50% normal and 50% unbalanced sperm. Furthermore, an increase in the disomy rate for chromosome 6 was also observed. In the screening for ICE, 10,007 spermatozoa were analyzed. The disomy rate for the sex chromosomes and chromosome 18 were not significantly different from those found in our controls, suggesting no evidence of interchromosomal effects in this patient. The use of FISH in decondensed sperm nuclei has proved once more to be an accurate approach to determine the chromosome anomalies in sperm and could help to better establish a reproductive prognosis.     

Clinical and cytogenetic manifestations of subtelomeric aberrations: Report of six cases

BACKGROUND: Fluorescent subtelomeric probes for the 41 different subtelomeric regions (the p arms of the acrocentric chromosomes were excluded) have been developed over the last 10 years. These probes can detect deletions, duplications, and translocations in the gene-rich subtelomeric regions of human chromosomes, regions where crossing over frequently occurs and where a high number of abnormalities have been found. Recently, commercially produced probes have become available, which has led to the detection of subtelomeric abnormalities in 7.4% of patients with moderate to severe mental retardation (Knight et al., 1999). CASES: We evaluated 43 dysmorphic children with developmental delay and/or mental retardation of unknown etiology and/or autism who were previously assessed for chromosome abnormalities, metabolic disorders, or recognizable dysmorphic syndromes, all of which were ruled out. Of the 43 children tested, 6 (14%) were found to have subtelomeric aberrations. CONCLUSIONS: We recommend that patients with dysmorphic features and mental retardation of unknown etiology who also have a normal standard chromosome analysis should have subtelomeric FISH testing performed earlier in their clinical workup.     

Analysis of chromosome segregation during preimplantation genetic diagnosis in both male and female translocation heterozygotes

Individuals carrying translocations suffer from reduced fertility or spontaneous abortions and seek help in form of assisted reproductive technology (ART) and preimplantation genetic diagnosis (PGD). While most translocations are relatively easy to detect in metaphase cells, the majority of embryonic cells biopsied in the course of in vitro fertilization (IVF) procedures are in interphase. These nuclei are thus unsuitable for analysis by chromosome banding or painting using fluorescence in situ hybridization (FISH). Thus several methods have been devised to detect translocation imbalance through FISH in single cells for purpose of PGD, among them polar body chromosome painting, interphase FISH with combination of subtelomeric and centromeric probes, breakpoint spanning probes, and cell conversion. Results with PGD indicate a significant decrease in spontaneous abortions, from 81% before PGD to 13% after PGD. They also indicate very high rates of chromosome abnormalities in embryos from translocation carriers, 72% for Robertsonian translocations and 82% for reciprocal translocations. Sperm analysis was found to be a good predictor of IVF and PGD outcome, with samples with more than 60% abnormal forms indicating poor prognosis. Similarly, the predictability from first PGD cycle results for future cycles was 90%. In summary, PGD can help translocation carriers to achieve viable pregnancies, but the success of the process is conversely related to the baseline of unbalanced gametes.     

Cryptic terminal chromosome rearrangements in colorectal carcinoma cell lines detected by subtelomeric FISH analysis

Epithelial tumour karyotypes are often difficult to study by standard cytogenetic methods because of poor chromosome preparation quality and the high complexity of their genomic rearrangements. Subtelomeric fluorescence in situ hybridisation (FISH) has proved to be a useful method for detecting cryptic constitutional chromosomal rearrangements but little is known about its usefulness for tumour cytogenetic analysis. Using a combination of chromosome banding, multicolour karyotyping and subtelomeric FISH, five colorectal cancer cell lines were characterised. The resulting data were compared to results from previous studies by comparative genomic hybridisation and spectral karyotyping or multicolour FISH. Subtelomeric FISH made it possible to resolve several highly complex chromosome rearrangements, many of which had not been detected or were incompletely characterised by the other methods. In particular, previously undetected terminal imbalances were found in the two cell lines not showing microsatellite instability.     

Cryptic Subtelomeric Rearrangements and X Chromosome Mosaicism: A Study of 565 Apparently Normal Individuals with Fluorescent In Situ Hybridization

Five percent of patients with unexplained mental retardation have been attributed to cryptic unbalanced subtelomeric rearrangements. Half of these affected individuals have inherited the rearrangement from a parent who is a carrier for a balanced translocation. However, the frequency of carriers for cryptic balanced translocations is unknown. To determine this frequency, 565 phenotypically normal unrelated individuals were examined for balanced subtelomeric rearrangements using Fluorescent In Situ hybridization (FISH) probes for all subtelomere regions. While no balanced subtelomeric rearrangements were identified, three females in this study were determined to be mosaic for the X chromosome. Mosaicism for XXX cell lines were observed in the lymphocyte cultures of 3 in 379 women (0.8%), which is a higher frequency than the 1 in 1000 (0.1%) reported for sex chromosome aneuploidies. Our findings suggest that numerical abnormalities of the X chromosome are more common in females than previously reported. Based on a review of the literature, the incidence of cryptic translocation carriers is estimated to be approximately 1/8,000, more than ten-fold higher than the frequency of visible reciprocal translocations.     

FISH studies on the telomeric regions of the T-cell acute lymphoblastic leukemia cell line CCRF-CEM

So far, the problem of an influence of translocations on the telomeres of the involved chromosomes has not been addressed yet in human cells. Therefore, the telomeres of a karyotypically rather well characterized T-cell acute lymphoblastic leukemia (T-ALL) cell line (CCRF-CEM) with several marker chromosomes were examined using peptide nucleic acid (PNA) telomere FISH probes to compare the telomere length of these markers with that of the chromosome arms of their origin. In addition, chromosome libraries, centromeric probes, and subtelomeric DNA probes were used to further define the marker chromosomes. Two markers could be newly defined and a concise karyotype of the cell line could be obtained by these detailed examinations: 42-47,X,-X,del(5) (q35?),t(5;15)(q14;q13.2),t(8;9)(p11;p24),del(9)(:p13-->qter)/inv(9)(pter-->p12::q21-->p12::q21-->qter),+13,+20,+der(22)(p+ [HSR?])[cp]. The relative telomere length of all chromosomes showed considerable interchromosomal, intercellular, and inter-passage variation. However, it could be shown, that in four different passages of the examined cell line the observed differences between relative telomere lengths of the markers and the chromosomes of their origin, with two exceptions (short arms of del/inv9 and der22), were not significant. On the other hand, because of its mentioned variability, telomere length alone is not sufficient to reliably define the derivation of markers.     

An efficient screening for terminal deletions and translocations of barley chromosomes added to common wheat

As a prerequisite to determine physical gene distances in barley chromosomes by deletion mapping, a reliable, fast and inexpensive approach was developed to detect terminal deletions and translocations in individual barley chromosomes added to the chromosome complement of common wheat. A refined fluorescence in situ hybridization (FISH) technique subsequent to N-banding made it possible to detect subtelomeric repeat sequences (HvT01) on all 14 chromosome arms of barley. Some chromosome arms could be distinguished individually based on the number of FISH signals or the intensity of terminal FISH signals. This allowed the detection and selection of deletions and translocations of barley chromosomes (exemplified by 7H and 4HL), which occurred in the progeny of the wheat lines containing a pair of individual barley chromosomes (or telosomes) and a single so-called gametocidal chromosome (2C) of Aegilops cylindrica. This chromosome is known to cause chromosomal breakage in the gametes in which it is absent. Terminal deletions and translocations in barley chromosomes were easily recognized in metaphase and even in interphase nuclei by a decrease in the number of FISH signals specific to the subtelomeric repeat. These aberrations were verified by genomic in situ hybridization. The same approach can be applied to select deletions and translocations of other barley chromosomes in wheat lines that are monosomic for the Ae. cylindrica chromosome 2C.     

Six cases of cryptic subtelomeric translocations in four families: the use of subtelomeric FISH probes as a diagnostic tool

Finding the diagnosis in children with mental retardation and a normal karyotype, whether or not associated with dysmorphic features, is important for defining an eventual syndrome and for genetic counselling of the families. Telomeric re-arrangements may be a common and underestimated-to-date cause of non-syndromic mental retardation. Using a FISH-based approach combining subtelomeric probes, we report the detection of 4 cases of cryptic translocations t(2;10)(p25.3;q26.3), t(4;17)(p16.2;q25), t(4;20)(p16.2;q13) and t(5;7)(p15.3;q36) associated with MR and dysmorphic features. We discuss the usefulness of subtelomeric FISH in children with unexplained delayed psychomotor development, when the genetic cause remains unknown and the karyotype is normal.     

Subtelomeric chromosomal rearrangements detected in patients with idiopathic mental retardation and dysmorphic features

Subtelomeric chromosomal rearrangements detected in patients with idiopathic mental retardation and dysmorphic features: Cryptic aberrations involving the subtelomeric regions of chromosomes are thought to be responsible for idiopathic mental retardation (MR) and multiple congenital anomalies, although the exact incidence of these aberrations is still unclear. With the advent of chromosome-specific telomeric Fluorescence In Situ Hybridization (FISH) probes, it is now possible to identify submicroscopic rearrangements of distal ends of the chromosomes that can not be detected by conventional cytogenetic methods. In this study, cryptic subtelomeric chromosomal aberrations were detected in two of ten patients with idiopathic MR and dysmorphic features by using FISH probes of subtelomeric regions of all chromosome arms. A cryptic unbalanced de novo translocation was detected between the subtelomeric regions of the chromosome 10p and 18p in a patient with severe mental retardation, sensorineuronal deafness and several dysmorphic features. In the other patient, with mild mental retardation and dysmorphic features, a de novo subtelomeric deletion of chromosome 2q was found. In conclusion, in both familial and sporadic cases with idiopathic MR and dysmorphic features, the detection of chromosomal aberrations including subtelomeric rearrangements is of great importance in offering genetic counseling and prenatal diagnosis.     

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed ^{1, 2}. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope ^{3, 4}. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies ^{5, 6}. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes ^7-10.The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (http://www.spectral-imaging.com/).SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia ^11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models ¹⁴. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).     

Isolation of the human chromosome 22q telomere and its application to detection of cryptic chromosomal abnormalities

A number of human telomeres have been successfully cloned using a modified yeast artificial chromosome (YAC) vector (half-YAC) cloning strategy, but to date, human chromosome 22q has not been identified by this approach. We used an alternative approach of genomic walking, starting from a subtelomeric sequence, TelBam3.4, present on a number of human chromosomes including 22q. This approach was successful in the development of a cosmid contig representing the terminal 140 kb of human chromosome 22q, providing telomeric closure of the genetic and physical maps for 22q. The most distal region of the contig contains subtelomeric repeats which crosshybridize to a number of chromosomes, while the proximal sequences are unique for 22q. The unique sequence cosmid was used as a 22qter-specific probe for fluorescence in situ hybridization (FISH) analysis, which confirmed that this cosmid was distal to the most telomeric marker previously available for chromosome 22. In addition, this cosmid was used to document a 22q terminal deletion that was not detectable by conventional cytogenetic analysis. Unique telomere-specific FISH probes such as this one will have significant diagnostic value in the detection of cryptic deletions and translocations in patients with unexplained mental retardation and other patient populations. Received: 21 November 1995     

Identification of de novo chromosomal markers and derivatives by spectral karyotyping

Despite major advances in molecular cytogenetics during the past decade and the important diagnostic role that fluorescence in situ hybridization (FISH) plays in the characterization of chromosomal abnormalities, the usefulness of this technique remains limited by the number of spectrally distinguishable fluorochromes or fluorochrome combinations. Overcoming this major limitation would allow one to use FISH to screen the whole human genome for chromosomal aberrations which, until recently, was possible only through conventional karyotyping. A recently described molecular cytogenetics technology, 24-color FISH using spectral karyotyping (SKY), permits the simultaneous visualization of all human chromosomes in 24 different colors. Most chromosomal aberrations detected during cytogenetic evaluation can be resolved using routine cytogenetic techniques alone or in combination with single- or dual-color FISH. However, some cases remain unresolved, in particular de novo supernumerary marker chromosomes and de novo unbalanced structural rearrangements. These findings cause particular diagnostic and counseling concerns when detected during prenatal diagnosis. The purpose of this report is to demonstrate the application of SKY in the characterization of these de novo structural chromosomal abnormalities. Eight cases are described in this report. SKY has considerable diagnostic applications in prenatal diagnosis because of its reliability and speed. The identification of the chromosomal origin of markers and unbalanced translocations provides the patient, physician, and genetic counselor with better predictive information on the phenotype of the carrier. Received: 2 June 1998 / Accepted: 16 June 1998     

Breakpoint mapping and haplotype analysis of three reciprocal translocations identify a novel recurrent translocation in two unrelated families: t(4;11)(p16.2;p15.4)

The majority of constitutional reciprocal translocations appear to be unique rearrangements arising from independent events. However, a small number of translocations are recurrent, most significantly the t(11;22)(q23;q11). Among large series of translocations there may be multiple independently ascertained cases with the same cytogenetic breakpoints. Some of these could represent additional recurrent rearrangements, alternatively they could be identical by descent (IBD) or have subtly different breakpoints when examined under higher resolution. We have used molecular breakpoint mapping and haplotyping to determine the origin of three pairs of reciprocal constitutional translocations, each with the same cytogenetic breakpoints. FISH mapping showed one pair to have different breakpoints and thus to be distinct rearrangements. Another pair of translocations were IBD with identical breakpoint intervals and highly conserved haplotypes on the derived chromosomes. The third pair, t(4;11)(p16.2;p15.4), had the same breakpoint intervals by aCGH and fosmid mapping but had very different haplotypes, therefore they represent a novel recurrent translocation. Unlike the t(11;22)(q23;q11), the formation of the t(4;11)(p16.2;p15.4) may have involved segmental duplications and sequence homology at the breakpoints. Additional examples of recurrent translocations could be identified if the resources were available to study more translocations using the approaches described here. However, like the t(4;11)(p16.2;p15.4), such translocations are likely to be rare with the t(11;22) remaining the only common recurrent constitutional reciprocal translocation.     

Spectral karyotyping and fluorescence in situ hybridization analysis of de novo partial trisomy 7p (7p21.2-->pter) and partial monosomy 12q (12q24.33-->qter)

An 8-year-old boy presenting with hypotonia, moderate mental retardation, developmental delay, and psychomotor retardation is reported. Magnetic resonance imaging of the brain at age 3 years revealed a Dandy-Walker variant. Cytogenetic analysis of the peripheral blood revealed a derivative chromosome 12 with unknown additional material attached to the distal region of the long arm of chromosome 12. The parental karyotypes were normal. Spectral karyotyping (SKY) using the 24-color SKY probes and fluorescence in situ hybridization (FISH) using the specific 7p, 7q, 12p, and 12q telomeric probes confirmed a duplication of distal 7p and a deletion of terminal 12q. The karyotype of the proband was designated as 46,XY.ish der(12)t(7;12) (p21.2;q24. 33)(SKY+, 7pTEL+, 12qTEL-). The present case provides evidence for the association of partial trisomy 7p (7p21.2-->pter) and partial monosomy 12q (12q24.33-->qter) with a cerebellar malformation and the usefulness of SKY and FISH in the identification of a de novo aberrant chromosome resulting from an unbalanced translocation.     

SKY and FISH analysis of radiation-induced chromosome aberrations: a comparison of whole and partial genome analysis

For a retrospective dose estimation of human exposure to ionising radiation, a partial genome analysis is routinely used to quantify radiation-induced chromosome aberrations. For this purpose, fluorescence in situ hybridisation (FISH) with whole chromosome painting probes for selected chromosomes is usually applied covering about 20% of the whole genome. Since genome-wide screening techniques like spectral karyotyping (SKY) and multiplex FISH (mFISH) have been developed the detection of radiation-induced aberrations within the whole genome has now become feasible. To determine the correspondence between partial and whole genome analysis of radiation-induced chromosome aberrations, they were measured comprehensively in this study using in vitro irradiated blood samples from three donors. We were able to demonstrate that comparable results can be detected with both approaches. However, complex aberrations might be misinterpreted by partial genome analysis. We therefore conclude that whole genome analysis by SKY is useful especially in the high dose range to correct aberration data for complex exchange aberrations.     

Dysmorphology and mental retardation: molecular cytogenetic studies in dysmorphic mentally retarded patients

In an institutionalised population of 471 mentally retarded adult residents (436 males and 35 females), 18 patients (16 males and 2 females) with dysmorphic features were selected to perform FISH studies by using subtelomeric probes to discover cryptic terminal deletions or duplications, undetectable with standard banding techniques. In the 13 investigated patients, no abnormalities were found with a selected battery of subtelomeric probes. The results of cryptic chromosomal rearrangement studies are variable but the frequency of positive diagnostic findings seems to be lower than previously expected.     

Development of arm-specific and subtelomeric region-specific painting probes for Chinese hamster chromosomes and their utility in chromosome identification of Chinese hamster cell lines

Arm-specific and subtelomeric region-specific painting probes for Chinese hamster chromosomes have been generated by microdissection and use of the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). Fluorescence in situ hybridization (FISH) analyses using these probes demonstrated their specificity. These probes painted every chromosome arm and a total of 15 subtelomeric regions, namely, both ends of chromosomes 1, 2, 3, 4, and 8 and one end of chromosome arms 5q, 6q, 7q, 9p, and Xp. Many cryptic chromosomal rearrangements in the CHO-9 and V79 cell lines that were not detectable with whole chromosome paints could be recognized when these newly developed probes were used.     

Assessing chromosomal abnormalities in two-cell bovine in vitro-fertilized embryos by using fluorescent in situ hybridization with three different cloned probes

The aim of this study was to assess the efficiency of fluorescent in situ hybridization (FISH) for detecting chromosomal abnormalities in in vitro-fertilized (IVF) bovine embryos as early as the 2-cell stage. Three different cloned probes were used, two derived from a unique sequence specific to the subtelomeric (D1S48) or subcentromeric regions (19C10) of chromosome 1 and the third (H1A clone) derived from a repetitive sequence that hybridizes to the subcentromeric regions of three other chromosomes (14, 20, 25). Our results show that the incidence of chromosomal abnormalities in 2-cell bovine IVF embryos varied from 28% to 44% according to the probes used for the analysis. Whereas the efficiency of FISH was high with somatic nuclei, it appeared to be highly variable with the 2-cell embryos. FISH efficiency depended firstly on the probe sequence (repetitive or unique sequence), secondly on the chromosomal target region (centromeric or telomeric regions), and thirdly on the embryo cell cycle phase. With a unique sequence probe (19C10) specific to the subcentromeric regions, FISH efficiency was better on nuclei in the S-phase cycle than on those in the G-phase. In S-phase 2-cell embryos, the overall incidence of chromosomal abnormalities was more accurately assessed. It reached 13% and was represented by 1n/2n mixoploidies.